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<pubnumber>842B94003</pubnumber>
<title>CWA Section 403:  Procedural and Monitoring Guidance</title>
<pages>352</pages>
<pubyear>1994</pubyear>
<provider>NEPIS</provider>
<access>online</access>
<operator>BO</operator>
<scandate>04/15/97</scandate>
<origin>hardcopy</origin>
<type>single page tiff</type>
<keyword>monitoring marine methods discharge water environmental usepa sampling species section fish sediment biological data sample samples analysis quality considerations benthic</keyword>
<author>  Tetra Tech, Inc., Arlington, VA.;Environmental Protection Agency, Washington, DC. Office of Wetlands, Oceans and Watersheds. United States. Environmental Protection Agency. Office of Water. Office of Wetlands, Oceans and Watersheds. ; Tetra Tech, Inc.</author>
<publisher>U.S. Environmental Protection Agency, Office of Water ,</publisher>
<subject>Water pollution monitoring; Ocean waste disposal; Aquatic ecology; Permits; Water pollution control; Marine biology; Biological accumulation; Environmental impacts; Environmental protection; Estuaries; Benthos; Water chemistry; Clean Water Act; Water pollution standards; Pollution regulations; Discharge(Water); Section 403; NPDES(National Pollutant Discharge Elimination System) Marine pollution--Sampling--Handbooks, manuals, etc ; Water--Pollution--Handbooks, manuals, etc</subject>
<abstract>The purpose of the document is to provide the Regions and NPDES-authorized States with a framework for the decision-making process to be followed in making a section 403 determination and to provide them with guidance for identifying the type and level of monitoring that should be required as part of a permit issued under the no irreparable harm provisions of section 403. Chapter 2 of the document presents an explanation of, and procedural guidance for, the overall process to be followed when issuing an NPDES permit in compliance with section 403 of the Clean Water Act. Chapter 3 discusses options for monitoring under the basis of no irreparable harm. Chapter 4 presents a summary of monitoring methods with potential applications to 403 discharges.  </abstract>

United States
Environmental Protection
Agency
Office of Water
(4504F)
EPA 842-B-94-003
March 1994
CWA Section 403:
Procedural and Monitoring
Guidance
                         Recycled/Recyclable
                         Printed with Soy/Canola Ink on paper that
                         contains at least 50% post-consumer recycled fiber
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CWA SECTION 403: PROCEDURAL AND
        MONITORING GUIDANCE
                 March 1994
      United States Environmental Protection Agency
      Office of Wetlands, Oceans and Watersheds
        Oceans and Coastal Protection Division
                Washington, DC
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                              DISCLAIMER
This document has been reviewed in accordance with U.S. Environmental Protection
Agency policy and approved for publication.  Mention of trade names or commercial
products does not constitute endorsement or recommendation for use.
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                             CONTENTS
                                                                 Page

TABLES	....	;.	.v:	....... ix

FIGURES.	xi

ACKNOWLEDGMENTS	xiii

ABBREVIATIONS	xv

GLOSSARY	xvii

EXECUTIVE SUMMARY	xxv

1.    INTRODUCTION	 1

     1.1  THE OCEAN DISCHARGE CRITERIA 	 2
     1.2  PURPOSE OF: THIS DOCUMENT	 3
     1.3  DOCUMENT FORMAT	 3

2.    SECTION 403 PROCEDURE	 5

     2.1  BACKGROUND	 5
          2.1.1   The Role of the Ocean Discharge Criteria
                 in NPDES Permit Issuance 	5
          2.1.2  Applicability of Section 403	5
          2.1.3  Individual or General Permit	7

     2.2  GENERAL PROCEDURE 	 8
          2.2.1   Request for Issuance/Reissuance of a
                 Section 402 Permit 	 8
          2.2.2  Determination of Information Requirements 	 10
          2.2.3  Determination of No Unreasonable Degradation	 11
          2.2.4  Decision to Issue/Reissue or Deny a Permit	 14
          .2.2.5  Insufficient Information	 14
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                          CONTENTS (continued)

     OPTIONS FOR MONITORING UNDER THE BASIS OF "NO
     IRREPARABLE HARM" 	
17
     3.1   BACKGROUND	 17

     3.2   CRITERIA FOR EVALUATING THE POTENTIAL
           FOR ENVIRONMENTAL IMPACT  	 18
           3.2.1  Major/Minor Discharges	... 18
           3.2.2  Discharges to Stressed Waters  	20
           3.2.3  Discharges to Sensitive Biological Areas	21
           3.2.4  Presence of Other Discharges in the Area 	25

     3.3   MONITORING REQUIREMENTS BASED ON PERCEIVED
           POTENTIAL ENVIRONMENTAL THREAT	 25
           3.3.1  Minimal Potential Threat 	25
           3.3.2  Moderate Potential Threat	27
           3.3.3  High Potential Threat  	29

     3.4   SUMMARY 	30

4.    SUMMARY OF MONITORING METHODS	 31

     4.1   PHYSICAL CHARACTERISTICS	 40
           4.1.1  Rationale	 40
           4.1.2  Monitoring Design Considerations	40
           4.1.3  Analytical Methods Considerations	 42
           4.1.4  QA/QC Considerations	48
           4.1.5  Statistical Design Considerations 	51
           4.1.6  Use of Data 	52
           4.1.7  Summary and Recommendations	52

     4.2   WATER CHEMISTRY 	 56
           4.2.1  Rationale	56
           4.2.2  Monitoring Design Considerations	56
           4.2.3  Analytical Methods Considerations	58
           4.2.4  QA/QC Considerations	63
           4.2.5  Statistical Design Considerations	66
           4.2.6  Use of Data 	67
           4.2.7  Summary and Recommendations	68
Iv
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                                         Section 403 Procedural and Monitoring Guidance
                           CONTENTS (continued)

4.3   SEDIMENT CHEMISTRY	 71
            4.3.1  Rationale  	 71
            4.3.2  Monitoring Design Considerations	71
            4.3.3  Analytical Methods Considerations	79
            4.3.4  QA/QC Considerations	83
            4.3.5  Statistical Design Considerations 	88
            4.3.6  Use of Data 	 89
            4.3.7  Summary and Recommendations	90

      4.4    SEDIMENT GRAIN SIZE	 93
            4.4.1  Rationale  	93
            4.4.2  Monitoring Design Considerations	93
            4.4.3  Analytical Methods Considerations	98
            4.4.4  QA/QC Considerations	 100
            4.4.5  Statistical Design Considerations 	 101
            4.4.6  Use of Data 	 102
            4.4.7  Summary and Recommendations	 102

      4.5    BENTHIC COMMUNITY STRUCTURE 	 105
            4.5.1  Rationale  	 105
            4.5.2  Monitoring Design Considerations	 106
            4.5.3  Analytical Methods Considerations	 114
            4.5.4  QA/QC Considerations	 121
            4.5.5  Statistical Design Considerations 	 123
            4.5.6  Use of Data 	 124
            4.5.7  Summary and Recommendations	 124

      4.6    FISH AND SHELLFISH PATHOBIOLOGY  	 127
            4.6.1  Rationale	 127
            4.6.2  Monitoring Design Considerations	 129
            4.6.3  Analytical Methods Considerations	 130
            4.6.4  QA/QC Considerations	 135
            4.6.5  Statistical Design Considerations 	 135
            4.6.6  Use of Data 	 136
            4.6.7  Summary and Recommendations	 139

      4.7    FISH POPULATIONS	 143
            4.7.1  Rationale  	 143
            4.7.2  Monitoring Design Considerations	 144
            4.7.3  Analytical Methods Considerations	 146
            4.7.4  QA/QC Considerations	 150
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                          CONTENTS (continued)

           4.7.5   Statistical Design Considerations	 150
           4.7.6   Use of Data 	 151
           4.7.7   Summary and Recommendations	 152

     4.8   PLANKTON: BIOMASS, PRODUCTIVITY, AND COMMUNITY
           STRUCTURE/FUNCTION	 154
           4.8.1   Rationale 	 154
           4.8.2   Monitoring Design Considerations	 155
           4.8.3   Analytical Methods Considerations	 157
           4.8.4   QA/QC Considerations	 160
           4.8.5   Statistical Design Considerations	 161
           4.8.6   Use of Data	 161
           4.8.7   Summary and Recommendations	 162

     4.9   HABITAT IDENTIFICATION METHODS	 165
           4.9.1   Rationale 	 165
           4.9.2   Monitoring Design Considerations	 166
           4.9.3   Analytical Methods Considerations	 167
           4.9.4   QA/QC Considerations	 171
           4.9.5   Statistical Design Considerations  	 171
           4.9.6   Use of Data 	 172
           4.9.7   Summary and Recommendations	 172

     4.10  BIOACCUMULATION	 175
           4.10.1  Rationale 	 175
           4.10.2  Monitoring Design Considerations	 176
           4.10.3  Analytical Methods Considerations	 184
           4.10.4  QA/QC Considerations	 187
           4.10.5  Statistical Design Considerations 	 188
           4.10.6  Use of Data 	 189
           4.10.7  Summary and Recommendations	 189

     4.11  PATHOGENS 	 192
           4.11.1  Rationale	 192
           4.11.2  Monitoring Design Considerations	 193
           4.11.3  Analytical Methods Considerations	 194
           4.11.4  QA/QC Considerations	200
           4.11.5  Statistical Design Considerations 	201
           4.11.6  Use of Data 	201
           4.11.7  Summary and Recommendations	201
vi
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                                       Section 403 Procedural and Monitoring Guidance
                          CONTENTS (continued)

      4.12  EFFLUENT CHARACTERIZATION	 204
           4.12.1  Rationale	204
           4.12.2  Monitoring Design Considerations	;.	206
           4.12.3  Analytical Methods Considerations	206
           4.12.4  QA/QC Considerations	208
           4.12.5  Statistical Design Considerations	209
           4.12.6  Use of Data	209
           4.12.7  Summary and Recommendations	210

      4.13  MESOCOSMS AND MICROCOSMS	:	 212
           4.13.1  Rationale	212
           4.13.2  Monitoring Design Considerations	213
           4.13.3  Analytical Methods Considerations	213
           4.13.4  QA/QC Considerations	214
           4.13.5  Statistical Design Considerations	215
           4.13.6  Use of Data 	215
           4.13.7  Summary and Recommendations	216
5.
LITERATURE CITED	 219
APPENDIX A: MONITORING METHODS REFERENCES	A-1

APPENDIX B: OCEAN DISCHARGE CRITERIA	B-1
                                                                       vii
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                                 TABLES
 Table
Page
2-1.   Ocean Discharge Guidelines	6

3-1.   Information Sources on Sensitive Marine and Coastal Environments:
      NOAA, FWS, and MMS	23

3-2.   Characterization of Section 403 Discharges Based on the Potential for
      Causing Environmental Impacts	26

4-1.   Sampling Method Categories	32

4-2.   Matrix Illustrating Relationship of Method Types to the
      Section 403 Ocean Discharge Guidelines 	  33

4-3.   Key Topics to Be Addressed in Marine Monitoring
      Quality Assurance Plans 	35

4-4.   QC Sample Types  	36

4-5.   List of Methods and Equipment	43

4-6.   Recommended Sample Preservation and Storage Requirements 	49

4-7.   Recommended Analytical Methods	49

4-8.   List of Analytical Techniques	60

4-9.   USEPA Organic Contamination Detection Techniques 	61

4-10.  Sample Preservation and Storage Parameters	64

4-11.  Definitions for Selected Limits of Detection	66

4-12.  Sampling Containers, Preservation Requirements, and Holding
      Times for Sediment Samples 	73

4-13.  Summary of Bottom Sampling Equipment 	  74
                                                                           IX
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                              TABLES (continued)

  Table                                                                  page


 4-14.  List of Analytical Techniques	 80

 4-15.  USEPA Organic Contaminant Detection Techniques	81

 4-16.  Summary of Quality Control Analyses	86

 4-17.  Summary of Warning and Control Limits for Quality Control Sample 	87

 4-18.  Summary of Bottom Sampling Equipment	94

 4-19.  Sediment Grain Size: Withdrawal Times for Pipet Analysis as a
       Function of Particle Size and Water Temperature 	100

 4-20.  Biological Indices	115

 4-21.  List of Pathobiological Terms	128

 4-22.  Biological Indices	 147

 4-23.  List of Analytical Methods	 167

 4-24.  List of Terms	„	 175

 4-25.  Highest-Ranking Candidate Fish for Use as Bioaccumulation
       Monitoring Species	 178

 4-26.  Recommended Large Macroinvertebrate Species for
       Bioaccumulation Monitoring 	180

 4-27.  List of Analytical Techniques	185

 4-28.  Microorganisms Responsible for Causing Adverse
       Human Health Efffects	 195

4-29. Laboratory Procedures for Bacterial Indicators	198

4-30. List of Terms Used with Effluent Characterization	205
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                                FIGURES
2-1.   Applicability of Section 403 Requirements	7

2-2.   Section 403 Decision Process	9

3-1.   Elements of Designing and Implementing a Monitoring Program	19

4-1.   Examples of Acceptable and Unacceptable Samples	84

4-2.   Generalized SAB Diagram of Changes Along a Gradient
      of Organic Enrichment	117

4-3.   Diagram of Changes in Fauna and Sediment Structure Along a
      Gradient of Organic Enrichment	119

4-4.   Examples of Acceptable and Unacceptable Samples	122
                                                                          XI
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                         ACKNOWLEDGMENTS
This work was completed through the efforts of Brigitte Farren, National 403 Program
Coordinator; Deborah Lebow, Chief, OCPD's Marine Discharge Section; Barry Burgan,
EPA's Assessment and Watershed Protection Division; and the 403 Regional Ocean
Discharge Coordinators.  For their help in the development and review of this document,
special thanks are extended to staff from EPA's Office of Research and Development,
Office  of Science and  Technology, and  Office  of  Wastewater  Enforcement and
Compliance.   This document was  prepared by Tetra Tech, Inc., under EPA Contract
Number 68-C7-0008.
                                                                         XIII
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                           ABBREVIATIONS
BOD    -   Biochemical oxygen demand
COE    -   United States Army Corps of Engineers
CWA    -   Clean Water Act
CZMA   -   Coastal Zone Management Act
CZMP   -   Coastal Zone Management Plan
EIS     -   Environmental Impact Statement
EPA    -   United States Environmental Protection Agency
MGD    -   Million gallons per day
MMS    -   Minerals Management Service
NESDIS -   National Environmental Satellite, Data and Information Service
NMFS   -   National Marine Fisheries Service
NPDES  -   National Pollutant Discharge Elimination System
NRC    -   National Research Council
NOAA   -   National Oceanic and Atmospheric Administration
NWI    -   National Wetlands Inventory
ODCE   -   Ocean Discharge Criteria Evaluation
OWEC  -   Office of Wastewater Enforcement and Compliance
PCS    -   Permit Compliance System
POTW  -   Publicly-owned treatment works
                                                                      XV
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                               GLOSSARY
Acute - A stimulus severe enough to rapidly induce an effect; in aquatic toxicity tests,
    an  effect  observed in  96 hours or less typically  is considered acute.  When
    referring to aquatic toxicology  or human health, an acute  effect is not always
    measured in terms of lethality.

Acute-to-chronic ratio (ACR) - The  ratio of the acute toxicity of an effluent or a
    toxicant to its chronic toxicity.  ACR is used as a factor for estimating  chronip
    toxicity on the basis of acute toxicity data, or for estimating  acute toxicity on the
    basis of chronic toxicity data.

Alternatives assessment - An assessment of the relative impacts (economic, social,
    and  environmental) of  on-site  effluent discharge versus discharge to  an
    alternative site, land disposal, or another waste disposal alternative.

Ambient - Of  or relating to a condition of the environment, such as temperature or
    pressure, that surrounds a body or object.

Anadromous  fish -  Fish that spend their adult  life in  the sea but swim upriver to
    freshwater spawning grounds to reproduce.

Analyte - The substance being identified and measured in an analysis.

Anoxic - Lack of oxygen.

Anthropogenic - Caused or influenced by the activities of humans.

Baseline - Defines the landward boundary of the territorial seas.

Benthic - Of, relating to, or occurring at the bottom of a waterbody.

Benthic biota - A form of  aquatic plant or animal  life that is found on  or near the
    bottom of a body of water.

Bioaccumulation  -  Process by which a  compound  is taken up  by  an  aquatic
    organism, both from water and through food.
                                                                           XVII
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 Glossary
   Bioassay - A test used to evaluate the relative potency of a chemical or a mixture of
       chemicals  by comparing  its effect  on a  living organism with the effect of a
       standard preparation on the same type of organism.


   Biocriteria -  Narrative  expressions or numeric values of the biological characteristics
       of aquatic communities based on appropriate reference conditions.


   Biochemical oxygen  demand (BOD)  - A measure of the amount of oxygen
       consumed  in the biological processes that break down organic matter in water.
       The greater the BOD, the greater the degree of pollution.

   Biogeographical - The geographical distribution of animals and plants.

   Biomass - A quantitative  estimate  of the entire assemblage of living organisms
       considered  collectively and measured in terms of mass volume or energy in
       calories.


   Calibration - The determination of any equipment deviation from a standard source
       so as to ascertain the proper correction factors.
                      i
   Chronic - A stimulus that lingers or continues for a relatively long period of time, often
       one-tenth of the life span or more. Chronic should be considered a relative term
       depending on the life span of an organism. The measurement of a chronic effect
       can be reduced growth, reduced reproduction, etc., in addition to lethality.

   Coastal zone - Lands and waters adjacent to the coast that exert an  influence on the
       uses of  the sea and its ecology or,  inversely, whose uses and  ecology are
       affected by the sea.

   Compensation depth  - The depth at which there is just enough light to allow the
       amount of oxygen  produced by phytoplankton through photosynthesis to balance
       the amount consumed by their respiration.


   Composite sample - A single effluent sample collected over a 24-hour period, on
       which only one toxicity test is performed.


  Contiguous zone - Defined in section 502(9) of the Clean Water Act to be "the entire
       zone established or to be established by the United States under Article 24 of the
       Convention of the Territorial Sea and the Contiguous Zone."
xvlii
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                                         Section 403 Procedural and Monitoring Guidance
CTD instruments -  Sampling instruments that measure conductivity, temperature,
    and depth.

Cytogenetic - Referring to chromosomal aberrations.

Data Quality Objectives - Specific,  integrated statements and goals developed for
    each data or information collection activity to ensure that the  data  are  of the
    required quantity and quality.

Drogue - A small object attached to a surface buoy to measure currents.

Effect concentration  - A point estimate of the  toxicant concentration that would
    cause an observable adverse effect (such as death, immobilization, or serious
    incapacitation) in a given percentage of the test organisms.

Effluent - Wastewater—treated or untreated—that flows out of a  treatment plant,
    sewer, or industrial outfall.  Generally refers  to wastes discharged into surface
    waters.

Effluent limitation -  Any restriction on quantities, rates, or concentrations of chemical,
    physical, biological, and other constituents that are discharged from point sources
    into waters of the United States,  including navigable waters of the contiguous
    zone or the ocean.

Epifaunal - Living in  the surface of the sediment.

Euphotic zone - The  upper layers of a lake or ocean into which sufficient sunlight
     penetrates for photosynthesis (usually down to about 80 meters).

Fecal coliform bacteria - Bacteria found in the intestinal tracts of animals.  They are
     an indicator of possible pathogenic contamination.

Grab samples - Samples collected over a very short period of time and on a relatively
     infrequent basis.   A separate toxicity test must  be performed on  each  grab
     sample.

Habitat - The place where a population lives and its surroundings, both  living and
     nonliving.

Hard-bottom habitats - Those benthic habitats such as coral reefs,  rocky subtidal
     areas, and "live  bottom" sponge/gorgonian habitats.
                                                                             XIX
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Glossary
  Hermatypic coral - Reef-building coral.

  Hypoxic conditions - A deficiency of oxygen.

  Indicator species - A  species whose characteristics show the presence of specific
      environmental conditions.

  Indigenous species  - A species native to or occurring naturally in a particular region
      or environment.

  Industrial  source - For  the purpose of  this report, a nonmunicipal source  of
      wastewater discharges.

  Infaunal - Living in the sediment.

  Irreparable harm - Significant undesirable effects occurring after the date of permit
      issuance that will not be reversed after cessation or modification of the discharge.
      (40CFR125.121(a))

  In situ - In the normal or natural position.

  Lipids - Fats or fat-like substances that contain aliphatic hydrocarbons, are water
      insoluble, and are easily stored in the body for use as fuel.

  Lowest-Observed-Adverse-Effect Level (LOAEL) - The  lowest concentration of an
      effluent or toxicant that results in statistically significant adverse health effects as
      observed in  chronic  or subchronic human  epidemiology studies or animal
      exposure.

  Marine waters - Territorial seas, the contiguous zone, and the oceans.

  Mixing zone - An area where an effluent discharge undergoes initial dilution and is
      extended to cover the secondary  mixing in the ambient waterbody.  A mixing
      zone is an allocated impact zone where water quality criteria can be exceeded as
      long as a number of provisions are maintained.

  Municipal source - A public source of wastewater discharges.
XX
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                                        Section 403 Procedural and Monitoring Guidance
National  Pollutant  Discharge  Elimination  System (NPDES) -  The  national
    permitting program for issuing,  modifying, revoking and reissuing, terminating,
    monitoring, and  enforcing permits,  and imposing and enforcing pretreatment
    requirements,  under  sections 301, 307, 318,  402, 403, and 405 of the Clean
    Water Act.

No-Observed-Adverse-E-ffect Level (NOAEL) - A tested dose of an effluent or a
    toxicant below which no adverse biological effects are observed, as identified
    from  chronic or subchronic  human epidemiology studies  or animal  exposure
    studies.

No-Observed-Effect Concentration (NOEC) - The highest tested concentration of an
    effluent or a toxicant at which no adverse effects are observed on the aquatic test
    organisms at a specific time of observation. Determined using hypothesis testing.

Ocean  Discharge Criteria -  40 CFR Part 125, Subpart M,  which  establishes
    guidelines for issuance of an NPDES permit for the discharge of pollutants from a
    point source into the territorial seas, the contiguous zone, and the oceans.

Ocean Discharge  Guidelines - Ten narrative guidelines listed at 40 CFR 125.122 of
    the  Ocean  Discharge Criteria  regulations for determination of  unreasonable
    degradation of the marine environment.

Ocean Discharge  Factors - Seven narrative factors listed at section 403(c)(1)(A)-(G)
    of the Clean  Water  Act for  determination of the degradation  of  the marine
    environment.

Pathogens  -  Microorganisms  that  can cause disease in other organisms or in
    humans, animals, and plants.

Phytoplankton - A type  of plant plankton that is the basic source of food in many
    aquatic and marine ecosystems.

Plankton - A collective term  for the wide variety of plant and animal organisms, often
    microscopic in size, that float or drift freely in the water because they have little or
    no ability to determine their own  movement.

Pretreatment - The reduction of the amount of pollutants, the elimination of pollutants,
    or the alteration of the nature of pollutant properties in wastewater prior to or in
    lieu of discharging or otherwise introducing such pollutants into a POTW.   The
                                                                           XXI
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Glossary
      reduction  or alteration may be  obtained by  physical,  chemical, or biological
      processes, by process changes,  or by other means, except as prohibited by 40
      CFR Part 403.

  Publicly-owned treatment works (POTW) - A treatment works, as defined in section
      212(2) of the Clean  Water Act, that is owned by  a State,  municipality,  or
      intermunicipal or interstate agency.

  Pycnocline - The stratifying vertical density gradient in the water column caused by
      differences in temperature and/or salinity.

  Sediment quality criteria - Elements of sediment quality standards, expressed as
      constituent concentrations, levels, or narrative  statements representing a quality
      of sediment that supports a particular use.  Sediment quality criteria reflect the
      use  of available  scientific  data  to assess  the  likelihood  of significant
      environmental effects on benthic  organisms from chemicals in the sediment and
      to derive regulatory requirements  that will protect against these effects.

  Soft-bottom habitats - Those benthic habitats composed of a significant benthic
      biological community consisting of organisms that burrow into the substratum.

  Spatial - Of or relating to space; involving  relations or measurements that have to do
      with space.

  Stormwater discharge - Precipitation that does not infiltrate the ground or evaporate
      because of impervious land surfaces but instead flows onto adjacent land  or
      water areas and is routed  into drain/sewer systems.

  Stressed waters - Waters that have been impaired by pollutants.

  Technology-based treatment requirements - NPDES permit requirements based on
      the application of pollution treatment or control technologies including (under 40
      CFR Part  125) best practicable technology (BPT), best conventional technology
      and  secondary   treatment  for  POTWs  (BCT),  best  available technology
      economically achievable (BAT), and new source performance standards (NSPS).

  Temporal - Of or relating to time; involving relationships or measurements that have
      to do with time.
XXII
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                                          Section 403 Procedural and Monitoring Guidance
Territorial seas - Defined in section 502(8) of the Clean Water Act to be the belt of
     the seas measured from the line of ordinary low water along that portion of the
     coast which is  in direct contact with the  open sea and the line  marking the
     seaward limit of inland waters, and extending seaward a distance of 3 miles.

Toxic - Harmful to living organisms.

Toxicity characterization - A determination of the specific chemicals responsible for
     effluent toxicity.


Toxicity test - A procedure to determine the toxicity of a chemical or an effluent using
     living organisms.  A toxicity test measures the degree of effect on exposed test
     organisms of a specific chemical or effluent.

Unreasonable degradation - Significant adverse changes in  ecosystem  diversity,
     productivity, and stability of the biological community within the area  of discharge
     and surrounding biological communities; threat to human health through direct
     exposure to pollutants  or through consumption  of exposed aquatic organisms;
     loss of aesthetic, recreational, scientific, or economic value that is unreasonable
     in relation to the benefit derived from the discharge. (40 CFR 125.121 (e))

Volatile organic compounds (VOCs) - Any organic compound that participates in
     atmospheric photochemical reactions.

Water quality-based  toxics control  -  An integrated  strategy  used in NPDES
     permitting to assess and control the discharge of  toxic pollutants to surface
     waters: the whole-effluent approach involving the use of toxicity tests to measure
     discharge toxicity and the chemical-specific approach involving the use of water
     quality criteria or State standards to limit specific toxic pollutants directly.

Water quality criteria - Elements of State water quality standards, expressed as
     constituent concentrations, levels, or narrative statements, representing a quality
     of water that supports a particular use.  When criteria are met, water quality will
     generally protect the designated use.

Water quality standards  - Provisions of State or Federal law  that  consist  of a
     designated  use  or uses for the waters of the United States  and water quality
     criteria for such waters based on such uses.  Water quality standards are to
     protect the public health or welfare, enhance the quality of water, and serve the
     purposes of the Clean Water Act.
                                                                           xxm
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Glossary
  Whole-effluent toxicity - The aggregate toxic effect of an effluent measured directly
      by a toxicity test.

  Zone of initial dilution (ZID) - The region of initial mixing surrounding or adjacent to
      the end of an outfall pipe or diffuser ports.

  Zooplankton - A type of animal plankton.
XXIV
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                          EXECUTIVE SUMMARY
THE PURPOSE AND IMPLEMENTATION OF THE 403 PROGRAM

The  Clean Water Act (CWA, or the Act), Public Law 95-217, was enacted in  1972.
Throughout the  years  it has  been periodically  amended,  with  the  most extensive
amendments being adopted in 1977 and the most recent in 1987. The Act is the single
most important and comprehensive piece of legislation dealing with the environmental
quality of the Nation's waters, covering both marine and freshwater systems.

Section  402 of  the  CWA  established  the National  Pollutant  Discharge Elimination
System  (NPDES).  This section of  the Act requires that  any direct discharger of
pollutants to the surface waters of the United States obtain an NPDES permit before the
discharge can  take place. To obtain an NPDES permit, a discharger must demonstrate
compliance with all applicable requirements of the Act. In the case of discharges to the
territorial sea,  the contiguous zone, or the ocean,1 these requirements include section
403 of the Clean Water Act.

Section  402 permits are intended to control the release of pollutants into waters of the
United States  from point source discharges such as  municipal  and industrial outfalls.
some stormwater discharges are also covered.  Dischargers are required to meet the
Act's minimum technology-based treatment requirements.  Discharges to State waters
are also required to comply with State  water quality  standards.  In addition to  these
requirements, discharges to marine waters are also subject to section 403 of the Clean
Water Act, which sets forth  criteria to prevent unreasonable degradation of the marine
environment and authorizes imposition of any additional effluent  limitations, including
zero discharge, necessary to protect the  receiving waters to attain  the objectives of the
Clean Water Act.

To implement  section 403,  EPA has  established 10 ocean discharge guidelines that,
along with other provisions of  40  CFR Part 125,  Subpart  M, provide the  basis for
determining whether a discharge will cause unreasonable degradation of the marine
environment.   Under the regulations,  if it is determined that a discharge would cause
unreasonable  degradation,  the  permit will be denied; discharges  that will  not cause
unreasonable  degradation may be permitted and the permit may include  conditions
necessary to ensure that unreasonable degradation will not occur during the time of the
permit.    These  determinations are  made  using  available  information,  including

1 For ease of reference, the waters of the territorial sea, contiguous zone, and oceans will hereinafter be referred to as "marine
 waters."
                                                                            XXV
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 Executive Summary
 information provided by the permit applicant, relevant environmental impact statements,
 section 301 (h) or other variance applications, existing technical and environmental field
 studies, and EPA industrial and municipal waste surveys.

 In  those  cases  where there  is  insufficient  information  to  determine  whether
 unreasonable degradation will occur, under 40 CFR 125.123(a) and (b), a permit will not
 be issued  unless (1) it can be determined that no irreparable harm will result from the
 discharge, (2)  there  are no  reasonable  alternatives to the  discharge,  and  (3)  the
 discharge will comply with certain permit conditions specified in the regulations. These
 conditions include bioassay-based  discharge limitations and  monitoring  to assist in
 determining whether and to what extent further limitations are necessary to ensure that
 the discharge does not cause unreasonable degradation.

 Because of the case-specific nature of permit decision making, the review and extent of
 required monitoring associated  with NPDES permits issued  under the provisions of
 section 403 are variable.  This is due to the  wide range of receiving environments and
 the variability of the discharge.  This document provides guidance on determining the
 nature and extent of monitoring studies needed to assess the impact of the discharges
 on the marine environment.

 USE OF THIS DOCUMENT

 This document is designed to provide the EPA Regions and NPDES-authorized States
 with a framework for  the decision-making process for section 403  evaluations and to
 provide guidance on the type and level of monitoring that should be required as part of
 permit issuance under the "no  irreparable harm" provisions of section 403.  (Generally,
 ambient monitoring is not required if a determination of "no unreasonable degradation"
 is made.)  The decision-making aspects of the program, such as determination of
 information   requirements  and  sufficiency  of  information,  determination   of  no
 unreasonable degradation, and the decision to issue/reissue  or deny a permit,  are
 described.  Options for monitoring under  the basis of no irreparable harm, including
 criteria  for  evaluating  perceived  potential impact  and  establishing  monitoring
 requirements to assess actual impacts, are discussed. Finally, summaries of monitoring
 methods for evaluating the following parameters are provided:

      •  Physical characteristics, such as temperature, salinity, density, depth,
         turbidity, and current velocity and direction, to characterize the water column,
         to verify hydrodynamic models, and to indicate spatial and temporal
         variations;

      •   Water chemistry to evaluate the quality of receiving waters;

      •  Sediment chemistry to determine pollutant levels in sediments; '.

      •  Sediment grain size to describe spatial and temporal changes in the benthic
         community;
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                                           Section 403 Procedural and Monitoring Quittance


      •  Benthic community structure to detect and describe spatial and temporal
         changes in community structure and function;

      •  Fish and shellfish pathobiology to provide information regarding damage or
         alteration to organ systems of fish and shellfish;

      •  Fish and shellfish populations to detect and describe spatial and temporal
         changes in the abundance, structure, and function of fish and shellfish
         communities;
      •  Plankton characteristics including biomass, productivity,  and community
         structure and function, to identify the dominant species, detect short- and
         long-term  spatial and temporal trends, and examine the  relationship between
         water quality conditions and community characteristics;

      •  Habitat identification to determine whether pollutant-related damage will
         cause long-lasting harm to sensitive marine habitats;
      •  Bioaccumulation to provide the link between pollutant exposure and effects;

      •  Pathogens to assess water conditions in the vicinity of discharges and
         surrounding areas and to assess relative pathogen contributions from
         permitted effluent discharges;
      •  Effluent characterization to predict biological impacts of an effluent prior to
         discharge; and
      •  Mesocosms and microcosms to assess ecological impacts from marine
         discharges.
Each method section contains an explanation of why the measurement of the parameter
of concern might be included as part of a 403 monitoring program, and  a discussion of
monitoring design considerations, analytical  methods, statistical design  considerations,
the use of data generated, and quality assurance/quality control considerations.
                                                                             XXVII
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                             1.  INTRODUCTION
The  Clean Water Act (CWA, or the Act),  Public Law 95-217, was enacted in  1972.
Throughout the  years  it has  been periodically  amended,  with the  most extensive
amendments being adopted in 1977 and the most recent in 1987. The  Act is the single
most important and comprehensive piece of legislation dealing with the environmental
quality of the Nation's waters, covering both  marine and freshwater systems.

Section  402 of  the  CWA  established the National  Pollutant  Discharge Elimination
System  (NPDES).  This section of the Act requires that  any direct discharger of
pollutants to the surface waters of the United States obtain an NPDES permit before the
discharge can take place. To obtain an NPDES permit, a discharger must demonstrate
compliance with all applicable requirements of the Act, including section 403 (for marine
discharges). Section 402 permits are intended to control the release of pollutants into
the navigable waters of the United States  from all point sources, including municipal,
industrial, and some stormwater discharges.

The  regulatory framework  established  under section 402  provides  a  two-pronged
approach  for   controlling   point  source   discharges.   The   first,  reliance  on
"technology-based"  standards, consists  of national minimum treatment requirements
based  on an  assessment  of  the achievability  of control technologies by individual
categories of dischargers.  The second, a "water quality-based" approach, stresses
water quality criteria, water quality standards,  and  the setting of pollutant effluent
limitations intended to maintain receiving surface water quality at a level sufficient to
protect classified designated uses of the receiving waterbody (e.g., fishable/swimmable).

Discharges  to  marine  waters  are  subject  to additional  regulatory  requirements
established under section 403 of the CWA. Section  403 applies to marine discharges
under NPDES  permits and allows for more stringent controls when necessary to protect
the environment.  It is not restricted by engineering attainability,  nor is it limited by
rigorous cost or economic restrictions when determining permit conditions. It includes
consideration of sediment as well as water column effects. It not only  protects aquatic
species but also places special emphasis  on unique, sensitive, or ecologically critical
species.  Under section 403, EPA or an NPDES-authorized State can impose discharge
limitations or other conditions needed to attain compliance with section 403.

The  Ocean Discharge  Criteria  implementing section 403  are intended to  prevent
unreasonable degradation of the marine environment and to  authorize imposition of
effluent limitations, if necessary, to achieve this goal. The  Ocean Discharge Criteria
were promulgated on October 3, 1980, 45 FR 65942 (see Appendix  B).  The Ocean
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Monitoring Options
Discharge Criteria provide flexibility for permit writers to customize permit application
requirements, effluent limitations, and reporting requirements to the specific conditions
of each individual discharger's situation in order to ensure compliance with section 403.
The criteria also ensure consistency by  imposing minimum requirements in situations
where the long-term impacts of the discharge are not fully understood.
1.1
THE OCEAN DISCHARGE CRITERIA
Section 403(c)(1) of the Clean Water Act specifies seven factors that are to be included
in guidelines for determining the potential for degradation of marine waters.  These
seven factors are the foundation for the 10 ocean discharge guidelines contained in the
EPA regulations and  discussed  in Section 2.2.3 of this document.   Under these 10
guidelines and the other provisions of the Ocean Discharge Criteria, no NPDES permit
may be issued that will cause unreasonable degradation of the  marine environment.
Prior to permit issuance; the director (defined as the EPA Regional Administrator or the
State Director where there is an  NPDES-authorized State program, or an authorized
representative)  is required to evaluate whether a proposed discharge will cause such
degradation.  To make this determination, the director considers the provisions specified
in 40 CFR 125.122(a) and (b).

In cases where sufficient information is available for the director to make a determination
whether unreasonable degradation of the marine environment will occur, the director is
governed  by 40 CFR ^25.123(3)  and (b).  Discharges that  cause unreasonable
degradation are prohibited; other  discharges may  be  permitted  under conditions
necessary to ensure that unreasonable degradation will not occur during the term of the
permit.

In cases where the director is unable to determine whether unreasonable degradation
will occur, 40 CFR 125.i23(c) applies. Under this provision no discharge will be allowed
unless the director can determine that:

      •  No irreparable harm will result from the discharge;
      •  There are no reasonable alternatives to the discharge; and
      •  The discharge will comply with certain permit conditions,  including
         bioassay-based discharge limitations and monitoring requirements. These
         conditions will assist in determining whether and to what extent further
         limitations are necessary to ensure that the discharge does not cause
         unreasonable degradaton. Pursuant to 40 CFR 125.123(d)(4), if on the basis
         of new data the director determines that continued discharges may cause
         unreasonable degradation of the marine environment, the permit must be
         modified or revoked.
The  Ocean Discharge Criteria encourage  the use of any  available information,  in
addition to  information supplied by the permit applicant, in determining the degradation
of marine  waters as  a result  of  a  discharge.  Therefore, the director may make a
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                                          Section 403 Procedural and Monitoring Guidance
determination using information contained in relevant environmental impact statements,
section 301 (h) or other variance applications, existing technical and environmental field
studies, or EPA industrial and municipal waste surveys.
1.2
PURPOSE OF THIS DOCUMENT
Because of the difficulty and uncertainty inherent in predicting the precise impacts of
discharges on marine waters, ocean discharge permits may be issued/reissued under
the "no irreparable harm" provisions of 40 CFR 125.123(c) and, as a result, will require
monitoring studies to more precisely assess the impact of these discharges on  the
marine environment. The purpose of this document is to provide the Regions and
NPDES-authorized States with a framework for the  decision-making process to be
followed in making a section 403 determination and to provide them with guidance for
identifying the type and level of monitoring that should be required as part of a permit
issued under the "no irreparable harm" provisions of section 403.
1.3
DOCUMENT FORMAT
Chapter 2 of this document presents an explanation of, and procedural guidance for, the
overall process to be followed  when  issuing an NPDES permit in compliance with
section 403 of the Clean Waiter Act.  Chapter 3 discusses options for monitoring under
the basis of "no  irreparable harm."  Chapter 4 presents a summary of monitoring
methods with potential applications to 403 discharges.
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                      2. SECTION 403 PROCEDURE
2.1
BACKGROUND
2.1.1     The Role of the Ocean Discharge Criteria in NPDES Permit issuance

The Ocean Discharge Criteria (40 CFR  Part  125,  Subpart M)  are  used  in  the
development of NPDES permits for the discharge of pollutants into marine waters. The
criteria are designed  to  protect marine resources from the  impact of wastewater
discharges and  to  prevent  unreasonable  degradation  of  the marine  environment.
Although referred to as the Ocean Discharge Criteria, they are promulgated regulations
that establish minimum requirements on  discharges to marine waters.   The 10
guidelines presented in the Ocean  Discharge Criteria  that must be considered when
reviewing a permit request under section 403 are presented in Table 2-1.

The regulations  promulgated pursuant to section 403 are not a substitute for other
authorities of the Clean Water Act, but are a component of section  402 NPDES permits
and apply in  addition to other applicable Clean Water Act requirements.  Permittees
subject to section 403 must still comply with all  other provisions  of the  Act including
sections 301 (technology-based effluent limitations), 303 (water quality standards), 306
(national  standards of performance), and  307 (pretreatment).  Permittees also  may be
subject to  section 311  (oil  and hazardous substances) and section  312  (marine
sanitation devices).
The NPDES program is administered by EPA's Office of Wastewater Enforcement and
Compliance  (OWEC); the section  403  program is administered by the Office  of
Wetlands, Oceans and Watersheds (OWOW).  OWOW is responsible for developing the
criteria and guidelines for ocean discharges that are  subject to section 403.  OWEC is
responsible for administering  the NPDES  permitting program.  The actual NPDES
permits are issued by EPA Regional offices or,  in States that have been granted NPDES
permitting authority, by the designated State environmental agency.

2.1.2     Applicability of Section 403

Point source discharges to waters of the United States are subject to the requirement to
obtain a section 402 NPDES permit. Permits for discharges seaward of the baseline of
the territorial seas must comply with the requirements of section 403. The "baseline"
typically follows the line of ordinary low water along the portion of the coast that is in
direct contact with the open sea although closing lines may be drawn straight across the
mouths of bays, in certain cases.
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Monitoring Options
                     Table 2-1.  Ocean Discharge Guidelines
    (1)   Quantities, composition, and potential bioaccumulation or persistence of
          the pollutants to be discharged;

          Potential transport of the pollutants by biological, physical, or chemical
          processes;
(2)


(3)
           Composition and vulnerability of potentially exposed biological
           communities, including

           -   unique species or communities,
           -   endangered or threatened species, and
           -   species critical to the structure or function of the ecosystem;
     (4)    Importance of the receiving water area to the surrounding biological
           community, e.g.,

           -   spawning sites,
           -   nursery/forage areas,
           -   migratory pathways, and
           -   areas necessary for critical life stages/functions of an organism;

     (5)    The existence of special aquatic sites, including (but not limited to)

           -   marine santuaries/refuges,
           -   parks,
           -   monuments,
           -   national seashores,
           -   wilderness areas, and
           -   coral reefs/seagrass beds;
           Potential direct or indirect impacts on human health;
(6)

(7)

(8)


(9)


(10)
           Existing or potential recreational and commercial fishing;

           Any applicable requirements of an approved Coastal Zone Management
           Plan (CZMP);

           Such other factors relating to the effects of the discharge as may be
           appropriate; and

           Marine water quality criteria.
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                                           Section 403 Procedural and Monitoring Guidance
Section 403 requirements apply only to point source discharges beyond the baseline of
the territorial sea (represented in Figure 2-1 with a heavy black line).  This limits the
number of land-based  discharges subject to  section 403.   For example,  only the
discharges numbered 3 through 9 in Figure 2-1 would be subject to the provisions of
section 403.

EPA's permit regulations (40 CFR 122.21) require that permit applicants list the latitude,
longitude, and  name of  the receiving waters for each outfall.  Where the permitting
authority is uncertain as  to whether the outfall is within the waters covered by section
403, guidance should be requested from EPA Headquarters. In some waters where the
baseline location is  unsettled, e.g.,  where the coastline is highly irregular,  EPA will
contact the  Department of State, which chairs an interagency group  responsible for
defining the boundaries of the territorial seas.
2.1.3
Individual or General Permit
Most NPDES  permits are issued to individual facilities with one or more outfalls.  A
number of conditions exist, however, that may warrant the issuance of a "general permit"
covering multiple sources (40 CFR 122.28).  These conditions include the following:

      •  When there are expected to be a number of the same or substantially similar
         discharges;
                      Ocean and Estuarine Discharges
                       Estuary    V Sewage
                               Treatment
                                            jrritoria! .' Contiguous '. Open Ocean '
                                            Seas  ;  Zone
                                            Nautical   to 12
                                            Miles • Nautical Miles •
                                 Baseline
               Figure 2-1. Applicability of Section 403 Requirements
                                                                               7
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Monitoring Options
      •  When such discharges are expected to have the same effluent limitation
         requirements; and
      •  When, in the opinion of the director, the discharges would be more
         appropriately controlled by a general permit.

General  permits  may  be written  only if  the area receiving the  effluent  is  of  a
homogenous nature;  if it is circumscribed by  the  same  geographical  or political
boundary; if the uses of the receiving waters are of similar value; and if the area is not
significantly distorted by oceanographic anomalies.  Examples of discharges covered
under a general permit include offshore oil and gas operations and seafood processing
operations.   Exceptions to provisions  of  a general permit can  be  controlled  as
subcategories within the permit as long as distinguishing features of the subcategory are
easily identified (e.g., water depth, lease block numbers).

Dischargers requesting coverage under a general permit must only notify EPA of their
intent to be covered and provide the necessary information to establish similarity to the
permitted discharges.  In writing a general permit, however, EPA is the permit applicant
and must fulfill all the necessary data requirements.
2.2
GENERAL PROCEDURE
The discussion that follows presents guidance on the procedure to be followed by permit
writers when  reviewing an NPDES permit request  under section  403 and deciding
whether to issue such a permit and under what conditions. Once a determination has
been  made that section 403 applies to a particular discharge, a general progression of
decisions is required to  complete an Ocean Discharge Criteria Evaluation (ODCE).
Figure 2-2 presents the decision process that is undertaken in the review of a section
402 permit request involving ocean discharges.  The discussion that follows describes
each  of the steps in this process.  Much of this discussion is also presented in the
preamble to the section 403 regulations (45 FR 65942, October 3,1980).

2.2.1      Request for Issuance/Reissuance of a Section 402 Permit

Any point source discharger to the navigable waters of the United States that meets the
criteria defined in section  402  of the Clean Water Act must obtain a section 402 NPDES
permit prior to discharging.   In addition, any  permit issued  under  section 402 for
discharges to  marine waters must also comply with the requirements of section 403.  A
permit application  is  submitted to the State in which the discharge occurs if the  State
has been granted NPDES permitting authority by EPA.   EPA processes  the permit
application if the State in which the discharge occurs is not authorized or if the discharge
occurs in Federal waters (waters beyond the territorial seas).
 8
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                                                             Section 403 Procedural and Monitoring Guidance
                                                Applicant submits request for issuance/
                                                reissuance of permit and Information
                                                necessary to evaluate potential Impacts
                                                of discharge on the marine environment
                                                         40 CFR 125.124
                                                 Evaluation to determine unreasonable
                                                  degradation based on 40 CFR 125.122
               Issue/Reissue Permit
               May Require

               - Effluent Limits
               - Effluent Monitoring
               - Environmental Monitoring
               - Bioassays
               - Best Management Practices
               - Special Conditions
                                                    Issue/Reissue Permit
                                                    Must Require

                                                    - Effluent Limits
                                                    - Effluent Monitoring
                                                    - Environmental Monitoring
                                                    - Bioassays

                                                    - Best Management Practices

                                                    - Special Conditions
                                                          Permit Expiration
1 Unreasonable Degradation is:
  (1) Significant adverse changes in ecosystem diversity, productivity and  stability of the biological community within the area
      of discharge and surrounding biological communities.
   (2) Threat to human health through direct exposure to pollutants or through consumption of exposed aquatic organisms; or
   (3) Loss of aesthetic, recreational, scientific, or economic values which is unreasonable in relation to benefit derived
      from the discharge.
2 Irreparable harm is significant undesirable effects which will not be  reversed after cessation or modification of the discharge.
3 Assuming other applicable requirements are met.
                         Figure 2-2.  Section 403 Decision  Process
                                                                                                                         9
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Monitoring Options
2.2.2    Determination of Information Requirements

Prior to the issuance of an NPDES permit for a discharge subject to section 403, the
director must determine that "no unreasonable degradation" of the marine environment
will occur as a  result  of the discharge.  Such a determination is to  be made  after
consideration of  the effects of pollutant discharge on human health and welfare, marine
life,  and   aesthetic,  recreational,  and   commercial  values;  the  persistence  and
permanence of  these  effects;  the  effects of varying disposal  rates;  the alternative
disposal or recycling options available; and the effect on alternative uses of the ocean.

Before any decision regarding the degradation of the marine environment can be made,
the director must first determine what information is necessary to evaluate the effects of
the discharge based on the guidelines presented in the Ocean Discharge Criteria.  The
director should survey  the currently available information about the discharge and the
area in which the discharge would  occur.   This information  would include the  data
contained in the permit application form, as well as data available from other Agency
reports and studies.

Following a survey of currently available information, the director should determine what
additional information might be required from the applicant for the ODCE under 40  CFR
125.124.  The permitting authority can require the discharger to supply information
necessary  to make  a  determination of "no unreasonable  degradation,"  including the
following:
       •  Analysis of chemical constituents of the discharge;
       •  Bioassays necessary to determine limiting permissible concentrations for the
         discharge;
       •  Analysis of initial dilution;
       •  Process modifications that will reduce the quantities of pollutants that will be
         discharged;
       •  Analysis of the locations where pollutants are sought to be discharged,
         including biological communities, and physical description of the discharge
         facility; and  ;
       •  Evaluation of available alternatives to the discharge of pollutants, such as
         land-based disposal.

The applicant is  responsible for collecting the additional information and submitting it to
the director for review.
10
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                                           Section 403 Procedural and Monitoring Guidance
2.2.3    Determination of No Unreasonable Degradation

The section 403 regulations define unreasonable degradation as

      ... significant adverse changes in ecosystem diversity, productivity and
      stability of the biological community within the area of discharge and
      surrounding biological communities, threat to human health through direct
      exposure to pollutants or through consumption of exposed aquatic
      organisms, or loss of aesthetic, recreational, scientific or economic values
      which is unreasonable in relation to the benefit derived from the discharge.

The determination of  no unreasonable degradation  is to  be  made based  on  a
consideration of the 10 guidelines in 40 CFR  125.122.  Discussion of these guidelines
also appears in the preamble to the section 403 regulations (45 FR 65942, October 3,
1980).  The following discussion is intended  to provide basic technical guidance that
might be used in determining compliance with the section 403 criteria.

     (1)    Quantities, composition, and potential bioaccumulation or persistence
           of pollutants to be discharged. An assessment of the potential effects
           of a discharge on the marine environment should carefully consider the
           potential presence of persistent or toxic pollutants, especially when
           present in significant quantities relative to marine water quality criteria
           developed pursuant to CWA section 304(a).  The potential for
           bioaccumulation or persistence of pollutants  in the environment is of
           particular importance. In areas that do not contain sensitive species or
           unusual biological communities or  are not important for surrounding
           biological communities, discharges containing primarily conventional
           pollutants may be of lesser concern if data indicate that there will be
           significant mixing with the receiving waters based on the flow of the
           discharge and the physical characteristics of the discharge site, such
           as water depth, density gradient, and turbulence (USEPA, 1980).  An
           assessment of the effluent characteristics should include the types,
           sources, amounts, and temporal characteristics of the effluent, as well
           as its physical, chemical, and toxicological properties and known or
           demonstrated environmental impacts.

     (2)    Potential transport of the pollutants by biological, physical, or chemical
           processes.  An assessment of receiving water characteristics important
           to the transport and fate of pollutants should  include an evaluation of
           the physical, chemical, hydrodynamic, and sedimentologic features of
           the waterbody. An assessment of the biological processes that may
           impact the fate and transport of contaminants and their daughter
           products also may be necessary and would include consideration of
           degradation through physicochemical processes, metabolic  processes,
           bioturbation, and potential for transport through the food chain.
                                                                              11
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Monitoring Options
    (3)    Composition and vulnerability of potentially exposed biological
           communities. An assessment should be made of the presence of, and
           potential for, effects of a discharge on the species and biological
           communities that might be impacted by the discharge. Particular
           attention should be given to the possible presence of species identified
           as endangered or threatened pursuant to the Endangered Species Act,
           and species critical to the structure or function of the ecosystem, such
           as species in food chain relationships.

     (4)    Importance of the receiving water area to the surrounding biological
           community. The permitting authority should consider the vulnerability
           of the area of discharge and  its role in the larger biological community.
           Environmentally significant or sensitive areas such as spawning sites,
           nursery or forage areas, migratory pathways or areas necessary for
           other functions or critical stages in the life cycles of organisms, areas of
           high productivity, or areas under stress due to biological or climatic
           conditions or discharges from other sources would be of most concern.

     (5)    Existence of special aquatic site. The potential for impacts on special
           aquatic sites near or adjacent to the discharge should be considered.
           Special aquatic sites include both those of special biological
           significance and those of aesthetic or recreational importance.
           Examples presented in the Ocean Discharge Criteria include:

              •   Marine sanctuaries,
              •   Refuges,
              •   Parks,
              •   National and historic monuments,
              •   National seashores,
              •   Wilderness areas, and
                      i
              •   Coral reefs.
     (6)    Potential direct or indirect impacts on human health.  Under the 403
           criteria, the permitting authority is to consider the potential impacts of
           the discharge on human health, either directly through physical contact
           or indirectly through the food chain.  The location of the discharge, the
           type and volume of the discharge's effluent, and the nature of the
           surrounding biological communities should  all be considered in
           performing this assessment.

     (7)    Existing or potential recreational and commercial fishing. In
           determining whether unreasonable degradation will occur, the 403
           crtieria call for consideration  of potential impacts on recreational and
12
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                                          Section 403 Procedural and Monitoring Guidance
           commercial fishing. This will involve identification of existing and
           potential fishery resources that might be adversely affected by the
           discharge and consideration of the nature and extent of these effects.

     (8)    Any applicable requirements of an approved Coastal Zone
           Management Plan (CZMPt.  The Ocean Discharge Criteria specifically
           identify the need to assess the applicable requirements of an approved
           State Coastal Zone Management Plan. Once a State has an approved
           Coastal Zone Management Plan, the Federal consistency provisions of
           section 307(c)(3) of the Coastal Zone Management Act (CZMA)
           generally require that applicants for Federal licenses or permits obtain
           a certification from the  State as to consistency with the approved plan.
           This obligation applies to NPDES permits (see 40 CFR 122.49(d)).

     (9)    Such other factors relating to the effects of the discharge as may be
           appropriate. Permitting authorities are given the prerogative to
           evaluate factors other than those specifically defined in the Ocean
           Discharge Criteria when making a determination of "no unreasonable
           degradation." These factors could include other statutory
           requirements, impacts on other  uses of the ocean such as navigation
           or resource exploitation, or potential biological impacts not previously
           described. The permitting authority should determine what additional
           information is appropriate under given discharge circumstances and
           request such information from the applicant.

    (10)   Marine water quality criteria. Discharges to the territorial seas are
           subject to and must comply with any applicable State water quality
           standards.  In the absence of such State standards, or in waters
           beyond a State's jurisdiction, EPA marine water quality standards
           developed under section 304(a) of the CWA should be considered
           when evaluating discharges. Although not binding like State water
           quality standards under section 303, the EPA section 304 marine water
           quality standards provide important technical information that should be
           considered in performing an ODCE.

Following a review of existing information related to the 10 guidelines described above,
the permitting authority prepares an ODCE to compile and explain the information base,
conclusions, and regulator/ determinations  and requirements related to a section 403
review.  The  ODCE should specifically address each  of  the 10 guidelines set  out in 40
CFR  125.122 and, in  general,  may be  organized  under five  broad categories  of
information:
    (1)    Characterization of the effluent and receiving water

           -   discharge,
           -   receiving water,
           -   fate and transport;
                                                                              13
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Monitoring Options
     (2)    Potential effects

           -  aquatic life,
           -  human health,
           -  socio-economic;

     (3)    Other statutory/regulatory factors

           -  CZMA,
           -  Endangered Species Act,
           -  others (State regulations, treaties, administrative directives, etc.);

     (4)    Findings/Conclusions; and

     (5)    Regulatory outcome or actions.


A copy of the ODCE should be made available to NPDES permitting staff for inclusion as
part of the  Administrative  Record for  the  associated NPDES permit.   One of three
determinations is possible after an evaluation of a discharge based  on a review and
assessment of the Ocean Discharge Criteria as presented in an ODCE: (1) unreasonable
degradation  will  not occur; (2)  unreasonable  degradation will occur;  or  (3) insufficient
information is available to make a determination.

2.2.4    Decision to Issue/Reissue or Deny a Permit

A determination  of "no unreasonable degradation" during  the permit application  review
process will allow the permit development and issuance process to proceed. The permit
may or may not impose further data-gathering requirements (i.e., monitoring), discharge
limitations,  or other  special conditions  deemed  necessary to prevent any  future
unreasonable degradation of the marine environment.  It  may also include a reopener
clause. In practice, virtually all permits subject  to section 403 have included reopener
clauses.  If during the permit application  review process a determination is made that
unreasonable  degradation of the  marine environment will  occur, the permit will be
denied (40 CFR 125.122(b)).
2.2.5
Insufficient Information
In those cases where there is insufficient information to make a determination  of "no
unreasonable  degradation,"  under 40 CFR  125.123(c)  the  discharge  cannot  be
permitted unless three criteria are met:

    (1)    No irreparable harm will result from the discharge;

    (2)    There are no reasonable alternatives to onsite disposal; and

    (3)    The discharge will comply with certain mandatory permit conditions
           necessary to ensure that unreasonable degradation will not occur.
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                                            Section 403 Procedural and Monitoring Guidance
No Irreparable Harm

The  first  determination that must be made  following  a decision  of  "insufficient
information" is that the discharge  will not cause irreparable  harm  to  the marine
environment during the period for which the discharge will be permitted.

The Ocean Discharge Criteria define irreparable harm as "significant undesirable effects
occurring after the date of permit issuance which will not be reversed  after cessation or
modification of the discharge."  In evaluating  the potential for irreparable harm,  the
permitting authority  will need to  make a  determination  of whether the discharger,
operating  pursuant to  its permit conditions, will not cause permanent and significant
harm to the environment during the period in which further data on  the effects of the
discharge are collected.  Certain  factors are particularly  significant  in assessing  the
likelihood  of irreparable harm,  including  the quantity  of pollutants expected to be
discharged and their potential for persistence in the marine environment. An additional
factor is the sensitivity of the area into which the discharge is proposed.  For example, a
discharge could cause irreparable harm to unusual and interdependent communities,
such  as coral reefs and  associated  communities.   Data on the  effects of similar
discharges in similar areas are directly relevant to the determination of irreparable harm.
Information demonstrating the timely recovery of the environment after the cessation of
discharges from similar facilities would be a strong indication that irreparable harm is not
likely to  occur (USEPA,  1980).  In addition to considering the permanence of  the
impacts, consideration must also be given to the significance of the anticipated impacts.
This would involve consideration of such  factors  as the areal extent of impacts,  the
ecological or  economic  significance of affected resources, and potential impacts on
human health.

Reasonable Alternatives

If it is determined that no irreparable harm will occur as a result of a discharge, then the
second determination that inust  be made prior to permitting the discharge  is that there
are no reasonable alternatives (as defined in 40 CFR 125.121 (d)) to on-site disposal.
Such alternative sites  would include disposal facilities located on land and discharge
points within internal waters.   In determining whether a site is a reasonable alternative
to on-site disposal, the permitting authority should consider its distance from the location
of the proposed discharge and whether its use would  cause unwarranted economic
impact on the discharger.  The amount of material requiring disposal  should  also be
considered along  with  the availability of existing  land-based  disposal sites  within a
reasonable  distance from the  point of discharge and the  estimated  uncommitted
capacity of such sites.  A second basis for evaluating the feasibility of alternative sites is
the  relative   environmental  harm  of  disposal  associated  with   each  alternative.
Alternative sites  are not considered "reasonable alternatives"  if on-site disposal is
judged to be environmentally  preferable.
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Methods
Other Mandatory Permit Conditions

The final criterion for authorizing discharges for which there is insufficient information to
make a  determination of  "no  unreasonable degradation" is that the discharge be in
compliance  with  all permit conditions  established  pursuant  to  §125.123(d)  of the
regulations,  including a bioassay-based  discharge limitation {similar to those of EPA's
ocean dumping regulations, 40 CFR Part 227) and  monitoring requirements.  These
permit conditions are specified to assist in determining whether and to what extent
further limitations  are  necessary to ensure  that  the  discharge does  not cause
unreasonable degradation (USEPA, 1980).

Monitoring is  required as  part of a  permit issued  under  the  "no irreparable harm"
provisions of section 403.  One of the principal purposes of the monitoring program is to
collect sufficient information to determine whether unreasonable degradation will occur
as a result of the discharge. Such information is then used to make a decision whether
to reissue the  permit during the next  round of permitting.  In addition, it is vital that the
monitoring program should also be designed to ensure that no irreparable harm to the
marine environment will occur during the life of the current permit.  In order to do this,
the monitoring program that is put in place must be sufficient to assess the impact of the
discharge on  water, sediment, and biological quality and should  include,  where
appropriate, analysis of the bioaccumulative and/or  persistent impact on aquatic life.
This monitoring program may include effluent  analysis, bioassay analysis,  and field
studies (USEPA, 1980).

It is not possible to make an a priori determination as to what constitutes an  acceptable
and cost-effective monitoring  program.  Site-specific conditions such as the volume of
waste discharged, the types of pollutants discharged, and the location of the discharge
will play  a role in determining what monitoring will be required of a discharger.  These
considerations are discussed further in Chapter 3 of this document.

In addition to bioassay and monitoring requirements, the permitting authority may also
specify other permit conditions under 40  CFR 125.123(d). Seasonal restrictions on the
volume of wastes discharged  can be required where such restrictions are needed to
ensure protection of the marine environment.  Bioaccumulation testing of the liquid
and/or suspended particulate phase of the discharge can  be  required  where the
potential  for bioaccumulation exists, based on the nature of the pollutants discharged.
Process  modifications, such as the substitution of less hazardous chemicals for those
that are potentially harmful, can be required, as can process changes that would favor
the recycling and reuse of potentially harmful pollutants.

If the data gathered  pursuant to 40 CFR 125.123(d), in particular the monitoring data,
indicate that continued discharge may cause unreasonable degradation of the  marine
environment, under 40 CFR 125.123(d) the permit will be revoked or modified to include
additional limitations as necessary.
16
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         3.  OPTIONS FOR MONITORING UNDER THE BASIS OF
                        "NO IRREPARABLE HARM"
3.1
BACKGROUND
The  purpose  of  this chapter is to provide guidance  to  the  EPA  Regions and
NPDES-authorized States for determining the general types and level of monitoring that
should be required as part of a 402 permit issued or reissued under the section 403
provisions of "no irreparable harm." The guidance presented in this chapter is intended
to be used as a general assessment tool. The determination of the specific monitoring
requirements needed for a. particular discharge involves a case-by-case  determination
based  on the  potential environmental threats posed  by the  particular discharge and
consideration  of existing  information  concerning  site-specific conditions.  Particular
consideration  should be given to the  proximity of a discharge to sensitive ecological
zones, the existence of known observed biological stress based on available baseline
data for the area, discharges that  have high mass emission rates and/or concentrations
of priority pollutants and other toxic substances, and the presence of other discharges in
the vicinity of the discharge in question.

A summary of methods  available  for  conducting monitoring programs  for various
environmental  parameters is presented  in  Chapter  4.   This methods summary is
presented as  a guide for determining what methods are  available  or  are  being
developed. The discussion that follows in this chapter identifies only the  general types
of monitoring  that might  be appropriate as  part of  a permit issued under the  "no
irreparable harm" provisions of  section 403.  The  actual  monitoring  requirements
specified in a 402 permit should be determined based on site-specific environmental and
discharge conditions.  The references cited in Appendix A, as well as other sources,
should be referred  to in  the actual development and implementation  of monitoring
programs.

As pointed out by the National Research Council (NRC, 1990), despite the considerable
efforts and expenditures associated with environmental monitoring, most programs fail
to provide the information needed  to understand the condition of the marine environment
or to assess the effects of human  activity on it.  In its report Managing  Troubled Waters,
the NRC (1990) identified several  factors critical to the design and implementation of an
effective monitoring  program. EPA suggests that the recommendations made by the
NRC be incorporated into any monitoring program required under section 403.  These
recommendations include the following:
       •   The  goals and objectives of the monitoring program need to be clearly
          articulated in terms that  pose questions that are meaningful to the public and
          that provide the basis for scientific investigation.
                                                                            17
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Monitoring Options
      •   Not only must data be gathered, but attention must also be paid to their
          management, synthesis, interpretation, and analysis.
      •   Procedures for quality assurance are needed, including scientific peer review.
      •   Because a well-designed monitoring program results in unanswered
          questions about environmental processes or human impacts, supportive
          research should be provided.
      •   Adequate resources are needed not only for data collection but also for
          detailed analysis and evaluation over the long term.
      •   Programs should be sufficiently flexible to allow for their modification where
          changes in conditions or new information suggests the need.
      •   Provision should be made to ensure that monitoring information is made
          available to all interested parties in a form that is useful to them.
The central elements involved in designing and  implementing  an effective monitoring
program, as presented in the NRC's report (1990), are illustrated in Figure 3-1.

3.2       CRITERIA FOR EVALUATING THE POTENTIAL FOR
          ENVIRONMENTAL IMPACT

To assess the potential impact posed by a discharge and thus the types and level of
monitoring that might be necessary as part of a permit issued under the "no irreparable
harm" provisions of  section  403, the following site-specific determinations should be
made:
      •   Is the discharge in question a "major" or a "minor" discharge?
      •   Does the discharge occur in stressed waters?
      •   Does the discharge occur in the vicinity of sensitive biological areas?
      •   What are the number and types of other discharges in the vicinity of the
          discharge in question?

3.2.1      Major/Minor Discharges

It  is recommended  that  the Office  of  Wastewater Enforcement and Compliance's
(OWEC) classification of major/minor discharges  be  used  for according such status to
discharges of concern.  OWEC classifies industrial  discharges as  "major" or "minor"
based on an evaluation of the potential for toxic pollutant discharge, traditional pollutants
in  the effluent, potential human  health impacts, flow  rate of effluent, and various water
quality factors. Municipal  discharges are classified as "major" if ownership is public, the
facility is active, the  flow rate is 1 million or more gallons per day or a  population of
10,000 is served, or the discharge causes significant  water quality impacts. In addition,
each  EPA  Region,  in consultation   with  the   environmental  agency   of   an
NPDES-authorized State, is allowed to designate  a  certain percentage of  its total
18
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                               Section 403 Procedural and Monitoring Guidance
         No
                           Step"!
                           Define
                    Expectations and Goals
                           Step 2
                           Define
                       Study Strategy
                            I
                           Step 4
                          Develop
                      Sampling Design
                        Can Changes
                        Be Detected?
                           Steps
                      Implement Study
                            I
                           Steps
                     Produce Information
                        Is Information
                         Adequate?
                                Yes
                          Step?
                   Dessiminate Information
                            I
                       Make Decisions
                                                           StepS
                                                     Conduct Exploratory
                                                      Studies if Needed
Figure 3-1. Elements of Designing and Implementing a
           Monitoring Program (NRC, 1990)
                                                                       19
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Monitoring Options
permits as "discretionary addition" major permits. This classification has been created
for those permits that the Region or State believes should be accorded major status, but
for some reason were not classified as such by OWEC.

OWEC's Permit Compliance System (PCS) is a data base management  system that
tracks individual facility permit conditions.  The PCS Permit Facility Data File includes
the major/minor classification for NPDES discharges.

3.2.2     Discharges to Stressed Waters

As part of the permit development process, a determination  of whether  a discharge
occurs in or near stressed waters should be made, and the presence of stressed waters
should be considered when determining the type and level of ambient monitoring that
should be required of a discharger.  Such ambient monitoring should  be  designed to
determine whether and to what extent the permitted discharge is contributing to stressed
conditions and whether permit limitations are helping to alleviate such stress.

Two sources of information can be used to assess whether a discharge of concern  is
occurring in stressed waters: State 305(b) reports and 304(1) lists.

Section 305(b) of the Clean Water Act requires States to report to EPA on the  extent to
which their surface waters are meeting the goals of the Act and to recommend how the
goals can be achieved.   Each State, territory, and Interstate  Commission develops a
program to  monitor the quality of its surface water and  groundwater and to report the
current status of water quality to  EPA  in biennial Water Quality Assessment reports.
Among other things, these 305(b) reports allow EPA to:
       •   Determine the status of water quality;
       •   Identify water quality problems and trends;
       •   Evaluate the causes of poor water quality and the relative contributions of
         pollution sources; and
       •   Determine the effectiveness of control programs.

In 1990, 11 States and two territories  reported on the  degree to which their coastal
waters support the uses for which they have been designated.  This represented 4,230
coastal miles or only 22 percent of the Nation's estimated  19,200 miles of coastline.
Efforts are  being made, however, to improve the 305(b)  reporting  process. These
improvements hopefully will result in a  higher percentage of States reporting on their
coastal water quality conditions.
20
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                                          Section 403 Procedural and Monitoring Guidance
Another source of information on stressed coastal waters is the State 304(1) "long list."
This is a comprehensive list of waters of the State that are impaired by point or nonpoint
source discharges of toxic, conventional, and nonconventional pollutants.  Although
section  304(1)  of the Clean Water Act was identified as a one-time listing of waters,
States are encouraged to  maintain updated  information  on the status of their near
coastal  waters.  Unfortunately,  as  was the case with 305(b) reports, not all coastal
States have assessed the condition of their offshore waters.

For those coastal waters that have not been assessed by the States, a determination of
whether a  discharge  occurs in  stressed waters should be  made based on the best
professional judgment of the  permitting authority  using other sources of existing
information.   Sources of additional  information on  potentially stressed waters include
EPA's 301 (h) and  National Estuary  programs, the  Environmental  Monitoring  and
Assessment   Program  (EMAP),  and  the   National  Oceanic   and  Atmospheric
Administration's   (NOAA)  Strategic  Assessment   Program,  as   well   as  other
biological/ecological assessments conducted by the Federal Government (e.g., Minerals
Management  Service (MMS)  and U.S.  Geological  Survey (USGS)  of  the U.S.
Department of the Interior,  and the  U.S. Army Corps  of Engineers (COE))  and State
governments.

3.2.3     Discharges to Sensitive Biological Areas

The determination of what constitutes a  sensitive  biological  area is subject  to  the
discretion of the permitting authority,  but at a minimum should include the following:

      •  Spawning sites,
      •  Nursery areas,
      •  Migratory pathways,
      •  Marine sanctuaries/refuges,
      •  Coral reefs/seagrass beds, and
      •  Other areas necessary for critical life stages/functions of important marine
         organisms.

Numerous  sources   of  information concerning  the  location of sensitive  marine
environments  are available.  These include previous  surveys conducted by State or
Federal environmental agencies. In addition to specific State environmental agencies,
major Federal sources of such information include NOAA and the U.S.  Fish and Wildlife
Service (FWS)  and  MMS  of the  U.S.  Department of the  Interior.   As part  of  the
information-gathering  effort for  an ODCE conducted for a 403 review, the discharger
should  be  required to investigate the existence of  applicable information on sensitive
                                                                              21
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 Monitoring Options
 marine environments developed by these and other sources.  Table 3-1  lists various
 sources in NOAA,  FWS, and  MMS for information regarding  sensitive marine
 environments.

 Several programs within NOAA compile information on important and sensitive marine
 habitats.   NOAA's  National Environmental Satellite,  Data and  Information Service
 (NESDIS)  maintains an  archive of worldwide data on the  physical and  chemical
 properties of the ocean.  Over the past decade, NESDIS has  received large amounts
 of physical, chemical, and biological data collected within the U.S. Exclusive Economic
 Zone.   These data  derive primarily from programs organized to study the effects of
 offshore oil development, ocean dumping, and  other human  activities on marine
 ecosystems.   NOAA's  National Marine Sanctuary program has  the responsibility of
 preserving and restoring the conservation, recreational, ecological, or aesthetic values
 of designated marine sanctuaries.  The program presently manages seven national
 marine sanctuary sites.  The National Marine Fisheries Service (NMFS) of NOAA can
 provide information concerning  the  presence of spawning  and  nursery  sites  for
 important commercial and recreational fisheries, as well as information on catch of
 particular  species  in  State  and  Federal offshore waters.    NMFS  also  has
 responsibility  for the management of some marine  mammals under the Marine
 Mammal Protection  Act of 1972;  the FWS has management responsibility for others,
 including  endangered  species.   Together,  these  two  agencies  have  compiled
 considerable  information  on  marine  mammal populations, life  cycles,  and habitat
 requirements.

 The FWS  collects and interprets diverse information  on fish and wildlife species,
 populations, and habitats.  Included under the management responsibility of the FWS
 are anadromous fish  species and endangered species,  as  well  as some marine
 mammals.  The FWS publishes ecological characterization studies and species profiles
 for numerous  marine/estuarine fishery  species.   The  FWS  is  also  responsible  for
 producing detailed wetland maps for the contiguous United States.   The mapping of
 coastal wetlands has been an ongoing effort of the National Wetlands Inventory (NWI)
 program at FWS. Other habitat types, including submerged aquatic vegetation, rocky
 shores, reefs, and intertidal softbottoms, are also depicted on NWI maps although they
 are not covered as completely as the wetlands.

 As part of its offshore oil and gas  leasing program, the  MMS has conducted numerous
 environmental  impact studies and other special studies to assess the environment and
 potential impact of offshore oil and gas activities on marine  resources in leasing areas.
These  studies  include in-depth evaluations of the lease area, the affected environment,
 and the potential environmental consequences of offshore oil and gas activities.
22
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                                       Section 403 Procedural and Monitoring Guidance
Table 3-1. Information Sources on Sensitive Marine and Coastal Environments:
                          NOAA, FWS, and MMS
NQAA
Strategic Environmental
 Assessment Division
U.S. Department of Commerce
6001 Executive Boulevard
Rockville, MD 20852-3806
301/443-8843
Alaska Region
National Marine Fisheries Service/NOAA
P.O. Box21668
Juneau, AK 99802
907/586-7221
Marine and Estuarine Management Division
NOAA/N/ORM2
U.S. Department of Commerce
Washington, DC 20235
Northeast Region
National Marine Fisheries Service/NOAA
14 Elm Street, Federal Building
Gloucester, MA 01930
813/893-3141
National Oceanographic Data Center
NOAA/NESDIS E/OC21
2001 Wisconsin Ave., NW
Washington, DC 20235
202/634-7500

Northwest Region
National Marine Fisheries Service/NOAA
7600 Sand Point Way, NE
BIN C15700-Bldgl
Seattle, WA 98115-0070
206/526-6150
Southeast Region
National Marine Fisheries Service/NOAA
9450 Koger Boulevard
St. Petersburg, FL 22703
813/893-3141

FWS

Region  1
U.S. Fish and Wildlife Service
Eastside Federal Complex
911 NE  11th Avenue
Portland, OR 97232-4181
503/231-2122
Southwest Region
National Marine Fisheries Service/NOAA
300 S. Perry Street
Terminal Island, CA 90931
213/514-6196
Region  2
U.S. Fish and Wildlife Service
500 Gold Avenue, SW
Albuquerque, NM 87103
505/766-2321
                                                                        23
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 Monitoring Options
                             Table 3-1 (continued)
  Region 4
  U.S. Fish and Wildlife Service
  Richard B. Russell Federal Building
  75 Spring Street, SW, Room 1246
  Atlanta, GA 30303
  404/331-3594
Minerals Management Service
Branch of Environmental Studies (644)
Washington, DC 20240
202/343-7744
 Region 5
 U.S. Fish and Wildlife Service
 One Gateway Center, Suite 700
 Newton Corner, MA  02158
 617/965-5100
Minerals Management Service
Atlantic OCS Regional Office
1951 Kidwell Dr., Suite 601
Vienna, VA 22180
703/285-2165
 Region 7
 U.S. Fish and Wildlife Service
 1011 E.Tudor Road
 Anchorage, AK  99503
 970/786-3542
Minerals Management Service
Gulf of Mexico OCS Regional Office
1201 Elmwood Park Boulevard
New Orleans, LA 70123
504/736-2896
 National Wetlands Inventory Program
 U.S. Fish and Wildlife Service
 18th and C Street, NW
 Washington, DC  20240
 202/235-2760
Minerals Management Service
Pacific OCS Regional Office
1340 W. 6th Street
Los Angeles, CA 90017
213/894-7120
 Division of Endangered Species
 U.S. Fish and Wildlife Service
 4401 North Fairfax Drive, Room 452
 Arlington, VA 22203
 703/358-2171
Minerals Management Service
949 E. 36th Avenue, Room 110
Anchorage, AK 99508-4302
907/261-4620
 Office of Management Authority
 U.S. Fish and Wildlife Service
 Division of Endangered Species
 4401 North Fairfax Drive, Room 432
 Arlington, VA 22203
 703/358-2093
24
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                                           Section 403 Procedural and Monitoring Guidance
3.2.4    Presence of Other Discharges in the Area

The permitting authority must determine whether the presence of other discharges in the
vicinity of the discharge of concern is likely to increase the potential for environmental
impact and thus provide justification  for additional monitoring  requirements.   This
determination will depend on the number of other discharges in the area, their effluent
flow, and the concentration of contaminants being discharged. Although individually the
discharges in the area may not pose a significant threat to the marine environment, the
cumulative effect of numerous discharges to the same receiving waterbody could result
in a potentially  significant  impact on the marine  environment  in  the  vicinity of the
discharges. Consideration of the presence of other discharges occurring in the vicinity
of the discharge in question is particularly important in light of EPA's current emphasis
on watershed management.

3.3      MONITORING REQUIREMENTS BASED ON PERCEIVED POTENTIAL
         ENVIRONMENTAL THREAT

Based on an evaluation of the criteria presented above, discharges  can  be placed into
one of three categories based on their perceived potential for causing  environmental
impacts (Table 3-2):

      •  Minimal potential for causing environmental impacts,
      •  Moderate potential for causing environmental impacts, or
      •  High potential for causing environmental impacts.
The level and type of environmental monitoring required of a discharger will depend on
its potential for causing environmental impacts. If sufficient information is not available
to place a discharge into  one of these general categories, a  potential worst-case
scenario should be  assumed  (i.e.,  the  discharge  poses  a  high  potential   '
environmental impacts).
                                                                   for
3.3.1
Minimal Potential Threat
Dischargers perceived to pose a minimal potential threat to the marine environment are
those "minor" dischargers that do not discharge to stressed waters or sensitive biological
areas and also do not discharge in the vicinity of other discharges where the combined
effects of effluents  could  potentially cause  unacceptable  cumulative  impacts.  The
permitting authority  will  have to determine the potential for cumulative impacts from
multiple discharges on a case-by-case basis.

It is recommended that discharges perceived to be of minimal potential environmental
concern  be  required  to  conduct  effluent chemical  characterizations,  including
whole-effluent toxicity testing, and  to meet aquatic life and human health water quality
criteria or standards. In addition, sediment contamination, which can involve deposition
of toxicants over long  periods of time, is responsible for water quality impacts in many
                                                                             25
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 Monitoring Options
          Table 3-2.  Characterization of Section 403 Discharges Based on the
                         Potential for Causing Environmental Impacts
 Potential for Causing
Environmental Impacts
Major/Minor
 Discharge
         Location
               Rationale
Minimal
Minor
 Does not occur in stressed
 waters;
 Does not occur in the vicinity
 of sensitive habitats;
 AND3
 Does not occur in the vicinity
 of other discharges where
 the potential for cumulative
 impacts exists.	
Excludes high-volume and/or highly toxic
discharges.
Unreasonable degradation does not
currently appear to be a problem.
Small likelihood of impacts to recreational,
commercially, economically, or ecologically
important species.
Small likelihood of contributing to
cumulative impacts.	
Moderate
Minor
                        Major
 Occurs in stressed waters or
 in the vicinity
 of sensitive habitats
 ORb
 Occurs in the vicinity of other
 discharges where the
 potenital for cumulative
 impacts exists.
Excludes high-volume and/or highly toxic
discharges.
In spite of low-volume and relatively nontoxic
nature of the discharge, it may be contributing
to currently stressed conditions.
May impact habitats of recreationally,
commercially, economically, or ecologically
important species that occur in the vicinity of
the discharge.           !
May be contributing to cumulative impacts.
             - Does not occur in stressed
               waters;
             - Does not occur in the vicinity
               of sensitive habitats;
               AND3
             - Does not occur in the vicinity
               of other discharges where
               the potential for cumulative
               impacts exists.
                              May include high-volume and/or highly toxic
                              discharges.
                              Unreasonable degradation does not appear
                              to be occurring although because of the
                              volume and nature of the discharge, the
                              potential exists.
                              Depending on the volume, fate, and transport
                              of the discharge and the toxicity and
                              persistence of contaminants in the effluent,
                              the potential exists for far-field impacts to
                              recreationally, commercially, economically, or
                              ecologically important species.
                              Small likelihood of contributing to cumulative
                              impacts.	
High
 Major
- Occurs in stressed waters or
  in the vicinity of sensitive
  habitats
  ORb
- Occurs in the vicinity of other
  discharges where the
  potential for cumulative
  impacts exists.	
May include high-volume and/or highly toxic
discharges.
May be contributing to the unreasonable
degradation (i.e., stressed conditions) of the
environment.
May be contributing to cumulative impacts.
 1 All conditions must be true.
 ' Either one or the other condition must be true.
  26
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                                           Section 403 Procedural and Monitoring Guidance
areas.  Therefore,  for  discharges  posing a minimal  potential  threat to the marine
environment, it is recommended that near-field sediment contamination monitoring, in
the form of sediment toxicity tests,  be  a requirement for 402 permits issued with 403
provisions or conditions.
3.3.2
Moderate Potential Threat
Both major and minor discharges can be considered to pose moderate potential threats
to the marine environment. Minor discharges that are discharged to stressed waters or
in the  vicinity of  sensitive biological areas or are discharged in the vicinity of other
discharges where the combined effects of effluents could potentially cause unacceptable
cumulative impacts should  be considered to pose moderate threats to the marine
environment.  Major facilities that do  not discharge to stressed waters or sensitive
biological areas and that also do not discharge in the vicinity of other discharges where
the  potential for cumulative Impacts exists  should also be considered to pose moderate
threats to the marine environment.

For discharges posing  a moderate  potential threat, field monitoring (in addition to
sediment toxicity  and effluent chemical characterization  requirements for discharges
presumed to cause minimal environmental impacts) should be a conditional part of 402
permits issued with section 403 provisions for "no irreparable harm."   Such monitoring
efforts  should include  physical transport studies as well as near-field measurements of
contaminant  concentrations  in  the water   column, sediment,  and  benthic  biota.
Biosurveys and bioassessments should also be used to directly evaluate the overall
biological integrity (structure and/or functional characteristics) of the aquatic community.

Current measurements and  dye studies are  recommended for tracking  oceanic
wastewater plumes.  The  purpose of such studies is to estimate the distance from the
outfall that contaminated particles  may travel before accumulating on the bottom  and to
evaluate the movement and spatial extent of the plume, the likelihood that the plume will
reach  the  shoreline or other nearby areas, and the recirculation  potential  under
short-term influences for existing discharges as well as the  general circulation patterns
in the  vicinity of  discharge sites.  Seasonal  current  measurements  and dye studies
should be conducted  to detect changes in current strength and  direction  (particularly
wind-driven currents).   The  effects  of a storm  event on transport  should  also be
investigated.  Results of such studies can be used to identify areas potentially impacted
by the discharge  and to  identify existing and potential  problem  areas  for fish  and
sensitive life stages of marine organisms.  Data from physical transport studies can also
be used to calibrate and validate dispersion plume models.

Near-field  monitoring of chemical concentrations  can provide a  spatial and temporal
record  of contamination in the water column.  Water quality chemistry measurements
should be made as part of a  monitoring program to assess the effectiveness of  permit
                                                                              27
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Monitoring Options
limitations in meeting water quality standards and to identify discharges that may not be
in compliance with those standards.  Water quality data can also be used to calibrate
and verify mathematical models.

As discussed in  the  revised  Technical Support  Document for  Water Quality-based
Toxics  Control (USEPA, 1991c),  EPA  encourages  States  to  develop and adopt
biological criteria  in their water quality standards  to fully protect  aquatic habitats and
provide more comprehensive assessments to determine the nonattainment of aquatic
life uses.  Biocriteria are  numerical measures or narrative descriptions of the biological
integrity of unimpaired natural systems.  The biological communities in these waters
become a reference and represent the best attainable conditions.  The reference site
then becomes the basis for developing biocriteria for major surface water types (in this
case, coastal or marine waters).  An assessment of biological integrity should include a
measure of the structure and function of a community or species within a specific
habitat.  The specific  definition of biological integrity selected  by  a State will form the
basis for comparing impacted sites to an established reference condition.

For those discharges that pose a potential moderate threat to the marine environment,
near-field biosurveys  should be conducted and  the  results  compared to  reference
conditions.   Such  biosurveys  will  provide a  useful  monitoring of both aggregate
ecological impact and overall temporal trends  in  the condition of an aquatic system.
Biosurveys can detect aquatic life impacts that other available assessment methods
may miss, such as impacts caused by pollutants that are difficult to identify chemically or
characterize  toxicologically  and  impacts from  multiple  or  unexpected exposures.
Biological surveys should be used together with toxicity testing and chemical-specific
analyses to assess the  potential for nonattainment of designated aquatic life uses.
Some acceptable  protocols are currently available  for  developing biocriteria and
conducting biosurveys for streams and rivers (Plafkin et al., 1989).  EPA is planning to
publish guidelines for developing biological criteria for wetlands and near coastal waters.
These and other techniques developed by EPA's Office of Water and several States can
be used as guidance to support biosurveys and bioassessments (Karr et  al., 1986; Ohio
EPA, 1987; Lenat, 1988;  Schackleford, 1988; Maine DEP, 1987; Weber,  1973;  USEPA,
1991 a, b).

EPA has recommended  in  its Technical Support Document for Water Quality-based
Toxics Control (USEPA,  1991c) that  sediment  criteria be  adopted by  States in their
water quality standards.  EPA has developed draft sediment criteria for five nonionic
contaminants that are currently under Science Advisory Board review, and  more criteria
are being developed.  For a discharge posing a moderate potential threat to the marine
environment, it is recommended that near-field (i.e.,  within the zone  of deposition)
sediment chemical characterization  (together  with sediment toxicity  testing)  be a
requirement for 402 permits issued with section 403 provisions for "no irreparable harm."
Comparison of field measurements to sediment criteria could then be a reliable method
for providing early warning of a potential problem. Such an early warning would offer an
28
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                                           Section 403 Procedural and Monitoring Guidance
opportunity to take corrective action before adverse impacts occur. For those numerous
chemicals for which sediment criteria have not been developed, sediment chemistry
monitoring can  still be used to detect the presence of unsuspected "hot spots" and to
assess the need for remedial action.

3.3.3     High  Potential Threat

Discharges that may pose a high potential threat to the marine environment are "major"
discharges that occur in stressed waters or  near sensitive biological areas or that occur
in  the vicinity of other  discharges where  the  potential  for unacceptable cumulative
impacts exists.  It is recommended that, in addition to effluent and near-field monitoring,
far-field monitoring studies  be required  for such discharges.   The  specific  type of
monitoring required will be dependent on site-specific circumstances.

Plume tracking  studies should be conducted to estimate  environmental concentrations
of discharged contaminants and to determine potential exposure pathways and locations
of  field monitoring stations.  Such studies  may include measurements of  effluent
constituents in  the receiving water, tracer studies, aerial surveys, measurement of
sediment accumulation, or even indirect measures such as those provided by drogue or
current-meter studies.   These studies should be conducted on a  seasonal  basis to
accurately assess changes that may occur in current strength and direction. In addition,
a plume-tracking system using a new state-of-the-art technique, acoustical tracking, has
been  used in southeast Florida to indicate plume dynamics and  to quantify mixing
characteristics.   The results of plume tracking  should  be used both directly and in
combination with transport and fate modeling results to select far-field monitoring station
locations.   Such studies should  also be  used to assist in identifying  the  relative
contribution  of  the discharge of interest to observed  effects in  near- and  far-field
environments.

Both near-field  and far-field biosurveys, sediment chemistry monitoring, and sediment
toxicity testing should be required.  The location of monitoring stations should be based
in  part on the results of plume-tracking studies.  When sensitive biological areas  are
present in  the  vicinity  of the  discharge,  however, biological and sediment quality
measurements   should   be  conducted  in  these areas  regardless   of  whether
plume-tracking  and modeling studies indicate a potential threat to these environments.
The specific type of monitoring required will be dependent on the resources at risk as
well as the chemical characteristics of the  effluent.  For stressed waters, the type of
monitoring required will also be dependent on the type of stress observed (high levels of
contaminants in fish tissues, hypoxic or anoxic conditions, coral bleaching, etc.).
                                                                              29
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Monitoring Options
3.4
SUMMARY
The preceding discussion has presented an overall approach for evaluating the level
and general  type  of  monitoring activities required  as part  of a permit  issued to a
discharger subject to  403 monitoring requirements.  This approach relies on the best
professional judgment of the permitting authority in making some  of the determinations
required  in establishing monitoring requirements; for example, whether  a discharge
contributes to cumulative impacts resulting from multiple discharges.  EPA will modify
and expand upon  the guidance presented here as improved tools for making these
determinations are developed.

It should be noted that this guidance is  designed to be applied as a  tiered approach.
That is, if initial monitoring results indicate that the potential environmental threat posed
by a discharge may be greater than that originally suspected, then additional monitoring
requirements may be required of the discharger.  The discharger would be subject to the
requirements of 40 CFR 122.62 or 122.63, whichever is applicable.  For!example, as a
result of  sediment  toxicity tests, the monitoring requirements of a. discharger originally
thought to pose a  minimal potential environmental threat  may be increased to require
monitoring activities defined for a  discharger posing  a moderate potential threat.  As
specified in 40 CFR 125.123(d)(4) of the Ocean Discharge Criteria, a permit may be
modified  (or revoked) at any time if the permitting authority determines that a discharge
may cause  unreasonable degradation  of the  marine environment.   Therefore,  the
permitting authority can add  monitoring requirements to a permit  if  the  permitting
authority deems it necessary to ensure that unreasonable degradation is not occurring,
or will not occur, as a result of the discharge.
30
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               4.  SUMMARY OF MONITORING METHODS
This chapter presents a review and evaluation of a number of monitoring and analytical
methods that  are available for  use as  part  of  CWA section 403 environmental
assessments.  Some of these methods are commonly used in monitoring and analytical
programs,  while  others  have  not  been widely used  or  may  even  be  in  the
developmental/evaluation stage.  The purpose of this effort is to provide EPA program
managers and permit writers in the Regions and States with  a basic understanding of
methods available  for use in 403  environmental assessments,  as  well  as an
understanding  of the  potential benefits  and limitations  of these methods.   The
information presented  in this document  is designed to assist program managers and
permit writers in selecting monitoring and analytical program components appropriate for
use in  environmental  assessment programs.   The appropriateness of a particular
method  as part of a monitoring program will  depend in part on the level of detailed
information required, which will vary with  the perceived threat of a discharge to the
marine environment.

The  most  important component of  a  well-designed  monitoring program is  having
competent, knowledgeable personnel conducting the collection and analysis of samples
and  data.   This  document is  designed to  provide  only general guidance for
environmental  assessments, and  not specific  monitoring  techniques or ecological
information for each unique situation. This information can be provided only by technical
personnel who are knowledgeable of monitoring and analytical techniques as applied to
the discharge area they are charged with monitoring.

Numerous sources of information were  accessed in the preparation of this  methods
evaluation.  Sources contacted included EPA's Office of Research and Development,
the 301  (h)  and National  EEstuary programs, the National  Oceanic and  Atmospheric
Administration, the U.S. Fish and Wildlife Service, and university research laboratories.

The methods described in this document can be  grouped into six categories: water
quality,   sediment  quality,  biological  resources, human   health  risks,   effluent
characterization, and microcosms/mesocosms (Table 4-1).  The relationship of each
method  to the section 403 ocean discharge guidelines is presented in Table 4-2.
                                                                            31
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Methods
                    Table 4-1. Sampling Method Categories
                           Water Quality

                              1.   Physical Characteristics
                              2.   Water Chemistry

                           Sediment Quality

                              3.   Sediment Chemistry
                              4.   Sediment Grain Size

                           Biological Resources

                              5.   Benthic Community Structure
                              6.   Fish and Shellfish Pathobiology
                              7.   Fish Populations
                              8.   Plankton
                              9.   Habitat Identification Methods

                           Human Health  Risks

                              10. Bioaccumulation
                              11. Pathogens

                           Effluent Characterization

                              12. Effluent Characterization

                           Mesocosms and Microcosms

                              13. Mesocosms and Microcosms
For each of the method sections, a brief description of the method is presented, followed
by the rationale for use of the method described in terms of how the method addresses
the section 403 ocean discharge guidelines, what information is generated, and how that
information can be used in the 403 decision-making process. Each section describes the
major monitoring design issues associated with each measurement type,  as well as
32
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                            Section 403 Procedural and Monitoring Guidance
Table 4-2. Matrix Illustrating Relationship of Method Types
      to the Section 403 Ocean Discharge Guidelines
























Measurement Parameter
Physical Characteristics
Water Chemistry
Sediment Chemistry
Sediment Grain Size
Benthic Community Structure
Fish and Shellfish Pathobiology
Fish Populations
Plankton
Habitat Identification Methods
Bioaccumulation
Pathogens
Effluent Characterization
Mesocosms and Microcosms
Ocean Discharge Guidelines
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Methods
available laboratory and data analysis methods. The essential elements of appropriate,
method-specific quality assurance and quality control programs and statistical design
considerations are described, and an assessment of how the data generated can be
used  in a 403 assessment is provided.   All of the  information  presented  for each
monitoring  program component is summarized at the end of the  section.  Numerous
references  are also presented in Appendix  A to provide the reader with additional
sources of more detailed information concerning the use of the various methods.

This methods  chapter is also intended to provide a summary of available information
and  to  address  the  most important  issues  associated  with  the  design  and
implementation of the  monitoring  program. Issues common to all  monitoring methods
include quality assurance/quality control (QA/QC) and statistical design.

Quality Assurance/Quality Control Considerations -  A QA/QC  plan should be
developed  to  ensure  the  reliability and compatibility of data collected  during  the
monitoring program. Quality assurance  (QA), implemented at the management level,
focuses  on  guidelines  and  procedures,   such  as  delegation  of authority  and
responsibility,  to ensure data quality.  Quality control (QC) centers on  the  technical
activities, such as calibration or interlaboratory studies, needed to achieve specific data
quality.   QA is a discipline that begins with effective and conscientious work planning
and ends with a carefully constructed set of checks and balances designed to ensure
that uncertainties have;been reduced to a known practical minimum (USEPA 1987c).
QA is achieved by adopting specific guidelines and procedures to be followed during
sample collection, sample analysis, data handling, and data analysis. These guidelines
and procedures provide program quality control.  QC includes the use of blanks and
replicate samples to check field and laboratory sampling and handling techniques and to
examine natural variability in contaminant concentrations.               ;

Establishment of data quality standards is fundamental to the selection  of monitoring
methods and ensures comparability  among data collected. A quality assurance plan
should be developed by each monitoring program to standardize fundamental aspects of
data collection and handling.  Although the topics to  be  considered  in  a  quality
assurance  plan will  vary somewhat  among  programs, examples of  some key
considerations are provided in Table 4-3.

Data Quality Objectives -  A major part of the QA effort is the establishment of Data
Quality  Objectives (DQOs).  DQOs are specific,  integrated statements and  goals
developed for  each data or information collection activity to ensure that the data are of
the required quantity  and  quality.  DQOs  should specify the desired  sensitivity of
sampling methods, timing of sampling, and numbers of samples to be collected.  One
function of establishing DQOs is to ensure that monitoring/data collection studies yield
data adequate to meet their intended uses.  DQOs (or data performance criteria) are
also used to  delineate  QA/QC programs specifically geared to  the data collection
34
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                                          Section 403 Procedural and Monitoring Guidance
          Table 4-3. Key Topics to Be Addressed in Marine Monitoring
                       Program Quality Assurance Plans

            •   Instructions for proper sample preservation and storage

            •   Chain of custody documentation

            •   Source of standard reagents

            •   Source or preparation of quality control samples

            •   Frequency of analysis of quality control samples

            •   Data Quality Objectives

            •   Analysis of QC samples

            •   Review of results

            •   Corrective action when unsatisfactory results are obtained in the
               analysis of QA/QC samples

            •   Calibration and maintenance of equipment
activities to be undertaken. Development of DQOs usually consists of three processes:
(1) decision definition, (2) data use and needs identification, and  (3) data collection
program design.  DQOs should be continually reviewed during data collection activities
so that any  needed corrective action  may  be  planned  and executed  to  minimize
problems before they become significant.

DQOs play an important role in the selection of sample collection, sorting, and analysis
methods.  In fact, the selection of monitoring methods should be driven by DQOs.  Any
future modifications to monitoring protocols should be considered only after these new
methods meet established data performance criteria.  Data collected using different
methods should not  be compared  unless  information  exists  that  supports such
comparisons.
                                                                             35
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Methods
The methods sections that follow provide a discussion of existing monitoring methods.
These methods provide a starting point for considering monitoring methodologies. New,
cost-effective methods are sure to be developed for certain disciplines in the coming
years.  A key consideration  when  determining  whether new methods are  to  be
incorporated into the monitoring program is whether they meet the performance criteria
of the QA/QC program.  QA/QC considerations in the methods sections focus primarily
on sample collection, processing, storage, and analysis.

QC Samples  - Additional  samples, either collected  in  the field or  prepared from
standard reference materials, are used to check for field  or laboratory contamination,
test laboratory  accuracy, test field or  laboratory  reproducibility,  and assess  natural
variability.  Four basic types of QC samples  should be  submitted with each  set of
sediment or water quality samples (Table 4-4).  Although the frequency with which these
samples are collected will vary among programs, each should be collected at least once
for every 20 samples or sampling event, whichever is more frequent.

Data  from other existing monitoring  programs will often be  used in a  monitoring
program. Therefore, data will frequently be collected by a number of agencies following
different sampling and analytical strategies.  Under such conditions data comparability
becomes a major QA/QC consideration. Data  comparability must be evaluated  based
on both field and laboratory sources of variability.  Individual  programs will need to
address this aspect of QA on a case-by-case basis.
                          Table 4-4.  QC Sample Types
        Travel (trip) Blank


        Rinsate Blank


        Method Blank


        Field Split
Assess cross-contamination during handling
and transport

Assess contamination due to incomplete
cleaning of sample equipment

Assess sample contamination due to
reagents/sample handling in the laboratory

Assess variability in sampling technique and/or
laboratory handling

36
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                                           Section 403 Procedural and Mon/foring Guidance
Statistical Design Considerations - Prior to the collection of data, how the data will be
used to generate pertinent information essential to making sound decisions should be
specified. It is further recommended that analytical performance criteria (e.g., minimally
acceptable Type I and Type II error) be defined to establish quantitative expectations for
the monitoring  program.  The link between data, performance, and  decision-making
should be specified a priori to ensure that appropriate data are obtained and that spatial
and temporal variations are addressed by the monitoring plan.

Selection of the number of replicates is an  important component of program  design.
The use of power analyses, examining alternative sampling  and compositing strategies,
will lead to an effective monitoring design strategy.  These statistical  techniques may
mitigate  the high  costs  of collecting  and processing  samples.    As  a  general
recommendation, samples of equal  size should be collected whenever possible to
simplify statistical analyses.

Composite Sampling - Composite sampling consists of mixing two or more replicates
and/or samples. The chemical analysis of a composite sample provides an estimate of
an  average  contaminant concentration for  locations and/or times  composing  the
composite sample (see space and time bulking below).  Advantages of the composite
sampling strategy are that it provides a cost-effective strategy when individual chemical
analyses  are expensive and it results in  a more  efficient estimate of the mean at
specified sampling locations.

Because  of  the  reduced sample variance, composite sampling  may result in  a
considerable  increase in statistical power.   If the  primary objective  of a  monitoring
program is to determine differences in contaminant  concentrations among sampling
locations, composite sampling is an appropriate strategy.

Space-bulking consists of sampling from several locations and  combining samples  into
one or more  composite samples.  Time-bulking involves  taking multiple samples over
time from a single  location and compositing these samples.  The use of space- and/or
time-bulking  strategies should  be considered  with  the  awareness  that  significant
information concerning spatial and temporal heterogeneity may be lost.

Compositing of  tissues provides a means to  analyze bioaccumulation when the tissue
mass of an individual is insufficient for the analytical protocol.- If mixing species, tissue
composites are likely to be composed of different proportions of species and different
numbers, ages, and sexes of individuals.  These differences among composites  tend to
confound the question of whether  patterns of tissue residue concentrations are due to
differences in locations or interspecific differences in bioaccumulation. Mixing of species
is not recommended because of these confounding effects.
                                                                             37
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Methods
Composite sampling is not recommended if the objective of the monitoring program is to
determine compliance with specified sediment contaminant concentration limits since
this  sampling method  does not  detect the  true  range of sediment  contaminant
concentrations in the environment. The adoption of composite strategies will depend on
the objectives of individual monitoring programs.

Statistical Power - The large degree of temporal and spatial variability  observed for
many ecosystem state and  rate variables requires collection of sufficient replicate
samples to  ensure an  accurate description of the measure of interest.   However,
increases in replication increase sample processing costs.  Power analyses assist in the
allocation of sampling  resources (stations,  replication, and frequency) with  regard to
program finances and design (Sokal and Rohlf, 1981).

Power analyses  may  be applied  to determine the appropriate  number of  sample
replicates and/or subsamples in a replicate composite required to detect a specified
difference (USEPA, 1987d).  Statistical power increases  with the  increase of  sample
replicates. However, there is a point of diminishing  return  of statistical power with the
addition of successive sample replicates (USEPA, 1987d).  The number of replications
required to detect a specified minimum difference is a function of the statistical power
and  the variance in the  data.  Power analyses require  a priori knowledge of the
variability in  the data. A best guess or, preferably, variation observed in historical data is
often used initially in the design of the monitoring program.

For composite samples, statistical power increases with an increase in the number of
subsamples in  each replicate composite sample (USEPA, 1987a); however, with the
addition of successive subsamples to each composite, a diminished return of statistical
power exists. For composites of greater than  10 replications, the increase of power is
negligible given typical levels of data variability.

To improve the power of a statistical test while keeping the significant level constant, the
sample size should be increased; however, because of constraints in cost and time, this
option may  not be available. Power analyses have shown that, for a fixed  level of
sampling effort, a monitoring program's power is generally increased by collecting more
replicates at fewer locations.   The  number  and distribution  of sampling locations
required to evaluate the effectiveness of the monitoring program will depend on the size
and complexity of the site under investigation.

Power-Cost Analyses - The relative power of one design with respect  to another is
more meaningful when the relative costs of implementing alternative designs are taken
into  consideration.  Power-cost analyses  are fundamental in  selecting appropriate
sample/replicate  number, sample location, and  sampling frequency (Bros and Cowell,
1987; Cuff and Coleman, 1979; Ferraro etal., 1989; Millard and Lettenmaier, 1986).
 38
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                                           Section 403 Procedural and Monitoring Guidance
A cost function that describes the cost per replicate sample is required.  Power-cost
formulations for parametric statistical analyses are of the form:

                           Power-cost j = (1 - p j) / (Ci x ni)

where i is a replicate sample collection and  analysis scheme, c is the cost per replicate
sample, n is the  number of replicate samples, and p is  the Type II error.  Costs will
depend on sample collection, sorting, analysis equipment, and protocols.  Type II error
(p)  is a function of the number of replicate samples and the variance detected by a
sampling and analysis scheme.   The measured variance may be influenced by the
sampling equipment used and the unit replicate sample size  (e.g., as the unit replicate
area approaches patch size, variance increases).

Since total costs are usually fixed,  iterations of power-cost formulations for various
sample  processing  scenarios (e.g., sample collection, sorting, and analysis schemes)
and their comparisons will  result  in the  selection  of the appropriate monitoring
methodology.  Sokal and Rohlf (1981) provide a series of formulas for calculating the
most efficient sampling design for a given cost.
                                                                              39
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Methods
4.1  PHYSICAL CHARACTERISTICS

Physical characteristics are typically measured in situ and include temperature, salinity,
density, depth, turbidity, and current velocity and direction.  Physical data are used to
characterize the water column, verify hydrodynamic models, and indicate spatial and
temporal variations.
4.1.1
Rationale
Of the 10 guidelines used to determine unreasonable degradation or irreparable harm,
the following can be assessed by evaluating physical characteristics:

      .   Potential transport of the pollutants by biological, physical, or chemical
          processes and

      •   Marine water quality criteria.

Most chemical and biological processes in the marine environment are affected, either
directly or indirectly, by physical characteristics of the marine environment (Thomann
and Mueller, 1987). Consequently, physical characteristics are used to determine plume
and  sediment movement (hydrodynamics and fate  and transport modeling) and to
provide ancillary information to interpret other water chemistry variables. In addition, to
ensure   protection  of the  characteristic  indigenous   marine  community,  national
temperature criteria have been established in the "Gold Book" (USEEPA, 1986d).

All   biological  monitoring  should  include  consistent  physical  habitat  variable
measurements and  physical habitat matches between  sampling sites and reference
sites  or identified  conditions  in order to accurately assess the  condition  of biological
resources in the  area of sampling.  Physical measurement data such as grain  size,
depth,  salinity,  temperature,  dissolved oxygen,  and  vegetative  cover should  be
measured along with biological variable measurements.

4.1.2     Monitoring Design Considerations

The main uses of physical data  are to characterize the  area in the vicinity of the
discharge site and to serve as  ancillary information  for other variables.  As a result,
temperature, salinity, density, turbidity, and depth (depth of sample and depth to bottom)
should  be collected at every station location.   The cost  of the various methods  is
controlled by the  level of automation and, in comparison, physical data collection  is
relatively inexpensive.  In many  instances, the sampling location and frequency will be
controlled by other more expensively collected variables. The number and location  of
current measurements should be based on the complexity of the circulation patterns and
effluent characteristics.
 40
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                                           Section 403 Procedural and Monitoring Guidance
If  a mathematical  model will  be used  to  assess the  effects  of  ambient physical
characteristics and  the  fate and  transport of the discharged effluent, the monitoring
program should  be  designed to provide the  model with the required forcing boundary
condition data.  It may also be necessary to collect  meteorological data (e.g., wind
speed and direction, temperature, pressure)  for modeling efforts since plumes can be
affected by wind  effects.

General recommendations for sampling locations include the following:

      •  To predict potential transport of the  pollutants by biological, physical, or
         chemical processes, sampling stations need to be located in the vicinity of the
         zone of initial dilution (ZID) boundary, at control sites, and in impacted areas
         to allow adequate correlations to be made between water quality,
         oceanographic measurements, toxic substances, and biological data
         (USEPA, 1982a). Other locations may include the shoreline in swimming and
         shellfishery areas and within the ZID (Koh, 1988).

      •  Additional monitoring stations should be located near other pollutant sources,
         as applicable, to allow the effects of the subject discharge to be distinguished
         from these sources.

      •  Several samples should be taken at various depths across the waste field as
         the plume crosses the ZID boundary.

      •  Sampling  depths are generally acceptable if taken at standard depths (Pond
         and Pickard, 1983); however, features of water masses observed in profiling
         for salinity, temperature, and turbidity should take precedence in establishing
         sample depths. When using in situ methods (e.g., CTD), temperature and
         salinity measurements should be taken at 1-meter intervals over the entire
         depth profile (to within 1  meter of the surface and bottom). In areas of high
         stratification, a smaller interval would be appropriate.

      •  As a practical minimum when using  bottle sampling techniques, samples for
         temperature, salinity, and turbidity should be taken at four depths in the
         vertical profile: (1) 1 meter below the surface, (2) 1 meter above the bottom,
         (3) 1 meter above the pycnocline, and (4) 1 meter below the pycnocline.  If
         the waters are too shallow or no stratification occurs, it would be appropriate
         to take the latter two samples at  evenly spaced distances between the top
         and bottom samples.  In many cases, these depths will coincide with
         variables described in  other sections.

General recommendations for sampling frequency include the following:

      •  Sampling frequency should be high  enough to differentiate between
         high-frequency natural events (e.g.,  tides, storms) and mean conditions.
                                                                             41
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Methods
      •  To establish meaningful estimates of the plume behavior, it is essential that
         the monitoring program encompass the range of conditions that might occur.
         In particular, to obtain seasonal variations, measurements should extend for
         at least 2 years.

      •  Sampling frequency of temperature and salinity should be selected to provide
         data for critical periods (e.g., time of seasonal maximum and minimum
         stratification, low net circulation, time and duration of coastal upwelling, and
         exceptional biological activity).

      •  In monitoring the other physical characteristics of the receiving water,
         sampling events and frequencies should be timed to provide information
         during critical environmental periods.

      •  More frequent sampling in fish and shellfish harvesting areas, and in other
         biologically sensitive areas, may be required.

Moored instrument arrays provide  a cost-effective  method of continual sampling over
long periods  of time (from days to months) for many physical characteristics.  More
commonly used at remote offshore  locations, subsurface arrays can provide unattended
long-term data collection  in  estuarine  environments for  both  physical and  chemical
parameters.  Water depth, velocity  (speed and direction), temperature, and conductivity
or salinity  are  the most commonly measured characteristics.    Estimates of total
suspended  solids  and chlorophyll can  be recorded  also, using  turbidometers,
transmissometers, or fluorometers.  Dissolved  oxygen concentrations can also be
measured.  Sensors capable of in situ measurements of nutrient and trace contaminant
concentrations are under development.

Moored  arrays  have the potential  to provide synoptic coverage  at predetermined
intervals, which can range from minutes to days. The longer the interval between data
recordings, the longer the array can function without replacing the power supply (usually
batteries) or  the  recording  medium  (cassette tape  or  computer  memory  chips).
Real-time telemetry of data to a shore station is possible  with the more sophisticated
(and more expensive) instrumentation packages.  More commonly, the instrument array
is retrieved at regular intervals and the collected data post-processed. Moored arrays
that are deployed for long periods of time are susceptible to many types of interference.

4.1.3    Analytical Methods Considerations

Methods available for measuring physical characteristics include instruments that range
from simple mercury-filled thermometers that provide one observation of temperature to
state-of-the-art  CTD  (conductivity-temperature-depth)  meters that  provide vertical
profiles of salinity and temperature. Methods and/or equipment discussed in this section
are listed in Table 4-5.
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                                                  Section 403 Procedural ancf Monitoring Guidance
                       Table 4-5.  List of Methods and Equipment
Temperature
                                       Turbidity
Salinity
Density
Depth
mercury-filled or dial-type
centigrade thermometer
thermistor
reverse thermometer
bathythermograph
CTD

Knudsen titration
conductivity cells (CTD instruments)
           direct (hydrometers, pycnometers,
           magnetic float densimeters)
           indirect (function of salinity,
           temperature, and pressure)
           fathometer
           meter wheel
           hydrostatic pressure
              bourdon tube
              strain gauge
                                                            light transmissometer
                                                            Secchi disc
                                                            nephelometer
                                                            turbidimeter
                                                  Current Measurements
Eulerian Method (speed and
direction)
    mechanical
    electromagnetic
    acoustic
Lagrangian Method
    drogues
    surface drifters
    seabed drifters
                                       Dye Studies
                                                 fluorometer
                                                                                          43
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Methods
Temperature

Temperature  measurements may be made  with any good grade of mercury-filled or
dial-type centigrade thermometer, or a thermistor (USEPA, 1983a, Standard Method
170.1).  Surface temperatures (from bucket samples) may be  taken using an ordinary
mercury thermometer, taking care not to expose the bucket to the sun (heating) or to the
evaporating influence of the wind (cooling).

For measuring subsurface temperatures, the basic instrument is the "protected reversing
thermometer," a mercury-in-glass thermometer that  is attached to the water sampling
bottle.   When the sample bottle is closed, the thermometer is inverted.  The mercury
"breaks" at a particular point and runs to the other end of the capillary, thereby recording
the temperature of the water at the depth  where the sample was taken (UNESCO,
1988).

A bathythermograph  (BT) consists of a  thermistor  and  an  electronic  pressure
transducer. Wire extending from the BT to the vessel relays measures of temperature
and pressure. BTs may be operated from stationary or slow-moving (<4 knots) vessels.
Expendable  bathythermographs (XBTs)  are  based  on  tracking  an  expendable,
free-fa||jng temperature sensor. The system  is designed to operate from a stationary or
moving vessel (<15 to 20 knots) typically in deep waters.  Wires extending from the
free-falling transducer to the vessel  relay temperature and depth recordings.  The wires
are cut when the probe reaches its maximum depth.  BTs and XBTs do not  record
measures of  salinity.  If it has been determined that density  is not related to salinity,
bathythermographs  may  be a  cost-effective tool.   Monitoring  of  temperature and
pressure using XBTs is higher in cost since the probe is lost after each vertical profile.

Conductivity-temperature-depth meters (CTDs) are the tool  of choice since density
measures are typically dependent  on salinity, temperature,  and  depth  (see Density
below). Measures of density are important in identifying and tracking the movements of
waterbodies  and in determining the depth  at which  a water mass  will settle at
equilibrium. CTDs coupled with a computer can continuously integrate temperature and
conductivity measurements with depth.  The  CTD/computer combination provides
real-time  readouts that allow immediate modifications in  CTD position  and ensures
comprehensive water column coverage during the survey.

Salinity

The Knudsen titration method determines chlorinity by titration with a standard silver
nitrate solution,  and salinity is  then computed from a standard formula (Pond and
Pickard, 1983).  This titration method is practical but not convenient to  use on board a
ship (APHA, 1989).
44
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                                           Section 403 Procedural and Monitoring Guidance
Conductive cells are used for in situ measurements, and a variety of such instruments
are now available from several manufacturers (Pond and Pickard, 1983).  A CTD unit
consisting of conductivity, temperature, and depth sensors is lowered through the water
on the end of an electrical conductor cable, which transmits the information to recording
units on board ship (UNESCO, 1988).

Density

Direct measurement of density is generally not accurate enough for most oceanographic
considerations (Pond and Pickard, 1983). Nevertheless, devices such as hydrometers,
pycnometers, and magnetic float densimeters are sometimes used when great accuracy
is not required.

In the absence of adequate means to measure seawater density accurately and quickly,
it is common practice to determine density indirectly from salinity, temperature, and
pressure.  The density (or the specific gravity) of seawater depends on the temperature,
salinity, and (as a result of the slight compressibility of water) pressure.  In practice,
temperature, salinity, and pressure are measured and the density is  read from tables
that have been prepared from  laboratory determinations (e.g.,  Eugene,  1966).
Algorithms for automated data processing have been developed by UNESCO  and are
applicable for salinities as low as 2 ppt (Fofonoff and Millard, 1983). For lower salinities,
see Hill etal. (1986).
The simplest method to determine depth  is to pass the wire that is attached to the
instrument over a  meter wheel with a known circumference and a counter.  In calm
conditions and negligible currents, the length of wire used will be the actual depth.

Depth may also be estimated using a bourdon tube. The tube moves either the slider of
an  electrical  potentiometer or a strain-gauge pressure transducer, and the resulting
current corresponds to the depth of the instrument.

The total water depth at a  particular station  is typically determined with acoustic
transponders, which are widely available, are relatively inexpensive, and are accurate to
within 0.3 to 0.7 foot (Clausner et al., 1986).

Turbidity

To  determine the transmission of visible  light through the water,  the simplest device is
the Secchi disc, a  black-and-white plate  about 30  cm in diameter. The Secchi disc is
lowered into the sea, and  the depth at which it is lost to sight is recorded (Pond and
Pickard, 1983). Measurements of Secchi disc depth are probably the most widely used
means of estimating light penetration, and they can be used to estimate the attenuation
                                                                              45
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Methods
coefficients for collimated and diffuse light. As a result, the depth of the euphotic zone
may  be estimated.   Secchi  disc  readings vary with the  observer  because of
interpersonal differences in visual ability; therefore, caution must be exercised when
comparing Secchi disc readings taken by a variety of observers.

Turbidity is the measure of particulate matter (plankton and suspended sediments) in
the  water  column.    Measurements,  either  in  situ  or  remote,  are  made  by
spectrophotometric  methods.    This involves passing a light of known intensity and
wavelength through a water sample of precise length. The amount of light received by a
receptor is the percentage of light that has not been absorbed or reflected within the
path length. In remote measurements, water samples are obtained from discrete depths
and then analyzed on deck.  This method of  sampling is quick and accurate, but it is
labor-intensive and is restricted to sampling only at  those depths where water samples
were taken. In situ sampling involves passing a light transmissometer (usually as part of
a CTD system) through the water column. This provides for much higher sampling rates
and less labor.  To correlate data taken from different instruments, all values should be
expressed as a percent transmittance for a standard  path length.

Current Measurements

The two basic ways to describe fluid flow are the Eulerian method, in which the velocity
and direction are stated at every point in the fluid, and the Lagrangian method, in which
the path followed by each fluid particle is stated as a  function of time.

Eulerian Method

Mechanical meters include Savonious rotors,  ducted impellers, drag inclinometers, and
propeller-type meters.  Savonious rotor current meters use a unidirectional rotor on a
vertical axis of rotation and a vane that senses the horizontal direction of flow.  Although
these meters have been used for many years in oceanographic work, they are not suited
for operation in  shallows-water environments,  which are exposed to  wave and  swell
activity.  Ducted impeller current meters have been designed to reduce the  problems
associated with current measurements in the presence of waves (Brainard and Lukens,
1975). Davis-Wellar propeller-type meters are  also designed to operate in the presence
of waves.

Electromagnetic  current  meters measure the instantaneous horizontal  and vertical
velocity components at a flow sensor that contains a wire coil and two orthogonal  pairs
of electrodes.  The coil produces a magnetic field, and the electrodes measure the
voltage gradient across the coil, which is induced by the water as it flows through the
field.  The characteristics of these meters make them suitable for use in the presence of
waves and shallow water (McCullough, 1977).  The most recent electromagnetic current
meters are equipped with several probes to measure other physical characteristics of
the receiving water as well.
46
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                                          Section 405 Procedural and Monitoring Guidance
Acoustic current meters determine the current velocity by emitting a short acoustic pulse
and measuring the Doppler frequency shift of the backscattered signal. Small particles
in the water column (i.e., plankton and suspended sediments)  that float passively with
the current act as reflectors of the acoustic signal.  If the particles are moving toward the
sound source, the frequency is shifted higher, whereas it is shifted lower if the particles
are moving away. The frequency shift is proportional to the relative velocity between the
source and  the reflectors.  By range gating, return signal velocity  measurements at
discrete depths can be made. This lets one instrument take remote samples throughout
the water column from a depth  of 3 meters to over 1,000 meters. Another advantage of
this technology is that the current meter can be placed on a ship  and sampling can be
conducted while the ship is under way.  This allows for a synoptic measurement of the
current field over a large area with the use of only one instrument.  Acoustic current
meters  are  suitable for use  in the presence of waves, but the  accuracy of  the
measurements  decreases close to the sea  surface.   These instruments  are  also
expensive and require highly trained technicians to maintain them.

Another technology  employs  the use  of land-based radar systems that send  out
electromagnetic waves  and measure the reflected energy from the surface waves
(Appell and Curtin, 1990; Appell and Woodward, 1986).  Since  this system is set up on
the shore, it works out to only a few dozen kilometers offshore and measures only the
current  velocity in  the top meter of the water column.  This  system does have the
advantage that a ship is not needed to deploy or retrieve the instrument.

Lagrangian Method

Most drogues are drogue-buoy systems consisting of a small  marker buoy that is
tracked  at the surface and a larger submerged drogue portion that is set at the desired
depth by a connecting line between the two portions (USEPA, 1982a).  The drogue
portion  must be weighted and ballasted  so that the drogue assembly has  sufficient
negative buoyancy to keep both the drogue and connecting line  in their intended vertical
orientation and to keep the buoy mast upright.  Drogues are intended to passively drift
with the currents at a specified depth. In reality, some error is introduced in the drogue
trajectories by wind drag on the exposed portion  of the marker  buoy; by the relative
surface  current drag on the submerged  portion  of the  surface  buoy;  and,  for deep
drogues with long lines, by the relative current drag on the connecting line.  If possible, it
is best to avoid drogue studies under high-wind conditions, especially when measuring
lower current speeds in deeper waters. Drogues can be used for periods ranging from a
couple of days to many months and can be tracked by satellite.

Most drogues can  be classified into one of the following four categories: (1) parachute
drogues, (2) cruciform drogues, (3) window shade drogues, and (4) cylindrical drogues.
Of these types, parachute and cruciform drogues are the most widely used  (USEPA,
•1982a).
                                                                             47
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Methods
Surface drifters are used to measure the average path of currents at the surface.  Drift
bottles and drift cards are the types most commonly used.  Vertical drift bottles and drift
cards are useful for evaluating  the movement of effluent that reaches the surface.
Horizontal drift cards, which float on the surface, may be used to determine the potential
movement of surface slicks due to oils or other floatables that form a surface  film.
Because these movements  are  influenced largely by the wind, horizontal drift cards
should be released under several different conditions (Grace, 1978).

Seabed drifters measure the average  path of currents  near the sea floor.   They are
useful  for determining the  fate  of  waste  materials subject to transport  by bottom
currents.  This  includes settleable solids and any portion of the effluent that remains
near the bottom.  The drifters provide information on the net movement of a waste field
along the bottom, including where the waste field may reach the shoreline, and a rough
estimate of how long it may take. If a sufficient number of drifters are recovered,  they
may indicate areas of possible shoreline contamination (Grace, 1978).

Dye Studies

Dye studies involve the  continuous or plug injection of a tracer (rhodamine WT or
rhodamine B) into a waste stream before  it is discharged. Fluorometers are used to
estimate the  concentration of  the sample  containing a fluorescent substance.   The
intensity of the fluorescent  light is compared  with readings for samples  with known
concentrations (standards) under the same conditions (Wilson, 1986). The advantages
of dye tracing are (1) low detection and measurement limits and (2) simplicity and accuracy
in measuring dye tracer concentration techniques.

Acoustic technology is  currently being  tested  to characterize and track oceanic
wastewater plumes.  Results from several tests conducted off the coast of Florida and
dye release experiments have been published (Demmann et al., in press).

4.1.4    QA/QC Considerations

All environmental monitoring programs should have a written and approved quality
assurance project plan (USEPA,  1987c). .Tables 4-6 and  4-7 list sample preservation
and  storage  requirements  and  recommended  analytical methods for salinity  and
temperature.  When several methods  are available, the selection should be made by
comparing the accuracy and precision of the candidate methods for the parameter range
expected  at the  site.   All  monitoring  programs  should   follow  manufacturers'
recommended calibration procedures.  In addition to these general considerations, the
following specific recommendations should be adopted as appropriate:

      •  Each temperature-measuring  instrument should be calibrated against a
         precision thermometer, certified by the National Bureau of Standards, at least
         every week (USEPA, 1987c).  It is recommended that calibration be calculated
         daily when temperature violation is suspected. Temperature probes may, at
48
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                                             Section 403 Procecfura/ and Monitoring Guidance
   Table 4-6.  Recommended Sample Preservation and Storage Requirements


                                                                    Holding Time
Parameter
Volume
Required (ml_)
Container     Preservative
Salinity
                240
Temperature
                1,000
                    G (with
                    paraffined
                    corks)
                    P,G
             None
             None
1 hour
(longer if
properly sealed
airtight)

Immediate
Note:     P = polyethylene, G = glass

SOURCES: APHA, 1989; USEPA, 1983.
                   Table 4-7.  Recommended Analytical Methods
Parameter
Salinity
Temperature
Method
Induction salinometer
or titration. Method
2520 (APHA, 1989).
Bathythermograph or
thermometric. EPA Method
170.1 (USEPA, 1983a).
Precision
titration:
±0.05 ppt
±0.05°C
Detection
Limit
Desired
1ppt

Significant
Figures
Desired
4
3
Note:     The water column, being a 3-D medium, requires greater numbers of samples to be collected
         (versus a 2-D medium) in order to be adequately described.
                                                                                  49
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Methods
         best, be accurate to within one-tenth of a degree Celsius. Temperature
         probe systems are rarely linear over large temperature ranges and must be
         checked against research-grade laboratory thermometers (APHA, 1989).

         Salinity probe systems offer moderate accuracy but should be cross-checked
         by discrete water samples analyzed by induction-type laboratory
         salinometers (APHA, 1989). Two standard determinations should be made
         before the start of each series of samples.  In addition, one standard sample
         should be analyzed to monitor instrument drift. It is recommended that
         duplicate determinations be made for at least 10 percent of the samples
         analyzed.

         Many multiprobe in situ measurement systems incorporate depth
         measurements by use of pressure transducers.  The accuracy and precision
         of such systems must be periodically checked. A pressure calibration can be
         done in a laboratory pressure chamber. Daily calibration may be done in
         calm waters of a known depth (e.g., inside a lagoon or a marina). Precision
         is determined by multiple measurements at the same depth.  Accuracy is
         evaluated by comparison to measurements made with a heavily weighted line.

         Instruments that measure turbidity require regular calibration. In situ
         instruments are most accurately calibrated by the use of primary standards,
         usually a carefully prepared suspension of Formalin or other approved
         standard. Transmissometer-type instruments can be calibrated  by
         comparison to solutions of known total suspended solids. These solutions
         should approximate the turbidity of the water that is to be sampled.  For other
         instruments, including those nephelometers or turbidity meters that measure
         discrete samples, either on deck or  in the laboratory, calibration  using an
         approved primary standard suspension is recommended (APHA, 1989).
         Calibration in all cases should be conducted at the start of each  series of
         analyses and after each group of 10 successive samples. Duplicate
         analyses should be conducted on at least 10 percent of the total number of
         samples (U.S. EPA, 1987c).

         Transmissometers can be calibrated in air and must be strictly cleaned.

         A standard suspension of Formalin, prepared under closely defined
         conditions, is used to calibrate the nephelometer (USEPA, 1983a, Method
         180.1). The nephelometer should be calibrated at the start of each series of
         analyses and after each group of 10 successive  samples. Duplicate
         analyses should be conducted on at least 10 percent of the total number of
         samples (USEPA, 1987c).

         Current meters should be calibrated before  and after each major deployment
         of instruments.

         UNESCO (1988) presents details on the calibration of CTD instruments.
so
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                                           Section 403 Procedural and Monitoring Guidance
      •  Fluorometer calibration performed prior to use with a series of prepared
         concentrations, blanks, and spikes is recommended as a quality control
         check (Wilson, 1986).

4.1.5    Statistical Design Considerations

Temporal Analyses

In general,  one  would  expect to find temporal  and spatial  patterns  in  physical
characteristics. As a result, the primary purpose of measuring physical characteristics is
to describe what these patterns are (e.g., range, seasonal variations) and to determine
the primary physical mechanisms affecting  the monitored area.  As a result, statistical
comparisons between sampling locations and sampling periods are not common except
for turbidity.  In this instance, the reader is referred to Section 4.2, Water Chemistry, for
analysis approaches that would be appropriate.  All raw data should have error limits
associated with them to indicate the level of confidence in the data.

Tidal and seasonal variability in the distribution and magnitude of water column physical
characteristics is  typically observed (Day et al., 1989).  Time  series  analyses (e.g.,
temporal autocorrelation  and  spectral  analyses) may be necessary to examine the
effects of the cyclical influences and/or filter out these cyclic forcing functions in order to
examine long-term temporal trends.

Graphical Representation

The  physical characteristics data described in this section are typically summarized in
graphical form. The most common summary graphs include  temperature-depth profiles,
salinity-depth profiles, and temperature-salinity plots. Physical oceanographers typically
analyze characteristic diagrams of water properties.  The  most common curve is a
temperature-salinity (T-S) plot.

Alternatively,  horizontal sections (e.g., contour maps) of  properties  are  useful  for
displaying geographical distributions. Multiple physical characteristics may be displayed
by displacing one graph above the other.  More details about analyzing characteristic
diagrams can be found in Pickard and Emery (1982).

Current data can be displayed in numerous fashions including stick plots, a time series
of rectangular components, and horizontal maps (Pickard  and  Emery, 1982). These
figures are typically plotted after  periodic  components have been removed through
spectral analysis, leaving only the residual nonperiodic components.
                                                                              51
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Methods
4.1.6
Use of Data
Data  describing the physical parameters  can be  used to determine water  column
stability, characterize oceanographic conditions in the discharge area, determine initial
dilution, assist in the prediction of plume behavior, identify the area impacted  by the
discharge, and identify existing and potential problem areas for fish  and sensitive  life
stages of marine organisms (e.g., upwelling areas).

Salinity and temperature can be used, together with current speed and direction, to aid
in describing mass movement of diluted waste  plumes to far-field sites where impacts
must be assessed (Wright, 1988).  Furthermore, salinity, temperature, and currents are
used to assist in evaluating the results of benthic and other biological responses.

Current-speed data may be used  to determine the distance from the outfall that the
sediment will travel before accumulating on the bottom.

Dye studies are particularly useful to evaluate the movement of the plume, the likelihood
that the  plume  will  reach the shoreline or nearby biologically sensitive  areas, the
potential  for  re-entrainment of previously discharged effluent,  and the recirculation
potential  under short-term influences  for existing discharges, as well as the  general
circulation patterns in the vicinity of discharge sites (USEPA, 1982a).

Physical  characteristics data  are  used to  calibrate and  validate hydrodynamic and
dispersion plume  models (Davis,  1988;  Leighton et al., 1988; USEPA, 1982a).  For
relocated or  proposed  discharges, numerical circulation and transport models are the
most  useful  methods for assessing the effects of ambient currents and stratification on
dispersion  and  transport  of  the  waste  field  and  for  estimating  the  potential  for
recirculation of previously discharged effluent. Statistical analysis can be performed to
forecast plume direction under varying physical conditions.

Turbidity data can be used to estimate the reduction in light transmittance.  Reduction of
the depth to which sunlight penetrates,  due to an increase in turbidity, will cause a
decrease in the compensation depth of phytoplankton,  resulting in reduced biological
growth (Thomann and Mueller, 1987).

4.1.7     Summary and Recommendations
Rationale
          Most chemical and biological processes in the marine environment are
          affected, either directly or indirectly, by the physical characteristics of the
          marine environment.
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                                          Section 403 Procedural and Monitoring Guidance
      •  Physical data can be used to determine plume and sediment movement
         (hydrodynamics and fate and transport modeling) and to provide ancillary
         information to interpret other water chemistry variables.

      •  National temperature criteria to ensure protection of the characteristic
         indigenous marine community have been established in the "Gold Book" for
         temperature (USEPA, 1986d).

Monitoring Design Considerations

      •  In general, data on temperature, salinity, density, turbidity, and depth (depth
         of sample and depth to bottom) should be collected at every station location.
         Sufficient data should be collected in the vertical profile for temperature and
         salinity. Common practice is to collect one data point per meter of depth.

      •  The number and location of current measurements should be based on the
         complexity of the circulation patterns and the effluent characteristics.
         Stations should be located in the vicinity of the zone of initial dilution (ZID)
         boundary, at control sites, and in impacted areas.

      •  If a mathematical model will be used to assess the effects of ambient physical
         characteristics and the fate and transport of the discharged effluent, the
         monitoring program should be designed to provide the model  with the
         required forcing boundary data.

      •  Data should be  collected at  a frequency sufficient to characterize seasonal
         patterns and critical periods (e.g., time of seasonal maximum  and minimum
         stratification, low net circulation, time and duration of coastal upwelling, and
         exceptional biological activity).

      •  Estimates of the error should be made with each variable.

Analytical Methods Considerations

      •  Temperature measurements may be made with:

         -  Mercury-filled or dial-type centigrade thermometer
         -  Thermistor
         -  Reverse thermometer
         -  Bathythermograph
         -  CTD instrumentation

      •  Salinity measurements are preferably made with:

         -  Conductivity cells (CTD  instruments)
                                                                              53
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 Methods
       •   Density measurements are preferably made with:

          -   Indirect methods (function of salinity, temperature, and pressure)

       •   Depth measurements are preferably made with:

          -   Hydrostatic pressure techniques

       •   Turbidity measurements may be made with:

          -   Light transmissometer
          -   Secchi disc

       •   Current measurements may be made with:

          -   Eulerian methods - propeller-type meters, electromagnetic meters,
             acoustic-doppler meters, etc.
          -   Lagrangian methods - drogues, surface drifters, etc.

       •   Plume studies may be performed with:

          -   Fluorescent dyes injected into the waste stream
          -   Emerging acoustic technologies

QA/QC Considerations

       •   A multistep QA/QC program must be implemented to verify that all
          calibrations were conducted and properly applied to data and, where
          possible, to ensure historical verification.

       •   All equipment must be calibrated regularly.

       •   Blank, spike recovery, and replicate analyses are recommended quality
          control checks for those parameters to which they can be applied.

       •   For replicates, a minimum of three repetitions is required for statistical
          significance.

Statistical Design Considerations

       •   The primary purpose is to describe the patterns of physical characteristics
          (e.g.,  range, seasonal variations) and to determine the primary physical
          mechanisms affecting the monitored area. As a result, statistical
          comparisons between sampling locations and sampling periods are not
          common except for turbidity.

       •   Common graphical summaries include:
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                                            Section 403 Procedural and Monitoring Guidance
            Temperature-depth profiles, salinity-depth profiles, and
            temperature-salinity plots.
            Horizontal sections (i.e., contour maps) of properties.
            Time series data, which are often displayed as a function of time with
            multiple physical characteristics displayed by placing one graph above the
            other.
            Current data, which can be displayed with stick plots, a time series of
            rectangular components, and horizontal maps.
Use of Data
          Physical characteristics are used to determine water column stability, to
          characterize oceanographic conditions in the discharge area, to determine
          initial dilution, to assist in the prediction of plume behavior, to identify the area
          impacted by the discharge, and to identify existing and potential problem
          areas for fish and sensitive life stages of marine organisms (e.g., upwelling
          areas).

          Statistical studies can be performed to forecast plume direction under varying
          physical conditions.

          Dye studies are particularly useful to evaluate the movement of the plume,
          the likelihood that the plume will reach the shoreline or nearby biologically
          sensitive areas, the potential for re-entrainment of previously discharged
          effluent, and the recirculation potential under short-term influences for
          existing discharges, as well as the general circulation patterns in the vicinity
          of discharge sites.

          Physical characteristics data are used to calibrate and validate hydrodynamic
          and dispersion plume models.

          Turbidity can be used to estimate the reduction of light transmittance.
          Reduction of the depth to which sunlight penetrates, due to an increase in
          turbidity, can reduce biological community growth.
                                                                                55
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Methods
4.2
WATER CHEMISTRY
Monitoring of  water chemistry  parameters  is conducted  to  evaluate the quality of
receiving waters.   Commonly employed EPA methods were  intended for use  in the
detection of pollutants at high concentrations relative to those usually found in natural
waters. Much attention has been devoted recently to the need for the development of
more appropriate  methods, but  evaluation and validation of these methods have not
been conducted on a wide scale.  Until these and other QA measures are developed,
the traditional  EPA methods are generally the methods of choice for the analysis of
water  quality.   Recommended  Protocols  for  Measuring  Selected Environmental
Variables in Puget Sound (USEPA,  1986-1991)  contains  discussions of QA/QC
concerns, sampling considerations,  and recommended instrumental methods.  The
Compendium of Methods  for Marine and Estuarine Environmental  Studies (USEPA,
1990b) includes a  number of method variations for nitrogen species, phosphorus, and
chlorophyll.
4.2.1
Rationale
Of the 10 guidelines used to determine unreasonable degradation or irreparable harm,
the following can be assessed by evaluating the water chemistry:

      •   Potential transport of the pollutants by biological, physical, or chemical
          processes;

      •   Potential direct or indirect impacts on human health; and

      •   Marine water quality criteria.

The  presence of toxic material in marine  waters due to point source discharges can
have adverse ecological and human health effects.  Near-field monitoring of chemical
concentrations can provide a spatial and temporal record of contamination in the water
column. Water quality chemistry measurements should be made as part of a monitoring
program to assess the effectiveness of the permit guidelines in preventing unreasonable
degradation of or irreparable harm to the marine environment.

4.2.2     Monitoring Design Considerations

SelectiorLof Analytes

The  chemicals that should be included in the monitoring program  are those chemicals
known or suspected to be in the discharge, as well as possible by-products.  Even
though potentially toxic  compounds in  the discharge  may not be fully known,  the
objectives of the program must be clearly defined. It may be necessary at the beginning
of the program to conduct an analysis for all  chemicals on the EPA-defined  Priority
Pollutant, Hazardous Substance, or Target Compound/Analyte Lists to determine which
are present; however, detection  in the  environment does not always correlate with
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                                          Section 403 Procedural and Monitoring Guidance
biological risks  and effects.  Furthermore, many of the chemicals that  pose risks to
marine systems are hydrophobia Water column monitoring for hydrophobic chemicals
may indicate concentrations that are several orders of magnitude below concentrations
in sediments and biota.  Unless compelling site-specific conditions warrant otherwise,
water column monitoring for hydrophobic chemicals is not recommended.  Limitations in
analytical  methodology, modeling   techniques,  and  toxicological  data restrict  the
usefulness  of the resulting  data.  In some  cases, levels of contaminants that are of
concern to  the health of organisms are below the lowest limit of detection of the best
available analytical methodology.

Sampling Considerations

Selection of sampling locations and  choice of sampling techniques are crucial decisions
to  be  made before the sampling  effort is initiated.  Recommended  Protocols for
Measuring  Selected Environmental Variables in Puget Sound (USEPA,  1986-1991)
contains discussions of  quality assurance requirements and sampling considerations
that are applicable to water quality monitoring. Sampling  must incorporate sites within
the zone of initial dilution, extending out to areas of low contaminant concentration and
farther to unaffected areas to address the complete spatial variability.

Water column  sampling  design  must consider not only the horizontal location of the
sampling stations, but also the vertical location within the water column and the time of
sample  collection.   Because the majority  of chemical analyses are performed, by
necessity, on discrete samples, consideration should be given to the number of depths
at which samples are collected. Obviously, such decisions will be based on site-specific
characteristics, such as water depth, contaminants of concern, suspected sources, and
the sensitivity or importance of  local biota.  Costs related  to the laboratory analyses
should also be weighed against the number of samples desired.

Water Column Sampling Equipment

Water column samples are frequently collected using bottle samplers. These are simple
devices, usually consisting  of cylindrical tubes with stoppers at each  end  and a closing
device that  is activated by an electrical signal.  Each bottle samples a discrete parcel of
water at a designated depth. Multiple samplers are fixed on a rosette frame in order that
several depths may be sampled during one  cast and/or that replicate samples may be
collected at a particular depth.  The most commonly used bottle  samplers include the
Kemmerer,  Van Dorn,  Niskin, and Nansen samplers (USEPA, 1987c). Alternatively, a
pump may be used to sample the water column (USEPA, 1987c).

Depending  on the depth of the water at the sampling site, samples should be taken at a
minimum of four depths in the vertical profile: (1)  1 meter below the surface, (2) 1 meter
above the  bottom,  (3) 1 meter above the pycnocline, and (4)  1  meter  below  the
                                                                             57
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Methods
pycnocline.   If the  waters are too shallow or no stratification occurs,  it would be
appropriate to take the latter two samples at evenly spaced distances between the top
and bottom samples.

Caged Organisms

The  California Mussel Watch  Program  and the National Oceanic and Atmospheric
Administration (NOAA) Status and Trends Program have employed the use of caged
transplanted mussels to monitor bioaccumulation of toxic chemicals over space and time
(Ladd et al.,  1984;  Goldberg  et  al.,  1978).   Caged  sentinel  species  offer several
advantages:

      •  They concentrate contaminants (up to 102-105 fold vs. ambient waters),
         facilitating laboratory analyses of contaminant concentrations.

      •  They provide a means of temporally integrating water quality conditions.

      •  They provide an assessment of biological availability of water column
         contaminants.

      •  They may be deployed and maintained in a number of diverse locales.

However,  the disadvantages of caged  organisms  include  the cost of transplanting
organisms, the possibility of loss  of  the cage/buoy system,  and the  possibility  of
introducing a "nuisance" species. Species have different bioaccumulation potentials for
various contaminants. It is recommended that multiple sentinel species be deployed to
ensure that a number of contaminants are sufficiently evaluated and a comprehensive
characterization of water contaminants is conducted.  Unfortunately, formulations that
would allow comparisons  of bioaccumulation data between different species  and/or
different tissues types have not yet been developed and comparison of tissue burdens
among the species is not acceptable.  Thus, it is essential that monitoring design
elements be standardized to allow for comparisons among studies.

4.2.3    Analytical Methods Considerations

Questions  to be  considered during the choice of  an appropriate analytical  method
include the parameters of interest, desired detection  limits, sample size requirements or
restrictions, methods of preservation, technical and  practical holding times, and matrix
interferences (especially from saline water).  D'Elia et al. (1989) discuss the common
analytical problems encountered during monitoring analyses of water samples.

Selection of appropriate methods should  be based on a compromise between full-scan
analyses,   which  are  economical but  cannot  provide  optimal  sensitivity for  all
compounds, and alternative methods that are more sensitive for specific compounds but
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                                          Section 403 Procedural and Monitoring Guidance
can result in higher analytical costs. A list of analytical techniques is presented in Table
4-8.  Table 4-9 shows which technique is appropriate for detecting a specific organic
contaminant.

Dissolved Oxygen

The titrimetric, or Winkler, method is the first method of choice for the measurement of
dissolved oxygen  (USEPA,  1983a).   The membrane  electrode method (360.1) is
recommended for samples containing interferents such as sulfur compounds, chlorine,
free iodine, color, turbidity, or biological floes, and also when continuous monitoring is
planned (USEPA, 1983a).

Nutrients

Nutrients such as ammonia nitrogen, total Kjeldahl nitrogen, nitrate-nitrite nitrogen, and
phosphorus are determined by spectrophotometric  measurements using a segmented
continuous  flow  analyzer, where samples and  reagents are  continuously added in
sequence separated by air bubbles and pumped through glass tubing (USEPA, 1983a).
Successive analyses can  be accomplished in  less time than would be required by
manual  methods because each analysis is  not carried to completion, but is brought to
the same stage of development and exposure by the timing of the stream flow through
the system.  Possible interferences include suspended solids, metal ions, and  residual
chlorine.

Ammonia nitrogen is determined  by treating the  samples with alkaline phenol  and
hypochlorite to produce indophenol blue, which is intensified with sodium nitroprusside.
Standard solutions should be made up using substitute sea water to approximate the
matrix of the samples (USEPA, 1983a). Total Kjeldahl nitrogen is defined as the sum of
free ammonia and organic nitrogen compounds, which  are converted  to ammonium
sulfate under the conditions of digestion (USEPA, 1983a).  Sulfates react with nitrogen
compounds of biological origin, but may not convert nitrogenous compounds of some
industrial wastes.  For the determination of nitrate-nitrite nitrogen, a filtered sample is
passed  through a granulated copper-cadmium column to reduce any nitrate to nitrite
(USEPA, 1983a).  The nitrite is then transformed to a highly colored azo dye, which is
measured spectrophotometrically.

Total phosphorus is determined by heating the samples in the presence of sulfates, then
cooling and measuring spectrophotometrically (USEPA, 1983a).

The fluorometric method for chlorophyll a is more sensitive than the spectrophotometric
method.  The sample is subjected to an excitation wavelength, and the fluorescence is
measured at a second emission wavelength (APHA, 1989).   The greatest uncertainty in
the method  is   the  choice of  reference  standard.     High  performance liquid
                                                                            59
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Methods
                     jUjtiJilL..*     *~t t v

                      Table 4-8. List of Analytical Techniques
DISSOLVED OXYGEN

   •   Winkler Titration

   •   Membrane Electrode

NUTRIENTS

   •   Continuous Flow Spectrophotometry
       - Ammonia Nitrogen
       - Total Kjeldahl Nitrogen
       - Nitrate-Nitrite Nitrogen
       - Total Phosphorus

   •   Fluorescence Spectrometry
       - Chlorophyll a

   •   High Performance Liquid Chromatography (HPLC)

TRACE METALS

   •   Atomic Absorption Spectrophotometry (AAS)
       - flame
       - graphite furnace (GFAAS)
       - cold vapor
       - gaseous hydride (HYDAAS)

   •   Inductively Coupled Plasma Emission
       Spectrometry (ICP)

ORGANICS

   •   Gas Chromatography
       - with electron capture detection (GC/ECD)
       - with mass Spectrometry (GC/MS)

   •   Liquid Chromatography
       - with mass Spectrometry (LC/MS)
USEPA method 360.2

USEPA method 360.1
USEPA method 350.1
USEPA method 351.2
USEPA method 353.2
USEPA method 365.4
Standard Method 10200
USEPA 200 series methods
USEPA method 200.7
USEPA 500/600 series methods
Chromatography (HPLC) is the most accurate of the methods used for the analysis of
chlorophyll (since all forms can be separately quantitated); however, it is also the most
expensive.
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                                           Section 403 Procedural and Monitoring Guidance
         Table 4-9.  USEPA Organic Contamination Detection Techniques
USEPA Method3
   Contaminant Type
 Method
601
602
603
604
606
608
609
610
611
614
624
625
F'urgeable Halocarbons
F'urgeable Aromatics
Acrolein/Acrylonitrile
F'henols
F'hthalate Esters
F'CBs and Organochlorine Pesticides
Nitroaromatics and Cyclic Ketones
Polycyclic Aromatic Hydrocarbons
Haloethers
Organophosphorous Pesticides
Volatile Organics
Semivolatile Organics
GC-ELCD
GC-PID
GC-FIDandPID
GC-FID
GC-ECD
GC-ECD
GC-FID-ECD
HPLC-UV-FL or GC-FID
GC-ECD
GC-FPD or NPD
GC-MS
GC-MS
 1 USEPA, 1983a.
Trace Metals

The choice of analytical method for trace metal analysis is determined by the required
detection  limit.  Inductively coupled plasma emission spectrometry (ICP) allows the
simultaneous measurement of several elements; however, the achievable detection
limits are  usually not as low as those  obtained by graphite furnace or hydride atomic
absorption spectrophometry (AA). The  combination of AA and ICP is the recommended
analytical  method for detection of metals since no technique is best  for all elements.
Cold vapor AA is the recommended technique for the analysis of mercury.

Graphite furnace AA is more sensitive than flame AA or  ICP  but is more subject to
matrix and spectral interferences.  Such interferences result in  potential quality control
problems  during the analyses and uncertainties in the resulting data.   Because of the
lower concentrations that can be seen by graphite furnace AA, particular caution  must
be taken with regard to laboratory contamination.  The concentration of each element is
determined   by  a separate analysis,   making the  analysis  of  a large  number of
contaminant  metals both labor-intensive and relatively expensive compared  to ICP. AA
                                                                               61
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Methods
methods may, however, be cost-effective for the analysis of a few metals over a large
number of samples.   It should  be noted that "ultraclean" sampling and analytical
techniques should be used for trace metal samples in open ocean waters.

Semivolatile Organic Compounds

Analysis of semivolatile organic compounds involves a solvent extraction of the sample,
cleanup of the extract, gas chromatographic (GC) separation, and quantitation. There
are  two  gas chromatography/mass spectrometry (GC/MS)  options  for detecting
extractable organic compounds:  internal standard  and isotope dilution.  The isotope
dilution  technique,  which requires  spiking  the sample  with a  mixture  of stable
isotope-labeled analogs of the analytes, is recommended because  reliable recovery
corrections can be made for each analyte with a labeled analog or a chemically similar
analog.  This method is more  expensive and less  widely  employed  than the internal
standard method, which is the  current method of choice in EPA's Contract Laboratory
Program.  Mass spectrometry provides positive compound identification by comparison
of both retention time and spectral patterns with standard compounds.

Many organic compounds can be identified by gas chromatography/electron capture
detection  (GC/ECD)  analysis.  GC/ECD provides  greater  sensitivity (lower detection
limits) relative to GC/MS; however, GC/ECD  does not  provide positive compound
identification.  Since the identity  of a compound  is determined solely  by matching
retention time with that of a standard, confirmation of the initial identity of the compound
on a second GC column is required for confidence in the reliability  of the qualitative
identification  of the  compound.   When  a compound is  present  at a high  enough
concentration, its identity should be confirmed by the use of GC/MS.  GD/ECD is the
method  of choice for pesticides and PCBs.  Glass capillary GC achieves a much higher
degree of resolution  in the analysis of the  PCB congeners than the  standard packed
column  methods; however, few labs  regularly employ this specialized technique.  The
liquid chromatography/mass spectrometry (LC/MS)  method is being developed for the
detection and quantitation of nonvolatile organic compounds.

Volatile Organic Compounds

The  purge-and-trap  GC/MS  technique is commonly employed for detecting volatile
organic compounds in water.  As in the case of semivolatiles, GC/ECD may be used to
achieve lower detection limits, although this introduces a  level of uncertainty to the
qualitative identification of compounds. The isotope dilution technique is recommended
if the Data  Quality  Objectives (DQOs)  of the  monitoring program  require  accurate
quantitation of each  compound. This technique, however,  carries additional analytical
time  and expense.
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                                          Section 403 Procedural and Monitoring Guidance
4.2.4
QA/QC Considerations
Measurement data are no better than the planning that goes into setting data quality
objectives,  choosing appropriate sampling and analysis methodologies, and  setting
quality control  criteria.  An  expert chemist and the references cited in this document
should be used to develop a comprehensive QA plan.

Sample Collection

The primary criterion for an adequate sampling device is that it consistently collect
undisturbed and  uncontaminated samples.  Water column sampling devices should be
inspected for wear and tear leading to sample leakage upon ascent.  It is prudent to
have a backup sampler on board the survey vessel in case the primary sampler is found
to be unsuitable during the cruise.

In the field, sources of contamination include sampling gear, lubricants and oils,  engine
exhaust, airborne dust, and ice used for cooling samples. If samples are designated for
chemical analysis, all sampling equipment (e.g., siphon hoses, scoops, containers) must
be made of noncontaminating material and must be cleaned appropriately prior to use.
Potential airborne contamination such  as stack gases and cigarette  smoke should be
avoided.  Furthermore, samples and sampling containers should not be touched with
ungloved fingers.  Sampling containers and preservatives are summarized in Table
4-10.

Splitting  water  samples  for   ultraclean  chemical  analysis  (for  open  ocean
samples—especially for metals) and the analyses themselves should be conducted with
noncontaminating tools under "clean room"  conditions.   Recommended container
materials, sample sizes, preservation techniques, and analytical holding times should be
determined before the sampling effort is undertaken.

Various types of  blanks can be prepared and analyzed to identify possible sources of
sample contamination.   Field blanks are  aqueous  samples manipulated in the field.
Rinsate blanks are aqueous samples that are poured over the sampling equipment and
collected.  Filtration blanks are aqueous samples drawn through the filtration apparatus.
Trip blanks are packed in the sample coolers to detect cross-contamination of volatile
organic compounds.

Blind duplicates,  treated and identified  as separate samples, may be sent to the same
laboratory for analysis,  or one of the pair may be sent to a  "reference" laboratory for
comparison. Standard reference material may  be prepared and sent as a performance
check to the laboratory.  Depending on how these types of samples are handled, they
may be used as a check for accuracy and/or precision.
                                                                            63
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Methods
Table 4-10. Sample Preservation and Storage Parameters
Sample
Analyte
Nutrients
Ammonia
Nitrate
Nitrate + Nitrite
Nitrite
Total or dissolved metals
(except Hg)
Total or dissolved Hg
Particulate metals
Semivolatiles
Volatiles
Container3

P,G
P,G
P,G
P,G
P,G,TFEC
G.TFE
P,G
G
G
Size

500 mL
100mL
200 mL
100ml_
500mL
250 mL
1 gal
1 L
40 mL
Storage
Preservative

H2SO4, pH<2b
HaSCH, pH<2b
refrigerate
H2SO4, pH<2b
H2SO4, pH<2b
HNOs, pH<2
HNOs, pH<2
_d
Cool, 4°C
extraction
Cool, 4°C
Lifetime

28 days
48 hours
28 days
48 hours
6 months
28 days
6 months
7 days to
7 days
a P = linear polyethylene, G = borosilicate glass, TFE = tetrafluoroethylene.

b It is recommended that samples be analyzed as soon as possible.

0 If aliquot for Hg taken from this sample, cannot use linear polyethylene.

d Samples should be filtered as soon as possible and always within 24 hr.
Laboratory Analyses

Laboratory performance standards as  measured by method  QC protocols  should be
used to evaluate  and  select appropriate analytical methods.   Changes in selected
laboratory protocols should only be considered and/or approved if proposed procedures
meet or exceed established performance criteria.
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                                           Section 403 Procedural and Monitoring Guidance
QA reports should describe the results of quantitative QC analyses, as well as other
elements critical to accurate interpretation of analytical results. It is recommended that
these reports be recorded and stored in a data base for future reference.

Trip blanks indicate whether any contamination occurred in the field or during shipping
of samples.  Rinsate  blanks are used  to check for contamination due to inadequate
cleaning of field equipment.

Field  splits, treated and identified as separate samples, may be sent to  the same
laboratory for analysis or one  sample may be sent to a  "reference"  laboratory for
comparison.  Standard reference material may be  placed in a sample container at the
time of collection and sent "blind" to the laboratory.

Calibration standards  should  be analyzed  at the beginning of sample analysis and
should be verified at the end of each 12-hr shift during which analyses are performed..
The  concentrations of  calibration standards  should bracket the expected  sample
concentrations, or sample dilutions or sample handling modifications  (i.e., reduced
sample size) will be required.

Analysis of method or  reagent blanks should be conducted to demonstrate the absence
of contamination from sample handling in the laboratory. At  least one method blank
must be included with  each batch of samples and should constitute  at least 5  percent of
all samples analyzed.  All  blanks should be free  of detectable concentrations of the
target compounds, or steps should be taken to remove the source of contamination
before sample analysis proceeds.

Spike recovery analyses are required to assess method performance for the particular
sample matrix.  Spike  recoveries serve as an indication of analytical accuracy, whereas
analysis of standard  reference  materials (SRMs)  can measure extraction  efficiency.
Commonly recommended control limits include 75-125 percent recovery for spikes and
80-120 percent recovery for the analysis of SRMs.

Replicates must be analy2:ed to monitor  the  precision  of laboratory  analyses.   A
minimum of 5 percent of the analyses should be laboratory replicates. Field duplicates
should also be collected  where practical.   Common control limits are ±20 relative
percent difference between duplicates.

Currently,  no universally accepted convention for reporting detection limits of analytical
procedures exists.   Table  4-11 lists definitions of various detection limits used by the
American  Chemical Society's Committee on Environmental Improvement  (CEI).  The
IDL does not address  possible blank contaminants or matrix interferences and is not a
good  standard  for  complex environmental matrices.  The LOD and LOQ account for
                                                                             65
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Methods
blanks, but not matrix interferences. The MDL provides high statistical confidence but,
like the LOQ, may be too stringent.  Detection limits must be specified and reported to
ensure adequate data quality and comparability between protocols.

4.2.5     Statistical Design Considerations

The statistical analysis of water chemistry data typically involves calculating summary
statistics and testing for spatial and temporal trends.  Summary conditions are useful for
spatial displays (e.g., plume contours), load estimations, and general reporting. Testing
for trends is useful for evaluating whether water chemistry conditions have improved or
degraded  over  time  or  space.    In  comparison to  other  variables  (e.g.,  see
Bioaccumulation Methods), it  is  not  common to collect replicated or  composite data
except for  QC procedures.   This is the  usual  case  since most  monitoring  designs
typically opt for collecting water chemistry data more frequently or at more stations.

Summary Statistics

To summarize data, the analyst typically estimates statistics for central tendency (e.g.,
mean, median) and variability (e.g., standard deviation, interquartile range) of grouped
data.  Uncertainty should be indicated by reporting estimates with  confidence limits or

              Table 4-11. Definitions for Selected Limits of Detection
  Instrument Detection Limit (IDL)
  Limit of Detection (LOD)
  Limit of Quantitation (LOQ)
  Method Detection Limit (MDL)
The smallest signal above background noise that an
instrument can detect reliably.

The lowest concentration level that can be determined
to  be statistically different  from the blank.   The
recommended value for LOD is  3a, where a is the
standard deviation of the blank in replicate analyses.

The level above which quantitative  results may be
obtained with a specified degree  of confidence.  The
recommended value for LOQ is 10a, where a is the
standard deviation of the blank in replicate analyses.

The minimum concentration  of a  substance that can
be identified, measured, and  reported with 99 percent
confidence that the analyte  concentration is greater
than  zero.  The MDL  is determined from  seven
replicate analyses of a sample  of  a given  matrix
containing the analyte.
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                                           Section 403 Procedural and Monitoring Guidance
percentiles (Ward and Loftis, 1986). Alternatively, data may be summarized graphically
with plots (e.g., box and whisker plots, time series plots, scatter plots, contours, profiles,
etc.).

When  developing  summary statistics, it  is important to use comparable data.  It is
recommended that data collected or analyzed with different methods not be combined
unless a study comparing the different methods  has  been performed.  An appropriate
study would establish the level of comparability between methods.

For the purposes  of estimating summary statistics, it is generally  recommended that
statistics not be used on left-censored data (i.e., data less than or equal to the detection
limit).  Porter et al. (1988) and Gilliom and Helsel  (1986) provide a discussion on the
implications of analyzing left-censored data and alternative procedures for  estimating
summary statistics when the data are censored.

Trend  Detection

Prior to the collection of data, the statistical test (and significance level) used  to analyze
the data  should be specified. By specifying the test to be used a priori, the  monitoring
plan can be designed to ensure  that the  correct type of data (e.g.,  spatial versus
temporal coverage) is collected.

Monitoring  programs can be evaluated using power analysis.  The  relative power of a
sampling is more meaningful when the relative costs of implementing alternative designs
are  taken  into  consideration.   Power-cost analyses  are fundamental  in  selecting
appropriate sample/replicate  number, sample location, and sampling frequency (Ferraro
etal.,1989).

For  allocating  samples  and  resources,  Hirsch (1988)   provides  an approach  for
evaluating alternative sampling strategies for two-sample designs.  This approach would
be appropriate when comparing concentrations of a water  chemistry variable from two
different locations (e.g., an impaired and control area) or two different time periods (e.g.,
two  different cruises) at the same location.   As  a  general  recommendation, equal
resources (e.g., equal number of samples) should be allocated for each sampling period
for two-sample designs (Hirsch, 1988).
4.2.6
Use of Data
Results of the chemical analyses can be used to monitor levels of pollutants both in the
zone of initial dilution and in areas only minimally affected by the discharge. Sampling in
this manner establishes spatial trends in the accumulation and transport of discharged
pollutants.
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Methods
The data can also be used as boundary and initial conditions for modeling work that may
be used to map the transport and fate of various chemicals throughout the ecosystem
under varying discharge scenarios. The modeling can lead to developing water quality
standards and discharge  guidelines.  During the monitoring program, water chemistry
data can be used to identify discharges that are not in compliance and to ensure that
water quality standards are being maintained.

Monitoring  of  pollutant levels  in  the water column is a widely-accepted  means  of
measuring the condition of the  aquatic habitat.  However, the singular use of pollutant
loading data to assess the condition of the water column or to guide the decision-making
process is not recommended. Data acquired from monitoring water contaminant levels,
in conjunction with the water's  physical properties, may be used to assess  the health
risks to human populations  and the ecological risks  to individuals, populations, and
communities living in the water column.  Monitoring of water column chemistry remains
a powerful tool in the  evaluation of spatial and temporal effects of anthropogenic and
natural disturbance.

4.2.7    Summary and Recommendations

Rationale

      •  Monitoring water quality parameters will provide information on ambient water
         conditions and the potential for transport and persistence of contaminants
         from permitted discharges.

Monitoring Design Considerations

      •  The analytical methods must show appropriate selectivity, specificity, and
         sensitivity for the contaminants of concern.

      •  Sampling  and sample-handling procedures should be well-defined and
         consistent so as not to compromise the integrity and representativeness of
         the samples.

      •  Sampling  numbers and locations should be appropriate to the level of
         information required.

Analytical Methods Considerations

      •  The Winkler (titrimetric) method is the first method of choice for the
         measurement of dissolved oxygen unless the samples contain interferents
         such as sulfur compounds, chlorine, free iodine, color, turbidity, or biological
         floes, or when continuous monitoring is planned.  In those instances, a
         membrane electrode should be used.
68
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                                           Section 403 Procedural and Monitoring Quittance
      •  Nutrients such as ammonia nitrogen, total Kjeldahl nitrogen, nitrate-nitrite
         nitrogen, and phosphorus are determined by spectrophotometric
         measurements using a segmented continuous flow analyzer.

      •  The usual method for chlorophyll determination is fluorometric; however, the
         choice of method is determined by the need to separate the different forms.
         HPLC is the most accurate method, but it is also the most expensive.

      •  A combination of AA spectroscopy and ICP spectrometry is the
         recommended method for the detection of metals.

      •  Cold vapor AA spectroscopy is the recommended protocol for the detection of
         mercury.

      •  For monitoring programs where the most accurate quantitation is important,
         the isotope dilution GC/MS method is recommended for volatile and
         semivolatile organic compounds.  GC/ECD is the method of choice for many
         organic chemicals (e.g., pesticides and RGBs).

QA/QC Considerations

      •  Calibration standards and blank, matrix spike, and replicate analyses are
         recommended quality control checks.

      •  A report describing the objectives  of the analytical effort; methods of sample
         collection, handling, and preservation; details of the analytical  method;
         problems encountered during the analytical process; any necessary
         modifications to the written procedures; and results of the QC analyses
         should be included with the quantitative data.

Statistical Design Considerations

      •   The analyst typically estimates statistics for central tendency (e.g., mean,
         median) and variability (e.g., standard deviation,  interquartile range) of
         grouped data.  Uncertainty should be indicated by reporting estimates with
         confidence limits or percehtiles.

      •   When developing summary statistics, it is important to use comparable data.
         It is recommended that data collected or analyzed with different methods not
         be combined unless a study comparing the different methods has been
         performed.

      •   For the purpose of estimating summary statistics, it is generally
         recommended that statistics not be performed on left-censored data.

      •   Prior to the collection of data, the statistical test (and significance level) used
         to analyze the data should be specified.
                                                                             69
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Methods

      •  Monitoring programs can be evaluated using power analysis for detecting
         long-term (monotonic) trends in nutrient data.  In general, the approach
         described there can be applied for other variables and statistical tests (e.g.,
         monotonic or step-trend test).
Use of Data
      •  Monitor ambient levels of pollutants in the environment.
      •  Establish spatial and temporal trends in the accumulation and transport of
         pollutants discharged into ambient waters.
      •  Calibrate and verify mathematical models.
      •  Develop water quality standards for receiving waters.
      •  Identify noncompliant discharges.
 70

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                                           Section 403 Procedural and Monitoring Guidance
4.3
SEDIMENT CHEMISTRY
Monitoring of pollutant levels in sediments is a widely-accepted means of measuring the
condition of the benthic habitat and is a powerful tool in the evaluation of spatial and
temporal effects of anthropogenic and natural disturbance.  Sediment pollutant loading
data should be used, however, in conjunction with other tools to assess the condition of
the benthic habitat or to guide the decision-making process.
4.3.1
Rationale
Of the 10 guidelines used to determine unreasonable degradation or irreparable harm,
the following can be assessed by evaluating sediment chemistry:

      •  Quantities, composition, and potential bioaccumulation or persistence of the
         pollutants to be discharged;

      •  Potential transport of the pollutants by biological, physical, or chemical
         processes; and

      •  Potential direct or indirect impacts on human health.

The sediments represent a potential sink for many chemical contaminants in the marine
environment (USEPA,  1989e). The objective of monitoring bulk sediment chemistry is to
detect sediment toxicity and  to describe spatial and temporal changes in sediment
pollutants.  The results may be used to monitor rates of change  following discharge
initialization, to evaluate the condition of benthic habitats,  and to provide early warnings
of potential impacts to the marine ecosystem.

4.3.2     Monitoring Design Considerations

Selection of Analytes

The chemicals that should be included in the monitoring  program are those chemicals
known or suspected to be in the discharge, as well as possible by-products. Although
potentially toxic compounds in the discharge may not be  fully known, the objectives of
the program must be clearly defined.  It may be necessary  at the beginning  of the
program  to conduct an analysis for all chemicals on the EPA-defined Priority Pollutant,
Hazardous Substance,  or Target  Compound/Analyte Lists to  determine  which are
present; however, detection in the environment does not always correlate with biological
risks  and effects.   Limitations in  analytical methodology,  modeling techniques,  and
toxicological data restrict the usefulness of the resulting data. In some cases, levels of
contaminants that are of concern to the health of organisms are below the lowest limit of
detection of the  best available analytical methodology.
                                                                             71
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Methods
Sampling Considerations

Selection of sampling locations and choice of sampling techniques are crucial decisions
to be made before the sampling effort is initiated.   Recommended Protocols  for
Measuring Selected Environmental  Variables in  Puget Sound (USEPA,  1986-1991)
contains discussions of quality assurance requirements and sampling considerations
that are applicable to sediment quality monitoring.   Sampling  must incorporate sites
within the zone of initial dilution, extending out to areas of low contaminant concentration
and further to unaffected areas to obtain the complete spatial variability. A summary of
sample containers and handling guidelines is presented in Table 4-12.

Sediment Sampling Devices

The protocols required to collect an acceptable surficial sediment sample for subsequent
measurement of chemical variables have generally been neglected.  In fact, sampling
crews have been given wide latitude in how samples are collected.  Because sample
collection protocols influence all subsequent laboratory and data analysis, however, it is
essential that sediment samples be  collected  using  acceptable and standardized
techniques.  Sediment sampling  devices are the  same regardless of whether the
samples  are  collected for chemistry,  grain  size, or benthic infauna  analyses.
Consequently, enough sediment may be collected  in the same grab or core for all three
analyses.   Equipment selection depends  in  large part on the  use  of  the  sample
collected.   Consideration should also be given to  water depth, currents, ocean bottom
characteristics, and degree of homogeneity and representativeness of sampling.

Collection of undisturbed sediment requires that the sampler:

      •  Create a minimal bow wake when descending;

      •  Form a leakproof seal when the sediment sample is taken;

      •  Prevent winnowing and excessive sample disturbance when ascending; and

      •  Allow easy access to the sample surface in order that undisturbed
         subsamples may be taken (ASTM, 1991).

Penetration well below the  desired sampling depth is preferred to prevent sample
disturbance as the device closes. It is best to use a sampler with a weight adjustment to
enable the modification of penetration depths.

Several types of devices can be used to collect sediment samples:  dredges, grabs, and
box corers (Mclntyre et al.,  1984). There are many variations to these types of sampling
device.  Many of these devices  sample the benthic  habitat in a unique manner (Table
4-13).  Accordingly, conducting comparisons  between data collected using different
devices is inadvisable.
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                                                  Section 403 Procedural and Monitoring Guidance
    Table 4-12. Sampling Containers, Preservation Requirements, and Holding
                               Times for Sediment Samples
                                          Container3
Contaminant

Acidity
Alkalinity
Ammonia
Sulfate
Sulfide
Sulfite
Nitrate
Nitrate-nitrite
Nitrite
Oil and grease
Organic carbon

Metals

Chromium VI
Mercury
Metals, except above

Organic Compounds
Extractables (including phthalates,        G, Teflon-lined cap
   nitrosamines, organochlorine pesticides,
   PCBs, nitroaromatics, isophorone,
   polynuclear aromatic hydrocarbons,
   haloethers, chlorinated hydrocarbons,
   and TCDD)
Extractables (phenols)

Purgeables (halocarbons and aromatics)

Purgeables (arolein and acrylonitrite)

Orthophosphate
Pesticides

Phenols
Phosphorus (elemental)
Phosphorus, total
Chlorinated organic compounds
                                      G, teflon-lined cap

                                        G, Teflon-lined
                                           septum
                                        G, Teflon-lined
                                           septum
                                            P,G
                                      G, Teflon-lined cap

                                            P,G
                                             G
                                            P,G
                                      G, Teflon-lined cap
Preservation     Holding Time
P,G
P,G
P,G
P,G
P,G
P,G
P,G
P.G
P,G
G
P,G
P,G
P,G
P,G
Cool, 4°C
Cool, 4°C
Cool, 4°C
Cool, 4°C
Cool, 4°C
Cool, 4°C
Cool, 4°C
Cool, 4°C
Cool, 4°C
Cool, 4°C
Cool, 4°C
Cool, 4°C


14 days
14 days
28 days
28 days
28 days
48 hours
48 hours
28 days
48 hours
28 days
28 days
48 hours
28 days
180 days
                                                            Cool, 4°C
             7 days (until extraction)
             40 days (after extraction)
 Cool, 4°C

 Cool, 4°C

 Cool, 4°C

 Cool, 4°C
 Cool, 4°C

 Cool, 4°C
 Cool, 4°C
 Cool, 4°C
 Cool, 4°C
 7 days (until extraction)
40 days (after extraction)
        7 days

        3 days

       48 hours
 7 days (until extraction)
40 days (after extraction)
       28 days
       48 hours
       28 days
 7 days (until extraction)
30 days (after extraction)
aPolyethylene (P) or glass (G)
SOURCE: ASTM, 1991.
                                                                                          73
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Methods

                 Table 4-13. Summary of Bottom Sampling Equipment8
        Device
         Use
      Advantages
       Disadvantages
  Fluorocarfaon plastic
  or glass
  tubs
Shallow wadable waters
or deep waters if
SCUBA available. Soft
or semiconsolidated
deposits.
Preserves layering and
permits historical sudy of
sediment deposition.
Rapid-samples immed-
iately ready for laboratory
shipment. Minimal risk of
contamination.
Small sample size requires
repetitive sampling.
  Hand oorer with
  removable
  fluorocarbon plastic
  or glass liners
Same as above except
more consolidated sedi-
ments can be obtained.
Handles provide for greater
ease of substrate
penetration. Above
advantages.
Careful handling necessary to
prevent spillage, Requires
removal of liners before repetitive
sampling. Slight risk of metal
contamination from barrel and
core cutter.
   Box corer
   Gravity corers, that
   is, Phleger corer
   Young grab (fluoro-
   carbon plastic- or
   Kynar-lined modi-
   fied 0.1m2 Van
   Veen)

   Ekman or box
   dredge
Same as above.
Semiconsolidated
sediments.
Collection of large
undisturbed sample
allowing for subsampling.

Low risk of undisturbed
sample contamination.
Maintains sediment  •
integrity relatively well.
Lakes and marine areas. Eliminates metal
                       contamination. Reduces
                       pressure wave.
Soft to semisoft
sediments. Can be
used from boat, bridge,
or pier in waters of
various depths.
Obtains a larger sample
than coring tubes. Can be
subsampled through box
lid.
                                                                      Hard to handle.
Careful handling necessary to
avoid sediment spillage. Small
sample, requires repetitive
operation and removal of liners.
Time-consuming.

Expensive. Requires winch.
Possible incomplete jaw closure
and sample loss. Possible shock
wave, which may disturb the fine
sediments ("fines"). Metal
construction may introduce
contaminants. Possible loss of
"fines" on retrieval.
   Ponar grab
   sampler
Useful on sand, silt,
or clay.
Most universal grab
sampler. Adequate on most
substrates. Large sample
obtained intact, permitting
subsampling.
Shock wave from descent may
disturb "fines." Possible
incomplete closure of jaws results
in sample loss. Possible
contamination from metal frame
construction. Sample must be
further prepared for analysis.
 74
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                                                     Section 403 Procedural and Monitoring Guidance
                                   Table 4-13.  (continued)
       Device
         Use
       Advantages
        Disadvantages
 BMH-53 piston
 corer
Waters of 4-6 ft depth
when used with
extension rod. Soft to
semiconsolidated
deposits.
Piston provides for greater
sample retention.
 Cores must be extruded on site to
 other containers. Metal barrels
 introduce risk of metal
 contamination.
Van Veen
BMH-60
Useful on sand, silt, or
clay.
Sampling moving
waters from a fixed
platform.
Adequate on most
substrates. Large sample
obtained intact, permitting
subsampling.
Streamlined configuration
allows sampling where
other devices could not
achieve proper orien-
tiation.
 Shock wave from descent may
 disturb "fines." Possible
 incomplete closure of jaws results
 in sample loss. Possible
 contamination from metal frame
 construction. Sample must be
 further prepared for analysis.

 Possible contamination from
 metal construction. Subsampling
 difficult. Not effective for sampling
 fine sediments.
Petersen grab
sampler
Shipek grab
sampler
Orange-peel grab
Smith-Mclntyre
grab
Useful on most
substrates.
Used primarily in
marine waters and large
inland lakes and
reservoirs.
Useful on most
substrates.
Scoops, drag buckets Various environments
                    depending on depth
                    and substrate.
Large sample; can
penetrate most substrates.
Sample bucket may be
opened to permit
subsampling. Retains
fine-grained sediments
effectively.

Designed for sampling hard
substrates.
Heavy, may require winch. No
cover lid to permit subsampling.
All other disadvantages of Ekman
and Ponar.

Possible contamination from
metal construction. Heavy, may
require winch.
                      Inexpensive, easy to
                      handle.
Loss of "fines." Heavy, may
require winch. Possible metal
contamination.

Loss of "fines" on retrieval
through water column.
aComments represent subjective evaluations.
 SOURCE: ASTM, 1991.
                                                                                                 75
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Methods
Grab samplers

Grabs are capable of consistent sampling of bottom habitats.  Depending on the size of
the device, areas from 0.02 to 0.5 m2 and depths  ranging from 5 to 15 cm  may be
sampled.  Limitations of grab samplers include :

      •  Variability among samples in penetration depth depending on sediment
         properties;

      •  Oblique angles of penetration, which result in varying penetration depths
         within a sample; and

      •   Folding of the sample, which results in the inability to section the sample and
         the loss of information concerning the vertical structure in the sediments.

The careful use of  these  devices, however,  will  provide  quantitative data.  Grab
samplers are the tools of choice for a number of marine monitoring programs (Fredette
etal., 1989; USEPA,  1986-1991).

Core samplers

Box corers use a  surrounding frame  to ensure  vertical  entry;  therefore,  vertical
sectioning of the sample is  possible (USEPA, 1986-1991). These devices are  capable
of maximum penetration depths of 15 cm and may collect volumes 5 to 10 times those
of grab samplers. Limitations of box corers include :

      •   Large  size  and weight, which require the use of cranes or winches and a
          large vessel for deployment;

      •   Higher construction expenses; and

      •   Lack of calibration studies to permit comparisons to grab samples.

The Hessler-Sandia box corer uses dividers  to section the core into subsections,
facilitating subsampling of the core. Box corers are recognized as the tools of choice for
maximum accuracy and precision when sampling soft-bottom habitats.

Collection of sediments and collection of benthic organisms should be done concurrently
to mitigate the costs of field sampling and to permit sound correlation, regression,  and
multivariate analyses.  Therefore, it is recommended that the sampling device also be
suitable  for benthic sampling.   Grab  and core sampling  devices  permit adequate
sampling of both sediment and benthic infaunal communities with one sampling device.
 76
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                                          Section 403 Procedural and Monitoring Guidance
Sample Depth

It is recommended that  the  upper 2 cm of the sediment column be examined to
characterize surficial sediments.  Although the 2 cm specification  is arbitrary, it does
ensure  that  relatively  recent sediments  are  sampled,  that adequate volumes of
sediments are readily obtained for laboratory analyses, and that  data from different
studies can be compared.

Sampling of depths other than 2 cm or vertical stratification of deeper sediment cores
may be appropriate, depending on the objectives of the monitoring program and the rate
of sediment accumulation. For example, if information concerning only the most recent
sediment contamination is required, examination  of the upper 1 cm may be appropriate.
Stratification of deeper cores will  provide historical data of sediment contaminant levels
and depositional events.  Comparison of data from studies analyzing different sediment
depths is not advised.  If the  potential for bioaccumulation  of contaminants in infaunal
organisms  is a concern, sampling to the depth  of  the  anoxic  layer is desirable.
Penetration well below the desired sampling depth  is  preferred  to prevent sample
disturbance as the device closes.

Total Organic Carbon and Acid  Volatile Sulfides Normalization

Total organic carbon (TOG) and acid volatile sulfides (AVS) are considered by many to
be the  most important parameters in  defining  organic  and metal concentrations in
sediments.  Toxic hydrophobic  contaminant concentrations have been found to be
related to the TOG content of the sediment  (Karickhoff et al., 1979).  Likewise, toxic
metals concentrations have been found to be related to the AVS concentration of the
sediment (DiToro et al., in press a, b). The AVS pool is available to bind metals, thereby
reducing their bioavailability.

Normalizations  of sediment contaminant concentrations to TOG and AVS have  been
conducted  to estimate  the   bioavailability of  inorganic  and  organic  contaminants.
Furthermore,  these  normalizations  have  been  made  to  account for some of the
variability  found in  bioaccumulation rates and biological assemblages.    TOC/lipid
normalized accumulation factors (AF)  have also been used to predict tissue residue
concentrations (Ferraro et al., 1990; Lake et al., 1987). AVS will be used in  a similar
manner to assist in the development of accumulation factors for metals.

Normalization based on AVS  concentrations  is considered a potentially useful tool for
assessing the bioavailability of certain divalent metals (e.g., Cd and Ni). However, only
a few laboratories  can perform these analyses at present  and the procedure has not
undergone any  round robin verification.  In addition, sampling for AVS is very subject to
error because contact with air can greatly affect results.  Although  AVS normalizations
represent a promising tool for evaluation of sediment  metals  data,  general use in
estuarine monitoring programs may not yet be practical.
                                                                             77
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Methods
These normalizations assume the following:

      •  Organic contaminants partition predominantly to sediment organic carbon;
         metal contaminants partition predominantly to sediment AVS.

      •  Rapid steady-state kinetics of contaminants.

Development of standardized methods for measuring TOC and AVS in the laboratory
are required before normalized contaminant concentrations may be compared among
studies.

The decision to normalize for  TOC and  AVS  will  depend on  monitoring  program
objectives.   For example,  if the objective is to identify the "footprint" of  a  discharge,
normalization may not be appropriate; if the  monitoring objective  is to identify "hot
spots"—i.e., those areas where bioavailable contaminants represent a risk to human
and ecological health—normalization may be justified.

Selection of Sampling Period

Sediment transport processes can vary on a seasonal  scale, while ocean discharges
are generally constant over time. Therefore, sediment contaminant levels may display a
seasonal pattern  in  accumulation  rates.   Sediment processes are  associated with
seasonal patterns of benthic turbulent mixing and sediment transport phenomena. The
frequency of sampling should take into account the expected rate of change in sediment
contaminant concentration.

Because of seasonal  variations, it is recommended that direct comparisons  between
samples  collected during different times of the year be  avoided. Studies investigating
interannual  variation  in the  concentrations  of sediment  contaminants should  be
conducted  during the  same  season  (preferably  the  same month)  each year.
Furthermore, it is  recommended  that  sediments be  sampled when  contaminant
concentrations are expected to be at their highest levels  in order to evaluate worst-case
scenarios.

Seasonal comparisons within stations, however, can be valid and  useful because
(1) seasonal water column changes can affect sediment chemistry (e.g., anoxia can
affect AVS and  metal  bioavailability);  (2) seasonal  fluctuations  in the  presence,
abundance, and activity of infauna can lead to  changes  in bioturbation and thereby the
chemistry of surficial sediments; and (3) seasonal changes in discharge activity can
lead to changes  in  sediment loadings (e.g., in  Alaskan waters, where  ice might
preclude discharge during winters, one might expect chemical concentrations  in
sediments  to be low in spring and higher in fall).
78
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                                           Serf/on 403 Procedural and Monitoring Guidance
4.3.3    Analytical Methods Considerations

Questions to be considered during the choice of an appropriate analytical method include
the parameters of interest, desired detection limits, sample size requirements or restrictions,
methods of preservation, technical and practical holding times, and  matrix interferences.
Several USEPA documents (e.g., 1986a and 1986-1991) discuss the common analytical
problems encountered during monitoring analyses of sediment samples.  It will frequently be
necessary to use methods other than those currently approved by EPA to achieve a desired
sensitivity in a marine environment. However, alternative methods should be considered
only  when they have been demonstrated to provide a  level of precision  and accuracy
equivalent to or exceeding that of the EPA-approved method.  Any proposed changes in
analytical methods should be reviewed by analysts with experience in marine waters.

Chemical Residue Analyses

Several  factors  determine achievable  detection limits  for  a specific  contaminant,
regardless of analytical procedure. These factors include:

      •  Sample size; 50-100 g (wet weight) with a minimum final dilution volume of
         0.5 ml_ is considered adequate (USEPA, 1986-1991).

      •  Presence of interfering substances.

      •  Range of pollutants to be analyzed; an optimal method may be developed
         without regard to potential effects on other parameters.

      •  Level of confirmation — qualitative (e.g., presence or absence) or quantitative
         (e.g., residue concentrations) analyses.

      •  Level of pollutant found in the field and in analytical blanks.

A  list of analytical procedures and  USEPA method numbers is given in Table 4-14.
Selection of appropriate  methods should be based on a trade-off between full-scan
analyses, which  are economical  but cannot  provide optimal  sensitivity  for some
compounds, and alternative methods that are more sensitive for specific compounds but
can result in higher analytical costs.  Table 4-15 lists which technique  is appropriate for
detecting a specific contaminant.

Metals and Metalloids

Trace element analyses  by inductively coupled plasma emission spectrometry (ICP)
allow for several elements to be  measured  simultaneously.  Detection limits of ICP for
most metals are generally comparable to those achieved by graphite furnace atomic
absorption spectrophotometry (GFAA); however, detection limits for several metals (e.g.,
arsenic,  selenium, and  mercury) are  significantly  lower using atomic  absorption
spectrophotometry (AA).
                                                                              79
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Methods
                    Table 4-14.  List of Analytical Techniques
                                (USEPA, 1986a)
METALS/METALLOIDS

   •  Atomic Absorption Spectrophotometry (AA)

      -  flame
         graphite furnace (GFAA)
         cold vapor
         gaseous hydride (HYDAA)

   •  Inductively Coupled Plasma Emission
      Spectrometry (ICP)

ORGANICS

   •  Gas Chromatography (GC)

      -  with electron capture detection (GC/ECD)
      -  with mass spectrometry (GC/MS)
                                                    USEPA method 7000 series

                                                    USEPA method 7470
                                                    USEPA methods 7060 and 7740

                                                    USEPA method 6010
                                                    USEPA method 8080
                                                    USEPA methods 8240 and 8270
                     Pollutants Tested for by Following Techniques:
                 ICP

Al, Sb, As, Ba, Be, B, Cd, Ca, Cr, Co, Cu, Fe,
Pb, Mg, Mn, Mo, Ni, K, Se, Si, Ag, Na, Tl, V, and
Zn
                                           Al, Sb, As, Ba, Be, Cd, Cr, Co, Cu, Fe, Pb, Mn,
                                           Hg, Mo, Ni, Se, Ag, Tl, Sn, V, and Zn
SOURCE: USEPA, 1986a.

The combination of AA and ICP is the recommended analytical method for detection of
metals and metalloids since no technique is best for all elements (USEPA, 1986a). Cold
vapor AA analysis  is the recommended technique for mercury  (USEPA, 1986a).
Digestion methods for sediment  samples are reviewed by  Plumb (1981).  The EPA
Contract Laboratory Program requires the use  of nitric  acid and hydrogen peroxide
(USEPA, 19901).

GFAA is more sensitive (i.e.,  lower detection limits) than flame AA or ICP, but it is more
subject to matrix  and spectral interferences.  GFAA requires particular caution with
regard to laboratory contamination. The concentration of each element is determined by
80
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                                            Section 405 Procedural and Monitoring Guidance
         Table 4-15. USEPA Organic Contaminant Detection Techniques

USEPA Method3  Contaminant Type                                 Method
8010
8015
8020
8021
8030
8040
8060
8080
8090
8100
8110
8140
8150
8240
8270
Purgeable Halocarbons
Purgeable Nonhalogenated Volatiles
Purgeable Aromatics
Purgeable Organics
Acrolein/Acrylonitrile
Phenols
Phthalate Esters
PCBs and Organochlorine Pesticides
Nitroaromatics and Cyclic Ketones
Polycyclic Aromatic Hydrocarbons
Haloethers
Organophosphorous Pesticides
Chlorinated Herbicides
Volatile Organics
Semivolatile Organics
GC-ELCD
GC-FID
GC-PID
GC-FID and GC-PID
GC-FID and PID
GC-FID
GC-ECD
GC-ECD
GC-FID-ECD  '
HPLC-UV-FL or GC-FID
GC-ECD
GC-FPD or NPD
GC-ECD or ELCD
GC-MS
GC-MS
'USEPA, 1986e.
a separate analysis, making the analysis of a large number of contaminant metals both
labor-intensive and relatively expensive compared to ICP. AA methods may, however,
be cost-effective for the analysis of a few metals over a large number of samples.

Semivolatile Organic Compounds

Analysis of Semivolatile organic compounds involves a solvent extraction of the sample,
cleanup of the characteristically complex  extract, and gas  chromatographic  (GC)
analysis and  quantitation.   There are two  gas  ehromatography/mass spectrometry
(GC/MS) options for detecting extractable organic compounds: internal standard and
isotope dilution. The isotope dilution technique, which requires spiking the sample with
a mixture of stable isotope-labeled analogs of the analytes, is recommended because
reliable recovery corrections  can be made for each analyte with a labeled analog or a
chemically similar analog.  The isotope dilution  method is more expensive and less
widely employed than the internal standard  method, which  is the current  method of
choice  in the  Contract  Laboratory Program.   Mass  spectrometry provides positive
compound identification by comparison of both retention time and spectral patterns with
                                                                               81
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Methods
standard compounds.  This technique, however, lacks the sensitivity to detect levels of
most trace contaminants in marine  systems.  Alternative methods  providing greater
sensitivity are available but are compound-specific and, therefore, more expensive.

The identification of many organic compounds can be made by gas chromatography/
electron capture detection (GC/ECD) analysis.  GC/ECD provides greater sensitivity
(lower detection limits) relative to GC/MS; however, GC/ECD does not provide positive
compound identification.  Since the identity of a compound is determined  solely by
matching retention time with that of a standard, confirmation of the initial identity of the
compound on a second GC column is required for confidence in the reliability of the
qualitative identification of the  compound.  When a compound  is present at a  high
enough concentration, its identity should be confirmed by the use of GC/MS.   GC/ECD
is the method of choice for pesticides and RGBs.  Glass capillary GC achieves a much
higher degree of resolution in the analysis of PCB  congeners than the standard packed
column methods; however, few laboratories regularly employ this specialized technique.

The detection and concentration of  PCBs can be more  accurately  determined using
comparison mixtures other than the standard industrial Aroclor mixtures.  Evaluations of
PCB assemblages in environmental samples by quantification as Aroclors or total PCBs
are of  limited accuracy due to environmental  degradation and differential affinities of
PCB congeners for  different   environmental  compartments.    A  more meaningful
evaluation can be accomplished by quantification by PCB isomer groupings (dependent
on the  number of chlorine atoms per molecules).  This has the advantage of indicating
the relative concentrations of the groups containing the most toxic and bioaccumulating
congeners (McFarland et al., 1986).  An alternative method of analysis is to test for the
individual  PCB congeners.   Using  congener-specific  methods instead of Aroclor
standard  mixtures  provides  more  accurate  identification and quantification  and
eliminates the necessity of subjective decisions on the part of the analyst (Eganhouse,
1990).  However, beside being more expensive and time-consuming, the compatibility of
these enhanced detection methods with the standard Aroclor method  is an  important
consideration for monitoring programs with existing historical  data.  All other organic
compound groups are recommended for analysis by GC/MS (USEPA,  1986a).

Volatile Organic Compounds

The  purge-and-trap  GC/MS technique is employed for  detecting volatile  organic
compounds in water. A successful variation for detection of volatile organic residues in
sediments involves a  device  that vaporizes  volatile organic  compounds  from the
sediment sample under vacuum and then condenses the volatiles in a super-cooled trap
(Hiatt, 1981). The trap is then transferred to a purge-and-trap device,  where it is treated
as a water sample. The heated purge-and-trap technique, employed  in the Contract
Laboratory Program, involves the addition of a weighed amount of sediment to a known
volume of water. As the chamber is heated, inert gas is bubbled through the suspension
to  sweep volatile   organics  out  of  the  sediment.  The isotope dilution  option is
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                                          Section 403 Procedural and Monitoring Guidance
recommended if the data quality objectives (DQOs) of the monitoring program require
accurate quantitation of each compound.  This option involves additional analysis time
and expense.

Sediment Toxicity Tests

Toxicity tests may be necessary if the sediment contains contaminants that could result
in an adverse impact on the benthic environment and/or the water column. Tests with
whole  sediment are used to determine  effects on benthic  organisms.  Tests with
suspensions of the sediment are used to determine effects on water-column organisms.
Test organisms should be representative of sensitive organisms existing in the vicinity of
the actual site. Mortality of a certain percent of the organisms in the laboratory test does
not imply that the population of the species around the site would decline by the same
percent;  however,  results  can  be  compared  with reference-sediment results  to
determine whether the site sediment has higher toxicity (EA and Battelle, 1990).

An introductory guide to toxicity testing is presented in Part 8000 of APHA (1989). Most
of the procedures currently used for sediment toxicity testing are based on USEPA/COE
(1977).  Additional discussion and  procedures can be found in the draft testing manual
for dredged material (EA and Battelle, 1990) and in USEPA (1986-1991) and Swartz et
al. (1982, 1985).

Acute toxicity tests are  conducted to determine the immediate  effects of short-term
exposures under specific conditions, providing little information about possible delayed
effects. It can be difficult to apply laboratory results to field situations because  of the
fact that motile organisms might avoid exposure when possible and toxicity to benthic
organisms is dependent on field-specific sediment characteristics, the  dynamics of
equilibrium partitioning,  and  routes of  exposure.   ASTM (1990) proposes  standard
guidelines for conducting 10-day static toxicity tests.
4.3.4
QA/QC Considerations
Measurement data are no better than the planning that goes into setting data quality
objectives,  choosing appropriate sampling and analysis methodologies, and setting
quality control criteria.  An expert chemist and the references cited in this document
should be used to develop a comprehensive QA plan.

Sample Collection

The primary criterion for an adequate sampler is that it consistently collect undisturbed
and uncontaminated samples.  The sampling device should be inspected for wear and
tear leading to sample leakage during ascent.  It is recommended that a backup sampler
be available on board the survey vessel in case the primary sampler is  lost or becomes
inoperable during the cruise.
                                                                             83
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Methods
In the field, sources of contamination include sampling gear, lubricants and oils, engine
exhaust, airborne dust, and ice used for cooling samples. If samples are designated for
chemical analysis, all sampling equipment (e.g., siphon hoses, scoops, containers) must
be made of noncontaminating material and must be cleaned appropriately prior to use.
Potential airborne contamination such as stack gases and cigarette smoke should be
avoided.  Furthermore, samples and sampling containers should not be touched with
ungloved fingers.

The following sample acceptability criteria should be satisfied (USEF3A, 1986-1991):

      •  The sampler is not overfilled with sample so that the sediment surface is
         pressed against the top of the sampler.

      •  Overlying water is present, indicating minimal leakage.

      •  The overlying water is not excessively turbid, indicating minimal sample
         disturbance.

      •  The sediment surface is relatively flat, indicating minimal disturbance or
         winnowing (Figure 4-1).
          Acceptable if Minimum
       Penetration Requirement Met
      and Overlying Water is Present
        Unacceptable
(Washed, Rock Caught in Jaws)
              Unacceptable
        (Canted with Partial Sample)
         Unacceptable
           (Washed)
           Figure 4-1. Examples of Acceptable and Unacceptable Samples
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                                         Section 403 Procedural and Monitoring Guidance
      •   The desired penetration depth is achieved.

If the sample does not meet ail the criteria, it should be rejected.

Splitting of sediment samples for chemical analyses should only be  conducted with
noncontaminating  tools  under  "clean  room" conditions.    Recommended container
materials, sample sizes, preservation techniques, and analytical holding  times should be
determined before the sampling effort is undertaken.

For analyses of metals, samples should be frozen and kept at -20°C. Although specific
holding times have not been recommended by EPA, a maximum of 6 months (28 days
for mercury) would be consistent with those for aqueous samples (Table 4-12).

For analyses of  volatile compounds,  samples should be stored in the dark at 4°C
(USEPA, 1986-1991; USEPA,  1987c).   Analyses of volatile compounds should be
performed within  7 days of  collection as recommended  by EPA.   If extractions  of
semivolatile compounds will not be performed within 7 days,  freezing of the samples at
-20°C is advised.  EPA has not established holding times for frozen samples. A general
guideline of a maximum of 6 months would be consistent with that for water samples
(USEPA, 1986-1991).

Samples for determination of TOC or AVS should be analyzed as soon as possible. If
not analyzed immediately,  TOC samples should be refrigerated  and their  pH brought
below 2 by addition of H?_SO4.  Acid volatile sulfides should  be stored in airtight
containers under an inert atmosphere and analyzed as soon as possible.

Various types of blanks can be prepared and analyzed to identify possible sources of
sample  contamination.   Field blanks  are aqueous samples manipulated in the field.
Rinsate blanks are aqueous samples that are poured over the sampling equipment and
collected.  Trip blanks are packed in the sample coolers to detect cross-contamination of
volatile organic compounds.

Blind duplicates, treated and identified as separate samples, may be sent to the same
laboratory for analysis, or one of the  pair may be sent to a "reference" laboratory for
comparison.  Standard  reference material may be prepared and sent as a performance
check to the laboratory. Depending on how these types of samples are handled, they
may be used as a check for accuracy and/or precision.
                                                                            85
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 Methods
 Laboratory Analyses

 Laboratory performance standards as measured by method QC protocols should be
 used  to  evaluate and  select appropriate analytical methods.   Changes in selected
 laboratory protocols should be considered and/or approved only if proposed procedures
 meet  or exceed established  performance criteria.  Tables 4-16 and  4-17 provide brief
 summaries of QC analyses for laboratory work.

 QA reports should describe  the results of quantitative QC analyses, as well as other
 elements critical to accurate  interpretation of analytical  results.  It is recommended that
 these reports be recorded and stored in a database for future reference.

                  Table 4-16.  Summary of Quality Control Analyses
       Analysis Type
    Recommended Frequency of Analysis
  Surrogate Spikes
  Method Blank
  Standard Reference
  Materials

  Matrix Spikes
  Spiked Method Blanks




  Analytical Replicates


  Field Replicates
Required in  every organic sample  -  minimum  3
neutral, 2 acid spikes, plus 1 spike for pesticide/PCB
analyses, and 3 spikes for volatiles.  Isotope dilution
technique (i.e., with all available labeled surrogates) is
recommended for full-scan analyses and to enable
recovery corrections to be applied to data.

One per extraction batch (semivolatile organics). One
per extraction  batch or  one  per  12-hour  shift,
whichever is more frequent (volatile organics).

<50 samples: one per set of samples submitted to lab.
>50 samples: one per 50 samples analyzed.

Not required if complete isotope dilution  technique is
used.
<20 samples: one per set of samples submitted to lab.
>20 samples: 5 percent of total number of samples.

As  many as  required  to establish  confidence  in
method before analysis of samples (i.e., when using a
method  for the first time or after any   method
modification).

<20 samples: one per set of samples submitted to lab.
>20 samples: 5 percent of total number of samples.

At the discretion of the project coordinator.

                                            toi
                                            .«. fa.
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                                             Section 403 Procedural and Monitoring Guidance
               Table 4-17.  Summary of Warning and Control Limits
                             for Quality Control Sample
Analysis Type
  Recommended
  Warning Limit
 Recommended
  Control Limit
Surrogate Spikes

Method Blank
   Phthalate,
   Acetone

   Other Organic
   Compounds

Standard Reference
Materials
Matrix spikes

Spiked Method Blanks

Analytical Replicates


Field Replicates

Ongoing Calibration
10 percent recovery
30 percent of the analyte
1 u.g total or 5 percent
of the analyte

95 percent
confidence interval
50-65 percent recovery

50-65 percent recovery
50 percent recovery


5 u,g total or 5 percent
of the analyte

2.5 (j.g total or 5 percent
of the analyte

95 percent confidence
interval for certified
reference material

50 percent recovery

50 percent recovery

 ±100 percent
 coefficient of variation
                            25 percent of
                            initial calibration
Calibration standards should be analyzed at the beginning of sample analysis and
should be verified at the  end  of each  12-hour shift during  which  analyses  are
performed. The concentrations of calibration standards should bracket the expected
sample concentrations,  or  sample dilutions or sample handling  modifications (i.e.,
reduced sample size) will be required.

Analysis  of methods or  reagent blanks  should  be conducted to demonstrate  the
absence  of contamination  from sample  handling in the laboratory.  At least one
method blank must be included with each batch of samples and should constitute at
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 Methods
 least 5 percent of all samples analyzed.   All blanks should be free of detectable
 concentrations of the target compounds, or steps should be taken to remove the source
 of contamination before sample analysis proceeds.

 Spike recovery analyses are required to assess method performance for the particular
 sample matrix. Spike recoveries serve as an indication of analytical accuracy, whereas
 analysis of standard  reference materials (SRMs) can  measure extraction efficiency.
 Commonly recommended control limits include 75-125 percent recovery for spikes and
 80-120 percent recovery for the analysis of SRMs.

 Replicates must  be analyzed  to monitor the precision  of  laboratory analyses.   A
 minimum of 5 percent of the analyses should be laboratory replicates.  Field duplicates
 should also  be collected  where practical.   Common control limits  are ±20 relative
 percent difference between duplicates.

 Currently, no universally accepted convention for reporting detection limits of analytical
 procedures exists.  Table  4-11 in  the  preceding section lists definitions of various
 detection limits used by the American Chemical Society's Committee on Environmental
 Improvement (CEI). The IDL does not address possible blank contaminants or  matrix
 interferences and is not a good standard for complex environmental matrices.  The LOD
 and LOQ account for blanks,  but not matrix interferences.  The  MDL provides high
 statistical confidence but, like the LOQ, may be too stringent.  Detection limits must be
 specified and reported to ensure adequate data quality  and comparability between
 protocols.

 4.3.5    Statistical Design Considerations

 Statistical strategies may  mitigate the  high  costs of collecting sufficient  samples of
 sediment. Power analyses can affect the strategy of compositing samples and can often
 lead to a cost-effective monitoring design strategy.

 Composite Sampling

 Composite sediment sampling consists of mixing samples from two or more replications
 collected at a particular location over time or collected at several locations at the same
time.  The analysis of a  composite  sample provides an  estimate of an average
contaminant  concentration  for  the locations  composing  the  composite  sample.
Advantages of the composite sampling strategy are:

      •  It provides a cost-effective strategy when individual chemical analyses are
         expensive.

      •  It results in a more efficient estimate of the mean at specified sampling
         locations.
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                                           Section 403 Procedural and Monitoring Guidance
Because of the reduced sample variance, composite sampling results in a considerable
increase in statistical  power.   If the  primary objective of a monitoring  program is to
determine  differences in  sediment  contaminant concentrations  among  sampling
locations, composite sampling is an applicable strategy.

Space-bulking consists of sampling from several locations and  combining sediment
samples into one or more composite samples.  Time-bulking involves taking multiple
samples over time from a single location and compositing these samples.  Space- and
time-bulking  strategies  should  be  used  judiciously  since significant information
concerning spatial and temporal heterogeneity may be lost.

Composite sampling is not recommended if the objective of the monitoring program is
based on water quality-based criteria with specified sediment contaminant concentration
limits since this  sampling method  does  not  detect  the true  range  of  sediment
contaminant concentrations in the environment.  The adoption of composite strategies
will depend on the objectives of individual monitoring programs.

Statistical Power

Statistical power increases with an increase in the number of sediment subsamples in
each replicate composite sample. However, with the addition of successive subsamples
to each composite, a  diminished return of statistical power exists.  For composites of
greater than 10 replications, the increase of power is negligible given typical levels of
data variability.

To improve the power of a statistical test, while keeping the significance level constant,
the sample size should be increased.  Because of constraints in cost and time imposed
by the monitoring program, however, this option may not be available.  Power analyses
have shown that for a fixed level of sampling effort, a monitoring program's power is
generally increased by collecting more replicates at fewer locations.  The number and
distribution of sampling locations required to evaluate the effectiveness of the monitoring
program will be depend on the size and complexity of the discharge.
4.3.6
Use of Data
The results may be used to monitor and evaluate the condition of benthic habitats and to
provide early warnings of potential impacts to the ecosystem. The data generated from
monitoring of the pollutant levels in estuarine sediments should be used in conjunction
with  those collected from  other physical  and  biological  monitoring.   Subsequent
analyses of health risks to human populations and  ecological risks to benthic individuals,
populations, and communities may be performed.
                                                                             89
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Methods
Sediment pollutant loading data should be used in combination with other monitoring
methods to assess the condition of the benthic habitat or to guide the decision-making
process. Data acquired from monitoring sediment contaminant levels,  in conjunction
with the sediment's physical properties, may be used to assess the bioavailability of
these pollutants. Subsequent analysis of this information along with biological data may
be used to  assess the impacts and risks of sediment pollutants to human populations,
benthic populations/communities, and the ecosystem.

Monitoring of pollutant levels in sediments is a widely-accepted means of measuring the
condition of the benthic habitat and is a powerful tool in the evaluation of spatial and
temporal effects of anthropogenic and natural disturbance.  However, for sediment
chemistry data to be useful in  interpreting toxicological response or in  predicting
environmental  risks from  sediment exposure, sediment  criteria are  needed.  The
Washington State  Department of Ecology (1991) has adopted sediment criteria, and
EPA has recently published a sediment compendium (USEPA, 1992).  Both of these
documents may provide useful guidance.   The ability to extrapolate from sediment
chemistry data to predictions of ecological or human health risks is, however, greatly
limited  by  a limited understanding of causal relationships  between  exposure and
response.

4.3.7     Summary and Recommendations

Rationale

       •  The sediments represent the ultimate sink for many chemical contaminants
         from offshore discharges.

       •  The objective of monitoring bulk sediment chemistry is to detect and describe
         spatial and temporal changes of these sediment pollutants.

Monitoring Design Considerations

       •  It is recommended that types of sampling gear and location and timing of
         sample collection be consistent to allow for comparisons among studies.

       •  Collection of undisturbed sediment requires that the sampler:

         -  create a minimal bow wake when descending;
         -  form a leakproof seal when the sediment sample is taken;
         -  prevent winnowing and excessive sample disturbance when ascending; and
         -  allow easy access to the sample surface in order that undisturbed
            subsamples may be taken.

       •  In general, analysis of the upper 2 cm is advised to examine recent sediment
         contamination events; however, site-specific conditions and program
         objectives should be considered when a sampling depth is selected.
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                                          Section 403 Procedural and Monitoring Guidance
      •  Penetration well below the desired sampling depth is preferred to prevent
         sample disturbance as the device closes.

      •  Total organic carbon and acid volatile sulfide normalizations are
         recommended to allow comparisons of chemical residue concentrations
         between locations. Normalizing the data will depend on the objectives of the
         individual monitoring program.

      •  It is recommended that sediments be sampled when contamination
         concentrations are expected to be at their highest levels.

Analytical Methods Considerations

      •  It is recommended that consistent analytical protocols be implemented to
         allow for comparisons between studies.

      •  Laboratories should perform acceptably on standard reference materials and
         intercalibration exercises before performing routine analyses.

      •  Periodic full-scan analyses should be undertaken to identify any new
         pollutants that should be monitored.

      •  Metals/Metalloids

         -   The combination of GFAA and ICP is the recommended method for the
            detection of metals and metalloids.
         -   Cold vapor AA is the recommended  protocol for mercury detection.

      •  Organics

         -   GC/MS in conjunction with isotope dilution is recommended for the
            detection of semivolatile and volatile organic compounds because it
            provides reliable recovery data for each analyte.
         -   A vacuum super-cooled trap in conjunction with a purge-and-trap device
            is recommended for the detection of volatile organics.

      •  Sediment Toxicity Tests

         -   May be necessary if the sediment contains contaminants that could result
            in an adverse impact on the benthic  environment and/or the water column.
         -   Results can be compared with reference-sediment test results to
            determine whether the site sediment has higher toxicity.

QA/QC Considerations

      •  The primary criterion for acceptable field sampling equipment and protocols is
         the collection of undisturbed and uncontaminated environmental samples.
                                                                            91
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Methods
      •  Adequate sampling containers and preservation techniques must be used
         and appropriate holding times followed to ensure the integrity of samples and
         their analyses.

      •  Blank, spike, and replicate analyses are recommended quality control checks.

      •  A report describing the objectives of the analytical effort; methods of sample
         collection, handling, and preservation; details of the analytical methods;
         problems encountered during the analytical process; any necessary
         modifications to the written procedures; and results of the QC analyses
         should be included with the analytical data.

Statistical Design Considerations

      •  Compositing of sediments consists of mixing two or more samples collected
         at a particular location and/or time period.

      •  Space-bulking (combining composites from several locations) and
         time-bulking (combining several composites over time from one location)
         should be used judiciously since information concerning spatial or temporal
         heterogeneity may be lost.

      •  Power analyses have shown that for a fixed level of sampling effort, a
         monitoring program's power is generally increased by collecting more
         replicates at fewer locations.

Use of Data

      •  The data gathered from monitoring of the pollutant levels in marine sediments
         should be used in conjunction with those collected from other physical and
         biological monitoring to monitor rates of recovery following environmental
         interventions, to evaluate  the condition of benthic habitats, and to provide
         early warnings of potential impacts to the estuarine ecosystem.

      •  Sediment pollutant loading data should be used in combination with other
         monitoring methods to assess the condition of the benthic habitat or to guide
         the decision-making process.
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                                          Section 403 Procedural and Monitoring Guidance
4.4
SEDIMENT GRAIN SIZE
The objective of monitoring sediment grain size composition is to help describe spatial
and temporal changes in the benthic community habitat. Grain size analysis measures
the frequency distribution of particle size ranges composing the sediment.  The size
categories (in descending order) are gravel, sand, silt, and clay (Folk,  1980;  Plumb,
1981). Sediment contaminants adsorb to small grain surfaces, and the availability of
sediment contaminants may be correlated with the grain size composition of the benthic
sediments.
4.4.1
Rationale
Of the 10 guidelines used to determine unreasonable degradation or irreparable harm,
the following can be assessed by evaluating sediment grain size:

      •  Quantities, composition, and potential bioaccumulation or persistence of the
         pollutants to be discharged and

      •  Potential transport of the pollutants by biological, physical, or chemical
         processes.

Results from sediment chemical monitoring can  be normalized according to sediment
characteristics to account for some of the variability found  in bioaccumulation rates.
Also,  grain size  information may be used  to aid in the analysis of the temporal and
spatial variability of benthic assemblages since the ability of infaunal organisms to build
tubes, burrow, capture food, and escape predation is affected by grain size.  The results
may be used to monitor rates of recovery following an event, to evaluate the condition of
benthic habitats, and to assist in providing early warnings of potential  impacts to the
marine environment.

4.4.2     Monitoring Design Considerations

Sediment Sampling Devices

Sampling for sediment grain  size is usually undertaken in conjunction with sediment
chemistry and  benthic  infauna  measurements.   Thus,  the  sampling devices and
techniques are similar for all three.  In the past, sediment samples have been collected
using  a  wide  range  of methods  and samplers.  Since  sample collection  protocols
influence all subsequent laboratory and data analysis, it is essential that sediment
samples be collected  using  acceptable  and  standardized techniques  (USEPA,
1986-1991).

A variety of devices can be used to collect sediment samples, and these fall under two
main categories:  grab samplers and core  samplers (Mclntyre et al., 1984; ASTM, 1991).
Each  device samples the benthic habitat in a different manner (Table 4-18); therefore,
comparisons among dat%collected using different devices is not recommended.
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Methods
                 Table 4-18. Summary of Bottom Sampling Equipment3
         Device
         Use
      Advantages
        Disadvantages
  Fluorocarbon plastic
  or glass
  tube
Shallow wadable waters
or deep waters if
SCUBA available. Soft
or semiconsolidated
deposits.
Preserves layering and
permits historical sudy of
sediment deposition.
Rapid-samples immed-
iately ready for laboratory
shipment. Minimal risk of
contamination.
Small sample size requires
repetitive sampling.
  Hand corer with
  removable
  fluorocarbon plastic
  or glass liners
Same as above except
more consolidated sedi-
ments can be obtained.
Handles provide for greater
ease of substrate
penetration. Above
advantages.
Careful handling necessary to
prevent spillage, Requires
removal of liners before repetitive
sampling. Slight risk of metal
contamination from barrel and
core cutter.
   Box corer
  Gravity corers, that
  is, Phleger corer
  Young grab (fluoro-
  carbon plastic- or
  Kynar-lined modi-
  fied 0.1 m2 Van
  Veen)

  Ekman or box
  dredge
Same as above.
Semiconsolidated
sediments.
Collection of large
undisturbed sample
allowing for subsampling.

Low risk of undisturbed
sample contamination.
Maintains sediment
integrity relatively well.
Lakes and marine areas.  Eliminates metal
                       contamination. Reduces
                       pressure wave.
Soft to semisoft
sediments. Can be
used from boat, bridge,
or pier in waters of
various depths.
Obtains a larger sample
than coring tubes. Can be
subsampled through box
lid.
Hard to handle.
Careful handling necessary to
avoid sediment spillage. Small
sample, requires repetitive
operation and removal of liners.
Tirne-consuming.

Expensive. Requires winch.
Possible incomplete jaw closure
and sample loss. Possible shock
wave, which may disturb the fine
sediments ("fines"). Metal
construction may introduce
contaminants. Possible loss of
"fines" on retrieval.
  Ponar grab
  sampler
Useful on sand, silt,
or clay.
Most universal grab
sampler. Adequate on most
substrates. Large sample
obtained intact, permitting
subsampling.
Shock wave from descent may
disturb "fines." Possible
incomplete closure of jaws results
in sample loss. Possible
contamination from metal frame
construction. Sample must be
further prepared for analysis.
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                                                     Section 403 Procedural and Monitoring Guidance
                                  Table 4-18.  (continued)
      Device
         Use
      Advantages
        Disadvantages
BMH-53 piston
corer
Waters of 4-6 ft depth
when used with
extension rod. Soft to
semiconsolidated
deposits.
Piston provides for greater
sample retention.
Cores must be extruded on site to
other containers. Metal barrels
introduce risk of metal
contamination.
Van Veen
BMH-60
Useful on sand, silt, or
clay.
Sampling moving
waters from a fixed
platform.
Adequate on most
substrates. Large sample
obtained intact, permitting
subsampling.
Streamlined configuration
allows sampling where
other devices could not
achieve proper orien-
tiation.
Shock wave from descent may
disturb "fines." Possible
incomplete closure of jaws results
in sample loss. Possible
contamination from metal frame
construction. Sample must be •
further prepared for analysis.

Possible contamination from
metal construction. Subsampling
difficult. Not effective for sampling
fine sediments.
Petersen grab
sampler
Useful on most
substrates.
Large sample; can
penetrate most substrates.
Heavy, may require winch. No
cover lid to permit subsampling.
All other disadvantages of Ekman
and Ponar.
Shipek grab
sampler
Used primarily in
marine waters and large
inland lakes and
reservoirs.
Sample bucket may be
opened to permit
subsampling. Retains
fine-grained sediments
effectively.
Possible contamination from
metal construction. Heavy, may
require winch. Tends to roll over
sample.
Orange-peel grab
Smith-Mclntyre
grab
Useful on most
substrates.
Designed for sampling hard
substrates.
Loss of "fines." Heavy, may
require winch. Possible metal
contamination.
Scoops, drag buckets Various environments
                    depending on depth
                    and substrate.
                       Inexpensive, easy to
                       handle.
                         Loss offines" on retrieval
                         through water column.
Vibratory Corer
Used primarily for
sandy unconsolidated
sediments
Operational at all depths.
Easily transported by truck
and ship. Can penetrate
deeper than conventional
corers.
Has rarely been used for the
study of environmental pollution.
aComments represent subjective evaluations.
SOURCE: ASTM, 1991.
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Methods
The collection of undisturbed sediment requires that the sampler:

      •  Create a minimal bow wake when descending;

      •  Form a leakproof seal when the sediment sample is taken;

      •  Prevent winnowing and excessive sample disturbance when ascending; and

      •  Allow easy access to the sample  surface so undisturbed samples may be
         taken.

Penetration well below the desired sampling depth is  preferred to  prevent sample
disturbance as the device closes.  It is best to use a sampler with a weight adjustment to
enable the modification of penetration depths.

Grab Samplers

Grabs are capable of consistent sampling of bottom habitats.  Depending on the size of
the device, areas from 0.02 to 0.5 m2 and depths ranging from 5 to 15  cm may be
sampled. Limitations of grab samplers include :

      •  Variability in penetration depth among samples depending on sediment
         properties;

      •  Oblique angles of penetration, which result in varying penetration depths
         within a sample; and

      •  Folding of the sample, which results in the inability to section the sample and
         the loss of information concerning the vertical structure in the  sediments.

The  careful use  of these devices, however, will provide  quantitative  data.  Grab
samplers are the tools of choice for a number of marine monitoring programs (Fredette
etal., 1989; USEPA, 1986-1991).

Core Samplers

Box  corers use a surrounding frame to ensure  vertical entry and, therefore, vertical
sectioning of the sample (USEPA, 1986-1991).  These devices are capable of maximum
penetration depths  of 15 cm  and may collect volumes 5 to 10 times those of grab
samplers. Limitations of box corers include :

      •  Large size and weight, which require the use of cranes or winches and a
         large vessel for deployment;

      •  Higher construction expenses; and

      •  Lack of calibration studies to permit comparisons to grab samples.
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                                          Section 403 Procedural and Monitoring Guidance
The Hessler-Sandia box corer uses  dividers  to  section the core into  subsections,
facilitating subsampling of the core. Box corers are recognized as the tools of choice for
maximum accuracy and precision when sampling soft-bottom habitats.

Sediment Profiling Camera

The sediment profiling camera allows vertical  in  situ  imaging  of the water-sediment
interface, from which grain size determinations may be conducted. Sediment grain size
determinations may be made at a maximum depth of 18 cm; however, penetration depth
of the viewing  prism may  be limited  because  of the physical  characteristics of the
sediment (i.e., penetration depths are greater in silt than in sand).

Distinctions  between finer  silts  and  clays are  not  possible.   Furthermore,  it is
recommended that quantitative calculations of sediment grain size from the image be
ground-truthed  (verified) by the results  of laboratory  analysis  of  collected sediment
samples.

Although the singular use of the sediment profiling camera  is not recommended, the
camera may be effective as a reconnaissance tool. Delineation of habitats with similar
physical characteristics may aid in the selection of appropriate sampling stations.

Recommendations

Collection of sediments and collection of benthic organisms should be done concurrently
to mitigate the costs of field sampling and to permit sound correlation, regression, and
multivariate analyses (see Section 4.5, Benthic  Community Structure).  Therefore, it is
recommended that the sampling device also be suitable for benthic sampling. Grab and
core sampling devices permit adequate sampling of both sediment and benthic infaunal
communities with one sampling device.

Sample Depth

For characterizing surficial sediments, it  is recommended that the upper 2 cm of the
sediment column be evaluated; however, a minimum penetration depth of 4-5 cm should
be achieved.  Although the 2 cm specification is arbitrary, it does ensure that relatively
recent sediments are sampled, that adequate volumes of sediment  can  be obtained
using readily available samplers, and that data from different studies can be compared.
Penetration depth is a function of sediment type and is greatest in fine  sediments and
least in coarse sediments.  Sampling devices generally  rely on either gravity or a piston
mechanism to penetrate sediments.  Ideally, the penetration depth can be varied by
adding or subtracting weight from  the sampler, but if the sampler cannot be made to
consistently achieve the desired depth, an alternative device should be  used (USEPA,
1986-1991).
                                                                             97
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Methods
Sampling of depths other than 2 cm or vertical stratification of deeper sediment cores
may be appropriate, depending on the objectives of the monitoring program and the rate
of sediment accumulation.  For example, if information concerning only the most recent
sedimentation events is required, examination of the upper 1  cm may be appropriate.
Stratification of deeper cores will provide historical data of sediment grain size and
depositional events.  If the potential for bioaccumulation of contaminants in infaunal
organisms  is  a concern,  sampling to the depth of the anoxic  layer is desirable.
Comparison of data from studies analyzing different sediment depths is not advised.
Sampler penetration well below the desired sampling depth is preferred to prevent
sample disturbance as the device closes.

Selection of Time of Sampling

Sediment grain size compositions  are often temporally stable, although some slight
seasonal variability may be present.  Changes are usually associated with seasonal
patterns of  benthic turbulent mixing and sediment transport phenomena. The frequency
of sampling should be related to the expected rate of change in grain size compositions.
A consistent sampling period is recommended  in  order that spatial  and temporal
comparisons may be made.

If seasonal  variations are exhibited,  it is recommended that direct comparisons between
samples  collected  during  different seasons be avoided.    Studies investigating
interannual variation in the percent composition of grain sizes should  be  conducted
during the  same  season (preferably the same month) each year.  Furthermore, it is
recommended that grain  size  be  sampled under all  discharge  conditions so that
changes in  the sediment composition due to discharge loading can be tracked.

4.4.3     Analytical Methods Considerations

Sediment grain size may  be expressed  in either millimeters  (mm) or  phi (<&) units.
These scales are related according to the equation:  <I> = -Iog2 (mm).  Data should be
converted to phi  units before  calculation of grain size parameters.  Sediments are
broadly classified  into three size classes:  silts and clays are less than  0.064 mm (4 <&)
in diameter, sands range from 0.064 mm (4  O) to 1 mm  (0 <&) in diameter, and gravels
are larger than 1  mm (Shepard, 1954; Folk, 1974). Grain size is normally reported as
the mean (Mz), although the median grain size (Md-) is sometimes used. Sorting is a
measure of the spread of the grain size distribution.  Buller and McManus (1979) provide
a good review of the methodological and statistical analysis of sediment samples.

Particle size determination can either include or exclude organic material.  If organic
material is removed prior to analysis, the "true" particle size distribution is determined. If
organic material is included in the analysis, the "apparent1 particle size distribution is
ascertained.  Most organic material  is in the silt/clay size range and can be removed
from the sediment either by acid washing or ashing.  If organic  material is left in the
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                                          Section 403 Procedural and Monitoring Guidance
sediments, it will tend to bias the results toward a smaller mean size.  Because true and
apparent distributions differ, detailed comparisons between samples analyzed by these
different methods are questionable.  It is therefore desirable that all samples within each
study and among different studies be analyzed using one of these two methods, but all
samples must be analyzed consistently. A standardized grain size analysis will allow all
comparisons between samples within each study and between different studies.

Samples can be collected in  glass or plastic containers.  A minimum sample size of
100-150 grams  is recommended. All samples should be stored at 4°C and can be held
for up to 6 months before analysis.  Samples cannot be frozen or dried prior to analysis
since this may change the particle size distribution.

Before the sample is analyzed,  it should be mechanically homogenized.  The sample
should be well-mixed during homogenization but should not be ground.  If the organic
fraction is to be removed, removal should be done by  the addition  of 10  percent
hydrogen peroxide and subsequent boiling. The next step is to wet-sieve the sample to
separate the  sand and gravel fraction (greater than  62.5 (im) and the silt and clay
fraction (less than  62.5 |im).   The gravel-sand fraction  can  be subdivided  by
mechanically  dry-sieving it through a graded series of screens. At minimum, the coarse
subsample can  be divided into the gravel  fraction (greater than 2 mm)  and the sand
fraction (less  than 2 mm)  using a single filter with a 2-mm mesh size.  A larger number
of sieves can  be used to subdivide these samples, with the number being determined by
the needs of the monitoring program.

The  silt-clay  fraction is  subdivided  using a  pipet technique  that depends on  the
differential settling  rates of particles of varying size.  The sample is suspended in a
1-liter  beaker of  distilled water, and subsamples are  removed  at  precise  times
(dependent on temperature) from a constant depth, as shown in Table 4-19 (USEPA,
1986-1991).   The subsample  is then dried  and  weighed to measure the  mass of each
size fraction.  At a minimum, clay particles (less than 3.9 fim) should be separated from
the silts  (greater than 3.9 (im but less  than  62.5  jim), but  the number of fractions
depends on the  requirements of the monitoring program (USEPA, 1986-1991).

An alternative method for the determination of grain size  for the sand fraction of the
sediment is the  use of a settling tube.  This technique is based on Stake's Law,  which
describes the sinking rate of a  particle relative to its diameter.  This technique requires a
much smaller sample size (0.5-1.0 gm) as compared to the 100-gm sample required for
dry sieving.   Furthermore, settling tube analysis is  relatively  rapid, and automated
settling tubes  that input the data directly to a personal computer are available.

It is  important  that sieving techniques and  the desired  number of subfractions  be
specified and  standardized to allow for comparisons between samples.
                                                                             99
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Methods
    Table 4-19.  Sediment Grain Size: Withdrawal Times for Pipet Analysis as a
                 Function of Particle Size and Water Temperature8
 Diameter Diameter
   Finer    Finer Withdrawal
Elapsed Time for Withdrawal of Sample, in
 Hours (h), Minutes (m), and Seconds (s)
man
(Phi)
4.0
5.0
6.0
7.0
8.0b
9.0
10.0
man
(um)
62.5
31
15
7
3
1
0
.2
.6
.8
.9
.95
.98
uepin
(cm)
20
10
10
10
10
10
10
18°C
20s
2m Os
8m Os
31m 59s
2h8m
8h32m
34h6m
19°C
20s
1m 57s
7m 48s
31m 11s
2h5m
8h18m
33h 16m
20°C
20s
1m 54s
7m 36s
30m 26s
2h2m
8h 6m
32h 28m
21 °C
20s
1m
7m
51s
25s
29m 41s
1h
7h
31h
59m
56m
40m
22°C
20s
1m 49s
7m 15s
28rn 59s
1h56m
7h44m
30h 56m
23°C
20s
1m 46s
7m 5s
298m 18s
1h53m
7h32m
30h 12m
24°C
20s
1m44s
6m55s
27m39s
1h51m
7h22m
29h30m
a It is critical that temperature be held constant during the pipet analysis.
b Break point between silt and clay.
SOURCE: Modified from Plumb (1981).
4.4.4     QA/QC Considerations

It is critical  that each  sample be  homogenized thoroughly in the laboratory before a
subsample is taken for analysis.  Laboratory homogenization should be conducted even
if  samples  were  homogenized  in  the field  because  sediments  differentially sort
themselves  during transport and handling.  In addition, after dry-sieving a sample all
material must be removed from the sieve.

The total amount of fine-grained  material used for pipet analysis should be 5-25 grams.
If more material is used, particles may interfere with each other during settling and the
possibility of flocculation may be enhanced; if less material is used,  the experimental
error in weighing becomes large relative to the sample size.  Once the pipet analysis
begins, the settling  cylinders must  not be disturbed  because  this will alter particle
settling velocities.
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                                           Section 403 Procedural and Monitoring Guidance
It is recommended that triplicate analyses be conducted on 1 of every 20 samples, or on
1 sample per batch if fewer than 20 samples are analyzed.  It is recommended that the
analytical balance, drying oven, and temperature bath be inspected daily and calibrated
at least once per week.

4.4.5    Statistical Design Considerations

Statistical  strategies may mitigate the high costs of collecting  sufficient quantities of
sediment.   Power analyses considering the strategy of compositing samples can often
lead to a cost-effective monitoring design strategy.

Composite Sampling

Composite sediment sampling consists of mixing samples from two or more replications
collected at a particular location at different times or at different locations at the same
time.  The analysis of a  composite sample provides  an  estimate of.an average
contaminant  concentration  for  the  locations  composing  the  composite  sample.
Advantages of the composite sampling strategy are:

      •  It provides a cost-effective strategy when individual analyses are expensive.

      •  It results in a more efficient estimate of the mean at specified sampling
         locations.

Because of the reduced sample variance, composite sampling results in a considerable
increase in statistical power.   If  the  primary objective of a monitoring program is to
determine differences in grain size between sampling locations,  composite sampling is
an appropriate strategy.

Space-bulking consists of sampling from  several locations and  combining sediment
samples into  one or more composite samples.  Time-bulking involves taking  multiple
samples over time from a single location and compositing these samples.  Space- and
time-bulking  strategies  should  be  used  judiciously  since significant  information
concerning spatial and temporal heterogeneity may be lost.  The adoption of composite
strategies will depend on the objectives of individual monitoring programs.

Statistical  Power

Statistical power increases with an increase in the number of sediment subsamples in
each replicate composite sample (USEPA,  1987d).   However, with the addition of
successive subsamples  to  each  composite, a diminished return of statistical power
exists.  For composites  of greater than  10 replications,  the  increase  of power is
negligible given typical levels of data variability.
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Methods
To improve the power of a statistical test, while keeping the significance level constant,
the sample size should be increased. Because of constraints in cost and time imposed
by the monitoring program, however, this option may not be available.  Power analyses
have shown that for a fixed level of sampling effort, a monitoring program's power is
generally increased by collecting more replicates at fewer locations. The number and
distribution of sampling locations required to evaluate the effectiveness of the monitoring
program will depend on the size and complexity of the discharge and  the surrounding
environment.
4.4.6
Use of Data
Results from sediment chemical monitoring can be normalized according to sediment
characteristics to account for some of the variability found in bioaccumulation rates.
Likewise,  grain size information  may explain  the temporal and  spatial  variability in
biological  assemblages.   These  results  may  be used  to monitor  rates of change
following the beginning of discharge, to evaluate the condition of benthic habitats, and to
assist in providing early warnings of potential impacts on the marine ecosystem.

The singular use of sediment grain size to assess the condition of the benthic habitat or
to guide the decision-making process is not recommended.  The data collected from
monitoring the physical characteristics of sediments should be used in conjunction with
those collected from chemical and  biological monitoring. Subsequent analyses of health
risks to human populations and ecological risks to benthic individuals, populations, and
communities may be performed. Sediment grain size provides evidence essential in the
evaluation of spatial and temporal effects of ocean discharges.

4.4.7     Summary and Recommendations

Rationale

      •   The objective of monitoring the physical characteristics of sediments is to
          detect and describe spatial and temporal changes in sediment grain size.

      •   Results may be used to monitor rates of recovery following environmental
          interventions, to evaluate the condition of benthic habitats, and to assist in
          providing early warnings of discharge impacts to the surrounding ecosystem.

Monitoring Design Considerations

      •   It is recommended that the types of sampling gear and the location and
          timing of sample collection be consistent to allow for comparisons among
          studies.

      •   Collection of undisturbed sediment requires that the sampler:

          -  create a  minimal bow wake when descending;
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                                          Section 403 Procedural and Monitoring Guidance
         -  form a leakproof seal when the sediment sample is taken;
         -  prevent winnowing and excessive sample disturbance when ascending; and
         -  allow easy access to the sample surface in order that undisturbed
            subsamples may be taken.

      •  Analysis of the upper 2 cm is advised in order to examine recent depositional
         events.

      •  Penetration well below the desired sampling depth is preferred to prevent
         sample disturbance as the device closes.

Analytical Methods Considerations

      •  Because true and apparent distributions differ, detailed comparisons between
         samples analyzed by different methods are questionable. It is therefore
         desirable that all samples within each study and among different studies in
         the study area be analyzed using only one method.

QA/QC Considerations

      •  Laboratory homogenization should be conducted even if samples were
         homogenized in the field.

      •  It is recommended that triplicate analyses be conducted on 1 of every 20
         samples, or on 1  sample per batch if fewer than 20 samples are analyzed.

      •  It is recommended that the analytical balance, drying oven, and temperature
         bath be inspected daily and calibrated at least once per week.

Statistical Design Considerations

      •  Compositing sediment sampling consists of mixing samples from two or more
         replications collected at  a particular location and time period.

      •  Space-bulking (combining composites from several locations) and
         time-bulking (combining several composites over time from one location)
         strategies should be used judiciously since information concerning spatial
         and temporal heterogeneity may be lost.

      •  Power analyses have shown that for a fixed level of sampling effort, the
         power of a monitoring program is generally increased by collecting more
         replicates at fewer locations.
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Methods
Use ofJData
         The singular use of sediment grain size monitoring to assess the condition of
         the benthic habitat or to guide the decision-making process is not
         recommended; data collected from monitoring the physical characteristics of
         sediments should be used in conjunction with those collected from chemical
         and biological monitoring.

         Results from sediment grain size monitoring can be used to normalize
         sediment pollutant results to account for some of the variability found in
         bioaccumulation rates.

         Grain size information may explain the temporal and spatial variability in
         biological assemblages.
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                                          Section 403 Procedural and Monitoring Quittance
4.5
BENTHIC COMMUNITY STRUCTURE
The objective of benthic monitoring  is the detection and description  of spatial and
temporal changes in community structure and function.  The results can assess the
condition or "health" of berithic habitats and  monitor the rates  of recovery following
environmental or human disturbances. Benthic fauna also represent  a significant food
source for  many marine organisms,  thereby  providing  an early warning of potential
impacts on  the marine ecosystem.

Benthic habitats may be broadly divided into hard- and soft-bottom substrates.  Those
habitats with soft-bottom substrates (e.g., sand, silt, mud) are composed of a significant
benthic biological community known as infauna. This community consists of organisms
that burrow into the  substratum itself.   The communities  found over  hard-bottom
substrata such as coral reefs, rocky subtidal  areas, and "live bottom" sponge/gorgonian
habitats are referred to as epifauna. There are numerous species of polychaete worms,
pelecypod molluscs, and sponges capable of penetrating the substratum; however, the
majority of the community is found on top of the substratum.  The latter hard-bottom or
"live bottom" communities of dense sessile epifauna are valuable because they attract
commercially and ecologically important species of fish (Darcy and Gutherz, 1984).
4.5.1
Rationale
Of the 10 guidelines used to determine unreasonable degradation and no irreparable
harm, the following can be assessed by evaluating the benthic community structure:

      •  Composition and vulnerability of potentially exposed biological communities and

      •  Importance of the receiving water area to the surrounding biological
         community.

Monitoring the abundance  of individual benthic organisms is a widely-accepted means
of measuring the condition  off the benthic habitat (Bilyard, 1987).  Benthic organisms are
exceptional indicators of benfhic conditions since:

      •  They are usually  sedentary; consequently, observed effects are in response
         to local environmental conditions, and their relative longevity of up to a year
         or more provides a  level of consistency to data gathered.

      •  They are sensitive to habitat disturbance; communities undergo dramatic
         changes in species composition and abundance in response to
         environmental perturbations.

      •  They often mediate the transfer of nutrients and toxic substances in the
         ecosystem (via bioturbation and as important prey organisms).
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Methods
Furthermore, monitoring benthic organisms is one of a handful of methods that provide
in situ measures of biotic health and habitat quality.  The assessment of benthic infaunal
and epifaunal community structure is a powerful tool  in the evaluation of spatial and
temporal effects of anthropogenic and natural disturbance.

4.5.2     Monitoring Design Considerations

The level of effort required to assess benthic community structure  is relatively high. A
field survey is required to record and/or collect organisms, and sorting, identifying, and
enumerating specimens require labor-intensive  and generally expensive processes.
Evaluations of benthic community structure  and  function may  also be costly and
time-consuming.

The  results of benthic monitoring  programs can vary substantially depending  on the
objectives  and  corresponding design specifications.   The  characteristics that  are
primarily responsible for the variability in the results are :

      •  Type of surveying and/or sampling gear;

      •  Sample sorting and/or data analysis protocols;

      •  Level of taxonomy; and

      •  Location and timing of sample collection.

The  object is to standardize  the  measurement techniques at all stages of the 403
monitoring  program to ensure that the data can be intercompared.  Efforts should be
made to try to match new  monitoring data  with historical  data, but differences in
techniques and sampling gear should be avoided.  Historical data  may be used only to
note trends in the benthic fauna and, in particular, to follow any succession occurring as
a result of increases in pollution.

Analyses of power-cost efficiencies  are essential  in selecting the appropriate survey
technique and/or sampling gear and sample/data processing protocols.  Ferraro et al.
(1989) provide an example of power-cost analysis.

Different techniques are available for surveying and  sampling organisms in hard- or
soft-bottom habitats. Since these protocols will influence all subsequent laboratory and
data analyses, it is essential that  benthic samples be collected using acceptable and
standardized techniques.  Furthermore, samples of sediments and benthic organisms
should  be  collected concurrently to mitigate the costs of field sampling and to permit
sound correlation and  multivariate analyses (see Sediment Chemistry).  Therefore, it is
recommended that soft-bottom  sampling devices also be suitable for sampling the
sediment (Table 4-18).
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                                           Section 403 Procedural and Monitoring Guidance
 Hard-Bottom Habitat Surveys
 In hard-bottom habitats, benthic surveys are used to document the  relative cover of
 sessile organisms on the substratum and species distributions.  Depending on weather
 and water conditions, these surveys can be performed by divers, or remotely operated
 vehicles (ROVs) or submarines can be used (Gamble, 1984; Holme, 1984). Specimens
 and sediment samples can then be directly collected by the diver  or submarine to
 provide vouchers for positive taxa identification and  for pathobiological  or chemical
 analyses.

 Types of Surveys

 Numerous methods  and techniques have been examined and compared to quantify
 hard-bottom community structure, particularly on coral reefs. These include simple line
 transects, quadrats,  and plotless distance or nearest neighbor techniques.  The data
 collected  on  species and organism coverage of the substratum allow calculations of
 species diversity, abundances, richness, and percent live tissue.  Both  randomly placed
 and "permanent"  sites (marked by pins cemented into the substratum to allow accurate
 monitoring of the same site over time) have been used;  each suffers from its own biases
 for statistical analyses.  Each technique also presents problems in quantification and
 interpretation of  data  (see Loya,  1978;  Gamble,  1984;  Brown,  1988).  Results  of
 comparative studies indicate that:

      •   Although line transects are popular, they are not really very helpful unless
          multiple, closely-spaced 10-meter transects along same-depth contours are
          evaluated (Dustan and Halas,  1987).

      •   The linear point intercept method appears to be quite efficient and yields
          species abundance estimates similar to the true habitat (Ohlhorst et al., 1988).

      •   One-meter-square mapped quadrats provide good data for colony counts and
          estimates of percent coverage (Weinberg, 1981), but 4-meter-square
          quadrats are better for detecting rarer species.

      •   Photographed belt quadrats (1x10 meters-square) may provide the  most
          information, although clear water conditions and long analysis time are
          necessary (Dodge et al., 1982).

 Examples of  line transects  include  a  straight weighted line  stretched  over  the
substratum, with the species and lengths of live tissue underlying the line measured by
tape, or the modification by Porter (1972), in which a chain of uniform links  is placed
over the corals and the number of links  is counted and converted to area of live tissue
cover.   Rogers et al. (1983) used the ratio of the straight line  to the length of chain
required to cover the same distance as an  estimate of the topographic relief of the
substratum.  Photogrammetry, in which still photographs are made of continuous areas
under the transect line, provides a permanent record that can be quantified by  computer
                                                                            107
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Methods
digitizing techniques (Bohnsack, 1979; Dodge et al., 1982; White and Porter, 1985).
Further developments in this area include computer automation for analyses and using
underwater video to record along transects. Although the latter technique is faster and
easier to perform either by divers or ROVs, problems with quantifying the observations
have not been worked out. However, video does provide an important visual record for
assessing changes overtime (Holme, 1984; Rogers, 1988).

For all of these methods, a number of trade-offs must be considered. Since  most of this
work must be performed by divers, diver safety is of the utmost importance, including
the amount of bottom time available, the skill levels of the divers, and the type and
degree of pollution that may  be encountered.  The types and amounts  of information
required for each species, as well as the length of time of the survey, will also affect the
study design and techniques to be used.  For many hard-bottom areas, there may not
be one  "best" method for  monitoring and sampling the epifauna.  Thus, two or more
methods may be required to provide an adequate assessment of benthic community
structure over time (see, for example, Dodge et al., 1982; Holme and Mclntyre, 1984;
Dayton, 1985; Witman, 1985; Rogers, 1988; Phillips etal., 1990).

Soft-Bottom Sampling Devices

In soft-bottom  habitats, several  types of devices can be used  to collect  benthic
macroinvertebrate samples from the  sediment: trawls, dredges, grabs, and box corers
(Mclntyre et al., 1984).  The organisms collected in each sample are then identified and
species distributions determined. Most of these devices  sample the benthos in a unique
manner. Accordingly, conducting  comparisons between data collected using different
devices is  inadvisable.  The same area and volume of sediment should  be sampled
since different species of benthic infaunal macroinvertebrates exhibit different horizontal
and vertical distributions (Elliot, 1971).

Collection of  an acceptable sediment sample for infaunal  analyses requires that the
sampler:

      •  Create a minimal bow wake when descending;

      •  Form a leakproof seal when the sediment sample is taken;

      •  Prevent winnowing and excessive sample disturbance when ascending; and

      •  Allow easy access to the sample surface in order that undisturbed
         subsamples may be taken.

Penetration well below the  desired sampling depth is preferred to prevent sample
disturbance as  the device closes.  It is optimal to use a sampler that has a means of
weight adjustment to allow modification of penetration depths.
 108
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                                          Section 403 Procedural and Monitoring Guidance
Trawls and Dredges

Trawls and dredges usually collect organisms over a variable and relatively large area
(USEPA, 1986-1991; Fredette et al.( 1989). The data collected using these devices are
qualitative since consistent and reproducible sampling of a consistent benthic area is not
possible.  Their design  and use preclude  the  collection of infaunal  organisms at
sediment depths greater than a few centimeters.  Although trawls and dredges may be
used   to   collect   samples  of  either  infaunal   or  epifaunal  specimens   for
chemical/pathobiological studies,  the specimens can be damaged by physical scrapes
or breakage during this type of collection and adherent sand or silt particles could
interfere with these analyses. However, these devices are suitable for reconnaissance
of potential sampling sites.

Grab Samplers

Grab samplers are capable of consistent sampling of bottom habitats; depending on the
size of the device, areas from 0.02 to 0.5  m2 and depths ranging from 5  to 15 cm may
be sampled. Limitations of grab samplers include:

      •   Variability among samples in penetration depth, depending on sediment
          properties;

      •   Oblique angles of penetration, which result in varying penetration depths
          within a sample; and

      •   Folding of the sample, which results in the inability to section the sample and
          the loss of information concerning the  vertical structure in the sediments.

However,  the careful use of these devices will provide quantitative data.  Grab samplers
are the tools of  choice for a number of marine monitoring programs because of their
ability  to  provide quantitative data at a  relatively  low  cost.  (Fredette et al.,  1989;
USEPA, 1986-1991).

Core Samplers

Core samplers use  a surrounding frame to ensure vertical entry; vertical sectioning of
the sample is possible (USEEPA,  1986-1991). These devices are capable of maximum
penetration  depths  of 15 cm and may collect volumes  5  to 10 times that of grab
samplers. Limitations of box corers include :

       •   Large size and weight, which require the use of cranes or winches and a
          large vessel for deployment;

       •   Higher construction expenses; and

       •   Lack of calibration studies to permit comparisons to grab samples.
                                                                             109
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 Methods
 The Hessler-Sandia box corer  uses dividers to section  the  core into subsections,
 facilitating subsampling of the core.

 Box corers are recognized as the tools of choice for maximum  accuracy and precision
 when sampling soft-bottom habitats. Smaller corers may also be handled by divers.

 Suction Samplers

 In situ suction devices use the flow of water to collect sediments and benthic organisms
 (Eleftheriou and Holme, 1984).   An open-ended suction  tube draws sediments and
 organisms into the flow of water,  and the organisms are trapped in a sieve or mesh bag
 at the end of the tube. Limitations of suction devices include :

       •  Little control of the volume of sediment collected;

       •  Collection of animals from surrounding sediments, therefore inflating
         abundances; and

       •  Abrading organisms during collection, making identification even more
         difficult.

 Fleishack et al. (1985) propose modifications to suction samplers that overcome some
 of these limitations.  Although  samplers can be operated remotely, most  must be
 controlled by  divers.  Diver-operated collections are  restricted to scuba  depths and
 conditions that permit safe  diving (e.g.,  relatively calm  waters, reasonable visibility).
 This restriction limits their use in monitoring programs.

 Sediment Profiling Camera

 The sediment  profiling camera is  a unique tool that allows vertical,  in situ imaging of the
 water-sediment  interface.   Photographs of  biological activity may  be made  at  a
 maximum depth of 18 cm; however, the penetration depth of the viewing prism may be
 limited because of the physical characteristics of the sediment (i.e., penetration depths
 are greater in silt than in  sand).

The sediment profiling  camera  system  can  give measurements  of  organism  tube
density, thickness of the pelletal layer, and infauna present in the  image area (Rhoads
and Germano, 1982).  Although this tool provides qualitative  information concerning
 benthos  activity, the sediment  profiling camera system cannot  provide quantitative
information on species  diversity, abundance, and  biomass.  Information concerning
species composition cannot be gained using this tool.  Consequently, characterizations
of the  benthic community structure cannot  be made from  the  data taken from these
images.
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                                          Section 403 Procedural and Monitoring Guidance
The  sediment profiling  camera  is  not  recommended  for the  collection of benthic
community structure data; however, it is an effective reconnaissance tool.  Delineation of
physically similar sampling sites may be determined through the use of this tool, aiding
in  the selection of sampling stations.  Ground-truthing of these images by means of
laboratory analyses of collected material is highly recommended.

If quantitative analyses of infaunal benthic community structure are required, a grab or
core sampler is  recommended.  Either of these sampling devices permits adequate
sampling of both sediment and benthic infaunal communities with one sampling device.

Sampling Area

The  numerous species  of benthic macroinvertebrates have varying scales of spatial
distribution both  horizontally and vertically  (Elliot, 1971; Livingston et at., 1976; Loya,
1978; Downing, 1979).  Field and laboratory costs increase with increasing sample size;
therefore, the determination of the optimal  sample  size must consider  spatial  and
temporal scales, as well as statistical power.  Preliminary community surveys should be
undertaken to determine the most appropriate technique for the site(s) and the optimal
balance between the types of information and/or samples needed and the time available
for each survey/sampling session and subsequent analyses. In particular, the limitations
and  requirements  of the biological indices used in  the data analyses to determine
community structure must be carefully considered to set the optimal sampling area.

Selection of Sampling Period

Benthic  infauna assemblages  are dynamic;  the  most common  temporal patterns
observed in benthic communities are those associated  with  seasonal  changes.
Seasonal variation in  benthic  assemblages may be  due to  changes  in physical,
chemical, and/or biological parameters, e.g., temperature, light transmissivity, dissolved
oxygen, predation, recruitment, etc.

Given  the seasonal variation characteristic  of benthic  assemblages in general,  it is
recommended that  direct comparisons between samples  collected during different
seasons be avoided. Studies investigating interannual variation in the characteristics of
benthic communities should be conducted during the same season (preferably the same
month) each year.

Data Collection Protocols

For both hard and  soft bottoms, the species present and numbers of individuals need to
be determined  to  characterize the benthic community structure.  During  hard-bottom
surveys, this information  may be collected  by the diver (recording the  data on an
underwater slate or waterproof paper, on which the format for either transect or quadrat
information has been preprinted to facilitate data recording), or it may be derived from
photographs (examined by light microscopy or computer scanning techniques) at a later
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 Methods
 date.  The organisms collected by grab or corer devices from soft bottoms must be
 rinsed from the  sediments, preserved,  and sorted for identification and enumeration.
 Several options  must be  considered when designing sorting protocols.  To ensure
 comparability  between  studies,  a  consistent set of sorting  procedures is  highly
 recommended.

 Sieving: Mesh Size and Location

 The use of different sieve mesh sizes limits the comparability of results between marine
 monitoring studies (Reish,  1959; Rees, 1984).  The major advantage of using a smaller
 mesh size is the retention of both juvenile and adult organisms as well as large- and
 small-bodied taxa. The major disadvantage is the concomitant increased cost of sample
 processing.  For example,  using a 0.5-mm mesh over  a 1.00-mm mesh could increase
 retention  of total  microfaunal organisms  by  130-180 percent; however,  costs  for
 processing the samples may increase as much as 200 percent (USEPA, 1986-1991).

 It is recommended that a standard mesh size be selected for all monitoring studies. The
 1.0-mm mesh size is commonly  used in impact assessments, and the 0.5-mm mesh
 size is sometimes used in recruitment studies.  The  issue of data comparability was
 resolved by recommending sequential screening and analysis  procedures (Becker and
 Armstrong, 1988).  By screening samples through both the 1.0- and 0.5-mm sieves, the
 1.0-mm fraction could be analyzed separately and the data compared with other impact
 studies.  The data for the  0.5-mm fraction of the samples could then be combined to
 yield information  on the complete sample for comparison with other recruitment data.

 Sieving can be  done either aboard the survey vessel or on shore after the cruise.
 Sieving occurs prior to fixation  (sample preservation) aboard the vessel, whereas
 waiting  until after the  cruise  requires  fixation  prior to sieving.    If inadequate
 concentrations of fixative are added and deterioration or decomposition  of  organisms
 occurs,  there may be a significant detrimental modification of the sample.  If large
 numbers of samples are  to be  collected, field sieving reduces the sample volume
 required for storage, as well as the modification/loss of data.

 Use of Relaxants

The use of relaxants prior to sieving and fixation reduces the tendency of organisms to
distort their shape or  autotomize,  and consequently facilitates taxonomic identifications
and morphometric measurements (USEPA, 1986-1991). Complete organisms, having a
"natural"  appearance, are easier to identify to a lower taxonomic group  than  are
distorted or fragmented specimens.
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                                           Section 403 Procedural and Monitoring Guidance
The magnitude to which relaxants can influence taxonomic identification, thereby limiting
comparisons  between monitoring studies, is  unknown.  It is not recommended that
comparisons be conducted between studies in which relaxants were used and those in
which they were not used.

Use of Stains

Vital stains—primarily rose bengal—are added to samples to facilitate sorting.  The stain
colors most infauna, enhancing their contrast with background debris.  Not all taxa stain
adequately,  and  some taxonomists  have found  that  staining interferes  with the
identification of certain taxa.  Although staining may aid the sorting process, a proper
quality control program should ensure that sorting efficiency  is maintained whether  or
not a stain is used.

Level of Taxonomy

The necessity to identify organisms to the level of species has recently been questioned
(Warwick, 1986). The liabilities of identifying organisms to the species level include :

      •  Identifications may be inaccurate and imprecise (Ellis, 1985).

      •  Identifications are time-consuming and costly; costs can be reduced as much
         as 30-50 percent by limiting identification of samples to higher taxonomic
         groups (USEPA, 1986-1991).

Identifications to higher taxonomic groups can provide gross characterizations of benthic
assemblages and may be sufficient to meet program objectives concerning detection of
community responses to anthropogenic disturbance.  Warwick (1986) contends that
detection of anthropogenic disturbances would be facilitated since these perturbations
affect community structure at taxonomic levels higher than the species level. Natural
disturbances, which generally affect community structure by species replacement, would
have little influence on analyses at higher taxonomic  levels.

However, identification to higher taxonomic levels may sacrifice the potential wealth  of
information available using  species-level identification such as  analyses of population
dynamics  and productivity.   Furthermore, it is generally  regarded that species is the
taxon most susceptible to  environmental stress; individuals tend to  be replaced by
another species within the  same genus  before  the genus  is replaced  (USEPA,
1986-1991).

The level  of  taxonomic identification  will depend  on  monitoring program objectives,
sample  size,  study sites, analytical measure, and  availability of trained  taxonomists.
Data based on lower taxonomic levels can be grouped for future comparisons with
higher level taxa.  It is strongly recommended that all samples identified only to higher
taxonomic levels be properly archived, since comparisons of lower taxonomic data may
                                                                             113
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Methods
be required later.  For in situ hard-bottom surveys, divers must have adequate training to
recognize the species being examined. When in doubt, or for species that are difficult to
identify in the field (e.g., soft corals, sponges), voucher specimens  must be collected,
properly labeled, and submitted to specialists for identification.

4.5.3    Analytical Methods Considerations

A variety of approaches are available to  assess the  effects of discharges  on benthic
soft- or hard-bottom communities.  Many assessment approaches are derived from
modifications of  Connell's  (1978)  Intermediate   Disturbance  Hypothesis.    Some
researchers have proposed that selected  community structure measures (e.g., species
composition, abundance) are predictable along  a gradient of environmental disturbance
(Pearson and Rosenberg, 1978; Gray and Mirza, 1979; Warwick, 1986).  A corollary to
this supposition is that  indicator species whose responses would epitomize  community
responses to habitat perturbations can be identified.

These assessment approaches may be grouped into three categories:

       •  Biological indices,

       •  Indicator species, and

       •  Multivariate analyses.

However, there has been little  consensus among biologists regarding the suitability of
various techniques for describing  community characteristics and/or for  assessing
impacts due to discharges. A critical evaluation of the  use of biological indices  to detect
environmental  change  is presented in USEPA, 1985d (Table 4-20).  In addition to
measures of change in the abundances of pollution-sensitive, pollution-tolerant,  and
opportunistic species, the indices shown in Table 4-20  are evaluated  on the basis of the
following criteria:

       •  Biological meaning,

       •  Ease of interpretation, and

       •  Sensitivity to community changes due to anthropogenic sources.

The  results of these evaluations and additional  information on other  analytical  methods
are summarized below.

Biological Indices

The  numbers of individuals and the numbers of species have been found to  be good
indicators  of anthropogenic disturbance,  as well as  of other environmental  stresses
(Table 4-20; Pearson  and Rosenberg,  1978;  Warwick, 1985).  Furthermore, these
simple biological indices are less ambiguous and are  often as informative as  diversity
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Section 403 Procedural and Monitoring


Index/Method
Biological integrity
Bray-Curtis
Dominance6
Infaunal index
Number of individuals
Number of species
Opportunistic and
pollution-tolerant species
Pollution-sensitive
species
Cover
Biomass
Margalef's SR
J
Shannon-Weiner H'

Table 4-20. Biological Indices
Biological
Characteristic Measured
Community structure
Dissimilarity
Community structure
Community structure
Total abundance
Total taxa
Community structure
Community structure
Living tissue
Standing crop
Diversity
Evenness
Diversity


Guidance
mm

Recommended
for Monitoring3
B
P,B
P,B
Bc
P,B
P,B
P,B
P,B
B
P,B
P,B
P,B
P,B













aP(plankton), and B(benthos) indicate those biological groups to which a given
         index may be applied.

bDefined as the minimum number of species required to account for 75 percent
         of the individuals in a sample (see Swartz et al., 1985).

°Where developed.
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Methods
indices (USEPA, 1985d;  Green,  1979;  Hurlbert, 1971).  Measures of  biomass have
inherent problems  in the collection  of the data, e.g.,  loss or gain of  weight due  to
preservative medium, drying times, and evaporative weight loss.  However, biomass,
used in conjunction with species composition and  abundance data, may demonstrate
the relative effects of enrichment on benthic communities  (Pearson and Rosenberg,
1978).

More  complicated  indices—e.g., species  diversity,  species richness,  dominance,
evenness—have found varying degrees of acceptance. Diversity indices, measures of
the distribution of  individuals among species,  have the  following  limitations  (Green,
1979):

      •  Often lack biological meaning;

      •  Are not robust empirical indicators of ecosystem "health";

      •  Do not incorporate information on form and function of resident species; and

      •  Are susceptible to biases associated with well-described taxa.

However, species diversity indices are a widely used measure of community structure
and provide a consistent level of data reduction for large-scale comparisons.

The dominance index  is a measure of the degree to which one  or a few  species
dominate the  community.  The  dominance index, herein defined as the minimum
number of species  required to account for 75 percent of the total number of individuals,
has been useful in describing community structure (Swartz et al.,  1985).  It is easily
calculated,  does not assume  an  underlying distribution of individuals among species,
and is statistically testable.

Several graphical analyses of these simple biological indices have been developed
although most require further  study before their effectiveness is known.   For example,
the use of Gray and Mirza's (1979) lognormal distribution of individuals per species and
Warwick's (1985) abundance-biomass comparison (ABC) methods, though  useful for
some habitats, have been found to be unpredictable for other habitats and other locales
(USEPA, 1989e;  Beukema,  1988).    However,  Pearson  and  Rosenberg's  (1978)
species-abundance-biomass (SAB) model appears  to be applicable to several forms of
environmental disturbance  and may be used  in conjunction with other analyses  to
assess spatial and temporal impacts due to disturbance (Figure 4-2). The SAB model
provides a means to assess the condition of the habitat based on species numbers, total
abundance, and total biomass.  Severely polluted areas are identified  by the lack of
organisms,  low species numbers, total abundance, and biomass.  Highly polluted areas
are identified by a  few, highly abundant populations of small-bodied, pollution-tolerant,
opportunistic species  (e.g.,  Capitella capitata); total  abundance  is high, although
biomass and species numbers remain low. Less  polluted areas (e.g.,  farther from a
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                                          Section 403 Procedural and Monitoring Guidance
                                                                B
                             Increasing organic input
                                PO = peak of opportunists
                                E  = ecotone point
                                TR = transition zone
                                S  = species numbers
                                B  = total biomass
                                A  = total abundance
    Figure 4-2. Generalized SAB Diagram of Changes Along a Gradient of Organic
                 Enrichment (from Pearson and Rosenberg,  1978)
point source, or over time following the cessation of organic input) may be identified by
high species numbers and biomass, as more competitive, larger-bodied species are
able to tolerate sediment conditions.  More pristine areas are characterized by a number
of small populations  of competitively  dominant species; species numbers remain
elevated, although biomass and total abundance are low. This approach, however,
requires the comparison of two or more sites along the pollution gradient.

For hard-bottom communities, some of the methods used  successfully for detection of
pollutant impacts in soft-bottom communities (Warwick, 1986) appear to have little direct
application  (Brown, 1988).  For example, the  sensitivity of  diversity indices to describe
change in coral communities may be less than that of other parameters such as coral
cover,  colony size, and  abundance, suggesting  that each parameter  requires careful
analysis based on the previous environmental history of the site, the nature of the
impact, and the community structure of the reef. Grigg and  Dollar (1990) have proposed
a stress index that measures net mortality of the organisms over time, partitioning the
mortality due to natural causes (N) and that due to man-made influence (I), thus allowing
these contributions to be compared. In terms of survival then:
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 Methods
                            Stress Index = 1.0-(N + I),

 where N +1 varies from 1 (total destruction) to 0 (zero mortality).

 Although there are problems in applying this index in  the field, development of a
 quantitative  index of  stress would assist  in  the assessment  of natural  versus
 anthropogenic contributions in hard-bottom benthic communities (Brown, 1988).

 Indicator Species

 Examination of abundances of individual indicator species are generally informative and
 may reduce the cost of the analysis.  The absence of pollution-sensitive species and the
 enhancement of opportunistic and pollution-tolerant species may assist in defining the
 spatial and temporal extent and  magnitude of impacts.  However, indicator variables
 must possess the following characteristics (Green, 1979):

       •   Must provide a sufficiently precise and accurate appraisal of:

          -   species of concern,
          -   anthropogenic disturbances to benthic communities, and
          -   presence/absence or magnitude of anthropogenic perturbation to the
             ecosystem.

       •   Must provide a cost-effective and statistically reliable alternative to monitoring
          all critical benthic community measures of habitat perturbation.

       •   Must be appropriate for the spatial and temporal scale demanded by the
          study objectives.

 Pearson  and  Rosenberg  (1978) demonstrated  that  along a  gradient  of  organic
 enrichment, a predictable community of benthic infauna could be observed (Figure 4-3).
 Communities  consist of opportunistic,  tolerant species in areas of severe pollution,
 giving  way  to less  tolerant  and  more competitively dominant species further from
 severely polluted areas (Figure 4-3). This model of indicator species composition and
 distribution has been found to be useful  in assessing both natural and anthropogenic
 disturbances.

 Several  indices,  derived from the  distributions  of  pollution-sensitive  species and
 opportunistic disturbance-tolerant species, have been developed to evaluate impacts to
 infaunal benthic community structures:  copepod/nematode ratio  (Raffaelli and Mason,
 1981),  infaunal trophic index (Word, 1978), and organism-sediment index (Rhoads and
 Germane, 1986).  The infaunal index, which was initially developed for  the Southern
 California Bight,  uses  the abundances  of  four  functional  groupings to  describe
community structure. Low values of the infaunal index indicate communities dominated
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                                           Section 403 Procedural and Monitoring Quittance
 Figure 4-3. Diagram of Changes in Fauna and Sediment Structure Along a Gradient of
              Organic Enrichment (from Pearson and Rosenberg, 1978)


by  deposit-feeding  organisms  that  are considered  to be  more  impacted  than
communities  dominated  by suspension  feeders  (high infaunal index values)(Word,
1978).

Polluted or disturbed conditions may be indicated by:

      •  High rates of the nematode/copepod ratio;

      •  Low values (A) of the infaunal index; and

      •  Low values (7) of the organism-sediment index.

However, further studies of the response patterns of infaunal species subjected to
anthropogenic perturbations are required in order to select appropriate indicators of
benthic community impact.  Again for hard-bottom communities, the use of epifaunal
indicator  species and other indices requires further research (Brown, 1988).

It is  recommended that  multiple pollution-tolerant and  pollution-sensitive indicator
species be selected by clearly defined criteria. Multiple indicator species often provide a
more complete representation of environmental conditions.
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Methods
Multivariate Analyses

Numerical classification encompasses a wide variety of techniques that have been used in
the analysis of benthic  data to  distinguish groups of entities  (e.g., sample locations)
according to similarity of attributes (e.g., species).  These techniques  differ from most
multivariate methods  in  that  no assumptions  are  made  concerning the underlying
distributions of the variables.  Detailed descriptions of numerical classification analysis can
be found in Pielou (1984), Romesburg (1984), Clifford and Stephenson (1975), Boesch
(1977), Sneath and Sokal (1973), and Anderberg (1973).  Boesch (1977)  is particularly
valuable  as an introduction and guide to the  use  of  numerical  classification analysis in
marine environmental  studies.  Guidance on the interpretation of classification results is
provided in an EPA Technical Support Document (USEPA, 1988d).

Ordination analyses have also been used to reduce the dimensionality of the data set while
maintaining the relationship among similar and dissimilar entities.  At present, no single
ordination technique has been shown to be clearly superior for the analysis of biological data
(USEPA, 1985d).

Multivariate analyses are effective heuristic tools. They generate visual representations
that often indicate where further analyses ought to be conducted.

Analytical Approach Recommendations

Some of the most informative measures of community structure are the simplest (Table
4-20):

      •  Number of individuals,

      •  Number of species,

      •  Dominance,

      •  Infaunal index,

      •  Abundances of pollution-sensitive species, and

      •  Abundance  of opportunistic and pollution-tolerant species.

These indices have proved to be useful over various  habitats and regions in assessing
changes to benthic community structures (USEPA, 1985d). Values of these indices may
be determined from  the  list of species  abundances generated during the taxonomic
identifications of collected specimens. Furthermore, the values of these six variables may
be easily tested  statistically using parametric or nonparametric  techniques.   It  is
recommended that no single index or analytical method be used  to  assess  impacts;
instead, the assessment of  impacts should incorporate information that each variable
and method contributes concerning benthic community structure.
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                                          Section 403 Procedural and Monitoring Guidance
Selection  of reference sites  is  key  to the  evaluation of  environmental  impact
assessment.  Results of analyses using  reference measures provide the means of
comparison by which anthropogenic  impacts are detected.  It is essential that selected
reference sites exhibit at least similar:
      •  Sediment characteristics (i.e., grain size or substratum type),
      •  Water depths,
      •  Flow characteristics,
      •  Salinity,
      •  Dissolved oxygen, and
      •  Temperature
compared to monitoring  program sampling sites.   Several  reference sites may  be
required to meet these criteria.
4.5.4     QA/QC Considerations
Sample Collection
Surveying and sampling equipment should be inspected for wear and tear to avoid loss
of data or sample leakage and loss upon ascent.  It is recommended that backup survey
and  sampling equipment be  available  on board  the vessel in  case  the primary
equipment breaks down or is lost during the cruise.
The  following infauna sample acceptability  criteria  should  be  satisfied  (USEPA,
1986-1991):
      •  Sediment is not extruded from the upper face of the sampler such that
         organisms may have been  lost.
      •  Overlying water is present,  indicating minimal leakage.
      •  The sediment surface is relatively flat, indicating minimal disturbance or
         winnowing (Figure 4-4).
      •  The entire surface of the sample is included in the sampler.
      •  The desired penetration depth is achieved.
If the sample does not meet all the criteria, it should be rejected.
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Methods
           Acceptable if Minimum
        Penetration Requirement Met
       and Overlying Water is Present
               Unacceptable-
        (Canted with Partial Sample)
        Unacceptable
(Washed, Rock Caught in Jaws)
        Unacceptable
          (Washed)
   Figure 4-4. Examples of Acceptable and Unacceptable Samples (USEPA, 1987c)
Appropriate QA/QC protocols for assessing the validity of hard-bottom surveys (e.g.,
multiple transects,  resurveying one or more transects or quadrats by different divers)
should also be followed.

Taxonomic Identification

A key QA/QC issue is taxonomic standardization.  Consistent taxonomic identifications
are achieved through interaction among taxonomists working on each major  group.
Participation of the laboratory staff in regional taxonomic standardization programs is
recommended to ensure regional consistency and accuracy of identifications.

Five percent of  all samples  identified by  one taxonomist should be reidentified by
another taxonomist who is also qualified to identify organisms in that major group.

It is advisable that at least three individuals of each taxon should be sent for verification
to recognized experts. These verified specimens should then be placed in a permanent
reference collection.   All specimens in the reference  collection should be stored in
labeled vials that are segregated by species and sample. Reference specimens should
be archived alphabetically within major taxonomic groups.
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                                           Section 403 Procedural and Monitoring Guidance
 It is also recommended that at least 20 percent of each sample be re-sorted for QA/QC
 purposes.  Re-sorting is the examination of a sample or subsample that has been sorted
 once and is considered free of organisms. Re-sorting should be done by someone other
 than the one who sorted the original sample. If a sample is found that does not meet the
 recommended 95 percent removal criterion, the entire sample should be re-sorted.  For
 epifaunal surveys, a portion of the photographed quadrats should be reanalyzed for
 comparisons.

 4.5.5    Statistical Design Considerations

 Consideration of statistical strategies will  mitigate the  high  costs of collecting and
 processing samples.

 Temporal Stratification of the Data

 The time of the year should be controlled or stratified in  the design; the use of annual
 averages is seldom a good practice. Temporal stratification of the data should not be
 attempted  until sufficient knowledge of long-term  natural cycles  is attained. Initially,
 simple regression analyses may be conducted on seasonally  stratified data  to identify
 monotonic  temporal trends.  Further examinations of whether conditions are  improving
 or degrading over time may be performed using various statistical time series analyses.

 Statistical  Power

 Selection of the number  of replicates is an important component of program design.
 The inherent patchiness of benthic communities requires collection of sufficient replicate
 samples to ensure an accurate description of the benthos.  However, increases in
 replication increase sample processing costs. Power analyses assist in the allocation of
 sampling resources (stations,  replication,  and frequency) with regard to program
 finances and design.

 Power analyses  may be applied to determine the  appropriate  number of sample
 replicates required to detect a specified difference (USEPA, 1987d).  The number of
 replications required to  detect  a specified minimum difference  is a function of  the
 statistical power  and the variance  in the data.   Power analyses  require a  priori
 knowledge  of the variability in the data.  A best guess or,  preferably, variation observed
 in historical data is often used initially in the design of the monitoring program.

To improve the power of a statistical test, while  keeping the significance level constant,
the  sample size (area sampled, number of replicates of grabs, quadrats, or  transects)
should be increased. Because of constraints in cost and time, however, this option may
not  be available.  Power analysis has shown that for a fixed level of sampling effort, a
monitoring program's power is generally increased by collecting more replicates at fewer
locations. Sampling should be conducted in a radiating pattern from the zone of  initial
discharge out to the distance where there will be no effects.
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Methods
4.5.6
Use of Data
Monitoring  of benthic community structure  provides in situ measures of the  benthic
habitat and is a powerful tool in the  evaluation of spatial and temporal effects of
anthropogenic and natural disturbance.  The presence or absence of certain organisms
is useful in indicating the previous condition of the environment (Bilyard, 1987; Tomascik
and Saunder, 1987).  Monitoring of benthic infaunal communities also provides data
required in  the design and validation of benthic community dynamics models (Pearson
and Rosenberg,  1978; Brown, 1988) and the selection of biological  indicators (Word,
1978).

In  addition,  monitoring of benthic communities  directly provides  accurate information
essential in assessing the effectiveness of  discharge reductions  (Bilyard, 1987).  For
example,  benthic  infauna  monitoring  provided  information  used  to assess the
effectiveness of pollution abatement plans in the recovery of  Southern California waters
(Reish,  1986; SCCWRP, 1988).  These studies  indicate  that  analyses  of  benthic
communities may be  effectively used to monitor the long-term health of the receiving
environment (Reish, 1986).
                             i
Currently, benthic communities have not proved useful for  identifying specific chemicals
or classes  of chemicals  present in the environment.  Further information concerning
specific responses to specific contaminants is required before infaunal  community
structure becomes useful in identifying specific contaminants (USEPA,  1989e).   In
addition, caution is recommended in the use of benthic community structure to predict
specific effects on potential predators.  Information on trophic relationships, competition,
and predation is  often not available. The capability to predict the effects of altered prey
communities on predators may improve with research on these topics. Factors  such as
prey  quality, distribution of  prey, and  interactions among  species  will  be important
components of this research.

However, benthic  invertebrates  do  serve as  effective  indicators  of  environmental
condition,  delineating  the   magnitude,  spatial  extent,   and  temporal  trends   of
anthropogenic and natural perturbations to the ecosystem  (Reish, 1986; Bilyard, 1987).
Monitoring   of benthic  infauna  and  epifauna  will provide  relevant accurate data
fundamental to achieving the objectives of most monitoring programs.

4.5.7    Summary and Recommendations
 Rationale
          The objective is to detect and describe spatial and temporal changes in the
          structure and function of benthic communities.
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                                          Section 403 Procedural and Monitoring Guidance
      •  Benthic monitoring provides in situ measures of habitat quality and is a
         powerful tool in assessing environmental impact.

Monitoring Design Considerations

      •  It is recommended that consistent types of surveying and sampling gear, data
         collection and sample sorting protocols, level of taxonomy, and location and
         timing of sample collection be implemented to allow for comparisons between
         studies.

      •  To reduce the variation due to seasonal differences, sampling should be
         conducted during the same season—preferably the same month—each year.

      •  Voucher specimens should be collected for in situ epifaunal surveys.

      •  For epifaunal surveys, data are collected on species and organism coverage
         of the substratum by diver, ROV, or submarine from specified transects or
         quadrats.

      •  For infaunal sampling, collection of undisturbed sediment requires that the
         sampler:

         -   create a minimal bow wake when descending;
         -   form a leakproof seal when the sediment sample is taken;
         -   prevent winnowing  and excessive sample disturbance when ascending; and
         -   allow easy access to the sample surface in order that undisturbed
            subsamples may be taken.

      •  Penetration well below the desired sampling depth is preferred to prevent
         soft-bottom disturbance as the device closes.

      •  Grab samplers and box corers are recognized as the tools of choice for
         maximum accuracy and precision when sampling soft-bottom habitats.

      •  Sorting through a standard sieve mesh size (i.e.,  0.5 mm) is recommended.
         Further sorting through other mesh sizes may be conducted in addition to
         sorting through this standard mesh size.

      •  Relaxants facilitate identification and morphometric measurements; however,
         standard procedures must be implemented to ensure valid comparisons
         among studies.

      •  Vital stains may facilitate sorting; however, a proper QA program should
         ensure that sorting efficiency is maintained.

      •  Identifications to higher taxonomic levels may be sufficient to meet program
         objectives; however, it is recommended that all samples be archived if
         comparisons to lower taxonomic levels will be required at a later date.
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 Methods
Analytical Methods Considerations

      •   It is recommended that simple measures of community structure be used to
          assess the condition of the benthos: number of individuals, number of
          species, dominance, infaunal index, abundance of pollution-sensitive
          species, abundance of pollution-tolerant species, and percent cover for
          hard-bottom indicator groups.

      •   Selected biological indices should retain biological meaning, be robust
          indicators of ecosystem "health," and incorporate species form and function.

      •   Indicator species should possess the following characteristics:

          -   sensitive to benthic perturbations of concern;
          -   cost-effective and statistically reliable alternative to measuring all species
             in a monitoring program;
          -   statistically reliable indicative measures of habitat perturbations; and
          -   appropriate for the spatial and temporal scale demanded by the study
             objectives.

      •   Selection of reference sites is key to the evaluation of environmental impact
          due to anthropogenic impacts; several reference sites may be required.

QA/QC Considerations

      •   Taxonomic standardization is essential to the analysis of community
          structure. Recommended protocols include consistent interactions among
          taxonomists, reidentification of selected samples, use of a reference
          collection, and re-sorting and analysis of selected samples or subsamples.

Statistical Design Considerations

      •   Power analyses  may be applied to determine the appropriate number of
          sample replicates required to detect a specified difference, thereby optimizing
          the high costs of collecting and processing samples.

Use of Data

      •   Data provide essential information in order to assess impacts due to
          anthropogenic perturbation, monitor recovery of the receiving environment,
          and validate community and population models.
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                                           Section 403 Procedural and Monitoring Guidance
4.6
FISH AND SHELLFISH PATHOBIOLOGY
Pathobiological methods provide information concerning damage to organ systems of
fish  and shellfish through an evaluation  of their structure,  activity, and function.
Anatomic pathology methods can give an indication of the nature of an altered state, for
example, by identifying the specific type of tumor present in an animal.  Reproductive
developmental studies examine the reproductive capacity of animals and can provide
information to aid in estimating and predicting population abundance and recruitment.
Biochemical/enzymological studies seek to detect differences in enzymatic activity as a
measure of biological condition.   Immunological  methods can demonstrate altered
immune response, an indicator of changes in bodily defense mechanisms and increased
susceptibility to disease.

Pathobiological methods should be  used  in concert to investigate cause-and-effect
relationships as a result of contaminant exposure. Anatomic pathology can serve as a
vital  link between observed effects on populations and communities in an estuary and
the changes in activity and function observed by other methods.
4.6.1
Rationale
Pathobiological methods can be used to examine adverse effects of pollutants on fish
and shellfish.  The presence of toxics in  water and sediments may not immediately
result in visible changes in these organisms.  Biomarkers offer a more sensitive and
reliable assessment  of  exposure  risks  than ambient  water  or  sediment  quality
monitoring.  Monitoring of pathobiological  effects provides  information necessary to
make determinations  of the existence of adverse  effects in animals  (e.g., tumors),
population productivity and stability (affected by reproduction and disease states), and
the loss of organisms  deemed valuable for ecological, aesthetic, recreational, scientific,
or economic reasons.  Table  4-21  outlines  some of the terms used to  describe
pathobiological methods.

Although the value  of these methods for establishing cause-and-effect links has  been
established during laboratory toxicity studies, some questions remain regarding their ability
to establish such links for field-collected organisms that are exposed to a variety of natural
environmental stresses and combinations of contaminants (Hinton and Couch, 1984; Couch
and Harshbarger, 1985; Mix, 1986;  Sindermann, 1990).  However, properly conducted
multidisciplinary monitoring  studies using these methods can provide regulatory agencies
with evidence of impaired health status in animals exposed  to contaminants in  estuarine
ecosystems.  This information can then be used to direct laboratory confirmation of the
cause, if necessary (see, for example, Buckley etal., 1985; Gardner et al., 1991). Continued
monitoring with these  methods  can be used to detect changes in a population's health
during and following environmental intervention.  Because  changes at the organismal
level  precede  changes in  population and  community characteristics, pathobiological
studies can provide an early indication of the effectiveness of management actions.
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Methods
biomarker




cytochrome P450



cytogenetics




genotoxic agents




hepatic

histopathology



hfstochemistry



immunoassay



immunology



inclusion bodies


macrophage


pathobiology
smooth endoplasmic
reticulum (SER)
Table 4-21. List of Pathobiological Terms


  any biological method used to detect the exposure of organisms to hazardous
  chemicals in the environment by measuring the response of the organisms to the
  contaminant through comparative molecular, biochemical, physiological, or
  anatomical observations of cellular dysfunction

  a protein in the microsomes of liver cells that is important in catalyzing the
  metabolism of steroid hormones and fatty acids and in the detoxification of a variety
  of chemical substances

  the study of cytology in relation to genetics, especially the study of chromosomal
  behavior in mitosis and meiosis. Modern cytogenetics has led to the identification of
  chromosomes as bearers of the genes and deoxyribonucleic acid (DMA) as the key
  molecule of the  gene

  chemical and physical agents that can produce genetic alterations at subtoxic
  concentrations and can result in altered heredity characteristics. Genotoxic agents
  generally possess specified chemical or physical properties that facilitate their
  interaction with nucleic acids

  of, or relating to, the liver

  pathologic histology; the science or study dealing with the cytologic and histologic
  (microscopic) structure of abnormal or diseased  cells, tissues, and organs in relation
  to their function

  the study of the  chemistry of cells and tissues using light and electron microscopy,
  special chemical tests, and special stains to determine the location of certain enzyme
  systems or reaction products in the cell

  measuring the protein and protein-bound molecules that are concerned with the
  reaction of an antigen with its specific antibody, as in the detection of hormones or
  other substances

  the study of being protected from a disease; the  study of the response of the body
  and its tissues to a variety of antigens, including  red cells, pollens, transplanted
  tissues, and even the individual's own cells

  bodies present in the nucleus or cytoplasm of certain cells in cases of infection by
  filtrable viruses or as the result of degenerative diseases or exposure to chemicals

  cells of the reticuloendothelial system having the ability to phagocytose particulate
  substances and to store vital dyes and other colloidal substances

  pathology, the study of disease, with emphasis more on the biological than the
  medical aspects of the essential nature, causes, and development of abnormal
  conditions,  as well as the structural and functional changes that result from the
  disease process

  a connecting network of tubules that course through the cytoplasm of the cell and can
  be viewed using the electron microscope; SER is essential to metabolic functions of
  the cells
SOURCES: Stedman's Medical Dictionary, 1982; Taber"s Cyclopedic Medical Dictionary, 1985.
EL'
   .<
   mSA
•:ir
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                                          Section 403 Procedural and Monitoring Guidance
4.6.2     Monitoring Design Considerations

A field survey to collect target organisms  and tissue samples may be required for
pathobiological monitoring (Hargis et al., 1984; U.S. EPA, 1987b).  In certain instances,
a large sample size may be needed to  establish statistical significance because of
normal variation from animal to animal, species and generic differences, and migratory
habits  of fish.  For these reasons, and the labor-intensive nature of pathobiological
methods, the availability and allocation of funds, time, equipment, and trained personnel
must be considered when planning to include pathobiological  methods in  monitoring
programs.

Selection of Target (Indicator) Species

A key component of any pathobiological monitoring program  is the selection of target
species.  The fundamental criterion is the ability to use the selected species to make
comparisons between sampling locations and sampling periods.  It is recommended that
the target species possess the following characteristics:

      •  Abundant enough, temporally and spatially, to allow for adequate sampling;

      •  Large enough to provide adequate amounts of tissue for analysis;

      •  Sedentary (nonmigratory) in nature to ensure that pathobiological
         abnormalities are representative of the study area; and

      •  Easily collected.

It  is also  recommended  that  an  existing database  documenting  exposures and
sensitivities of the target organism specific to contaminants of concern is available.

Fish provide sufficient tissue for analyses and may indicate potential threats to human
populations. However, fish are motile and pathobiological abnormalities detected may
not be representative of the study area (see Vogelbein et al., 1990).

Bivalve molluscs, either attached to the substratum  or burrowing  in sediments, have
been sampled extensively to examine the condition of these commercially important
estuarine species (e.g., Mussel Watch, National Status and Trends program). The most
common target species have been oysters (Crassostrea virginica) and mussels (Mytilus
spp.), although pathobiological and bioassay studies have also been performed on other
species, such as crabs and penaeid shrimp.

Secondary  considerations, based on economic importance and status as  a bioassay
organism, may be  applied to further winnow the list of candidate  target species to  a
practical number of species to be analyzed.
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Methods
Sampling Location

Appropriate locations of sampling stations depend on the objectives of the study.  For
example, to evaluate whether there is  a statistically significant increase in lesions,
stations should be located to collect specimens from contaminated and uncontaminated
(background  or  control) areas  for  statistical comparison.   It  is also  important to
demonstrate  a  dose-response relationship between  the pollutant concentration  and
incidence of tumors or other lesions  by locating sample stations along a contamination
gradient (i.e., from highly contaminated to moderately contaminated to uncontaminated).
It is recommended that stations be located in  areas where the geographic area of
contamination is large enough that sampled fish could reasonably be  expected to have
spent a considerable amount of time within the influence of the pollutant (U.S. EPA,
1987b). Nielsen and Johnson (1984) and Cailliet et al.  (1986)  may be consulted for
further fish  sampling  methodologies.  Information on sampling bivalves and other
invertebrates is contained in Couch  (1978), Yevich and Barszcz  (1983), Turgeon et al.
(1991), and other sources.

4.6.3    Analytical Methods Considerations

Anatomic Pathology Methods

Anatomic  pathology methods examine tissues with the naked  eye (gross  anatomic
pathology), the aid of a light microscope (LM methods), or the electron microscope  (EM
methods).  Gross anatomic methods are concerned with obvious adverse changes in
tissue that can  be observed in the  field  (Hunn,  1988; Hargis et  al.,  1984).   The
advantage of gross methods is  that large numbers  of specimens  can be examined
rapidly.  However,  the  methods are generally nonspecific  (i.e., it is not possible to
determine that a specific pollutant led to a specific disease).  An exception to  this is
cataracts  in  fish,  which have  been  linked with polynuclear  aromatic  hydrocarbon
pollution in the field (Hargis and Zwerner, 1988).

A  disadvantage of anatomic  pathology  methods is that they require specialized
personnel  and laboratories.  The methods are, however, generally standardized, routine,
and operational in existing Federal,  State, university, veterinary, and  private diagnostic
laboratories that specialize in aquatic animal pathology. Current  activities are aimed at
developing a field-to-laboratory response-diagnostic scenario in which adverse effects
are found in the field, diagnosis is made in the laboratory, and experimental studies are
initiated to verify results.   Refer to Yevich and  Barszcz (1980), Howard and  Smith
(1983), Johnson and Bergman (1984), Klontz (1985), Meyers and  Hendricks (1985), and
U.S. EPA (1987b) for more information on these methods and techniques.
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                                           Section 403 Procedural and Monitoring Guidance
Light Microscopy Methods

LM (histologic) methods  use the  light microscope to view cells and tissues.  These
methods can  detect  changes  such as  inflammatory  responses,  alterations  in the
appearance of cells, cancerous/precancerous lesions, and damage due to parasites
(USEPA, 1986c).  The advantages of LM methods are that they are organ-specific (i.e.,
the lesion can be localized to a specific organ) and can detect microscopic and subtle
cellular alterations that are not evident on visual inspection. However, LM methods are
more expensive (approximately $30/sample) and slower than gross methods.

Electron Microscopy Methods

EM methods use the  electron microscope to detect changes  in tissue at cellular and
subcellular levels, such as the identification of the nature of inclusion bodies or changes
in the amount of smooth  endoplasmic reticulum.  An advantage of EM methods is that
they can be highly specific because the pollutant can be localized within certain parts of
the cell.  These inclusions may contain the causative chemical agent; lead, gold, iron,
bismuth, uranium, beryllium, mercury, copper, and arsenic are a few of the metals that
can be deposited intracellularly (Sorenson and Smith, 1981).  Additionally, EM methods
can  be used to investigate subcellular mechanisms of pollutant action.   The  major
disadvantage of EM methods is that they are very expensive (minimum $400/fish) and
very slow, requiring highly skilled technical expertise.

Histochemical Methods

Histochemical  methods use the microscope in conjunction with special chemical tests
and  stains to localize specific enzyme systems or reaction  products in the cell. For
instance, histochemical assays  have  been used on liver tissue during and after tumor
formation to yield important information on the biochemistry of specific lesions (Hinton et
al.,  1988; Prophet et al., 1992; Sumner, 1988). Histochemical methods are reliable and
can be highly specific for  certain classes of organic compounds and metals.  However,
highly skilled technical expertise is required  to carry out the methods in the laboratory.

In Vitro Tests

In vitro tests are generally more sensitive than whole animal systems, less expensive to
carry out, and of shorter duration. However, in in vitro tests, defense mechanisms found
in the intact animal are missing.  In  vitro  systems offer the greatest flexibility for the
testing and study of environmental contaminants.  They can be designed to be relevant
to the species of interest  in a given area,  and multiple types of measurements can be
taken from a single test  system (e.g., metabolic products, mitotic activity, cytotoxicity
and genetic damage).  However, comparative in vitro and in vivo studies are needed  to
correlate and relate changes that occur in each system (Landolt and Kocan, 1983).
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Methods
Reproductive/Developmental Methods

Reproduction studies are  designed to examine a  number of parameters  in the
reproductive process:  gross examination of the egg and numbers of eggs,  embryo
viability,  the proportion  of fertilized  eggs  (i.e., fertilization  success),  and  larval
development and viability  (USEPA,  1986c).   Studies that  examine  the  egg itself
incorporate microscopic methods that are time-consuming and expensive (West, 1990),
but serve as a direct measure of reproductive success. Some methods deal with the
egg at the molecular level and analyze the action of chemical and physical  agents
whose toxicity is directed toward  genetic (DNA) components of the egg.

Different cytogenotoxic tests can be used to measure a diverse array of effects including
gene  mutation,  chromosome damage  (sister  chromatid exchange),  primary DNA
damage, or oncogenesis (i.e.,  tumor formation and development) (Brusick,  1980;
Landolt  and Kocan,  1983;  Klingerman, 1982; Shugart, 1990).   Another  area that is
currently being investigated as a  suitable, sublethal assay for toxicity of certain wastes in
the marine environment is the fin regeneration test (Weis et al., 1990). The information
collected using these methods can be used to assess the effects of pollutants on the
reproductive capacity of animals.  An understanding of these effects can be useful in
evaluating observed population and community-level changes relative to the occurrence
of specific pollutants.

Gonadotropic and steroidogenic hormones regulate the reproductive capacity of an
organism. The level of these hormones has been used to assess how pollutants affect
the reproductive  capacity of fish  (V. Varanasi, 1990, National Marine Fisheries Service,
NOAA,  personal communication).  The analysis is a  sensitive indicator  of exposure
affecting major  biological processes that impact the whole population.  However,
relatively detailed information about the normal reproductive cycle of  the  animals is
necessary to apply these methods in the field (V.  Varanasi, 1990, National  Marine
Fisheries Service, NOAA, personal communication).

Biochemical Methods

Biochemical methods have been used in field  studies to measure various indicators of
environmental contamination. These methods are inherently sensitive and may provide
basic information about early changes in response to  environmental contamination at
the cellular  level.  The development of a suite of indicators having both specific and
nonspecific responses can provide information on the type of stressors, mechanisms of
action,  extent   of  physiological  dysfunction,  and  potential   long-term  population
consequences (Thomas, 1990).

Fish can respond to generalized stress, contaminants being one type of stress, through
induction (increased synthesis)  of stress proteins (Pickering, 1981; Sanders, 1990).
Stress proteins  are  currently being investigated for use as  generalized biochemical
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                                           Section 403 Procedural and Monitoring Guidance
 indicators of stress  in fish, chemical-class pollutant indicators, and  mode of action
 indicators.   The  methods  for  detecting  stress proteins  involve  radioisotopic  and
 immunologic methods that measure the amount of stress protein present after a stress
 (i.e.,  exposure to a  pollutant) occurs.  At  present, cDNA  probes are being  used
 experimentally  to  measure  the correlation between stress  and induction  of stress
 proteins. These methods potentially afford a high degree of sensitivity.

 It has been suggested that  induction of the fish hepatic microsomal mono-oxygenase
 (MO) enzyme could serve  as a sensitive biological indicator  for certain classes of
 chemicals in water (Payne et al., 1987; Kleinow et al., 1987; Lech et al., 1982; Jimenez
 et  al.,  1990; Haux and Forlin, 1988).   Metallothioneins  (MT)  have  been under
 consideration for use as a monitoring tool for trace environmental metal pollution due to
 their induction as a result of exposure to certain metals (Engel and Roesijadi, 1987;
 Garvey,  1990;  Haux and Forlin, 1988).   However, additional  scientific research is
 required to  understand the basic biology of fish before the exact significance  of field
 studies using these techniques can be ascertained.

 A concern  when  measuring  biochemical  variables in fish  to  detect environmental
 pollutants is that their exact biological significance is rarely understood.  In addition, for
 most of the biochemical variables  studied,  the normal range for a particular  fish
 population and the factors influencing these variables are often  unknown (Neff, 1985).
 Even with  these  limitations,  biochemical  methods  hold  considerable  promise as
 sensitive  early indices of  exposure  to  environmental stressors  (Thomas,  1990).
 However, additional  research  is needed so  that simplified,  more cost-effective field
 methodologies can be developed.

 Immunoloaical Methods

 Immunological biomarkers are  simple, sensitive, reproducible,  and workable in the field
 (Weeks et al.,  1990; D. Anderson,  1990;  R.S.  Anderson, 1990).  These indicators
 provide supportive evidence  for linkage between a stressor (toxicant, etc.) and disease
outbreaks in fish and shellfish.  The immune response can be used to monitor a specific
antigen or microorganism responsible for pathological conditions in fish.  Biologists can
perform quick and sensitive  assays in the field or in their own diagnostic laboratories
because many immune assays are becoming available in kits (Rowley, 1990; Matthews
et al., 1990). Many immunological assays do not require sacrifice of the animal.  Blood
samples can be taken periodically to follow the kinetics of the effects of stress in a single
animal;  however, the effects  of handling  stress on aquatic species  must also be
considered in this case.  The immune response is physiologically similar among most
vertebrates and similar equipment and materials can be used to test all species of fish
as well as shellfish (see Anderson, 1987). There is a rapidly growing body of literature
on immunotoxicology from  veterinary and aquatic animal sciences.
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Methods
The selection of immune system indicators for the study of stress effects depends on
many  factors,  including  specific  study  objectives,  available  equipment,  training,
personnel,  and length and number of assays.  The most  sophisticated and sensitive
assays are costly and require highly trained personnel, whereas simple assays can be
performed by field biologists with only basic laboratory supplies.

A major limitation of immune indicators is that the response is sometimes too broad to
provide conclusive  evidence that the  observed reaction is actually due to a  specific
complex to be considered. Cross-reactions and heightened  responses to  nonspecific
factors may prevent the interpretation  of assays with absolute certainty.  The immune
response in fish or shellfish will be distinctive for a specific antigen or disease-causing
agent.  This will frequently make it difficult to know which immune indicator  is most
affected and which  immunological assay to apply. Expensive materials and laboratory
equipment are needed for sophisticated immunological assays.  Confirmatory assays
are advisable to be sure that a particular stressor is the only cause of a particular effect.
Many animals should be sampled because of natural variability among individuals.

Physiological Methods

Hematological methods have  been used by biologists for many years to  assess the
general health of fish in  hatcheries and research laboratories.  The procedures are
well-standardized (Blaxhall and Daisley, 1973; Wedemeyer and  Yasutake, 1977) and
are easy to carry out, even in the field.  Hematological measures may be affected by the
stress of capture, but are far less influenced than some other measurements,  such as
blood  glucose  (Larson  et al., 1985).   Hematologic methods have been successfully
implemented in the field and have been  rated as  the best  physiologic  method  for
evaluation of  pollutants (U.S. EPA, 1986c).   For the measurement of  hematological
factors in  fish,  techniques  similar to those used  in  human and veterinary clinical
laboratories are generally used  with minor modifications (Heath, 1987;  Bouck 1984).
Blaxhall and Daisley (1973), Wedemeyer and Yasutake (1977), and Ellis (1977) provide
practical guides to the adaptation of these methods for use on fish  blood.

The hematocrii/erythrocyte determination may not be as sensitive to pollution as is the
leucocyte count, at least as far as its  response to metals  is concerned (Larson et  al.,
1985).  It remains  to  be determined  how  sensitive the procedure is to  subtle
environmental changes.  In general, chemical and physical stressors cause a decrease
in the leucocrit, whereas infections produce the opposite response.  However,  it is also
possible  to obtain  elevations  in granulocytes  concomitant  with  a decrease in
lymphocytes, thereby yielding an unchanged leucocrit (Peters et al., 1980). Thus, the
method has limitations for the detection of chronic stress (Wedemeyer et al., 1983).
                                                                       t
Several obstacles, such as capture stress, limit the potential usefulness of physiological
tests in  field work.  Capture stress precludes the use of  sensitive, early indicators of
environmental  stress.  The use  of  physiological responses of wild fish to assess
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                                            Section 403 Procedural and Monitoring Guidance


 environmental  quality is difficult because responses due to toxicants often cannot be
 distinguished from those induced during handling of the wild fish (Bouck, 1984; Folmar,
 1993).

 4.6.4     QA/QC Considerations

 Pathobiological methods have a wide range of sensitivities and response times  (i.e.,
 when  a response  can  be detected).   For instance,  certain  immunologic, electron
 microscopic, and biochemical methods can detect early changes in cells and are  very
 sensitive.  Gross anatomic pathology methods, on the other hand, can detect cellular
 changes only after lesions can be seen and, therefore, are less sensitive.

 General considerations  for  QA/QC procedures have  been covered earlier in  this
 document.  With regard to  pathobiological methods, it should be noted that careful and
 consistent  handling  of  aquatic specimens  is  required  to  minimize  trauma  and
 confounding effects,  such as exposure  to air.   Organisms should be  held in the
 laboratory under conditions as near to those found at the site of collection as possible
 and for as  short a time  as practicable  before performing assays.  Fish and shellfish
 collected for histopathological examination must be properly fixed  (e.g., immersed  in a
 formaldehyde or glutaraldehyde solution) to stop metabolic activity.   This may require
 opening or sectioning the organisms to allow the fixative to rapidly penetrate all tissues
 and preserve the cellular structure in its existing condition.  The organisms must still be
 active or moribund, but not dead, before being fixed.  Failure to follow proper fixation
 procedures will  interfere with the interpretation of lesions in anatomic pathology studies.

 As with other monitoring methods, samples must be accurately labeled at the time of
 collection and  routine QA/QC  procedures should  be instituted, including  tracking
 samples, carefully recording methods used, using fresh solutions,  and treating both
 control and exposed samples equally.  Whenever possible, organisms for each group to
 be tested should be of the same species, age, and sex. Sections of tissues and organs,
 or for small  organisms, the whole  animal, should also be prepared as uniformly as
 possible with respect to homogeneity and orientation so that microscopic observations
 can be made on the same  organs and  areas  for each specimen.   Subsamples of
 sections should be  examined  (blind) by another pathologist to confirm the diagnoses.
 See  USEPA  (1987b)  for  additional   information  on  QA/QC  procedures  for
 histopathological examinations.

 4.6.5    Statistical Design Considerations

 Statistical strategies may mitigate the high costs of pathobiological monitoring methods.
As discussed earlier, power analyses considering the strategy of compositing samples
can often lead to a  cost-effective monitoring design strategy. Power-cost analyses  are
necessary in selecting the  appropriate sample/replicate number, sample location, and
sampling frequency.  If the primary  objective of a monitoring program is to determine
                                                                             135
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Methods
pathobiological differences between sampling locations, composite sampling may be an
appropriate strategy.  However,  a limitation of composite sampling is the inability  to
directly  estimate  the  range and variance of the population  of  individual samples.
Similarly, the use of space- or time-bulking strategies will severely limit a monitoring
program's ability to assess spatial and temporal heterogeneity of the samples.

Given that the monitoring program must accommodate a fixed level of sampling cost,
the best strategy for pathobiological monitoring is to collect more  replicates at fewer
locations.  For histopathology, from 25 to  100 animals per species may need to be
surveyed to detect low incidences of disease. The selection  of appropriate statistical
analyses must be made with these limitations in mind, as well  as the specific types of
statistical  analyses that can be  performed on different types  of data.  For example,
quantitative information from measurements of enzyme levels  or computerized image
analysis/morphometric programs  may be effectively analyzed by parametric statistics (if
the data fit the conditions for normal distributions, limitations of assumptions, etc.), but
histopathological  observations may  need to  be rated  on  presence/absence  or
categorized qualitative scales that will require  nonparametric techniques.   USEPA
(1987b) provides additional information on the types of sampling designs and statistical
tests that may be appropriate for these data.
4.6.6
Use of Data
Data Interpretation

Data interpretation for  pathobiological  methods may  be limited because there are
relatively  few trained  personnel,  facilities and  equipment  may be expensive, and
references for newly emerging techniques may be scarce.  However, because of the
recent interest in developing biomarkers for monitoring the effects of natural and
anthropogenic environmental stresses on aquatic organisms, many research programs
are under way at Federal, State, and academic facilities to develop, standardize, and
validate  the most promising  biomarkers for sentinel species  that  will  establish
cause-and-effect   links  for  pollutant  exposure.    Basic  laboratory  research  and
experimental studies must be conducted in conjunction with field work to elucidate the
relationships  between  contaminant  levels,  structural,  biochemical,  or functional
pathologies, and  population health (Johnson and Bergman, 1984; McCarthy, 1990).  A
critical concern is the coordination of efforts and creation of a multidisciplinary approach.

It is important to establish baseline data on selected species, as well as to demonstrate
the alterations in  the health of those species  due to contaminant exposures.  Methods
and techniques,   terminology,  and  interpretation  of  lesions  and  effects  must  be
standardized. In  addition to a rapidly expanding body of literature on baseline measures
of health, histological atlases for  several species  of fish and shellfish, and courses,
workshops, meetings, and special symposia (e.g.,  Responses of Marine Organisms to
Pollutants/Woods Hole,  MA, and Plymouth,  England; Annual Aquatic Toxicology
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                                            Section 403 Procedural and Monitoring Guidance
Workshop,  Canada),  several  professional societies  (e.g.,  Society  for  Invertebrate
Pathology, American Fisheries Society/Fish Health Section)  are facilitating training in
techniques and communication among investigators and laboratories.  Diseases of fish
and shellfish have  been reviewed by  several authors (e.g.,  Sparks,  1985; Ferguson,
1989;   Roberts,  1989;  Sindermann,  1990;  Couch  and  Fournie,  1993).    For
histopathological  studies of tumors in fish,  standardization  of  nomenclature will be
available in Dawe et al. (in press).

As for  any  monitoring program,  long-term studies of health and disease in aquatic
organisms will aid  in identifying and  interpreting observed pathobiological effects.
Furthermore, it will be important to archive data so that they will be available for future
comparisons.  The Registry of Tumors in Lower Animals at the National Museum  of
Natural History, Smithsonian  Institution, Washington, DC, houses over 3,500  cases of
neoplastic and nonneoplastic  lesions  representing a wide variety  of  host aquatic
species, pathogens,  and environmental  stresses from  field and laboratory studies
conducted around  the world.   The  Registry also contains the  Registry of Marine
Pathology, originally developed by the National Marine Fisheries Service (NMFS), and
the collection of  crustacean  histopathological material researched by Dr. Phyllis T.
Johnson,  NMFS.  Although primarily  serving as a clearinghouse for information on
neoplastic diseases and  research, the  Registry is also able  to direct inquiries for
information on nonneoplastic diseases, and the materials archived there are  available
for study by qualified investigators. Data collected from the long-term monitoring efforts
of NOAA's  National Status and  Trends  Program (NS&T) and  EPA's Environmental
Monitoring and Assessment Program (EMAP)  will also provide useful information for the
interpretation of the various pathobiological methods that are being employed in these
studies.

Data Integration

Measuring or evaluating the effects of stressors on fish and shellfish by pathobiological
indicators is difficult  primarily  because  of the  large  number  of variables  that can
influence biological response.  Variables including water temperature, nutritional status,
species, sex, reproductive and developmental stages,  and physiological functions can
render tests difficult to compare and evaluate.

Anderson (1990) outlined a plan  including four levels of study in a tiered approach to
investigate the effects of stress on the immune system and to quantify the  possible
contribution of these environmental and physiological variables on the stress response.
The plan, generally speaking,  appears to  be  applicable to all pathobiological  methods
and may be  adapted to them using the following four levels: (1) observations of fish and
shellfish populations in the field; (2) studies of caged fish or shellfish in the field; (3) in
vivo exposures in the laboratory; and (4) in vitro assays in the laboratory.
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Methods
The first level of study includes the collection of information from field observations on
relative  abundance, reduction in sport-fish catches, or decline in commercial harvests.
Reduced yields reported by anglers  in previously productive fishing grounds  may be
correlated with environmental stressors or pollutants occurring at these sites.  If sick or
moribund fish or shellfish are called to the attention of local biologists by anglers, gross
morphological descriptions might be made and isolation of the disease agent attempted.

The second level of investigation involves the use of caged fish or shellfish placed in the
field  to  test for the presence or action of environmental stressors.  Groups of fish or
shellfish can be placed in pens or cages at suspected sites and their responses (signs of
disease, acute mortality) can be compared with those of control fish or shellfish caged in
unstressful areas.  Organisms from the cages can be sampled at various intervals and
the intensity of various immune responses quantified as a function of the time the fish or
shellfish were  exposed.  Levels of immune responses can be compared by  injecting
specific disease agents into both control and test organisms, and recording disease and
death rates.

The  third level of assay is in vivo laboratory tests, by which immune response can be
evaluated in experiments with calibrated dilutions of specific contaminants and kinetic
measurements of the immune response at each dilution.  Challenges with  disease
agents can be more easily controlled  in the laboratory to provide information on how the
stressor makes the fish or shellfish more susceptible to specific pathogens.

A fourth level of investigation is the recently developed method for  testing the effects of
chemicals, drugs, and other stressors in vitro. Spleens and other immunopoietic organs
can  be removed from fish and placed  in tissue culture media and their reactions to
pollutants tested using the passive hemolytic plaque assay (Anderson et al., 1986). Use
of this method to measure the effects of stressors allows maximal control of the levels of
pollutants. Because the immune response is monitored under defined laboratory  and
environmental conditions, important information is obtained  about how specific stressors
affect the immune response.  In vitro methods require fewer fish than  in vivo  methods
because tissue and organ  samples can be divided  into  many sections, which also
reduces the variability of responses.

McCarthy (1990) presented  a research strategy to validate biomarkers and provide the
scientific  understanding  necessary  to  interpret biomarker responses.   An  evolving
monitoring program was proposed that focused broadly on evaluation of contamination
in an array of ecosystem types. The challenges and obstacles to be addressed in such
a program include the following:

       •  The quantitative and qualitative relationships between chemical exposure,
          biomarker response, and adverse effects must be established.
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                                           Section 403 Procedural and Monitoring Guidance
       •   Responses due to chemical exposure must be distinguishable from natural
          sources of variability (e.g., ecological and physiological variables,
          species-specific differences, and individual variability) if biomarkers are to be
          useful in evaluating contamination.

       •   The validity of extrapolating between biomarker responses measured in
          individual organisms and some higher-level effect at a population or
          community level must be established.

       •   The use of exposure biomarkers in animal surrogates to evaluate the
          potential for human exposure should be explored.


The plan is an ambitious multiyear research and development program involving the
formulation  of  a  long-term,  interagency,  interdisciplinary  activity  such  as  the
Environmental Monitoring and Assessment Program (EMAP) by EPA in cooperation with
other Federal agencies.

4.6.7     Summary and Recommendations

Rationale

       •   Pathobiological methods provide information concerning biological organ
          systems that, through an evaluation of organ structure, activity, and function,
          can be used to determine adverse effects of pollutants in the environment.

       •   Pathobiological methods should be used in concert so that cause-and-effect
          relationships can be evaluated.

Monitoring Design Considerations

       •   Sampling stations should be located along a contamination gradient (i.e.,
          from highly contaminated to uncontaminated). This type of sampling strategy
          will allow dose-response relationships to be evaluated.

       •   Fish pathobiological monitoring should be conducted only if the target
          species could reasonably be expected to have spent a considerable amount
          of time within the area of contamination.

       •   Large sample sizes will frequently be required for fish pathobiological
          monitoring due to natural variability among individuals and taxa.

Existing Analytical Methods

Anatomic Pathology Methods

      •   Tissues are examined with the naked eye or the aid of a microscope
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Methods
      •  Light microscopic (LM) methods
         -  detect changes at the cellular level
         -  organ and time-series specific
      •  Electron microscopic (EM) methods
         -  detect subcellular changes
         -  specific as to area of cell or site of pollutant action
      •  Histochemical methods
         -  detect enzyme systems or reaction  products in the cell
         -  specific to chemical class
Reproduction/Development Methods
      •  Reproductive capacity is reflected in population recruitment and abundance
      .  Methods that examine the egg itself provide a direct measure of reproductive
         effects from pollutants
      •  Cytogenetic (DNA) tests
         -  in vitro tests offer greatest flexibility in terms of sensitivity, expense, and
            time required and are frequently conducted for convenience and
            availability.
         -  sister-chromatid exchange (SCE) assays are DNA damage tests used as
            a screening tool and are dose-responsive and sensitive to low
            concentrations of pollutants.
         -  the aneuploidy technique is simple  and easy to use but is inaccurate for
            sublethal effects.
Biochemical Methods
      •   Several methods are specific for a certain compound class.
      •   Use a suite of indicators for environmental monitoring.
      •   Microsomal mono-oxygenase (MO) assays could serve as sensitive
          biological indicators for certain chemical classes.
      •   Metallothionein (MT) assays are nonspecific for metal exposure, but can
          provide information on the likelihood of a particular metal pool producing a
          pathological effect.
      •   Stress proteins are not pollutant-specific, but could result from generalized
          stress.
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                                          Section 403 Procedural and Monitoring Guttfance
Immunologic Methods
      •  Immunologic assays can provide information on pollutant-induced stress
         effects; however, they may require confirmatory assays.

      •  Many animals should be sampled because of natural variability among
         animals.

Physiologic Methods

      •  Serious "interferences" can be caused by stress induced during collection
         and may limit the potential usefulness of physiological tests because effects
         of toxicants cannot be distinguished from those induced during handling of
         the wild organisms.

      •  Hematologic methods for fish, such as measurements of hematocrit (packed
         cell volume), hemoglobin concentration, erythrocyte count, and leucocyte
         count (or volume), can be successfully used.  Although the fish are not
         immune to stress of capture, hematologic methods are influenced far less
         than some other measurements such as blood glucose.

      •  The leucocrit can be a more sensitive measure of metal pollution than the
         hematocrit.

QA/QC Considerations

      •  Pathobiological methods have a wide range of sensitivities.

      •  Organisms must be carefully handled and properly prepared for each method.

      •  The main areas of concern with regard to analytical QA/QC are precision,
         accuracy, representativeness, completeness, and comparability.

Statistical Design Considerations

      •  Composite tissue sampling consists of mixing tissue samples from  two or
         more individuals collected at a particular location and time.

      •  Space-bulking (combining composites from several locations) and/or
         time-bulking (combining several composites over time from one location)
         strategies should be used judiciously since information concerning  spatial
         and temporal heterogeneity may be lost.

      •  Power analyses have shown  that for a fixed level of sampling effort, a
         monitoring  program's power is generally increased by collecting more
         replicates at fewer locations.
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Methods
         The appropriate statistical tests must be performed and may vary depending
         on the type of data generated.
Use of Data
         Data use and interpretation may be limited by relatively few trained personnel
         and expensive equipment, but many research and training programs are now
         under way at Federal, State, and academic facilities to improve biomarker
         methods in sentinel species.

         Basic laboratory research must be conducted and biological methods must
         be tested in the field.

         Information communication

         -   need to coordinate efforts, communicate, and create a multidisciplinary
            approach to relate lab and field studies and establish cause-and-effect
            relationships.

         Data integration

         -   long-range research strategies should be followed to validate biomarkers
            and provide scientific understanding necessary to interpret biomarker
            responses.
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                                           Section 403 Procedural and Monitoring Guidance
4.7
FISH POPULATIONS
The objective of monitoring fish populations is  to  detect and describe spatial and
temporal changes in the abundance, structure, and  function  of fish communities.  To
attempt to protect  and preserve healthy fish  from  the possible  effects of pollutant
discharges, estimates of fish population abundance and detailed knowledge of fish life
histories are required.

Except in  the most severe  cases,  it is usually impossible  to directly  link pollutant
loadings to stock levels (i.e., there is a poorly established cause-and-effect relationship
between pollution and fish population responses).   Natural population fluctuations in
fishery stocks will usually be much greater than effects due to pollution. The effects of
discharges are obvious when fish are killed outright by high concentrations of toxic
substances (Rothschild, 1986).  However,  these  instances are rare compared to  the
prevalent situation,  in which fish are  exposed to lower pollutant levels that may not be
lethal but are thought to be harmful in some sense. Sublethal  pollutant loadings can be
expressed  in terms of increased body burden of toxicants or greater incidence of various
lesions. The effects of sublethal concentrations on growth, mortality, and reproduction
are not known (Rothschild,  1986)   As a  result  of  these limitations, fish population
measurement methodologies require further refinement and field validation before they
can be promoted for regular use in 403 monitoring or permit decisions.
4.7.1
Rationale
Under section  403 of the Clean Water  Act, permitters  must use  10 guidelines  in
determining whether a discharge results  in unreasonable degradation  or  irreparable
harm to the marine environment.  Fish population studies have generally not been used
to directly  link  a source  of pollution to observed responses.  However, some recent
studies have indicated that selection of resident bottom fish as indicators of pollution can
support other data linking sources of pollution to environmental effects, especially if such
studies are conducted in conjunction with benthic studies and parallels can be drawn
between the two.  In addition, such methodologies do address the following three 403
guidelines:

       •  Composition and vulnerability of potentially exposed biological communities;

       •  Importance of the receiving water area to the surrounding biological
         community; and

       •  Existing or potential recreational and commercial fishing.

Population  effects can include those caused by changed reproductive rates or changed
distribution and migration  patterns.   Effects of suppressed  reproductive rates  on
population density have been clearly demonstrated in the diminishing populations of the
striped bass  in San Francisco Bay.  The suppressed reproductive  rates have been
shown to correlate with toxic concentrations in the bay (Whipple et al., 1984). Several
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Methods
laboratory studies have demonstrated that certain fish can avoid toxics, including DDT,
endrin, and Duroban. There is, however, no proof that fish avoid toxicants in the field.  If
they do, "hotspots" would be avoided by such species and the population would suffer
less exposure to toxic effects.

One of the greatest problems in analyzing  effects of toxics on populations is separating
direct toxic  effects  from natural or non-pollution-related variations including climatic
changes and overfishing. Results of population studies must be evaluated together with
measurements   of  other  environmental  parameters  before  any  cause-and-effect
relationship can be established.

4.7.2    Monitoring Design Considerations

Specimen collection, analysis, and evaluation of fish community structure and function
are typically time-consuming,  labor-intensive, and  expensive  tasks.  A survey vessel
manned by an experienced crew and specially equipped with gear to collect organisms
is  required.   Expert taxonomists are needed to identify and enumerate collected fish
specimens.

The results of fish  monitoring  programs  can  vary substantially depending on the
objectives  and  corresponding design  specifications.   The  characteristics  primarily
responsible for the variability in the results are the following:

      •  Type of sampling gear,

      •  Volume sampled, and

      •  Location and timing of sample collection.

It is essential to understand the effects of these monitoring design characteristics on the
results and to standardize them as much as possible to ensure the comparability of
sampled data.   Analyses  of power-cost  efficiencies are  useful  in  selecting  the
appropriate sampling gear and  sample processing protocols.   Ferraro et al. (1989)
provide an example of power-cost analyses.

Sampling Devices

Sample collection protocols influence all subsequent laboratory and data analysis; it is
key  that fish  population samples be  collected using  acceptable and standardized
techniques.  Several types of  devices can be used to collect fish samples:  traps and
cages, passive nets, trawls (active nets), and  photographic surveys (Fredette et al.,
1989).  Many of these devices selectively sample  specific types of fish.  Accordingly,
conducting comparisons between data collected  using different devices and even
different nets of the same type  is inadvisable.
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                                           Section 403 Procedural and Monitoring Guidance
Traps and Cages

Traps and cages are usually designed to attract and capture specific organisms. They
are useful in studies examining the activities of a particular target organism in a given
area.  Traps and cages provide only qualitative measures of organisms in a particular
area.

Passive Nets and Trawls

Nets vary in their selectivity of the species that are captured and in the efficiency of
retaining captured specimens.  The size of the net, its configuration and orientation, and
the avoidance behavior of the target species should be considered when using any net.
The mesh size affects the speed at which the net can  be towed, as well as the size of
fish caught.  The slower the towing  speed, the more likely that some organisms will
either avoid or escape  the  net.  It  is highly recommended that the mesh size, net
opening, and duration, direction, and speed of towing be set in order to compare trawls.
In any  monitoring program,  otter trawl net  size must be kept constant to  ensure
intercomparison of sampling results.

Passive nets (e.g., gill  nets)  are deployed  at a fixed position; organisms become
entangled or trapped within the netted area.  Passive nets are used to collect selected
target  species  and  to  provide a qualitative means  of sampling fish  populations.
Limitations associated with passive nets include: .

      •  Nets, ordinarily, must remain in place for an extended period of time.

      •  Deployment and recovery of nets are typically time-consuming processes.

Trawls (active nets) are drawn through the water, and results are more immediate than
those obtained through  the use of passive nets.  Trawls are typically used to collect
large quantities of fish at various depths (Fredette et al., 1989).

Photographic Surveys

Photographic surveys are effective when the bottom topography is uneven or trawling is
not possible.  The utility of photographic surveys is limited by water clarity, difficulties in
identifying species, and  fish avoidance of the camera system.  In addition,  further
studies comparing photographic surveys to trawls are required before comparisons may
be made.  This method is also limited by the qualitative nature of the data  (Fredette et
al.,1989).

Volume Sampled

Different species  of fish have  different  scales of  horizontal  and  vertical  spatial
distribution (Gushing, 1975; Bond, 1979).  Costs of laboratory analysis of the sample
increase with increased  volume  sampled.  Analyses of spatial and  temporal scale,
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Methods
statistical power, and costs will assist in determining optimal sample volume. It is highly
recommended that a standard sample volume (same tow duration, tow speed, and net
opening area) be analyzed to ensure data comparability (Green, 1979).

Selection of Sampling Period

Fish assemblages are dynamic; the most common temporal patterns  observed  in fish
communities are those  associated  with  daily activity  patterns  (diurnal),  seasonal
changes, and life history strategies. To minimize energy expenditure, most fishes mimic
the diurnal activities of their prey.  These diurnal fluctuations typically occur  in the
vertical scale and should be considered to ensure collection of representative samples.
Seasonal variation in fish assemblages may be due to changes in physical, chemical,
and/or biological parameters: i.e., temperature, light transmissivity, dissolved oxygen,
predation, reproductive stage, recruitment, etc.

Given  the  seasonal variation characteristic  of fish assemblages in general, it is
recommended that direct comparisons  between  samples collected  during  different
seasons be avoided. Studies investigating interannual variation in  the characteristics of
fish communities should be conducted during the same season (preferably the same
month) each year.

4.7.3    Analytical Methods Considerations

There are a variety of approaches to assess the effects of anthropogenic perturbance on
fish communities of the marine environment.  These assessment approaches may be
grouped into three categories :

      •  Biological indices,

      •  Indicator species, and

      •  Multivariate analyses.

There has  been little consensus among biologists regarding the  suitability of various
techniques for describing community characteristics and/or for assessing  impacts. A
critical evaluation of the use of biological  indices to detect environmental change is
presented in an EPA Technical Support Document  (USEPA, 1985d).  The indices,
shown in Table 4-22, are evaluated on the basis of the following criteria  :

      •  Biological meaning,

      •  Ease of interpretation, and

      •  Sensitivity to community changes due to anthropogenic sources.

The results of these evaluations and additional information on other analytical methods
are summarized below.
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                                            Section 403 Procedural and Monitoring Guidance
                          Table 4-22. Biological Indices
         Index/Method
Biological Characteristic
      Measured
        Bray-Curtis (Curtis and Peterson, 1978)

        Dominance (Swartz et al.,1985)

        Number of individuals (USEPA,1985d)

        Number of species (USEPA.1985d)

        Biomass (USEPA,1985d)

        Margalefs SR (Margalef,1969)

        J (Pielou,1966)

        Shannon-Wiener H' (USEPA,1985d)
    Dissimilarity

    Community structure

    Total abundance

    Total taxa

    Standing crop

    Diversity

    Evenness

    Diversity
Biological Indices

The number of individuals and the number of species have been used as indicators of
anthropogenic disturbance, as well as other environmental stresses (USEPA,  1985d).
Furthermore, these simple biological indices are  less ambiguous and can  often be as
informative  as  diversity  indices  (USEPA,  1985d;  Green,  1979;  Hurlbert,  1971).
Measures of biomass have inherent problems in the collection of the data,  e.g., loss or
gain of weight due to preservative medium, drying times, or evaporative weight loss.

More   complicated indices-e.g.,  species  diversity,   species  richness,  dominance,
evenness-have found varying degrees of acceptance.  Because of this, these methods
should be  used in conjunction with other assessment  methods to help  verify results.
Diversity indices, which are measures  of the distribution of individuals among species,
have the following limitations (Green, 1984b):

      •  They often lack biological meaning;
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Methods
      •  They are not robust empirical indicators of any important correlates of
         environmental health;

      •  They do not incorporate information on form and function of resident species; and

      •  They are susceptible to biases associated with well-described taxa.

Even so, species diversity indices are a widely used measure of community structure.
Although diversity may not be able to pinpoint a single problematic discharge in an area
that contains many, it can be an indicator of degraded water quality.  Species diversity in
fish communities is noted to increase with increasing distance from a pollution source
(Armstrong et  al., 1970; Bechtel and Copeland, 1970; Tsai, 1968).  In fact, Bechtel and
Copeland (1970) found a linear relationship between the volume of toxic effluent
entering the Houston Ship Channel and the fish diversity of shallow stations.

The  dominance index is a measure of  the  degree to which one or a few species
dominate the  community.   The dominance  index,  herein defined  as the  minimum
number of species required to account for 75 percent of the total number of individuals,
has been useful in describing community structure (Swartz et al., 1985). Its  advantages
are  that it  is easily calculated, it  does not assume  an underlying distribution of
individuals among species, and it is statistically testable.

Indicator Species

The  evaluation of abundances of individual indicator species is generally informative and
may reduce the cost of the analysis. The  absence of pollution-sensitive species and the
enhancement  of opportunistic and  pollution-tolerant species may assist in defining the
spatial  and temporal  extent  and  magnitude  of  impacts.    A  preponderance  of
unspecialized  feeders indicates areas of stress.  However,  indicator variables must
possess the following characteristics (Green, 1984b):

      •  They must provide a sufficiently precise and accurate appraisal of:

         -  species of concern,
         -  anthropogenic disturbances  to communities, and
         -  presence/absence or magnitude of anthropogenic perturbance to the
            ecosystem.

      •  They must provide a cost-effective and statistically reliable alternative to
         monitoring all critical measures of habitat perturbance.

      •  They must be appropriate for the spatial and temporal scale demanded  by
         the study objectives.

Further studies  of  the  response patterns of fish species subjected to anthropogenic
perturbations caused by discharges are required in order to select appropriate indicators
of environmental impact.  If a suitable indicator species is identified, it is desirable to
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                                           Section 403 Procedural and Monitoring Guidance
monitor the status and trend of that species' population. This will also be true of certain
species having high economic or public value (e.g.,  striped bass).  A wide variety of
methods can be used to measure the size of fish populations and assess  population
structure. These include mark and recapture techniques and various techniques used to
determine population sex and age structure. Nielson and Johnson (1984) and Cailliet et
al. (1986) provide thorough discussions of these techniques and  their  applications.
Bicker (1975) provides  an excellent discussion of methods for the sampling of fish
populations.

Statistical Analyses

Numerical classification encompasses a wide variety of techniques that have  been used
in the analysis of fish data to distinguish  groups of entities (e.g.,  sample locations)
according to similarity of attributes (e.g., species). These techniques differ from most
multivariate  methods in  that  no assumptions are made  concerning  the  underlying
distributions of the variables.   Detailed descriptions of numerical classification analysis
can be found in Romesburg (1984), Clifford  and Stephenson  (1975), Boesch (1977),
Sneath and  Sokal (1973), arid Anderberg (1973). Boesch (1977) is particularly valuable
as an introduction and guide to the use of numerical classification analysis in marine
environmental studies.   Guidance on  the interpretation  of  classification  results  is
provided in an EPA Technical Support Document (USEPA, 1988d).

Ordination analyses have also been  used to  reduce the dimensionality of the data set
while maintaining the relationship between similar and dissimilar entities. At  present no
single ordination technique has  been shown  to be clearly superior for the analysis  of
biological data (USEPA, 1985d).

Multivariate  analyses are effective heuristic tools.  They generate visual representations
that often indicate where further analyses ought to be conducted.

Analytical Approach Recommendations

Some of the most informative measures of community structure are the simplest (Table
4-22):

       •   Number of individuals,

       •   Number of species,

       •   Dominance,

       •   Abundance of pollution-sensitive species, and

       •   Abundance of opportunistic and pollution-tolerant species.
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 Methods
These indices have proved to be useful over various habitats and regions in assessing
changes in fish population dynamics (USEPA, 1985d).  Values of these variables may
be determined from the list of species  abundances  generated  during the taxonomic
identification of collected specimens.  Furthermore, the values of these variables may be
easily tested  statistically  using  parametric or  nonparametric techniques.   It  is
recommended that no single index or analytical method be used to  assess impacts;
rather, the assessment of impacts should incorporate information that each variable and
method contributes concerning community structure.

Selection of reference sites is key to the evaluation of environmental impact assessment
of point source dischargers. Results of analyses using reference  measures provide the
means of comparison by which anthropogenic impacts are detected. It is essential that
selected reference sites exhibit similar habitat characteristics  compared to monitoring
program sampling sites. Several reference sites may be required to meet these criteria.

4.7.4     QA/QC Considerations

Sample Collection

Nets should be inspected for wear and tear leading to sample loss.

Taxonomic Identification

A key QA/QC issue is taxonomic standardization.  Consistent taxonomic identifications
are achieved through interaction among taxonomists working on each major group.
Participation of the laboratory staff in regional taxonomic  standardization programs  is
recommended to ensure regional consistency and accuracy of identification.

A sampling of the verified specimens should then  be placed in a permanent reference
collection.  All specimens in  the  reference collection should  be  stored  in  labeled
containers that are segregated by species and sample. Reference specimens should
be archived alphabetically within major taxonomic groups.

4.7.5     Statistical Design Considerations

Consideration of statistical strategies will mitigate the high  costs of collecting and
processing samples.   Power-cost  analyses are  necessary in  selecting  appropriate
sample/replicate number, sample location, and sampling frequency.

Temporal Stratification of the Data

The time of the year should be controlled or stratified  in the design; the use of annual
averages is seldom good practice.  Temporal stratification of the data should not be
attempted until sufficient knowledge  of long-term  natural cycles is  attained.  Initially,
simple regression analyses may be conducted on seasonally stratified data to identify
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                                           Section 403 Procedural and Monitoring Guidance
monotonic temporal trends.  Whether conditions are improving or degrading over time
may be further examined using various statistical time series analyses (e.g., temporal
autocorrelation, spectral analyses).

Statistical Power

Selection of the  number of replicates is an important component of program design.
The inherent patchy distribution of fish communities requires collection  of sufficient
replicate samples to ensure an accurate description of the area. However,  increases in
replication increase sample processing costs. Power analyses assist in the allocation of
sampling resources  (stations,  replication,  and frequency) with  regard  to program
finances and design.

Power analyses may be applied  to determine the appropriate  number of sample
replicates required to detect a specified difference (USEPA, 1987d).  The number of
replications  required to  detect a specified minimum  difference  is a function of  the
statistical  power and the variance  in the data.   Power  analyses  require  a priori
knowledge of the variability in the data. A best guess or, preferably, variation observed
in historical data is  often  used initially in the design of the monitoring program.

To  improve the power of a statistical test, while keeping the significance level constant,
the sample size should be increased.  Because of constraints in cost and time imposed
by the monitoring program, however, this option may not be available.  Power analyses
have shown that for a fixed level of sampling effort, a monitoring program's power is
generally increased by collecting more replicates at fewer locations.  The  number and
distribution of sampling locations required to evaluate the effectiveness of the monitoring
program will depend on the  size and complexity of the discharge and the surrounding
environment.
4.7.6
Use of Data
Monitoring of fish community structure provides a measure of the health of the marine
habitat and can be used as a tool in the evaluation of spatial and temporal effects of
marine discharges. The presence or absence of certain fish is useful in indicating the
condition  of the environment.   Monitoring  of fish communities  also provides  data
required in the design and  validation of fish population dynamics models and the
selection of biological indicators (USEPA, 1990d).

In addition, monitoring of fish communities may directly  provide accurate information
essential in assessing the effectiveness of pollution abatement programs.  Analyses of
fish communities may be effectively used to  monitor long-term change in the receiving
environment (Gushing,  1975).
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Methods
Fish serve as effective indicators  of overall environmental condition,  delineating the
magnitude,  spatial  extent,  and  temporal  trends  of  anthropogenic  and  natural
perturbations to the ecosystem.  Monitoring of fish will provide relevant accurate data
fundamental to achieving the objectives of many  marine monitoring programs. Such
measurements, however, will not provide information linking specific pollution sources to
observed effects.  The results of the fish stock monitoring program can also be unclear
as a result of the mobility of fish and their range of natural fluctuation. The data can be
used to augment other monitoring data to obtain a  clearer picture of the discharge
effects.

4.7.7    Summary and Recommendations

Rationale

      •  The objective is to detect and describe spatial and temporal changes in the
         size, structure, and function of fish communities to protect the economic,
         recreational, and aesthetic value of the resident fisheries.

      •  Monitoring provides in situ measures of habitat quality and is a powerful tool
         in assessing environmental impact.

Monitoring Design Considerations

      •  It is recommended that the types of sampling gear, the volume sampled, and
         the location and timing of sample collection be consistent to allow for
         comparisons among studies.

      •  To reduce the variation due to seasonal differences, sampling should be
         conducted during  the same season—preferably the same month—each year.

Analytical Methods Considerations

      •  It is recommended that simple measures of community structure be used to
         assess the condition of the marine fish: number of individuals, number of
         species, dominance, abundance of pollution-sensitive species,  and
         abundance of pollution-tolerant species.

      •  Indicator species should possess the following characteristics:

         -   sensitive to perturbances of concern;
         -   cost-effective and statistically reliable alternative to measuring all species
            in a monitoring program;
         -   statistically reliable indicative measures of habitat perturbance; and
         -   appropriate for the spatial and temporal scale demanded by the study
            objectives.
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                                           Section 403 Procedural and Monitoring Guidance
      •  Selection of reference sites is key to the evaluation of environmental impact
         due to discharges; several reference sites may be required to provide proper
         control for sampling sites.

QA/QC Considerations

      •  Taxonomic standardization is key to the analysis of community structure.
         Recommended protocols include consistent interactions among taxonomists,
         reidentification of selected samples, and use of a reference collection.

Statistical Design Considerations

      •  Power analyses may be applied to determine the appropriate number of
         sample replicates required to detect a specified difference, thereby optimizing
         the high costs of collecting and processing samples.

Use of Data

      •  Data provide essential information to assess impacts due to  anthropogenic
         perturbance,  monitor recovery of the receiving environment,  and validate
         community and population models.
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 Methods
4.8
PLANKTON: BIOMASS, PRODUCTIVITY, AND COMMUNITY
STRUCTURE/FUNCTION
Although  increased  primary production resulting from  intentional nutrient inputs has
been shown to  increase fish  stocks  in  some  experimental  systems,  the possible
increase in fisheries in naturally productive systems is considered insignificant. In fact, it
is more  likely that stocks will decrease due to  modification of the plankton  species
composition.  Biogeographical distributions of plankton  are  determined by  specific
environmental factors such as light, temperature, salinity, and nutrients.  Plankton vary
in size over a range of seven orders of magnitude.  Each predator species can utilize
only plankton within  two or three orders of magnitude in size,  at the most,  as a food
resource.  Additionally, the nutrient values of various prey differ so that a change in
species composition to less nutritive species may cause stress on the predator species.

The purposes of monitoring plankton characteristics are (1) to identify the dominant species;
(2) to detect short- and long-term  spatial and temporal trends  in overall  biomass and
productivity and species abundance, distribution, and composition; and (3) to examine  the
relationship between water quality conditions and these characteristics.

As was the case for fish population monitoring methods, measurements of plankton
biornass, productivity, and community structure/function are not sufficiently developed to
be used in a discharge-specific monitoring framework for section 403 assessments at
this time.  These measurements suffer from a lack of a specific cause-effect relationship
between pollution and plankton responses.
4.8.1
Rationale
Although  not  directly applicable  for use  in  monitoring the site-specific impacts of
individual point source discharges, plankton measurements can be used to evaluate 3 of
the 10 guidelines presented in the section 403  regulations for assessing the potential for
unreasonable degradation:

       •  Composition and vulnerability of potentially exposed biological communities;

       •  Existing or potential recreational and  commercial fishing; and

       •  Importance of the receiving water area to the surrounding biological
         community.

Changes in nutrient concentrations in coastal waters may result in the potential for
long-term  biological changes in the plankton community that may lead to changes in
species distribution and  abundance (both primary producers  and consumers).  A
concern of the section 403 program is that  excessive nutrient inputs can result in
excessive phytoplankton biomass, which in turn can lead to increased turbidity and/or
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                                            Section 403 Procedural and Monitoring Guidance
changes in phytoplankton  species  composition  and perhaps the trophic structure.  If,
based on water quality monitoring data, changes in trophic structure are suspected of
occurring, periodic monitoring of the plankton community may be required.

The plankton component of a monitoring program could be used to provide data necessary
to assess the effectiveness of management programs in mitigating potential adverse effects
due to changes in the plankton community biomass and structure/function.

4.8.2     Monitoring Design Considerations

Plankton

Plankton monitoring strategies should be able to delineate between natural  or seasonal
variability in plankton stocks and variations caused by changes in nutrient concentrations.
Characterization  of phytoplankton taxonomic abundance and  distribution  and  primary
productivity  provides indications of  water quality  conditions.   Monitoring  changes in
phytoplankton population composition and densities is critical for the  interpretation and
evaluation of long-term trends in water and habitat quality. Further understanding of the
causes  of excessive water  column  and sediment oxygen demand  requires tracking of
photosynthetic activity and metabolic rates over time  (Chesapeake Executive Council,
1988b).   Zooplankton  abundance  and distribution are  affected  by  both  changes in
phytoplankton and changes in predator populations. Therefore, population characteristics of
this group can indicate symptoms of water quality problems, fishing  pressure, and other
habitat problems for predator species  (Chesapeake Executive Council, 1988b).

Selection of Temporal Sampling Strategies

Because of  their  short  turnover times,  phytoplankton communities  may  respond  to
perturbations much more rapidly than  other biotic groups.  Therefore,  phytoplankton
samples should  be  collected  relatively  more frequently.   In  those  situations  where
phytoplankton communities  display pronounced seasonal  variations in  standing stock or
production, it may  be appropriate to use a temporally  stratified sampling approach.   For
example, in  the Maryland Chesapeake Bay Phytoplankton Monitoring Program, sampling
takes place once monthly from October through March and twice a month from April through
September (USEPA, 1989c).

The life span of zooplankton, on the other hand, is  longer than that of phytoplankton, so the
capacity for responding to perturbations is less than that of phytoplankton. Therefore, less
frequent  sampling  is required.  As  with the phytoplankton community, the  zooplankton
monitoring program should  consider the natural temporal  fluctuations in abundance and
species composition.
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Methods
In many cases, regular monitoring of the zoopiankton community may not be necessary
unless changes in the phytoplankton community that would induce changes in the herbivore
community are observed.  Because many zoopiankton graze on phytoplankton, in areas
where the phytoplankton community has been affected,  alterations  of the zoopiankton
community are a distinct possibility.  In other cases, monitoring of zoopiankton may be
desirable only when there is evidence of previous impact on the zoopiankton community or
in those situations where point and nonpoint source discharges are located in areas where
there  is  high potential  impact  on  zoopiankton (for  example,  in  environments  with
macroplanktonic larvae of important commercial or recreational species).

Sampling Methods

Phytoplankton

Phytoplankton samples should be  collected at a variety of depths throughout the water
column,  including  some above and some below  the pycnocline.   For example,
composite water column samples  collected for the Virginia  Chesapeake Bay Plankton
Monitoring  Program  are  taken  from five depths  above and five depths  below  the
pycnocline (USEPA, 1989c). Vertical collection of chlorophyll a (an  indirect measure of
phytoplankton standing stocks) can be examined at such stations through the collection
of water samples  with water bottles at various  depths followed  by fluorometric or
spectrophotometric determination of chlorophyll a.  If available, a pump station may be
used  with a flow-through fluorometer for a  continuous profile of  chlorophyll a
concentration with depth (Lorenzen, 1966).   Samples to be used for taxonomy analysis
should be collected with water bottles because agitation associated with pumping may
damage cells, making  them  unidentifiable.    Pumps should  be  used  only  for
determination of chlorophyll a concentrations.

Continuous vertical profiles of chlorophyll a concentrations can be obtained by attaching
a fluorometer to a conductivity-temperature-depth (CTD) system. An additional sensor
measuring dissolved oxygen  can  be attached to the CTD system.  This combined
instrument package will make direct measurements of chlorophyll  a levels and water
column stratification.  The  advantage of this measurement system is that instead of
pulling water up from depth to a measuring device on the deck of a  ship, the instrument
is lowered through the water  column, taking samples  in undisturbed water. Sampling
rates can be as high as 24 samples per seconds with a locating speed of 1 meter per
second.  Data are usually  collected by a computer-based  data acquisition system; in
advanced systems, data can  be displayed in real time as they are being collected. A
rosette water sampler can be lowered at the same time as the CTD package to collect
water samples at discrete depths.  These samples will be used for taxonomy analysis
and to verify and calibrate chlorophyll a measurements from the CTD fluorometer. The
calibration will be carried out using laboratory spectrophotometer methods.
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                                          Section 403 Procedural and Monitoring Guidance
Zooplankton

Because zooplankton possess  varying degrees of  swimming  ability,  they  have the
potential for aggregating in patches or in a narrow depth  strata.  This introduces
additional complications into quantitative sampling. It also means that zooplankton are
able to avoid certain types of gear.   Until recently, the role of the microbial loop, which
consists of bacteria,  flagellates, ciliates, and microzooplankton (<200 |im), has been
overlooked.  These organisms may represent a significant pathway in the reutilization
and conversation  of dissolved  organic carbon into larger  zooplankton and  benthic
organisms (Azam et al., 1983; Pomeroy, 1984).  The sampling methods to be used for
collecting zooplankton   will  vary  depending  on  the  size of  the  organisms.
Microzooplankton (size range of 20 - 200 ja,m) can be collected with water  bottles at
various depths similar to those used for phytoplankton (Jacobs and Grant,  1978), or
small  (for example 44 |o,m) rnesh nets can be used. Pumping systems can also be used
(Beers et al., 1967). These have the advantage of being able to take samples while the
ship is  under  way, but they may damage soft-bodied organisms and  they  are more
expensive and complicated than water bottles.  Triplicate samples should be collected
from each station depth, thus allowing for statistical analysis of intrastation variability.

For small mesozooplankton (greater than 200  |im),  nets are usually used (UNESCO,
1968).  Additional tows  may have to  be made with  larger nets in order  to  collect
representative samples  of  larger zooplankton and  larval fish.  All tows should be
replicated.  The number of  replicates necessary for the desired precision of estimation
should be determined during a preliminary or pilot sampling program.  A number of other
considerations, including net mouth diameter, towing speed, and shipboard handling of
samples, will affect sampling results. Some problems associated with the use of nets for
zooplankton  sampling  include  avoidance,  which  may  result in  underestimating
abundance and diversity  (McGowan and  Fraundorf,  1966; Wiebe and Holland, 1968),
and clogging and loss of filtration efficiency.

4.8.3     Analytical  Methods Considerations

Phytoplankton

Biomass and Productivity

Phytoplankton biomass can be indirectly measured through  the measurement of the
concentration  of chlorophyll  a  in  the  water.  This is done through  fluorometric or
spectrophotometric measurements.  Within these methods are differences in  extraction
techniques for  chlorophyll  determination, including  various  methods of  filtration,
solvents, temperature, and/or physical treatment (sonication or grinding)  (D'Elia et al.,
1986).
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Methods
The use of  chlorophyll  a  measurements,  especially  fluorometric,  has become
widespread primarily because the method is relatively fast,  simple, and reproducible.
With  both the fluorometric and spectrophotometric  determinations, however,  the
presence of accessory  pigments may interfere with determination of chlorophyll a.
Several researchers, nevertheless, have successfully used chromatographic procedures
to separate  interfering substances prior to determination  (D'Elia et al., 1986).  High
Performance Liquid Chromatography (HPLC) is  generally acknowledged as the most
accurate  (and  most   expensive)  method  for   chlorophyll  determinations  (C.F.
Zimmermann,  1990, Chesapeake Biological  Laboratory,  Solomons,  MD,  personal
communication).

Other methods for estimating phytoplankton biomass used in  earlier monitoring surveys
include  cell  counts, total cell volume estimates, protein  estimates, and dry weight.
These and other methods have certain disadvantages related to speed of the technique
or degree of accuracy (D'Elia et al., 1986).

Phytoplankton  primary productivity should be measured by the 14C  light-dark  bottle
technique (UNESCO, 1973). This technique is more  sensitive than, and requires shorter
incubation times than the  02  light-dark bottle  method.  However, the 02  method
measures gross primary production, net primary production, and  respiration,  using
inexpensive  laboratory reagents, while the 14C technique estimates only net primary
production and requires specialized training  and  equipment,  as  well as  relatively
expensive radioisotopes.

Taxonomic Analysis

Subsamples drawn from  water collected in water sampling  bottles should be preserved
for later microscopic analysis to determine phytoplankton community composition. The
choice of fixation will depend on the dominant types of phytoplankton known to inhabit a
given area.  (Buffered formaldehyde and Lugol's solution  are two common fixatives.)
Preserved phytoplankton samples normally must  be concentrated  for quantitative
microscopic analysis. Analysis should include identification of dominant phytoplankton
taxa and counts of individual species.  The description and comparison of phytoplankton
communities can be performed through the evaluation of species diversity, richness
(number of species), evenness, or numerous other parameters.  Alterations to  the
phytoplankton  community should be analyzed in relation to other potential  impacts on
other biological communities. These include but are not limited to:

      •    Food web impacts;

      •   Occurrence of toxic or nuisance phytoplankton; and

      •   Potential secondary impacts on zooplankton or fish communities (e.g., shift
         from  "more desirable" diatom-dominated communities to "less desirable"
         flagellate- or cyanobacteria-dominated communities).
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                                          Section 403 Procedural and Monitoring Guidance
Zooplankton

The taxonomic analysis should include the identification of dominant zooplankton taxa
and counts of individual species whenever possible. Particular attention should be given
to mesoplankton larvae  of commercially, recreationally, or  ecologically important
species.  Characteristics of the zooplankton community should include but not be limited
to:

      •  Species composition (richness and evenness),

      •  Abundance,

      •  Dominance, and

      •  Diversity.

Alterations in the zooplankton community should also be analyzed in relation to potential
impacts on other biological communities.  Such analyses should include, but not be
limited to, the structure and function of both larval  and adult zooplankton communities
and consideration of food web impacts.

Biological Indices

The numbers of individuals and the numbers of species  have been used as indicators of
anthropogenic disturbance, as well as other environmental stresses (USEPA, 1985b).
Furthermore, these simple biological indices are less ambiguous and can often be as
informative  as  diversity  indices  (USEPA,  1985b;  Green,  1979;  Hurlbert,  1971).
Measures of biomass have inherent problems in the collection of the data, e.g., loss or
gain of weight due to preservative medium, drying times, and evaporative weight loss.

More complicated indices  such as species diversity richness, dominance, and evenness
have found  varying degrees of acceptance.  Although diversity may not be able to
pinpoint a single  problematic discharge  in an area that contains many,  it can be an
indicator of degraded water quality.  Species diversity of phytoplankton (Patten, 1962)
and zooplankton (Copeland, 1966; Copeland and Wohlschlag, 1968; Odum et al., 1963)
communities is  noted to  increase  with  increasing distance from a pollution source.
Diversity indices, which are measures of the distribution of individuals among species,
have the following limitations (Green, 1984a):

      •  They often lack  biological meaning.

      •  They are not robust empirical indicators of any important correlates of
         environmental health.

      •  They do not incorporate information on form and function of resident species.
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Methods
      •  They are susceptible to biases associated with well-described taxa.

Because of this, these methods should be used in conjunction with other assessment
methods to help verify results.

The dominance  index is a  measure of the degree  to which  one or a few species
dominate  the community.   The dominance index,  herein defined  as the minimum
number of species required to account for 75 percent of the total number of individuals,
has been useful in describing community structure (Swartz et al., 1985). Its advantages
are that it is easily  calculated,  it does  not  assume an underlying distribution  of
individuals among species, and it is statistically testable.

Indicator Species

The evaluation of the abundance of individual indicator species  is generally informative
and may reduce the cost of the analysis.  The absence of pollution-sensitive species
and the enhancement of opportunistic and pollution-tolerant  species may assist  in
defining the spatial and temporal extent and magnitude of impacts. However, indicator
variables must possess the following characteristics (Green, 1984a):

      •  They must provide a sufficiently precise and accurate appraisal of:

         -   species of concern,
         -   anthropogenic disturbances to communities, and
         -   presence/absence or magnitude of anthropogenic perturbance to the
             ecosystem.

      •  They must be a cost-effective and a statistically reliable alternative to
         monitoring all critical measures of habitat perturbance.

      •  They must be appropriate for the spatial and temporal scale demanded by
         the study objectives.

Further  studies  of the  response  patterns  of  zooplankton  species subjected  to
anthropogenic perturbations caused  by discharges  are  required in order to  select
appropriate indicators of environmental impact.
4.8.4
QA/QC Considerations
Variability in measurements caused by field heterogeneity is quantitatively determined
by the analysis of replicate field samples. Replicate sampling should be conducted at all
field stations where measurements are to be used in comparisons. Analysis of replicate
sample data is necessary for assessing the reliability of such comparisons.
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                                           Section 403 Procedural and Monitoring Guidance
Laboratory performance and  calibration  should be  verified  at  the beginning and
periodically during the time analyses are performed. Commercially available chlorophyll
is available and recommended for use in calibration. Chlorophyll quality control samples
are  available from  EPA's  Environmental  Monitoring  and  Support  Laboratory  in
Cincinnati, Ohio.  Blind, split, or other control samples can  be  used to evaluate
performance. The Interim Guidance on Quality Assurance/Quality Control (QA/QC)  for
the Estuarine Field  and Laboratory Methods (USEPA, 1985b)  provides  a standard
operating  procedure for chlorophyll measurements.

4.8.5     Statistical Design Considerations

Temporal  stratification of the data should not be attempted until sufficient knowledge of
long-term  natural  cycles is attained.   Initially,  simple  regression  analyses may  be
conducted on seasonally stratified data to identify monotronic temporal trends.  Further
examinations of whether conditions are improving or degrading over  time may  be
conducted using statistical time series analyses (e.g., temporal autocorrelation, spectral
analyses,  etc.).

Information derived from water quality monitoring will be important  in interpreting the
results of  plankton sampling.  For example, phosphate concentrations may indicate the
cause of  a phytoplankton "bloom," while  measures  of dissolved oxygen levels may
describe the  effects of the "bloom" on other living estuarine resources.  Therefore, the
selection  of  water  quality  and  plankton sampling  strategies must  not  be done
independently.  These programs should be integrated to the fullest  extent possible to
allow correlation of observed responses to changes in water quality  parameters. Also,
alterations to the plankton community should be analyzed in relation to other impacts on
biological  resources such as food web impacts on fish communities.
4.8.6
Use of Data
The analyzed, collected data will be used initially to identify the prominent species and
relate  the temporal and spatial distribution  patterns to  water quality parameters.
Statistical techniques will be used to make these comparisons.   As the discharge
guidelines are implemented,  a plankton monitoring  program can be used to track the
effectiveness of maintaining nutrient levels.  As plankton levels fluctuate, their effects on
oxygen levels and fish and shellfish communities can be assessed. Plankton monitoring
strategies should be able to delineate between natural variability in plankton stocks and
variations caused by anthropogenic changes in nutrient concentrations.

Characterization  of phytoplankton  species  abundance,  distribution,  and  primary
productivity  provides indications of water quality conditions.  Monitoring  changes  in
phytoplankton community composition and densities is critical for the interpretation and
evaluation of long-term trends in water and habitat quality. Further understanding of the
causes of excessive water column and sediment oxygen demand requires  tracking  of
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Methods
photosynthetic activity and metabolic rates over time (Chesapeake Executive Council,
1988b).  Zooplankton  abundance and distribution are affected  by both changes in
phytoplankton  and  changes  in  predator   populations.     Therefore,  population
characteristics of this group  can indicate symptoms of water quality problems, fishing
pressure,  and other habitat  problems for  predator species (Chesapeake  Executive
Council, 1988b).

4.8.7    Summary and Recommendations

Rationale

      •  Track phytoplankton and herbivore populations if changes in trophic structure
         are suspected.

      •  Purpose of monitoring is to assess the effectiveness of the management
         programs in mitigating the potential impacts caused by excessive nutrient
         inputs and resulting changes in the planktonic community biomass and
         structure/function.

Monitoring Design Considerations

      •  Collect both phytoplankton and zooplankton samples at specific depths
         throughout the water column.

      •  Phytoplankton samples should be taken with water bottles and pumps used
         for chlorophyll concentrations.

      •  Zooplankton samples should be taken with bottles or nets of varying sizes.

      •  Monitoring programs should consider natural and temporal fluctuations in
         plankton biomass and species composition.

      •  Other components of the overall monitoring program, including water quality
         monitoring and fish and shellfish communities, should be analyzed relative to
         plankton community data to establish relationships and trends.

      •  Selected biological indices should retain biological meaning, be robust
         indicators of marine "health," and incorporate species form and function.

      •  Indicator species should possess the following characteristics:

         -  sensitive to planktonic perturbances of concern;
         -  cost-effective and statistically reliable alternative to measuring all species
            in a monitoring program;
         -  statistically reliable indicative measures of habitat perturbance; and
         -  appropriate for the spatial and temporal scale  determined by the study
            objectives.
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                                            Section 403 Procedural and Monitoring Guidance
      •  Selection of reference sites is key  to the evaluation of environmental impact
         due to anthropogenic perturbances; several reference sites may be requried
         to provide proper control for sampling sites.

Analytical Methods

      •  It is recommended that consistent types of sampling gear and location and timing
         of sample collection be implemented to allow for comparisons between studies.

      •  Phytoplankton biomass can be determined using fluorometric or
         spectrophotometric methods using a variety of filtration and extraction techniques.

      •  Phytoplankton productivity should be measured using the 14C light-dark bottle
         technique.

      •  Taxonomic analysis should include identification and counts of dominant species.

QA/QC Analysis

      •  Replicate samples should be taken at all field stations where applicable.

      •  Laboratory performance evaluations and calibrations must be done on a
         regularly scheduled basis.

      •  Standard chlorophyll samples, obtained from EPA, should be used for
         calibrations.

Statistical Design Considerations

      •  Power analyses may be applied to determine the appropriate number of sample
         replicates required to detect a specified difference.

      •  Temporal integration of the data should not be attempted until sufficient
         knowledge of long-term natural cycles is attained.
Data Use
         Identify dominant species.

         Detect short- and long-term spatial and temporal trends in overall biomass and
         productivity.

         Detect short- and long-term spatial and temporal trends in species abundance,
         distribution, and composition.

         Examine relationship between water quality condition and trends in plankton
         community characteristics.
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Methods
         Examine relationship between plankton community characteristics and impacts
         on other living resources (e.g., fish and shellfish communities).
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                                           Section 403 Procedural and Monitoring Guidance
 4.9
HABITAT IDENTIFICATION METHODS
 Sensitive marine habitats serve a vital purpose as spawning grounds, nursery grounds,
 forage areas, energy sources, shelter, and migratory routes and destinations for a
 multitude of marine organisms.  Pollution-related damage can cause long-lasting harm
 to these habitats to the extent of altering ecosystem diversity and function.

 For the purpose of this discussion, a sensitive habitat can be defined as a physical area
 used, at least for a significant portion of the year, by a disproportionate abundance of
 individuals and/or species, or as an area essential to the functioning of the ecosystem.

 Habitats need not be permanent entities but may expand and contract on a seasonal
 basis.  Examples of transitory  habitats include sea grass beds and  ice floes.  Marine
 habitats  can be  organized  in a  hierarchical structure  to  be considered  under  the
 discharge guidelines and can be grouped in the following manner:

      Subtidal
         Rock bottom
         Bedrock/rubble
         Coral
         Oyster/clam shell reef
         Unconsolidated bottom
         Cobble/gravel
         Sand
         Mud-rich organic
         Submerged aquatic vegetation (SAV)
         Sea grass (Zostera, Thalassia)
         Algal and other submerged plant communities
         Water column

      Intertidal
         Emergent vegetation (along exposed coasts)
         Marshes
         Mangroves
4.9.1
      Ice Floes
Rationale
The ocean discharge guidelines that relate to the presence of and impact on marine
habitats are the following;
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Methods
      •  Importance of the receiving water area to the surrounding biological
         community, e.g., spawning sites, nursery/forage areas, migratory pathways
         and areas necessary for critical life stages/functions of an organism;

      •  The existence of special aquatic sites, including (but not limited to) marine
         sanctuaries/refuges, parks, monuments, national seashores, wilderness
         areas, coral reefs/seagrass beds;

      •  Existing or potential recreational and commercial fishing; and

      •  Any applicable requirements of an approved CZMP.

The identification and delineation of these habitats should be made in both temporal and
spatial dimensions to account for  variations in  use  by the  biological community
throughout the year.   Both the aerial extent and the functional value to  biological
resources must be determined in considering the  identification  and importance of a
sensitive marine habitat.

Even though an effluent discharge may not directly alter the biological population in a
certain area, if the habitat essential for the survival of those individuals is destroyed, the
ecological results could be the same as if the population had been exposed directly.
The loss of critical habitat could cause the supported population to be either removed or
displaced to less suitable habitat.

Monitoring of discharge effects can help to ensure that long-term sublethal doses do not
accumulate  and become toxic.   It can also provide assurance that effects such as
increased turbidity or sedimentation do not reduce biological resource productivity below
sustainable levels.

During the  permit  review  process,  all important and sensitive habitats should be
identified and delineated to serve as a baseline for future monitoring activities.  Habitat
quality in terms of functional values for fish, bird, and marine mammal populations must
also be taken into account to gain a complete valuation of the resources that could be
affected by discharges. Some habitat monitoring activities overlap with other biological
monitoring activities described in other sections.  For habitats, the main concern is to
map the physical  boundaries and  to correlate  biological  populations  to changes in
habitat distributions and functional values.

4.9.2    Monitoring Design Considerations

Habitat monitoring is a two-part process. First, the aerial coverage and boundaries must
be determined; second, the importance or the functional value to biological  resources
must be determined.   The first determination can  be  made by using  charts, remote
sensing,  or submersible remotely operated vehicles. The second  analysis is carried out
by using quantification techniques to establish the value  of that habitat for particular
166
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                                          . Section 403 Procedural and Monitoring Quittance
 species.  The combining of these two techniques allows the identification of those
 habitats critical to the functioning of the ecosystem. The determination of the functional
 value of a habitat is much more subjective than delineation and must be done with care.
 The results of these  procedures  should be combined with data on trends in species
 distribution and water quality to provide a more complete assessment of the causes and
 potential effects of habitat degradation.   Habitat monitoring will also  indicate which
 environmental parameters and areas are critical to the ecosystem so that monitoring
 activities can be tailored to assess these needs.

 To conduct a  habitat trend analysis, the sampling design  must account for  natural
 variations caused by  extraordinary meteorological events and other phenomena. The
 recovery rate of habitat functions and its effects on the functional analysis described
 above must also be considered.  The primary concern of the habitat assessment is to
 accumulate an adequate set of baseline data from which trends can be established.
 During this initial phase, seasonal variations must be taken into account during sampling
 phases. Sampling and delineation should be carried out at times of maximum biomass
 or physical extent.  For example, SAV beds or ice floes go through seasonal declines (or
 disappearances) and  thus a finite period of time exists to conduct sampling.   Diurnal
 factors  such as tides and  weather conditions also affect biological populations, and
 sampling must be conducted taking these factors into account.

 4.9.3    Analytical Methods Considerations

 Habitats are  generally classified in a hierarchical scheme as  mentioned  above.
 Classification methods that can be used are referenced in Appendix A. The first course
 of action  in identifying,  delineating,  and evaluating  critical marine habitats  entails
 mapping out the boundaries of the habitat, while the second involves a determination of
 its value as an ecological resource.  Table 4-23 lists the methods that can be used to
 identify and evaluate sensitive habitats.
                      Table 4-23. List of Analytical Methods
   Areal Trends
       Functional Trends
Aerial Photography
Maps
Satellite Imagery
Remotely Operated Vehicles
Habitat Evaluation Procedure (HEP)
Habitat Suitability Modeling
Minimum Habitat Matrix
Wetland Evaluation Technique (WET)
                                                                             167
 image: 








Methods
Habitat Delineation

Aerial Photography

Various government agencies (NOAA and USGS among others) have used aircraft to
produce  aerial photographs of wide  areas for a  number  of  years.   The  use of
photographs in delineating marine habitats is of limited value since many features below
the low tide line cannot be viewed.  Aerial photographs can be produced in a large range
of scales and spatial resolutions depending on the area in question.  Fine detail can be
resolved for relatively small tracts, while large areas can be viewed at lower resolutions
to define gross features.  In some  regions there is an  extensive  record  of historical
photographs that can  be used to  spot trends  and to establish  a baseline.   New
overflights can be expensive and require ideal weather conditions, but  they can be of
value for intertidal mapping and for mapping some submerged aquatic  vegetation and
other shallow-water habitats.

Maps

As a preliminary method, maps and charts can be used to obtain a quick assessment of
the presence and delineation of sensitive habitats in the discharge area. Maps are the
least expensive spatial  data source and are also the most  simple. Nautical charts,
compiled by the Coast  and Geodetic  Survey of NOAA, contain bottom characteristic
information that could  indicate the  location  of areas  of biological  importance.  This
information is  generally accurate for shallow-water areas but may  be  fragmentary or
nonexistent for areas farther offshore.  The coordinated use of U.S. Geological Survey
topographic quadrangle maps, U.S.  Fish  and  Wildlife Service  National Wetlands
Inventory maps, and NOAA nautical charts can serve as an information base.

The principal advantages of using maps for delineating  habitats is their ease of use and
their low cost.   Disadvantages include the inaccuracies in the  plotting of habitat
boundaries and the fact that the maps in use may be outdated and thus do not reflect
current conditions.  In locations that have a history of human  use, there may be an
extensive list of earlier chart or map editions.' These earlier editions can  be an excellent
resource for spotting changes in habitat structure.

Satellite Imagery

Satellite imagery is a potential source of data when very large study areas are evaluated
and fine spatial detail  is not required.  This technique is useful to a maximum depth of
approximately 30 meters, and habitats can be resolved  only to a minimum of roughly 30
to 50 square meters.  For large study areas, the cost  of satellite images can be high,
especially when a long-term temporal series may be required for monitoring purposes.
If the area under investigation  contains large homogenous habitats, the resolution size
may not be a  constraining factor.  A major benefit of these images  is that the spectral
 168
 image: 








                                           Section 403 Procedural and Monitoring Guidance
properties and brightness values are recorded digitally.  This technology lends itself to
advanced spatial analysis methods that can be used to quantify productivity values.
These determinations are not possible with most aerial photographs.

Remotely Operated Vehicles

Remotely  operated vehicles   (ROVs)  are  small,  unmanned,  remotely operated
submersibles that are equipped with lights and cameras used to view portions of the
ocean bottom.   These  submersibles receive power and commands from a  support
vessel and are highly versatile for identifying possible habitats.  EPA Region 4 has used
ROVs to investigate  and  record the ocean bottom  immediately'surrounding  the
discharge outfalls of oil  and  gas drilling wells.  This methodology produces a precise,
nondestructive record of the nature and extent of any physical habitat. The  main liability
of this system is that unless vast amounts of time are spent, only small areas can be
investigated.   These systems are  also rather expensive  and require  well-trained
technicians to operate them. In addition, a ROV is limited by the length  of its umbilical
and by the visibility  of the water column in the search area.  The visibility problem may
be compensated for by the  addition of such technology as side scan sonar or sonar
attached to the ROV. Because of these constraints, the use of this system would be
confined to areas of most concern near the zone of initial dilution.

Functional Analysis

Functional analysis attempts to assess the use of the habitat by biological  resources and
to compare the value of one habitat to that of another.  Another aspect of functional
analysis is to calculate the change in a habitat's value over time.  These processes
entail site-specific studies that can involve a large amount of time and money. The data
required by this technique can be collected in connection with the techniques described
in the biological methods section of this report. The analysis of these data will center on
spatial and temporal variations at a particular habitat. Various methods  for conducting
functional analysis  are  referenced in Appendix A.   Some of the  most widely used
procedures are described below.

Habitat Evaluation  Procedures

Habitat evaluation procedures (HEPs) are procedures developed  by the U. S. Fish and
Wildlife Service to document the quality and quantity of  available habitat  for selected
wildlife species.  The data generated are used to compare two habitats at the same
point in time and a single habitat over a length of time. By combining these two sets of
information, the impact of proposed discharges can be quantified (Lonard and Clairain,
1986).
                                                                             169
 image: 








Methods
In the evaluation procedure, the habitat quality of selected species is documented based
on an evaluation of the ability of key habitat components to supply the life requisites of
the selected species.  The evaluation involves using the same key habitat components
to compare existing habitat conditions and the optimum conditions for the species of
interest (USFWS, 1980).

The limitations in using this approach involve the use of habitat quality as  an evaluation
parameter, which limits the methodology to those situations in which measurable and
predictable habitat  changes are an  important variable.   This approach  also forces a
long-term averaging type of analysis. There is no assurance that populations will exist
at the levels predicted by the analysis since all the environmental or behavior variables
that affect population levels may not be included.  In addition, socioeconomic or political
constraints not taken into account may prevent the actual populations from reaching
their predicted levels (USFWS, 1980).

Habitat Suitability Modeling

Habitat  suitability  modeling consolidates  habitat  use  information  into  a framework
appropriate for field application and is scaled to  produce an index between 0.0 (unsuitable
habitat)  and 1.0 (optimum habitat). Habitat suitability models are designed for use with the
habitat evaluation procedures described above (Toole et al.,  1987).  Multiple  regression
equations are used to  describe relationships between environmental characteristics and
productivity. Once the model has been validated, sensitivity analysis can be carried out to
project the results of varied discharge rates on the health of the site.

For modeling to make reliable  predications, the input data must accurately  reflect the
range of natural conditions observed. This may involve expensive and time-consuming
field studies in which the error limits and the QA/QC parameters must be well defined.
The reliability and the extent of the field data can be a major limitation in these studies.

Minimum Habitat Matrix

Minimum habitat guidelines for various species  are a  technique  developed with the
ultimate goal of reestablishing  balanced ecosystems in environmentally stressed areas.
This method is designed to provide information on the minimum habitat quality needed
by  a target species and  identifies  those  factors (both  environmental  and ecological)
required for the species.  This  information  is formatted into a habitat requirement matrix
that defines the habitat parameters needed for successful reproduction and survival of
the indicated species.  Such matrices can indicate the vital environmental parameters
that should be monitored, thus facilitating the development of monitoring programs.  This
process is used to estimate the feasibility, benefits, and potential costs of maintaining
and protecting  an estuarine environment  suitable for the successful reproduction and
survival of the indicated species (Chesapeake Executive Council, 1988a).
 170
 image: 








                                            Section 403 Procedural and Monitoring Guidance
 One potential problem with this method is that the primary indicated species and those
 organisms on  which the target species  depend  for food are both tracked with the
 intention  of maintaining habitat quality for both.   This method may not completely
 recognize the complex species interdependence within marine environments.

 Wetland Evaluation Technique

 The wetland evaluation technique (WET) was initially developed by the Federal Highway
 Administration and revised by the U.S. Army Corps of Engineers to assess the function
 and value of various components of habitats  and their suitability for specific fish and
 invertebrate  species.    WET  evaluates  functions and  values in terms  of  social
 significance,  effectiveness, and opportunity  (Adamus et al., 1987).  Social significance
 assesses the value of a site to society due to its special designation, potential economic
 value, and strategic location.  Effectiveness assesses the capability of a wetland to
 perform a function as a result of its physical, chemical, or  biological characteristics.
 Opportunity assesses the opportunity for a habitat to perform a function to its level of
 capability.

 WET assesses functions and values by characterizing a habitat in terms of its physical,
 chemical,  and biological processes and attributes  (Adamus  et  al.,  1987).   This
 characterization  is  accomplished  by  identifying threshold values  for  predictors.
 Predictors are  simple, or integrated, variables that directly or indirectly measure the
 physical,  chemical,  and biological processes  or attributes  of  a  habitat  and  its
 surroundings.

 4.9.4     QA/QC Considerations

 Since  sensitive habitat identification  is relatively  subjective, close controls  must be
 exercised during sampling and delineation. In the delineation of habitats, two individuals
 should independently define the boundaries of a single habitat area.  Any disagreement
 between the two should be rectified before a final determination is reached.  Sampling
 programs demand a high level of expertise on the part of the individuals conducting
 them.  All personnel must have an advanced  understanding of such topics as taxonomy,
 sampling techniques, and statistics. The sampling  strategy used will depend at least in
 part on the level of expertise or training available. The selection of a functional analysis
 method should be made after a review of the strengths and limitations of each method
 has been conducted.

 4.9.5      Statistical Design Considerations

 In designing  a habitat monitoring program, the researcher  must  first determine the
 resources available.  This will determine the extent of the monitoring program and the
 level of detail it can  provide.   Aerial assessment can be done with limited resources,
while  functional analysis  requires greater commitments of both finances and  time.
                                                                              171
 image: 








Methods
Functional  assessments require that the researcher decide which  functions are of
greatest  interest  in  the area of study or which will provide  the most  information
concerning the habitat attributes perceived to be of greatest value to the area.

Most habitat evaluation  techniques involve some form of ranking, either quantitative or
qualitative. The purpose of the monitoring program is to periodically compare  baseline
conditions to conditions after the permit has been issued. In terms of habitat monitoring,
this  means comparing  the  baseline acreage  or functional  rating of  the  habitat to
conditions some time after discharge has commenced. Therefore, the ability to  replicate
results is imperative.

4.9.6    Use of Data

The  object of the data set is to obtain a clear and precise spatial and temporal picture of
the physical extent of a sensitive habitat. An evaluation of the functional value of that
habitat is also made so that the effects of various impacts can be judged.  Sensitive
habitats should be clearly delineated  on nautical charts or topographic maps along with
a  complete  description of  their  composition.   The description  should  include  a
characterization of the  extent of  natural  seasonal variations in  the boundaries.  A
complete description of the functional value of the habitat to the biological community,
as well as  an estimate  of potential for loss of the resource from the effects of effluent
discharge,  should also be made.

These data will act  as a baseline for future monitoring programs that will track the
changes to the system.  The baseline study and the monitoring program should be able
to discriminate between natural variations and discharge-induced changes.

4.9.7    Summary and Recommendations
Rationale
          A sensitive habitat can be defined as a physical area utilized, at least for a
          significant portion of the year, by a disproportionate abundance of individuals
          and/or species, or as an area essential to the functioning of the ecosystem.

          Sensitive marine habitats should be identified and their boundaries delineated
          as part of an ongoing effort to assess the potential for impacts from effluent
          discharges on ecological communities.

          Monitoring programs that assess both the physical boundaries of the habitats
          and the support functions that they serve for biological communities should
          be conducted when sensitive habitats are present.

          Damage to habitats from discharge effluents can cause irreversible harm and
          create severe changes in ecosystem function and diversity.
 172
 image: 








                                           Section 403 Procedural and Monitoring Guidance
      •  Monitoring programs can provide assurance that effects such as increased
         turbidity or sedimentation do not reduce biological resource productivity
         below sustainable levels.

Monitoring Design Considerations

      •  Habitat monitoring consists of two parts:  (1) aerial coverage and boundary
         delineation and (2) functional analysis, which determines the value of the
         habitat to the system.

      •  Habitat delineation can be fairly straightforward, but functional analysis is
         rather subjective and must be done with care.

      •  Sampling strategies must account for variations caused by natural processes
         and must be done at times of maximum extent or biomass.

      •  A functional analysis technique should be selected after a review of the
         strengths and limitations of each method for each particular site.

Analytical Methods Considerations

      •  Sensitive habitats can be identified  using maps, charts, and photographic
         techniques.  ROVs can be used to investigate near-field habitats in the zone
         of initial dilution.

      •  Functional analysis determines the  importance of a habitat to the biological
         community.

      •  Functional analysis techniques are  based on theoretical relationships of the
         ecosystem; consequently, both the  theory and the data used should be
         vigorously evaluated.

      •  For habitat identification and delineations, the following techniques can be
         used:

         -  aerial photography,
         -  maps/charts,
         -  satellite imagery, and
         -  remotely operated vehicles.

      •  Functional analysis can be carried out using various methods. Those
         described are the following:

         -  habitat evaluation procedures (HEP),
         -  wetland evaluation technique  (WET),
         -  habitat suitability modeling,  and
         -  minimum habitat matrix.
                                                                              773
 image: 








 Methods
QA/QC Considerations

       •   Personnel identifying habitat should have advanced training, and two people
          should independently define the boundaries.

Study Design Considerations

       •   Ability to replicate results is important.

       •   The extent of the monitoring program can be based on funding level.

       •   Aerial assessments are inexpensive, while functional analyses are more
          expensive and more labor-intensive.

Use of Data

       •   Data  collected will consist of charts identifying the boundaries of sensitive
          habitats accompanied by complete descriptions.

       •   Functional analysis results will indicate which habitats are critical and the
          effects observed under various discharge scenarios.
174
 image: 








                                            Section 403 Procedural and Monitoring Guidance
4.10     BIOACCUMULATION

Bioaccumulation is the overall process of biological uptake and  retention of chemical
contaminants obtained from foods, water, sediments, or any combination of exposure
pathways. A number of expressions are used to describe the bioaccumulation potential
of contaminants.  These terms  are  explained in Table 4-24.   Bioaccumulation is a
consequence of an organism's physiological limitations to transform and excrete the
invading chemical substances. Potential consequences of bioaccumulation of chemical
contaminants in marine organisms include, but are not limited to:

      •   Significant mortality to susceptible organisms;

      •   Lethal or sublethal chronic toxic responses at later stages of the life cycle or
          under conditions of stress for susceptible organisms; and

      •   Transference of increasingly greater concentrations of toxic pollutants to
          organisms at higher trophic levels, including humans.

Bioaccumulation analyses  provide the link between exposure and effects  and thus can
generate important insights into ecological effects, human health  risks, and routes and
extent of pollutant exposure.

4.10.1    Rationale

The Clean Water Act section 403 point source discharge program requires  (among other
things) a determination of:
Bioavailability



Bioaccumulation


Bioconcentration


Biomagnification
      Table 4-24.  List of Terms

A measure of the physicochemical access that a toxicant has to the
biological processes of an organism. The less the bioavailability of a
toxicant, the less its toxic effect on an organism (USEPA, 1991c).

Uptake and retention of substances by an organism from its surrounding
medium and from food (USEPA, 1990a).

Uptake of substances by an organism from the surrounding medium through
gill membranes or other external body surfaces (USEPA, 1990a).

The process by which the concentration of a compound increases in different
organisms occupying successive trophic levels (USEPA, 1990a)
                                                                                175
 image: 








Methods
      •  The quantities, composition, and potential for bioaccumulation or persistence
         of the pollutants to be discharged;

      •  The potential transport of the pollutants by biological, physical, or chemical
         processes;

      •  The composition and vulnerability of potentially exposed biological communities;
         and

      •  Potential direct or indirect impacts on human health.

Each of these determinations is addressed to some degree through bioaccumulation
studies.

Monitoring  of bioaccumulation data  is  essential in  relating the presence of selected
chemical residues in marine waters and sediment to their transfer and accumulation in
marine  organisms and potential transfer to humans.  Toxics can occur in the water
column  at or near analytical  detection  limits;  however, over time, these contaminants
can  accumulate in  fish and shellfish tissue  to  measurable concentrations.  In areas
where water and sediments show measurable contaminant levels, it is difficult at best to
predict  rates of  uptake and bioaccumulation, although direct monitoring can provide
spatial and temporal records of the concentration and bioaccumulation of toxics. The
assessment of bioaccumulation in the  surrounding biological  community should be a
component  of a monitoring program  required in  a  permit  issued  under the  "no
irreparable harm" clause of section 403.

4.10.2   Monitoring Design Considerations

Typically, bioaccumulation studies are formulated according to the study objectives. If
the objective is  to  determine the  effects of bioaccumulation on human health, then
commercial and/or recreational fish and shellfish are tested.  If determining the effects
on the habitat or ecosystem is the objective, usually the benthic macroinvertebrates are
surveyed.  Unfortunately, comparisons between  studies are valid  only if  the study
designs and procedures are comparable.  Standardization of monitoring design would
allow for comparison between various studies.

A  common  deficiency in  many programs is the inability to collect sufficient  tissue
biomass of appropriate species across sampling locations throughout the study.  The
selection of appropriate species and tissues  must account for  natural fluctuations in
populations as well as changes due to anthropogenic perturbations. Indigenous species
initially present may not be available later, limiting temporal and spatial comparisons.
176
 image: 








                                          Section 403 Procedural and Monitoring Quittance
Selection of Target Species

A key component of any bioaccumulation monitoring program is the selection of target
species.  Concentrations of chemical  residues in tissues of target species serve as
indicators of contamination throughout the biological system.  The fundamental criterion
is  the  ability  to  use the selected  species  to make comparisons between sampling
locations and  sampling periods.

Target Species Characteristics

It is recommended that the target species possess the following characteristics (USEPA,
1985a):

      •  High bioaccumulation potential for selected contaminants of concern;

      •  Weakness or absence of metabolic regulation of selected contaminants to
         allow assessment of a "worst-case scenario";

      •  Abundant enough, temporally and spatially, to allow for adequate sampling;

      •  Large enough to provide adequate amounts of tissue for analysis;

      •  Sessile or sedentary in nature to ensure that bioaccumulation is
         representative of the study area;

      •  Easily collected; and

      •  Part of an existing data base of exposures and sensitivity.

Suggested fish and macroinvertebrate  target species for various  regions of the United
States are listed in Tables 4-25 and 4-26 (USEPA,  1985a).

Secondary considerations based on economic importance and status  as a bioassay
organism may be applied to further winnow the  list of candidate target  species to a
practical number of species to be analyzed.

Candidate  target  species  should  be  objectively  ranked  based  on  ecological
characteristics that would enhance their potential for bioaccumulation and facilitate
sampling and analytical procedures (USEPA, 1985a). The advantages of using benthic
macroinvertebrates as target species are:

      •  They are sendentary and therefore represent bioaccumulation in the study
         area.

      •  They represent a significant food source  for higher trophic levels and
         therefore indicate the existence of contaminants in the food web.
                                                                            177
 image: 








Methods
B
              Table 4-25.  Highest-Ranking Candidate  Fish for Use
                    as Bioaccumulation Monitoring Species
                                                    Secondary Selection Criteria
State
MASSACHUSETTS




















RHODE ISLAND


NEW YORK






NEW JERSEY



VIRGINIA






lE.iir' ^rjaSssi
Locality
Swampscott



Lynn


South Essex




Boston



Fall River

New Bedford


Newport


Upper East River


Lower East River


Lower Hudson River
Cape May



Portsmouth



Virginia Beach


1
Species
Winter flounder
Yellowtail flounder
Ocean pout
Windowpane
Winter flounder
Yellowtail flounder
Ocean pout
Winter flounder
Yellowtail flounder
Windowpane
American eel
Ocean pout
Winter flounder
Yellowtail flounder
Ocean pout
Windowpane
Winter flounder
Windowpane
Winter flounder
Scup
Summer flounder
Winter flounder
Scup
Weakfish
Winter flounder
Windowpane
Weakfish
Spot
Scup
American eel
Hogchoker
Spot
Red hake
Windowpane
Summer flounder
Spot
Summer flounder
Atlantic croaker
Hogchoker
Spot
Red hake
Summer flounder
tea *Jy,'XS >£«""!'»''*••* W»x A
eSst&'r XX^^X' .X •.'•£<^«*^'. "y«"*<iWfiS
Economic
Importance
Yes
Yes
No
No
Yes
Yes
No
Yes
No
No
No
No
Yes
Yes
No
No
Yes
No
Yes
No
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
No
No
Yes
No
No
Yes
Yes
Yes
No
No
Yes
No
Yes
* , #*v"*< £$•/£>>>* < < SoftiSw&jjy*'-??'?* ,
Bioassay
Species
Yes
Yes
No
No
Yes
No
No
Yes
No
No
No
No
Yes
No
No
No
Yes
No
Yes
No
Yes
Yes
No
No
Yes
No
No
Yes
No
No
No
Yes
No
No
Yes
Yes
Yes
No
No
Yes
No
Yes
yfAsffiW t.-s'^W-v?*' -. '-., ,v ,-W,
Xi * v'r-v *'""&?£%&*.??£?*
178
 image: 








Section 403 Procedural and Monitoring Quittance
* s , ->-.-•/>*•: ^"fj, ',-w, ''3&'j;?3&!
State
CALIFORNIA
(Northern)




CALIFORNIA
(Southern)








WASHINGTON
(^'•^tff^ff, , %J1 ^J'jJ^"** -fr"" V'*'*' **X %'"'****'• > JVJ , V^-Jifc, ^^^^K.^VX'-SSvMX.Xvs^A^'v^v..
' > *% A^V •." ' ' ' ' ^"'-s i /' * rti ' ,* Vv * •>•*«''* ^»i?v ^ - •• ^-V"V " ' * V»t"A' o' 5 > * >£ b^pw- v;
Table 4-25. (continued)
Locality
San Francisco
Oakland
Monterey
Santa Cruz
Watsonville
Goleta
Santa Barbara
L.A. County
Orange County
Hyperion
Oceanside
Escondido
San Elijo
San Diego
Central Puget Sound
^fy^ t°'Vv- •",, "

Secondary Selection Criteria
Economic Bioassay
Species Importance Species
English sole
Pacific sanddab
Big skate
English sole
Starry flounder
Pacific staghorn
sculpin
English sole
Curlfin sole
English sole
English sole
Curlfin sole
Dover sole
Pacific sanddab
Longspine combfish
Spotted cusk-eel
English sole
Pacific sanddab
Dover sole
Curlfin sole
English sole
Dover sole
Pacific sanddab
English sole
Dover sole
Longspine combfish
Big skate
California skate
Dover sole
Blackbelly eelpout
Pacific sanddab
English sole
Dover sole
English sole
Pacific sanddab
Longspine combfish
English sole
Dover sole
Rock sole
Spotted ratfish
Rex sole
C-O sole
Yes
Yes
No
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
No
Yes
Yes
No
No
Yes
Yes
Yes
Yes
Yes
No
No
No
Yes
No
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
No
Yes
Yes
Yes
No
No
Yes
No
No
Yes
No
Yes
Yes
No
No
No
No
No
Yes
No
No
No
Yes
Yes
Yes
Yes
No
No
No
No
No
No
No
Yes
No
Yes
No
No
Yes
No
No
No
No
No
                                     179
 image: 








Methods
           Table 4-26.  Recommended Large Macroinvertebrate Species
                          for Bioaccumulation Monitoring
             Region
    Recommended Species3
Massachusetts to Virginia
Alaska to California
Florida, Virgin Islands, and Puerto Rico

Hawaii
American lobster (Homarus americanus)
Eastern rock crab (Cancer irroratus)
Hard clam (Mercenaria mercenaris)
Soft-shell clam (Mya arenaris)
Ocean quahog (Artica islandica)
Surf clam (Spisula solidissima)
Edible mussel (Mytilus edulis)

Spiny lobster (Panulirus interruptus)
Dungeness crab (Cancer magistei1)
Rock crab (Cancer antennarius)
Yellow crab (Cancer anthonyi)
Red crab (Cancer productus)
California mussel (Mytilus californianus)

Spiny lobster (Panulirus argus)

Spiny lobster (Panulirus penicillatus)
a Additional species that may occur at specific discharge sites and are considered acceptable
bioaccumulation monitoring species include the American oyster (Crassostrea virginica) and the Pacific
oyster (Crassostrea gigas).
A disadvantage of macroinvertebrates is  the lack of  adequate  tissue  biomass for
analyses.  Adult fish are often used not only for their significant biomass, but also as a
measure of possible contaminants available to the human population.  Fish, however,
are motile and may not be representative of the study area.

Ideally, studies  should include samples of  numerous species from many phyla.  This
diversity will  ensure the  collection of  bioaccumulation  data for a wide  spectrum of
contaminants and will indicate a species' potential to bioaccumulate those contaminants.
For example, oysters and other bivalves are ideal for monitoring bioaccumulation of
PAHs because of their limited ability to metabolically transform these contaminants.  The
monitoring of a food chain could establish  the  potential  for transport from one trophic
level to another.  Species with extensive bioaccumulation data would be preferred.  A
fundamental criterion is the ability to use the selected species to make comparisons
between sampling locations and sampling periods.
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                                          Section 403 Procedural and Monitoring Guidance
Caged Indicator vs. Indigenous Species

The California Mussel Watch Program, the U.S. Mussel Watch Program, and the NOAA
Status and  Trends Program have employed the use of both  resident and caged
transplant mussels to monitor bioaccumulation of toxic chemicals  over space and time
(Goldberg etal., 1978; Boehm, 1984; Ladd etal., 1984). Caged indicator target species
offer several advantages over indigenous species :

      .  The biology and ecology of these indicator species are usually well described.

      •  Descriptions of culture and/or maintenance of the organisms under laboratory
         conditions are often available.

      .•  The method provides control of initial temporal and spatial variation of
         individuals and/or biomass.

      •  The method allows the use of a specific age, size, and/or genetic stock.

      •  The method ensures on-site bioaccumulation.

By controlling for initial conditions, the rate of change of tissue contamination may be
calculated.  In addition, the Long Island Sound National Estuary Program has shown
that  performing chemical residue analyses on caged indicator species appears to be  a
promising approach for identifying sources of pollution (USEPA, 1982c).

The  use of a caged indicator species indicates only uptake due to exposure to the water
column, and unfortunately methods for caging sediment-ingesting organisms are not yet
available.  The bioaccumulation of sediment-sorbed contaminants is thought to be
indicated by transfer through interstitial waters (Knezovich and  Harrison, 1987), and the
results often do not relate to species found on site. Therefore, caged organisms may
not provide a full-spectrum  profile of the contaminants that are bioavailable at any one
site.

The advantages of indigenous species are as follows:

       •   Results obtained will relate directly to those species which may be impacted
          and will provide a direct measure of potential risks to human health.

       •   There is no cost of transplanting organisms.

       •   There is no possibility of loss of cage/buoy system.

       •   There is no possibility of introduction of a "nuisance" species.

Unfortunately, common indigenous  species may not meet  the criteria for use as
bioaccumulation target species, negating any advantage of using a native species. The
most common  problem is  collecting sufficient tissue  biomass of appropriate species
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 Methods
 across sampling locations throughout the study.  The composition of benthic community
 species  is subject to large natural fluctuations,  as well as fluctuations in response to
 pollution (Pearson and Rosenberg, 1978); indigenous species initially present may not
 be available later, limiting temporal and spatial comparisons.

 Selection of Tissues

 The type and location of tissue analyzed will depend on the objectives of individual
 monitoring programs. It is strongly recommended that all analyses be conducted with
 the same species  and tissue  type  in  order that scientifically  and statistically valid
 comparisons may be made.

 In  fish, liver tissue is closely associated  with  regulation and storage of many toxic
 substances (Fowler, 1982).  Its high fatty tissue content tends to accumulate lipophilic
 contaminants.  Therefore, contaminant levels in the liver can be used to estimate the
 range of contaminants being assimilated.  For macroinvertebrates, hepatopancreas or
 digestive gland tissue performs functions analogous to those of fish liver tissue.

 Contaminants in edible muscle tissue represent those contaminants that are retained in
 a form that allows transfer to humans.  Sampling of muscle tissue is appropriate for
 human exposure assessments  and  quantitative health risk  determinations.  Within a
 fillet, contaminant concentrations may vary; therefore,  it  is  recommended  that a
 consistent location within the muscle tissue be analyzed (USEPA,  1989a).

 Whole-body analyses should be conducted when predators consume the whole body of
 the  target organism.   If organisms are not cleansed of materials  contained  in the
 digestive tract, contaminants  in the gut contents will be included in the analyses.  To
 provide the most accurate estimate of the total amount of  contaminants available  to
 most macroinvertebrate predators, this type of cleansing may  not be required.

 Total Organic Carbon and Acid Volatile Sulfides. and Tissue Lipid Normalization

 Toxic sediment concentrations  of hydrophobic contaminants have been  found to be
 related to the total  organic carbon content (TOC) of the sediment (Karickhoff  et al.,
 1979). The bioavailability of  metal contaminants has been found to be related  to the
 acid volatile sulfide (AVS) concentrations of the sediment (DiToro  et al., in press).  TOC
 and AVS normalizations  have  been conducted  to  estimate the  concentrations of
 sediment contaminants  that are bioavailable  over different  sampling   locations.
 Sediment TOC and AVS normalizations are recommended to account for the variability
 in bioavailable sediment contaminant concentrations between  locations.

 Lipids  appear to be a storage  site of organochlorines,  hydrocarbons, and  other
 hydrophobic contaminants in  a variety of organisms (de Boer, 1988).  In fact, just as
TOC and AVS are considered by many to be the most important parameters in defining
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                                           Section 403 Procedural and Monitoring Guidance
organic and metal concentrations in sediments, lipid content is considered the salient
parameter in defining hydrophobic residue concentrations found in tissues (Phillips and
Segar, 1986).  Likewise, tissue lipid normalizations have been suggested to account for
the variability in tissue contaminant concentrations between individuals.

Normalization  of  sediment  contaminant  concentrations  to  TOG  and  AVS  and
normalization of tissue contaminant concentrations to the tissue  lipid concentrations
have been made to permit comparisons of toxic chemical residues in tissues between
studies,  locations,  individuals,  and  tissue  types.    These  sediment and tissue
normalizations assume the following::

      •  Contaminants partition predominantly to sediment TOC, sediment AVS, and
         organismal lipids.

      •  Contaminants partition reversibly between the sediment particles and the
         organism.

      •  Rapid steady-state kinetics of contaminants are maintained.

      •  Sediment is the only source for the bioaccumulation.

Lipid normalization has been performed  in order that individuals with differing  body fat
levels may be compared.  Again, it is essential that all analyses be conducted with the
same tissue type from the same species to ensure that scientifically and  statistically
valid comparisons may be made.  TOC/lipid normalized accumulation factors (AF) have
also been used to predict tissue residue concentrations (Ferraro et al., 1990; Lake et al.,
1987).

Threefold differences in lipid concentration may result as a consequence of various lipid
analysis  techniques.   Development  of protocol  standardization or intercalibration
between lipid techniques is required  before  chemical residues in tissues  can be
compared  between studies,  Microtechniques have been developed for analyses of
lipids from single individuals (Gardner et al.,  1985). Finally, the decision to normalize for
TOC, AVS, and  percent  Ispid will depend on monitoring  program  objectives.  For
example, if the monitoring objective is to identify "hot spots"—i.e., those areas where
high body burdens present a risk to human and ecological  health—normalization may
not be appropriate.  If the objective  is to  identify temporal trends in bioaccumulation
rates, however, normalization may be justified.

Selection of Sampling Period

The timing of sampling  should  be based on biological  cycles that  influence an
organism's susceptibility to bioaccumulation.  For example, for crustaceans, just after
molting and before hardening of the integument occurs, restricting its permeability, there
is a significant increase in potential  for bioaccumulation  of toxic  contaminants.  The
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Methods
frequency of sampling should be related to the  expected rate of change in  tissue
concentrations of contaminants. A consistent sampling period is recommended in order
that spatial and temporal comparisons may be conducted.

For many aquatic vertebrate and invertebrate organisms, the reproductive cycle exerts a
major influence  on tissue concentrations of many contaminants, especially lipophilic
compounds  (Phillips, 1980). For many species, lipophilic contaminants are transferred
from the muscle  and liver to the eggs as lipids are mobilized and transported to the egg
during oogenesis (Spies  et al., 1988; Gardner et al., 1985).  There is evidence  that
lipophilic contaminants have deleterious effects on the developing egg and/or the  larvae
(Spies et al., 1988; Hansen et al., 1985; Niimi, 1983).  Transfer of contaminants from
adult to egg and  its  potential impacts on future fisheries recruitment and fisheries
production are under investigation.

It  is  recommended  that  target  species  be  sampled when  tissue  contaminant
concentrations are  expected to be at their highest levels in order to evaluate worst-case
scenarios. For those organisms where the body is consumed whole, contaminant levels
are usually at their  highest at or just before spawning. For organisms where the muscle
tissue is consumed, contaminants in muscle tissue usually reach a peak well before
spawning.

4.10.3   Analytical Methods Considerations

Factors to be considered  during the choice of an appropriate analytical method include
the parameters  of interest, desired detection limits, sample size  requirements or
restrictions, methods of preservation, technical and practical holding times, and matrix
interferences.  Several USEPA documents (1986b, 1987c, 1990c) discuss the common
analytical problems encountered during monitoring analyses of tissue samples.

Chemical Residue Analyses

Several factors  determine  achievable detection  limits  for  a specific contaminant,
regardless of analytical procedure. These factors include:

      •  Size of the tissue sample available for analysis.  In general, the more tissue
         available  for analysis, the better the detection levels that can be achieved; a
         minimum of 30 g (wet weight) is usually considered adequate (USEPA,
         1987c);

      •  Presence of interfering substances;

      •  Range  of pollutants to be analyzed—an optimal method may be developed
         without regard to potential effects on other parameters;

      •  Level of confirmation—qualitative vs. quantitative analyses; and
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                                            Section 403 Procedural and Monitoring Guidance
      •   Level of pollutant found in the field and in analytical blanks.

Selection of appropriate methods ought to be based on a trade-off between full-scan
analyses, which are  economical  but  cannot provide  optimal  sensitivity for  some
compounds, and alternative methods that are more sensitive for specific compounds but
can result in higher analytical costs. A list of analytical techniques is presented in Table
4-27.

The Computerized Risk and Bioaccumulation System (CRABS, Version 1.0) is an expert
system   designed  to  predict tissue residues of 15  neutral  organic  pollutants  in
sediment-dwelling  organisms  and  the  human   cancer risk from  consumption  of
contaminated  shellfish  (USEPA,  1990c).   Thermodynamic  partitioning,  first-order
kinetics,  or toxicokinetic models are used to predict  bioaccumulation  from bedded
sediment.  Steady-state tissue residues  are predicted from any of the models, whereas
the two kinetic models are used to predict either non-steady-state uptake or elimination.
The lifetime human cancer  risk is then predicted for the consumption of clams and other
nonmobile sediment-dwelling organisms containing the predicted  or measured tissue
residue.  The cancer risk is predicted for a single pollutant from a single-species diet.
Shellfish consumption rates can be estimated if site-specific rates are available.
            Table 4-27. List of Analytical Techniques (USEPA, 1986b)

METALS/METALLOIDS

      •  Atomic Absorption Spectrophotometry (AA)             USEPA 7000 series methods
            flame
            graphite furnace (GFAA)
            cold vapor
            gaseous hydride (HYDAA)
USEPA method 7470
USEPA methods 7060 and 7740
      •  Inductively Coupled Plasma Emission Spectrometry (ICP)  USEPA method 6010

ORGANICS

      •  Gas Chromatography (GC)
            with electron capture detection (GC/ECD)
            with mass spectrometry (GC/MS)	
USEPA method 8080
USEPA methods 8240 and 8270
SOURCE: USEPA, 1986b.
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Methods
This program is  now available for distribution.  All inquiries should be directed to the
Narragansett,  Rhode  Island,  Environmental  Research  Laboratory  (Hatfield Marine
Science Center, Newport, OR 97365, 503/867-4042).

Tissue residue data necessary for the evaluation of dredged materials and for ecological
and human risk  assessments are most directly derived using bedded sediments (i.e.,
deposited rather than suspended sediments) in bioaccumulation tests (USEPA, 1989d).
Sediments are the largest receiving grounds for toxics known to bind with particles, such
as organics with high octanol-water partitioning coefficients (e.g., PCBs  and DDT) and
many heavy metals. Previous techniques have varied to meet specific requirements for
the task at hand, and comparability of results is questionable.   USEPA  (1989d)
standardizes  an approach  for  conducting  sediment  bioaccumulation  tests  using
sediment-ingesting organisms  exposed to bedded marine sediments.   Guidance  is
presented for "routine" testing in the laboratory and has  not been tailored for specific
regulations or geographic locations.  Data can be generated by the 28-day test that is
applicable for quantitative ecological and human health risk assessments.  It should be
noted that this is a "living" document subject to revisions warranted by experience.

Metals and Metalloids

Nitric acid/perchloric acid digestions  should be  considered  for analysis of tissue
samples. Perchloric acid is especially useful for the dissolution of fat.

Trace element analyses by  inductively coupled plasma  emission spectrometry  (ICP)
allow for several elements to be  measured simultaneously.  However, the detection
limits of ICP are generally not as sensitive as those achieved by graphite furnace atomic
absorption spectrophotometry (GFAA).

The combination of atomic absorption spectrophotometry techniques (AA) and ICP is
the recommended  analytical method for detection of metals and metalloids since no
technique is best for all elements.  Cold vapor AA analysis is the only  recommended
technique for mercury.

GFAA is more sensitive than  flame AA,  but  is more subject to matrix and  spectral
influences. GFAA requires particular caution with regard to laboratory contamination. In
either case, the  concentration of each element is determined by a separate analysis,
making the analysis of a large  number of contaminant metals both labor-intensive and
relatively expensive compared to ICP.

Semivolatile Organic Compounds

Analysis of semivolatile organic compounds involves a solvent extraction of the sample,
cleanup  of the  characteristically complex  extract,  and gas  chromatography  (GC)
analysis and  quantification.  There  are two gas chromatography/mass spectrometry
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                                           Section 403 Procedural and Monitoring Guidance
 (GC/MS)  options  for  detecting extractable  organic  compounds:  internal  standard
 technique and isotope  dilution.   Isotope  dilution is  recommended  because reliable
 recovery corrections can be made for each analyte with a labeled analog or a chemically
 similar analog (Tetra Tech, 1986).

 The identification of pesticides and PCB can be made by gas chromatography/electron
 capture detection (GC/ECD) analysis.   GC/ECD provides greater sensitivity relative to
 GC/MS;  however,  GC/ECD does  not  provide  positive  compound  identification.
 Confirmation of pesticides and PCBs on an alternative  GC/ECD column or preferably by
 GC/MS, when sufficient concentrations occur, is recommended for reliable results (Tetra
 Tech, 1985).  All other organic compound groups  are recommended for analysis by
 GC/MS (Tetra Tech, 1985).

 Volatile Organic Compounds

 The purge-and-trap  GC/MS  technique is employed for  detecting volatile  organic
 compounds  in water. A successful variation for detection of volatile organic residues in
 tissues involves a device that vaporizes volatile organic compounds from the tissue
 sample under vacuum and  then condenses the volatiles in a super-cooled trap (Hiatt,
 1981). The  trap is then transferred to a purge-and-trap device, where it is treated as a
 water sample. The isotope  dilution option is recommended because it provides reliable
 recovery data for each analyte (Tetra Tech, 1986).

 4.10.4   QA/QC Considerations

 Analysis of blanks should be conducted to demonstrate freedom from contamination.  At
 least one method blank must  be included with each batch of samples and  must
 constitute at least 5 percent of all samples analyzed.

 Spike recovery analyses are  required  to assess  method performance on the sample
 matrix. This method serves as an indication of analytical accuracy, but not necessarily
 of extraction efficiency.

 Replicates must be  analyzed to monitor  the precision  of laboratory analyses.   A
 minimum of 5 percent of the analyses should be laboratory replicates.  Triplicates should
 be performed with each sample batch over 40 samples.

 Laboratory performance and calibration  should be verified at the beginning and end of
 each 12-hr shift during which analyses are performed.

 Reports delineating the  essential elements of the bioaccumulation component of  the
 program should be included with the quantitative QA/QC analyses. It is recommended
that these reports be recorded  and stored in a data base for future reference.
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Methods
4.10.5   Statistical Design Considerations

Composite Sampling

Composite tissue  sampling consists  of  mixing  tissue samples from  two or more
individual organisms, typically of a single species, collected at a particular location and
time period.  The analysis of a composite sample provides an estimate of an average
tissue concentration for the individual  organisms  composing  the composite sample.
Advantages of the composite sampling strategy are :

      •  It provides a cost-effective strategy when individual chemical analyses are
         expensive.

      •  It provides a means to analyze bioaccumulation when the tissue mass of an
         individual is insufficient for the analytical protocol.

      •  It results in a more efficient estimate of the mean at specified sampling
         locations.

Because of the  reduced sample variance, composite sampling results in a considerable
increase in statistical power.  If  the primary objective of a monitoring  program is to
determine differences  in contaminant  tissue among  sampling locations,  composite
sampling is an appropriate strategy.

Composite sampling is not recommended if the objective of the monitoring program is to
determine compliance with  specified tissue contaminant concentration limits since this
sampling method does not detect the true range of tissue contaminant concentrations in
the population.  Special considerations related to composite sampling  include :

      •  The range and the variance of the population of individual samples cannot be
         directly estimated.

      •  If species are mixed, tissue composites are likely to be composed of different
         proportions of species and numbers of individuals, confounding whether
         patterns of tissue residue concentrations are due to differences in locations or
         to interspecific differences in bioaccumulation.

Space-bulking consists of sampling of individual organisms from several locations and
combining tissue samples into one or more composite samples.  Time-bulking involves
taking multiple samples over time from a single location and compositing these samples.
The use of space- and/or time-bulking strategies should be carefully considered since
significant information concerning spatial and temporal heterogeneity may be lost. The
adoption of composite strategies will depend on the objective of individual  monitoring
programs.
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                                           Section 403 Procedural and Monitoring Guidance
Statistical Power

Tetra Tech (1987) demonstrated that the statistical power increases with the increase of
the number of individuals in each replicate composite sample.  However, a diminished
return of statistical  power exists  with the addition of successive individuals to each
composite.  For composites of greater than 10  individuals, the increase of power is
negligible given typical levels of data variability.

For moderate levels of variability in chemical residue data, 6 to 10 individuals composing
each of 5 replicate composites should be  adequate to detect a treatment difference
equal to 100 percent of the  overall mean among treatments (Tetra Tech, 1987).  For
analyses of individuals, a minimum of five organisms at each site is recommended.

To improve the power of a statistical test, while keeping the significance level constant,
the sample size should be increased.  Because of constraints in cost and time, however,
this option may not be available.  Power analyses have shown that for a fixed level of
sampling effort, a monitoring program's power is generally increased by collecting more
replicates  at fewer locations.  The  number and  distribution of sampling locations
required to evaluate the effect of a discharge will depend on the volume and transport of
the effluent.

4.10.6   Use of Data

Results of the bioaccumulation analyses can be used to :

      •  Establish spatial and temporal trends in the bioaccumulation of toxicants of
         selected marine fish and macroinvertebrates;

      •  Identify existing arid potential problem areas for fish and macroinvertebrate
         contamination; and

      •  Supply data that can be used to calculate the human health risk of
         consuming marine fish and shellfish.

4.10.7   Summary

Rationale

      •  Monitoring the accumulation of chemical residues in tissues of marine
         organisms will provide information essential in relating the presence of
         selected contaminants in marine waters and sediment to their transfer and
         accumulation in marine organisms.
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 Methods
Monitoring Design Considerations

      •  Target species should possess the following characteristics :

         -   high bioaccumulation potential for selected contaminants of concern;
         -   weak or absent metabolic regulation of selected contaminants;
         -   abundant enough, temporally and spatially, to allow for adequate
             sampling;
         -   large enough to provide adequate amounts of tissue for analysis;
         -   sessile or sedentary in nature to ensure bioaccumulation is representative
             of the study area; and
         -   easily collected.

      •  Caged indicator species

         -   Method allows for control of initial temporal and spatial variation of
             individuals and/or biomass.
         -   Method allows for the use of specific age, size, and/or genetic stocks.

      •  Indigenous species

         -   Results obtained will relate directly to those species that may be impacted.

      •  Target tissues

         -   Fish liver or macroinvertebrate hepatopancreas analyses may be used to
             estimate the range of contaminants being assimilated.
         -   Muscle tissue analyses are appropriate for human exposure assessments
             and quantitative health risk determinations.
         -   Whole-body analyses should to be conducted when predators consume
             the whole body of the target organism.

      •  TOC/Lipid Normalization

         -   Allow comparisons of chemical residue concentrations in tissues between
             locations,  individuals, and tissue type.
         -   Limitations in use due to differences in lipid analysis techniques.
         Time of Sampling

         -   Timing of sampling should be based on biological cycles that influence an
            organism's susceptibility to bioaccumulate.
         -   It is recommended that target species be sampled when tissue
            contamination concentrations are expected to be at their highest level.
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                                          Section 403 Procedural and Monitoring Guidance
Analytical Methods Considerations

      •  Metals/Metalloids

         -   A combination of AA and IGP is recommended for the detection of metals
            and metalloids.
         -   Cold vapor AA is the recommended protocol for mercury detection.

      •  Organics

         -   GC/MS in conjunction with isotope dilution is recommended for the
            detection of semi volatile organic compounds.
         -   Super-cooled trap, in conjunction with a purge-and-trap device, is
            recommended for the detection of volatile organics.

QA/QC Considerations

      •  Blank, spike recovery, and replicate analyses are recommended quality
         control checks.

      •  Reports delineating the essential elements of the bioaccumulation
         component of the program should be included with the quantitative QA/QC
         analyses.

Statistical  Design  Considerations

      •  Compositing tissue sampling consists of mixing tissue samples from two or
         more individual organisms collected at a particular location and time period.

      •  Space-bulking (combining composites from several locations) and
         time-bulking (combining several composites over time from one location)
         strategies should be used judiciously because information concerning spatial
         and temporal heterogeneity may be lost.

      •  Six individuals composing each of five replicate composites should be
         adequate to detect a treatment difference equal to 100 percent of the overall
         mean among treatments.

Use of Data

      •  Establish spatial and temporal trends in the bioaccumulation of toxicants of
         selected marine fish and macroinvertebrates.

      •  Identify existing arid potential problem areas for fish and macroinvertebrate
         contamination.

      •  Supply data that can be used to calculate the human  health risk of
         consuming marine fish and shellfish.
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Methods
4.11      PATHOGENS

Monitoring for pathogenic microorganisms is currently conducted by State environmental
and human health agencies in shellfish harvesting areas and bathing beaches.  National
Pollutant Discharge Elimination System (NPDES) pathogen  monitoring programs may
be under way at selected locations both to assess the condition of water in the vicinity of
discharges and surrounding areas and to assess relative pathogen contributions from
these permitted effluent discharges.

The National Shellfish Sanitation Program (NSSP) has been established as a joint effort
involving the Food and  Drug Administration  (FDA),  State agencies, and the shellfish
industry to set forth guidelines for the management of State shellfish programs.  As part
of the NSSP, the Food and Drug Administration (FDA) provides technical assistance to
States for studying specific pollution  problems, provides data to establish closure levels
for shellfish  harvesting, conducts applied research on various contaminants to assist in
developing standards and criteria, and evaluates the  effectiveness  of State shellfish
sanitary control programs. In addition, since 1966, data have been compiled periodically
by FDA  and the  National  Oceanic  and Atmospheric Administration (NOAA) on the
classification by States  of coastal and estuarine waters with regard to suitability  for
shellfishing activities.  In addition to classifying their waters as to their  suitability  as
shellfish  harvesting areas,  States also  issue beach closures.   These closures are
typically based on water quality criteria developed by the Federal government.

4.11.1     Rationale

Monitoring of pathogens  in the marine environment can be used in the CWA section 403
point source discharge program to address the following ocean discharge guideline:

      •   Potential direct or indirect impacts on human health.

Human  pathogens  found  in  the  marine  environment include  viruses,  bacteria,
protozoans,  helminth and parasites.  In the United States, viruses and bacteria are the
most important pathogens,  in terms  of both the  number of organisms released to the
environment and the severity of the diseases they cause (OTA, 1987).

Humans can be exposed to pathogens by direct contact with contaminated waters (e.g.,
swimming, surfing, diving) or indirectly through  ingestion of  contaminated food (e.g.,
molluscan shellfish). The preponderance of evidence indicates that the etiologic agents
for waterborne  outbreaks of acute  gastroenteritis (AGI)  are the  Norwalk-like  viruses
(Kaplan et al. 1982).  Hepatitis A has been  linked to the consumption  of raw or partially
cooked molluscan shellfish (Feingold, 1973; CDC, 1979; Ohara et al., 1983).  Bacteria
responsible for typhoid and cholera are known to be water- and seafood-borne. These
pathogens can  enter  the marine environment through the discharge of raw sewage,
wastewater effluent from sewage treatment plants, failing septic tanks, and the dumping
of sewage sludge (OTA,  1987).  Monitoring of human pathogens provides information
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                                          Section 403 Procedural and Monitoring Guidance
essential in relating the  presence of pathogens in marine waters and shellfish to
outbreaks of disease.   Monitoring data may be used to identify potential sources of
pathogens. The assessment of pathogen contamination should be a component of a
monitoring  program where pathogens may  present a risk to human health and the
economic vitality of the marine environment.

4.11.2    Monitoring Design Considerations

Water Column Sampling

Bacteria are not uniformly distributed throughout the water column (Gameson, 1983);
bacterial abundances, several orders of magnitude greater than underlying waters, can
be found in a thin microlayer on the surface of the water (Hardy, 1982).  If feasible, it is
recommended that samples of this microlayer and separate samples of the underlying
waters be collected. However, standardized  methods for sampling this microlayer have
not been established.  If it is not feasible to sample both  the microlayer and underlying
waters, the "scoop" method should be used to ensure that the surface  microlayer is
sampled (USEPA, 1978).

Water samples  for bacterial analyses are frequently collected using sterilized plastic
bags (e.g., Whirl-Pak) or screw-cap,  wide-mouthed bottles.  Several depths may be
sampled  during one cast, and/or replicate samples may be collected at a particular
depth by using Kemmerer or Niskin samplers (USEPA, 1978).  Any device that collects
water samples in unsterilized  tubes should  not be  used for collecting bacteriological
samples without first obtaining data that support its use. Pumps may be used to sample
large volumes of the water column (USEPA, 1978).

Sentinel Organisms

Analyses  of  sentinel  shellfish tissue (e.g., mussels   and  oysters)  offer several
advantages:

      •  They concentrate pathogens and may be useful viral analyses since viruses
         are often present in low numbers.

      •  They provide a means of temporally integrating water quality conditions.

      •  They may be direct measures of human exposure to pathogens (i.e.,
         consumption of  contaminated food).

      •  They may be deployed and maintained in a number of diverse locales.
However, the disadvantages of sentinel organisms include:

      •  Transplanting organisms may be costly;
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Methods
         There is a possibility of losing the cage/buoy system; and

         Species may not be tolerant of all test site conditions (e.g., low salinity, high
         turbidity).
It is essential that monitoring design elements be standardized to allow for comparisons
between marine studies. The selection of a standard sentinel species appropriate to the
locality is recommended; interspecies differences would not allow comparisons of body
burdens.

4.11.3   Analytical Methods Considerations

Many  pathogens  can  be present  in  wastes,  contaminated media,  and  infected
organisms. Human pathogens are of concern in marine waters because they are:


      •  Associated with major debilitating diseases (e.g., hepatitis, cholera);

      •  Infectious at low doses;

      •  Resistant to environmental stress; and

      •  Not readily enumerated due to low numbers.


Table 4-28 provides examples of pathogenic organisms known to cause adverse human
health effects. The alternative to  directly identifying pathogens of concern has been the
enumeration  of bacteria,  which are indicators  of human waste contamination or
indicators of human illness.

Indicators of Human Health Risks

Because of the inability to enumerate  pathogens of concern, indicators  of human
pathogen densities have been used to assess human health risks.  Indicators useful in
predicting infectious disease rates should have the following characteristics:


      •  High abundances should be consistently found in human fecal wastes.

      •  No significant extra-human fecal sources should be present.

      •  The indicators must provide temporally and spatially reliable and accurate
         appraisals of the  pathogen of concern.


Currently,  there is no consensus on which indicator organism, if  any, is specific for
human feces.
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                                                 Section 403 Procedural and Monitoring Guidance
    Table 4-28.  Microorganisms Responsible for Causing Adverse Human Health
                                         Effects3
Disease
         Pathogenic
         Organism
      Seafood
      Source
Hepatitis
Gastroenteritis
Hepatitis A virus
Non-A and non-B
hepatitis virus

Aeromonas hydrophilia
and Plesiomonas
shigelloides

Vibrio mimicus

Vibrio parahaemolyiicus
                                   Vibrio vulnificus

                                   Vibrio cholera, O group
                                   Vibrio cholera,
                                   Non-O group 1

                                   Norwalk virus
                                  Small round structured
                                  virus

                                  Campylobacterjejuni
Raw oysters
Steamed clams
Steamed and raw clams
Raw clams
Raw oysters
Oysters
Cockles
Raw molluscan shellfish

Raw molluscan shellfish
Shellfish
Raw oysters

Clams and snails
Raw oysters
Crab
Shrimp
Lobster

Raw oysters

Raw oysters
Boiled shrimp, boiled crab

Raw oysters
                                Raw oysters
                                Raw clams

                                Raw oysters
                                Raw clams
a Includes naturally occurring microorganisms as well as microorganisms associated with pollution.
SOURCE:  NOAA, 1988
                                                                                        195
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Methods
Coliform Bacteria

For decades, the concentration of coliform bacteria, either total or fecal coliform, has
been considered a reliable indicator of the presence and density of pathogens.  The use
of total coliform bacterial criteria to protect human health from disease associated with
contaminated water is widespread. Furthermore, total coliform concentrations have the
advantage of providing a basis for comparison with historical data (USEPA, 1988f).

However,  these indicators  and their corresponding water quality standards have not
been  related to incidences of disease through epidemiological studies.  Controversy
exists on  the efficacy of coliform bacteria to predict the  presence and inactivation  of
other  types of pathogens (Pederson,  1980).  In fact, recent studies indicate that fecal
coliform bacteria may not be a reliable indicator for predicting the  risks associated with
direct exposures to pathogens in the  marine environment (Cabelli et al.,  1979,  1982).
Fecal conforms are not pathogenic  and  are less  resistant to  environmental  stress
compared to many  pathogens (Borrego et  al., 1983).   Furthermore, fecal  coliform
bacteria are not specific to mammalian fecal pollution.   The lack of this specificity
prompted the development of methods for enumerating fecal coliform bacteria specific to
mammalian fecal pollution (e.g., Escherichia coli). However, with respect to recreational
water quality criteria, EPA has not recommended the use of E. coli tor marine waters.

Enterococci

Enterococci  are streptococcus bacteria  indigenous  to the intestines  of warm-blooded
animals.   Cabelli  et al. (1983)  found that  the  densities of enterococci were  highly
correlated with the  incidence of gastrointestinal (Gl) symptoms among  swimmers;
reported swimming-associated Gl symptoms were poorly correlated with fecal coliform
densities.  Enterococci also have the following advantageous characteristics:
         They are tolerant to high salinity and are of particular value in the analysis of
         marine waters.

         Taxonomic identifications are relatively simple and can reveal the origin of
         mammalian pollution (e.g., humans, livestock).

         Genetic fingerprinting techniques that can link these bacteria in the
         environment to specific sources of contamination have been developed.
State-of-the-art genetic fingerprinting techniques are very costly and require further
study in order to assess their reliability. EPA has adopted enterococci as an indicator of
microbiological water quality for recreational marine waters.
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                                           Section 403 Procedural and Monitoring Gurafance
Viruses
Viruses are being recognized as major etiologic agents for many outbreaks of human
illness as a result of swimming in contaminated water and consuming  contaminated
seafood. Viruses are excreted only by infected individuals; they are not normal flora in
the intestinal  tract.   Traditional indicators of microbial  contaminants appear to be
inadequate for predicting human health risks associated with both consuming molluscan
shellfish and swimming in waters that contain sewage-associated viruses.  However,
examination of water for enteric viruses is not recommended at this time, except in
special  circumstances,  because  of  methodology  limitations.  Even state-of-the-art
methods for concentrating viruses from water are still being researched and continue to
be modified and improved.   None of the available virus detection methods have been
tested adequately with  representatives  from  all of the virus groups of  public health
importance.  In addition, some of these methods require  expensive equipment and
materials for sample processing and all virus assay and identification procedures require
expensive cell culture and related virology laboratory facilities.

Laboratory Techniques

It  should  also be  noted  that no  single  procedure   is  adequate to  isolate all
microorganisms from water, and the presence of one microorganism does not signify the
presence or absence of any other (Table 4-29). A more detailed description of analytical
methods for a number of pathogens  is  presented in  Standard  Methods for the
Examination of Water and Wastewater (APHA, 1989).

Fecal Bacteria

Two standard  methods are presented  here  for the detection of fecal  bacteria:  the
membrane filter procedure and the multiple-tube fermentation procedure.

The membrane filter (MF) technique involves sample filtration followed by direct plating
for detection and enumeration of coliform bacterial densities. The MF technique can be
used  to  test  relatively large volumes of samples and yields numerical  results more
rapidly than does the multiple-tube  procedure.  The statistical reliability  of  the  MF
technique is greater than that of the Most Probable Number (MPN) procedures (APHA,
1989).  However, the MF technique  has limitations, particularly in testing waters with
high turbidity and noncoliforrn (background) bacteria.  The MF technique can be used to
measure bacterial densities of  Escherichia coll (E.  coif)  and enterococci  in ambient
waters (USEPA, 1985e) since EPA has approved this technique for use in seawater.

The  multiple-tube  fermentation technique  involves a series of fermentation tubes
containing growth media that are inoculated with the appropriate decimal dilutions of
water (multiples and  submultiples of  10  ml_),  based  on the probable  coliform density.
Following inoculation at 35  ± 0.5°C for 24 ± 2 hr, the tubes are examined for gas or
acidic growth  (distinctive yellow color).  If no gas or acidic  growth  has formed, the
                                                                             197
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Methods
            Table 4-29.  Laboratory Procedures for Bacterial Indicators
                                          Laboratory Procedures
Test Organisms
Fecal coliform
bacteria
Water
MPN tubes using A-l
broth (APHA, 1989)
(fecal coliform
bacteria/100 mL)
Sediment
MPN tubes using A-l
broth (APHA, 1989)
(fecal coliform
bacteria/100 mL)
Tissue
MPN tubes using EC
broth (APHA, 1989)
(fecal coliform
bacteria/1 00 mL)
Fecal coliform
bacteria/E.co//

Enterococci
C. perfringensb
mTEC (DuFour et al.,
1981)(E. co/^ 00 mL)

mE (Levin et al.,  1975)a
(enterococci/100 mL)

MPN tubes using iron
milk (St. John et al.,
1982) (C. perfringensl
100 mL)
MPN tubes using iron
milk (St. John et al.,
1982) (C. perfringensl
100 mL)
MPN tubes using iron
milk (St. John et al.,
1982) (C. perfringensl
100mL)
aThis method is a tedious process. EPA Region 2 and the State of New Jersey have developed a
modified mE isolation technique.

h'wo laboratory techniques are available for C. perfringens: mCP by membrane filtration for water
(Bisson and Cabelli, 1979) and sediment (Emerson and Cabelli, 1982), and iron milk tube using MPN
techniques (St. John et al., 1982). The latter method is recommended (pending comparative data)
because the procedure is simpler and less costly.
samples are reincubated and examined again at the end of 48 ± 3 hr. Production of gas
or acidic growth  on tubes  after this  time period constitutes a positive presumptive
reaction.  These samples are then submitted to the confirmed phase of testing, in which
the culture is transferred to a fermentation tube containing brilliant green lactose bile
broth. The tubes are then inoculated for 48 ± 3 hr at 35 + 0.5°C.  Formation of gas in
any amount of time constitutes a positive confirmed phase.  The Most Probable Number
(MPN) index is then used to estimate bacterial density.   This technique is commonly
used in assessing bacterial levels in shellfish.

The MPN index is an index of the number of coliform bacteria that, most probably, would
produce the results observed in the laboratory examination (APHA, 1989).  Values for
the  MPN  index can be obtained from  standard tables  based  on the  results  of the
multiple-tube fermentation technique or from Thomas's formula (APHA, 1989).
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                                            Section 403 Procedural and Monitoring Guidance
Viruses
 Detecting viruses in marine waters requires collecting a representative water sample,
 concentrating viruses in the sample, and identifying and estimating abundances of these
 concentrated viruses. Difficulties in detecting virus abundances include:


       •   Viruses are very small (20-100 nm).

       •   Virus concentrations in waters are spatially and temporally variable, and
          typically low.

       •   Dissolved and suspended material in the sample interferes with accurate
          virus detection.
Because virus concentrations may be very low, significant volumes of water may be
required (e.g., on the order of tens to thousands of liters).  Dose-response curves are
lacking for most pathogens; however, as few as  10 to  100 bacteria are capable of
inducing disease (OTA, 1987).

Three different techniques for concentrating and enumerating viruses are presented in
Standard Methods (APHA, 1989):


       •  Adsorption to and elution from microporous filters;

       •  Aluminum hydroxide adsorption and precipitation; and

       •  Polyethylene glycol hydroextraction-dialysis.


The adsorption and elution technique pressure-filters viruses on microporous filters and
then elutes them from the filter in a small liquid volume.  Generally, two types of filters
are  available:   electronegative  and electropositive  filters.    Currently,  insufficient
documentation  on  the  efficacy of electropositive  filters exists.   Limitations  of this
technique include:


       •  The adsorbent filter may be clogged by suspended material.

       •  Dissolved colloid material may interfere by competing with viruses for
         adsorption sites on the filter.

       •  Viruses adsorbed to suspended material may be removed during suspended
         material clean-up procedures.
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Methods
Methods for recovering solids-associated viruses are found in APHA (1989). In spite of
these limitations,  the  adsorption and elution technique  remains the most promising
method for detecting viruses.

Aluminum   hydroxide   adsorption   and   precipitation   and   polyethylene   glycol
hydroextraction-dialysis are used to reconcentrate viruses in proteinaceous and organic
buffer eluates.  These  methods may be used to concentrate viruses in waters having
high virus densities (e.g., wastewaters).  They may  also be used as a second-step
concentration  procedure   following  processing  of   large  fluid  volumes  through
microporous  filters.   However,  these two techniques  are impractical  for primary
processing of large fluid volumes (APHA, 1989).

New Techniques

Traditional techniques  lack the ability to detect apparently viable, but nonculturable,
microorganisms.  Recently developed monoclonal antibody and gene probe techniques
permit  the   detection  and  enumeration   of  both  culturable  and  nonculturable
microorganisms.   Since  these  methods  have the  ability to  detect  nonculturable
organisms, they may serve as more  precise techniques for monitoring microbial water
quality. Furthermore, these new techniques will allow direct monitoring of pathogens of
concern.  The  goal of monitoring  specific  pathogens, such as  Salmonella,  Shigella,
Giardia, or Legionella, may soon be realized.

Sample Handling

Preservation and  storage of water samples can be significant sources of error. Sample
bottles must be resistant to sterilization procedures.   Samples should be refrigerated
(1-4°C) during  transport to a laboratory and analyzed within  6 hours of  collection
(USEPA, 1978).

4.11.4    QA/QC Considerations

It is recommended that sterile distilled water be transported to the field, transferred to a
sample bottle, and processed routinely as a field blank to ensure  that samples were not
contaminated during collection and transport. It is also recommended that 10 percent of
the samples be analyzed in duplicate.  Furthermore, 10 percent  of the samples should
be split and  analyzed by two or more laboratories.

Intralaboratory and interlaboratory quality control practices should be documented, and
QC reports should be available for inspection. Further recommended quality assurance
guidelines for a  microbiology laboratory are available (Inhorn,  1977; Bordner et al.,
1978) and are also discussed in APHA (1989).
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                                           Section 403 Procedural and Monitoring Guidance
4.11.5   Statistical Design Considerations
Bacterial counts often are characterized as having a skewed distribution because of
many low  values  and a few high ones  (APHA,  1989).   Application  of  parametric
statistical techniques requires the assumption of symmetrical distributions such as the
normal curve.  An approximate normal distribution can  be obtained from positively
skewed data by converting numbers to their logarithms (APHA, 1989). Accordingly, the
preferred  statistic  for  measuring  central tendency  of microbiological  data is the
geometric mean.

Consideration  of statistical strategies will mitigate the high costs of collecting and
processing samples.   Power-cost analyses are necessary in  selecting appropriate
sample/replicate number, sample  location, and  sampling frequency (Ferraro et al.,
1989).

4.11.6   Use of Data

The assessment of pathogen contamination should be a component of a  monitoring
program where pathogens may present a risk to human health and the economic vitality
of an estuary.  Monitoring of human pathogens provides information essential in relating
the temporal and spatial distribution of infectious agents in marine waters and shellfish
to the epidemiology of pathogens of concern  (e.g.,  affected  human populations,
locations, and timing of the outbreak).

Furthermore, monitoring data can be used to identify discharges that may be significant
sources of pathogens  and to ensure that water quality standards are maintained.
Monitoring  data  can also be  used to verify fate  and transport, human health risk
assessment, and epidemiological modeling predictions.

4.11.7   Summary and Recommendations

Rationale

      •  The objective is to detect and describe spatial and temporal changes in
         abundances of indicators of human health pathogens.

      •  Monitoring indicators of human pathogens provides information essential  in
         relating the presence of infectious agents in marine waters and shellfish to
         the incidence of disease outbreaks and potential sources of these agents.
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Methods
Monitoring Design Considerations

      •  If feasible, samples of the surface microlayer should be collected separately
         from samples of underlying waters. If not feasible, samples containing both
         underlying and microlayer waters should be collected.  It is highly
         recommended that consistent types of sampling protocols be implemented to
         allow for comparisons between studies.

      •  Analysis of tissues of sentinel organisms (e.g., mussels and oysters) confers
         the following advantages:

         -   such organisms concentrate pathogens;
         -   such organisms provide a means of temporally integrating water quality
             conditions; and
         -   such organisms may be deployed and maintained in a number of locales.

Analytical Methods Considerations

      •   Indicators of human pathogens should have the following characteristics:

         -   should be consistently found in high abundance in human fecal wastes;
         -   should not have significant extra-human fecal sources; and
         -   must provide temporally and spatially reliable and accurate appraisals of
             the pathogen of concern.

      •   Fecal coliform bacteria densities.

         -   May not be a reliable indicator for predicting the risk associated with direct
             exposures to pathogens.
         -   This measure provides a means to compare current data to historical data.
         -   Laboratory analyses include membrane filter and multiple-tube
             fermentation techniques.

      •   Enterococci densities

          -   Densities are highly correlated with the incidence of gastrointestinal
             symptoms.
          -   Taxonomic identifications are relatively simple and can reveal the kinds of
             mammalian pollution.
          -   State-of-the-art genetic fingerprinting techniques can link bacteria in the
             marine environment to specific sources; however, these techniques are
             very costly and require further study to assess their reliability.
          -   Laboratory analyses include membrane filter technique.

      •   Virus densities

          -   Viruses are recognized as major etiologic agents for many outbreaks of
             human illness.
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                                           Section 403 Procedural and Monitoring Guidance
         -   Examination of water samples for enteric viruses is not recommended
             because of methodology limitations.
         -   Laboratory analyses include absorption to an elution from microporous
             filters, aluminum hydroxide absorption-precipitation, and polyethylene
             glycol hydroextraction-dialysis.

      •  Monoclonal antibodies and gene probes show promise in detecting and
         enumerating both culturable and nonculturable microorganisms.

QA/QC Considerations

      •  Sterile distilled water should be transported and transferred to sample bottles
         as field blanks to assess contamination during collection  and transport.

      •  Ten percent of the samples should be analyzed in duplicate.

      •  Ten percent of the samples should be split and analyzed at two or more
         laboratories.

Statistical Design Considerations

      «  The preferred statistic for measuring central tendency of  microbiological data
         is the geometric mean.

Use of Data

      •  Provide essential information to assess threats to human health;

      •  Establish temporal and spatial trends in pathogen densities and identify
         potential relationships between the presence of the indicator and incidence of
         human illness; and

      •  Provide information that can be used to verify fate and transport, human
         health risk, and epidemiology models.
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Methods
4.12    EFFLUENT CHARACTERIZATION

"End-of-pipe" effluent characterizations  based on  laboratory studies can  be used to
predict biological impacts of an effluent prior to a discharge.  The  Technical Support
Document  for Water Quality-based Toxics Control (USEPA,  1991c) presents (among
other things) an integrated approach for ensuring protection of aquatic life and human
health from  impacts caused  by  the release of toxics to surface waters.   For the
protection  of aquatic life,  the integrated  strategy involves the use of three control
approaches: the chemical-specific control approach, the whole-effluent toxicity control
approach,  and  the biological  criteria/bioassessment and biosurvey approach.  For the
protection of human  health, technical constraints do not yet allow for full reliance on an
integrated  strategy,  and thus primarily chemical-specific assessments  and control
techniques should be employed.  Table 4-30 contains a list of terms used in effluent
characterization.

Pollutant-specific criteria  developed pursuant  to CWA  section  304(a)(1)  present
scientific data and guidance on the environmental effects of pollutants that reflect the
latest EPA recommendations on acceptable limits for aquatic life  and  human health
protection.   These limitations  are generally  developed  from  laboratory-derived,
biologically-based numeric water quality criteria adopted within a State's water quality
standards.  Water quality criteria are adopted by a State for the protection of designated
uses of the receiving water. Criteria can be specific numeric limits or, in the absence of
specific numeric criteria for a chemical, biological, or physical parameter, can be based
on narrative criteria. EPA publishes chemical-specific  water quality criteria that may
provide the basis on which States adopt their criteria (USEPA, 1986-1988).   EPA has
published 25 chemical-specific saltwater aquatic life criteria (acute and chronic).

When an effluent's constituents are not completely known or when a complex mixture of
potentially  additive,  antagonistic, or synergistic toxic pollutants  is discharged,  a
whole-effluent  toxicity  limitation  can  be. implemented.   The whole-effluent toxicity
limitation can also be appropriate when more than one discharger is located in a specific
area and the potential exists for  effluent mixing and additive toxic effects, and when a
pollutant-specific evaluation is impractical because of a lack of information about the
toxic effects of a pollutant. Toxicity  evaluations such as these can be used as part of
403 monitoring.

4.12.1     Rationale

 Effluent characterization can  be  used in the CWA section 403 point source discharge
 program to address  the following  evaluation criteria:
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                                                    Section 403 Procedural and Monitoring Guidance
             Table 4-30.  List of Terms Used with Effluent Characterization
Acute
Acute-to-chronic ratio
(ACR)


Chronic
Composite sample
Effective concentration
Grab samples
A stimulus severe enough to rapidly induce an effect; in aquatic toxicity tests,
an effect observed in 96 hours or less typically is considered acute. When
referring to aquatic toxicology or human health, an acute effect is not always
measured in terms of lethality.

The ratio of the acute toxicity of an effluent or a toxicant to its chronic toxicity.
ACR is used as a factor for estimating chronic toxicity on the basis of acute
toxicity data, or the reverse.

A stimulus that lingers or continues for a relatively long period of time, often
one-tenth of the life span or more. Chronic should be considered a relative
term depending on the life span of an organism. The measurement of a
chonic effect can be reduced growth, reduced reproduction, etc., in addition
to lethality.

A single effluent sample collected over a 24-hour period, on which only one
toxicity test is performed.

A point estimate of the toxicant concentration that would cause an
observable adverse effect (such as death, immobilization, or serious
incapacitation) in a given percentage of the test organisms.

Samples collected over a very short period of time and on a relatively
infrequent basis. A separate toxicity test must be performed on each grab
sample.
Lowest-Observed-Adverse- The lowest concentration of an effluent or toxicant that results in statistically
Effect Level (LOAEL)       significant adverse health effects as observed in chronic or subchronic
                          human epidemiology studies or animal exposure.
No-Observed-Adverse-
Effect Level (NOAEL)


No-Observed-Effect
Concentration (NOEC)


Toxicity characterization

Whole-effluent toxicity
A tested dose of an effluent or a toxicant below which no adverse biological
effects are observed, as identified from chronic or subchronic human
epidemiology studies or animal exposure studies.

The highest tested concentration of an effluent or a toxicant at which no
adverse effects are observed on the aquatic test organisms at a specific time
of observation. Determined using hypothesis testing.

A determination of the specific chemicals responsible for effluent toxicity.

The aggregate toxic effect of an effluent (usually measured directly with a
toxicity test).
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Methods
      •   The quantities, composition, and potential for bioaccumulation or persistence
          of the pollutants to be discharged;

      •   The potential impact on human health through direct and indirect pathways; and

      •   Marine water quality criteria developed pursuant to section 304(a)(1).

Assessment  of these evaluation criteria  can be made through whole-effluent toxicity
testing (acute and chronic)  and analysis of the  chemical  composition of the  effluent.
This assessment would demonstrate potential impacts from  the effluent in question.

4.12.2    Monitoring Design Considerations

Determination of whether an effluent sample is typical of the wastewater may require the
collection of  a large number of samples.  Further, what constitutes a "representative"
sample is a function of the parameter of concern.  Guidelines for determining the
number and frequency of samples required to represent effluent quality are contained in
the  Handbook for Sampling and  Sample  Preservation  of Water and  Wastewater
(USEPA, 1982b).

Both quantitative  (change  in  concentration)  and  qualitative  (change  in toxicants)
variability commonly occur in effluents.   Changes  in effluent toxicity are the  result of
varying concentrations of individual toxicants, different toxicants, changing in-stream
water  quality  characteristics  (affecting  compound  toxicity),  and  analytical  and
toxicological error. Conventional parameters, BOD, TSS, and other pollutants limited in
the facility's permit will provide  an indication of the operational status of the treatment
system on the day of sampling. This information may be used in a determination as to
whether the system is operating properly,  requires repair, or needs to be upgraded.  For
industrial discharges, information on production levels and types of operating processes
may be helpful in determining the required magnitude of the monitoring program.

4.12.3    Analytical Methods Considerations

Sampling

The choice of grab or composite samples will depend on the specific discharge  situation
(e.g., plant retention time) and the  objectives of the test.   In toxicity characterization
testing, samples that are very different from one another give results that are difficult to
interpret. However, composite sampling has an averaging  effect, which tends  to dilute
toxicity peaks and may thus provide misleading results when testing for acute toxicity.
Composited samples, therefore, are more appropriate for  chronic toxicity tests where
peak toxicity of short duration is of less concern. If the toxicity of the effluent is  variable,
grab samples collected during peaks of effluent toxicity provide a measure of maximum
effect.  However, while grab sampling may provide information on maximum effluent
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                                            Section 403 Procedural and Monitoring Guidance
toxicity, it can be difficult to schedule sampling to coincide  with  peaks in toxicity.
Aeration during collection and transfer of effluents should be minimized to reduce the
loss of volatile chemicals.

Chemical-Specific Analysis

Pollutant-specific testing  for comparison  to  water quality criteria should  be done
following standard methods.  Choice of a particular method may vary depending on the
precision and accuracy required and the resources available for the study. A review of
water chemistry analytical methods is presented  in the section in  this report on water
quality and in Standard Methods for the Examination of Water and Wastewater (APHA,
1989).  However, it should  be noted that pollutant concentrations are considerably
higher in  effluent streams and the  precision required for adequate measurement is
lower.

Whole-Effluent Toxicity Analysis

Whole-effluent toxicity testing can be somewhat more complex than a pollutant-specific
approach. Upon  arrival of the sample in the laboratory, temperature, pH, hardness, and
conductivity should be measured.   Total residual  chlorine, total  ammonia,  alkalinity,
dissolved oxygen (DO), and organic carbon  measurements may also be appropriate.
These measurements  can  provide  necessary information should the toxicity of the
effluent change over time.

EPA recommends that for whole-effluent toxicity data generation the process should be
divided into three basic steps: initial dilution determination, toxicity testing procedures,
and triggers for permit limit development.  The dilution determination is an estimate of
the effluent dilution at the edge of the mixing zone and should take  any applicable State
mixing zone requirements into consideration.

Whole-effluent toxicity testing should entail both  acute and chronic testing.   An acute
toxicity test is defined as a test of usually less than 96 hours in duration in which lethality
is the measured endpoint. A chronic toxicity test is defined as a long-term test in which
sublethal effects, such as fertilization, growth, and reproduction are usually measured,
although in highly toxic effluents lethality may also result. Traditionally, chronic tests are
full-life-cycle tests or at least 30-day tests.  However, the  duration of most of the EPA
chronic toxicity tests has been  shortened to 7 days by focusing on the most sensitive
life-cycle stages.  For this reason, the EPA chronic toxicity tests are called  short-term
chronic tests.  Whole-effluent toxicity limits  can be inserted into permits as a means of
controlling pollutants when  chemical-specific criteria  have  not  been developed  or
synergistic effects are found to occur.
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 Methods
 For whole-effluent toxicity testing, EPA recommends that three species (a vertebrate, an
 invertebrate, and a plant) be tested quarterly for a minimum of 1 year.  This may be
 adjusted to require testing of only the most sensitive species. Conducting tests quarterly
 for 1 year is recommended to adequately assess the variability of toxicity observed in
 effluents.  The  use of three  of the five commonly used  marine organisms—inland
 silverside, sheephead minnow, mysid shrimp, Champia, and sea urchin—has generally
 been sufficient to measure the toxicity of any effluent for the purposes of projecting
 effluent toxicity impact and making regulatory decisions.  Specific test  methodologies
 can  be found in a variety of EPA manuals, including Methods for Aquatic Toxicity
 Identification Evaluations - Phases I, II,  and III (USEPA, 1988a, b, c), Methods for
 Measuring  the  Acute Toxicity of Effluents to Freshwater and Marine  Organisms
 (USEPA, 1985c), Short-Term Methods for Estimating the Chronic Toxicity of Effluents
 and Receiving  Waters to Marine and Estuarine Organisms  (USEPA, 1988e),  and
 Biomonitoring for Control of Toxicity in Effluent Discharges to the Marine Environment
 (USEPA, 1989b).  Numerous other sources of toxicity testing methodologies exist (see
 reference list) and may be more appropriate for the effluent of interest.

 Biological Criteria/Bioassessment

 To fully protect aquatic habitats, water quality criteria should address biological integrity.
 Biocriteria should be used as a supplement to existing chemical-specific criteria and as
 criteria where such chemical-specific criteria have not been established.  Biocriteria are
 numerical values or narrative expressions that describe the reference biological integrity
 of aquatic communities inhabiting  unimpaired waters.  The biological communities in
 these waters represent the best attainable conditions (USEPA, 1991c).

 Resident biota integrate  multiple impacts over  time and  can detect impairment  from
 known and  unknown causes.  Biocriteria can be used  to verify improvement in water
 quality in response to  regulatory efforts and detect continuing degradation  of waters.
 Numeric criteria  can  provide effective  monitoring criteria  for  inclusion  in  permits
 (USEPA, 1991).

 The  assessment of biological integrity should include measures of  the structure  and
 function  of  an  aquatic community of species  within a specified habitat.  Expert
 knowledge  of the  system  is  required  for the  selection  of  appropriate  biological
 components and measurements indices.  See Section 4.5 for  further discussion of
 benthic community structure.

 4.12.4   QA/QC Considerations

 Basic  sampling  and  laboratory  quality assurance  and  quality  control   (QA/QC)
 procedures for pollutant-specific characterization are addressed in the section on water
 chemistry. QC problems specific to effluent characterization include variability in effluent
 concentrations and toxicity response of test organisms, and changes in effluent toxicity
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                                           Section 403 Procedural and Monitoring Guidance
over time.  A quality assurance project plan (QAPj'P) should be developed and adhered
to.  The QAPjP should include quality verification, which entails a demonstration that the
proposed study plan was followed as detailed and that work carried out was properly
documented.  The  QAPjP  should increase communication  between clients, program
planners, field and  laboratory personnel, and data analysts. The QAPj'P must make
clear the specific responsibilities of each  individual.  The QC procedures involve
standardized guidelines, such as  the number of samples to be taken and the mode of
collection,  standard operating procedures  for analyses,  and spiking protocols.  QC
protocols for standard toxicity test  methods are described in USEPA (1985c).

4.12.5    Statistical Design Considerations

Statistical design of an effluent characterization  project must account for variability in
effluent and toxicity testing response.  However, the  design of any given experiment
must be weighed against the importance of the data and decisions to be based on the
data. The critical nature  of certain data will demand stringent controls, while statistical
rigor (and associated costs) can be lessened in  other experiments having less impact
(e.g., initial toxicity testing).

The choice of a statistical method to analyze toxicity test data and the interpretation of
the results of the analysis of the data from toxicity tests can be problematic because of
the inherent variability and  sometimes unavoidable anomalies in biological data.  The
assistance of a trained statistician is recommended  before selecting the method  of
analysis and interpretation  of the results.   The data should be plotted  to help  detect
problems and unsuspected  trends or  patterns in  the  responses and  to aid  in
interpretation of the results. The analysis of the data is dependent upon  the number of
replicates and  the distribution and homogeneity of the data.  A variety of  statistical
methods for the analysis of toxicity data (including  Dunnett's procedure, Bonferroni's
t-test, Steel's many-one rank test,  the Wilcoson Rank Sum test, and the Probit analysis)
are presented in USEPA (1988e).

4.12.6    Use of Data

Pollutant-specific effluent characterization can be used for comparison to State water
quality standards.  A chronic criterion is a level  at which aquatic organisms and their
uses  should not be affected unacceptably if the 4-day average  concentration  of the
pollutant is not exceeded more than once every 3 years on average.  An  acute criterion
is a limit at which the pollutant concentration should not be exceeded more than once
every 3 years on average. Care should be taken  when applying these criteria  to assess
potential impacts on locally important species that are very sensitive.

Whole-effluent toxicity testing data (both acute and chronic) may be  used to screen an
effluent for potential unreasonable degradation  of  the environment.   Whole-effluent
toxicity  is a useful parameter for assessing and  protecting  against impacts  on water
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Methods
quality and designated uses caused by the aggregate toxic effect of the discharge.
Thus, toxicity itself is used as the effluent parameter, and the toxicants creating the
toxicity need not be specifically identified or controlled unless a Toxicity Reduction
Evaluation (TRE) is required to ensure compliance with a toxicity limit in the permit.

If a threshold of toxicity is desired, then the results of whole-effluent toxicity testing may
be  used  to  derive  the  No-Observed-Effect  Concentration   (NOEC)  and  the
Lowest-Observed-Effect Concentration (LOEC). Whole-effluent toxicity testing can also
produce a concentration-dependent result of some amount of adverse effect called the
Effective Concentration (EC).  For example,  ECso  is the effluent concentration that
would affect 50 percent of the organisms tested. Interpretation of EC values, therefore,
requires the judgment of a toxicologist.

4.12.7   Summary and Recommendations

Rationale

      •  Effluent characterization may be used to assess potential impacts, both acute
         and chronic, of the effluent on the surrounding biological communities without
         interference from other sources.

Monitoring Design Considerations

      •  Effluent variability, including potential changes in contaminants and
         contaminant concentrations, should be considered when designing a
         monitoring plan.

Analytical Methods Considerations

      •  Grab or composite samples should be used depending on the objectives and
         type of test performed.

      •  Chemical-specific testing can be performed to characterize toxicity and
         ensure compliance with water-quality standards.

      •  Both acute and chronic tests can be used to assess whole-effluent toxicity.

QA/QC Considerations

      •  See section on water chemistry.

      •  QAPjP should be developed, adhered to, and documented.
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                                            Section 403 Procedural and Monitoring Guidance


Statistical Design Considerations

      •   Effluent variability and toxicity testing response must be accounted for in the
          monitoring plan.

Use of Data

      •   Chronic and acute toxicity levels can be set in permits.

      •   Toxicity Reduction Evaluations (TREs) can be required.

      •   Threshold and effective concentrations can be established to predict
          ecological risk.
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Methods
4.13     MESOCOSMS AND MICROCOSMS

The use of mesocosms and microcosms for the assessment of ecological impacts from
marine dischargers is considered to be premature. This monitoring approach  is the
focus of research and holds promise for future use.  It has been used to some extent by
EPA's Office of Pesticides to predict impacts from the use of pesticides.

Microcosms and mesocosms, on a small to medium size scale, respectively, attempt to
simulate the complex nature of natural environments. An effort is made to determine the
response  of  an  ecosystem to  perturbation  through  an accurate simulation of the
physical, chemical, and biological characteristics of an ecosystem and the  biological
interactions of the organisms in the ecosystem.  While there is no  absolute separation
between  a mesocosm  and a microcosm, in  practice  most mesocosms are outdoors
where temperature and light intensity  are  ambient for the parent community.   Most
mesocosm researchers try to realistically  scale for the factors they think  are  most
important in controlling  ecological relationships.  Most microcosms are incubated in the
laboratory and are subject to greater environmental control. Microcosms are usually
smaller, and more replicates are used.

4.13.1    Rationale

Of the 10 guidelines used  to determine unreasonable degradation or no irreparable
harm, several can be  addressed through the  use of mesocosms and microcosms
including:

      •  The potential  transport of such pollutants by biological, physical or chemical
         processes and

      •  The composition and vulnerability of potentially exposed biological
         communities.

Mesocosms  and  microcosms can be  used to gain  a realistic  characterization of
ecosystem-level impacts of marine discharges subject to section 403. With mesocosms
and  microcosms,  the   responses  of  complete  ecosystems to  pollutants  can  be
investigated.   Other advantages include the possibility of  experimentation, at near
natural conditions, with  addition of isotopic tracers to an ecosystem and the opportunity
to test numerical transport models under near natural conditions.

If  insufficient  information  exists to determine  whether a discharge will  result in
unreasonable degradation of the marine environment, EPA must make a determination
as to whether a  discharge will cause irreparable  harm during the period in  which
monitoring is undertaken.  The use of microcosms  allows for the incorporation of
ecosystem recovery (a  critical factor in making a determination of irreparable harm) as
an endpoint for future ecological risk assessment (Perez et al., 1990).
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                                          Section 403 Procedural and Monitoring Quittance
4.13.2    Monitoring Design Considerations

A microcosm should be designed to represent a miniature ecosystem  that responds
quickly to perturbation.   The  emphasis is on ecosystem-level  properties, although
species-level attributes such as fecundity and mortality can also be monitored if desired.
Most generic microcosms do not simulate a site-specific natural system, but function as
very generalized simulations of a large class of ecosystems.   However, the use of
undisturbed, natural aquatic, and benthic communities (through the use of a sediment
core) in a single system allows for the simulation of a natural system (USEPA, 1983b).
The design of the microcosm allows the system to be defined in terms of its physical and
temporal  boundaries,  its light and  temperature  regime,  its water composition, the
turbulence and turnover rate, the ratio of benthic surface area to seawater volume, the
sediment characteristics, and the water flow rate over the sediment surface.

The results of microcosm or mesocosm experiments are measured by the ecological
effects on the biota of the  system.  When selecting biota to include in the test, two
factors should be considered, both of which involve selection of the benthic component
of the system. If the natural system has more than one distinct benthic community, then
those organisms which are directly linked  to human consumption or those which are
important to the  economics and aesthetics of the area should  be  considered for
experimental use. Moreover, if some of the benthic communities contain species known
to be more  sensitive to environmental contaminants than others, these communities
should also be considered.

4.13.3   Analytical Methods Considerations

Microcosms for which ecosystem effects protocols have  been  developed are small,
static,  open ecosystems.    Kenneth Perez and  other  researchers at  the  EPA
Environmental  Research Laboratory in  Narragansett, Rhode Island,  developed an
experimental marine microcosm  test protocol that employs  a time frame of 30 days
(USEPA, 1983b).  The methodology was published in the Federal Register (volume 52,
number  187, pages 36352-36360)  for use  in  developing  site-specific data on the
chemical fate and ecological effects of chemical substances subject to regulation under
the Toxic Substances Control Act (TSCA) and could  be adapted to  the  403 ocean
discharge program. This protocol attempts to couple undisturbed pelagic  and benthic
communities within a single system,  the physical and chemical conditions of which are
equated  to those in the  natural  system  being simulated.  The protocol and  support
document (USEPA, 1990e)  describes the steps necessary to develop the experimental
microcosm (tanks, paddles,  benthic cylinders, pumps, and pump air supply) and support
equipment (room, water bath, light and turbulence fixtures, test compartments, and an
air  evacuation  system).   A diver-collected  sediment core is  used for  the  benthic
subsystem, and the test water  is collected by a nondestructive method and exchanged
at least three times a week, coinciding with biological and chemical sample collection.
Water flow, turbulence,  light  intensity, temperature, and  simulated tidal  flow are all
controlled to simulate the natural system for the 30-day time period.
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 Methods
 Other researchers contend that to study ecosystem-level effects it is essential that the
 microcosms function (1) as homeostatic, self-sustaining ecosystems capable of existing
 through at least a year and  (2) independent of outside subsidies except for light and
 replacement of evaporated water.  Each microcosm is considered a unique ecosystem
 and intraexperiment  replication among  microcosms  is  enhanced  by using defined
 chemical  media  and standard  physical conditions  (light,  temperature,  day length).
 Inoculation with well-developed  interactive  couplings of the organisms is essential to
 achieve these goals.  Selection of the exact species to be used should be based on the
 specific objectives of the study.

 During microcosm studies, effluent to  be tested should  be added on  a  gradient.  A
 typical experimental design might consist of six replicates in each of four treatment
 groups: a control, a single addition  of a low concentration,  a  single addition of a high
 concentration, and repeated additions of low concentrations.  Example protocol steps
 and the variables monitored for this type of generic microcosm are described in Taub
 (1984).

 The physical  structure  of  a mesocosm  system  determines  the ability  of water,
 organisms, and pollutants to move from compartment to compartment within the system.
 Coastal ecosystems and the fate and effects of pollutants within these systems are
 strongly  affected by vertical  mixing and the  interaction of  planktonic  and benthic
 processes.   Donaghay (1984) classified  mesocosm systems  by  their degree  of
 planktonic/benthic coupling into three  categories:  single well-mixed benthic coupled,
 single stratified,  and totally benthic decoupled.   Single well-mixed  benthic  coupled
 systems are composed of a well-mixed water column in direct contact with sediment and
 benthos.  This system has been used extensively at the  Marine Ecosystem Research
 Laboratory (MERL) with very good results validating field data; defining loadings, fates,
 and effects; and  manipulating natural systems to  define underlying processes.  The
 single stratified system design involves well-mixed top and bottom layers separated  by
 an  unmixed thermocline with  only the bottom layer in contact with the sediment.  The
 system  is used to study systems where stratification is important.  The single system
 without benthic coupling is intended to model fates and effects in stratified deep water
 systems without benthic coupling.

 4.13.4    QA/QC Considerations

 In some cases the accuracy of the results of  mesocosms and  microcosms  may  be
 limited by a number of factors.   The  size of  the systems  most  often excludes
 macrofauna such as fish and  large crustaceans.   The size of a microcosm can also
 affect the mixing rates of surficial sediments. An increase in the size of a microcosm will
 result in an increase in  exposure of sediment particles to the  overlying water column.
 Results  from smaller-sized systems have underestimated potential risks of complex
 effluent  (Perez et al., in press;  Pontasch et  al., 1989); however, attempts  are  being
214
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                                           Section 403 Procedural and Monitoring Guidance
made to develop "scaling laws" to describe this relationship.  These systems are also
limited to photo-stable test substances because the light intensity may be higher than
that which would occur naturally.

4.13.5   Statistical Design Considerations

The results of the microcosm and mesocosm experiments assist in determining the fates
and ecological effects of contaminants within a marine system.  The pelagic biota are
characterized by the number and species  composition of phytoplankton, zooplankton,
and transient larval forms.  The benthic community is characterized by the structural
composition (see Section 4.5, Benthic Community Structure).  The experimental marine
test protocol (USEPA, 1990e) recommended test design and statistical analysis allow for
the independent assessment of the solvent carrier (if  used) and the effluent for all
variables measured. Also,  the differences in the biotic response between the control
microcosms and the natural system will provide a measure  of the  validity of the test
response.  A  multivariate analysis of variance, followed by univariate analysis, and
regression techniques are recommended for the analysis of all data.

Statistical  design considerations of mesocosms must consider that the scientific and
financial resources for mesocosm studies are limited.  Therefore, efforts must be made
to maximize the  information gained and  the statistical rigor of the analysis  while
minimizing  the number of systems analyzed.  Four different experimental designs are
presented by Donaghay (1984): single system, paired  system,  replicated, and single
gradient. Each design has advantages and disadvantages, and each is appropriate for
specific purposes.

4.13.6   Use of Data

Mesocosm research and microcosm research serve somewhat different purposes. If a
researcher wants to have  maximum confidence in extrapolating back to a specific,
large-scale environment, the mesocosm provides greater realism in that it allows more
large-scale processes to be included in the system.  If simplification  is the goal—either
to semi-isolate certain components to determine their importance in  the degradation of
pollutants  or to provide more easily  analyzed ecosystems for test purposes—then
microcosms are more appropriate.

The use of microcosms and/or  mesocosms in an ocean discharge criteria evaluation as
mandated under section 403 would be most appropriate for general permits issued for a
whole category of discharges (e.g., regional oil and gas exploration/production activities)
mainly  because of the higher cost associated with implementing a  series of such
experiments. However, the use of microcosms  and mesocosms can be less expensive
than  implementing field  experiments  and can  provide  quantitative estimates of
processes and responses that can only be assessed qualitatively with field experiments.
In addition, Perez and Morrison (1985) showed that the monetary costs of environmental
                                                                            215
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Methods
assessments of a single chemical using a single microcosm system versus a series of
simple bioassay and physicochemical test systems are, at the worst case,  effectively
equivalent. Assessments of complex effluents performed using microcosms could result
in more accurate results with a potential cost savings.

The use  of  microcosms  and/or mesocosms  could  add significant  benefits to an
ecological risk assessment of an ocean discharge.  Microcosms and/or mesocosms can
be  used  to estimate  ecological  effects,  chemical  fate,  transport  mechanisms,
bioaccumulation, and  ecological  risk.  Where multiple discharges occur in  one area,
microcosms and/or mesocosms  could  be used  to assess  the  risk associated  with
individual  discharges or combinations of discharges.  Estimating ecosystem recovery
through the use of mesocosms in ecological risk assessments is a recent application
(Perez et al.,  1990) and could provide important information concerning the occurrence
of irreparable harm.

4.13.7   Summary and Recommendations

Rationale

      •  Mesocosms and microcosms can be used to gain a realistic characterization
         of ecosystem-level impacts of marine discharges subject to section 403(c).

      •  The transport and fate of pollutants to the complete ecosystems  can be
         investigated in near natural conditions.

Monitoring Design Considerations

      •  A microcosm should be designed to represent a miniature ecosystem that
         responds quickly to perturbation.

      •  Most generic microcosms do not simulate a site-specific natural system, but
         function as very generalized simulations of a large class of ecosystems.

      •  The design of the microcosm allows the system to be defined in terms of its
         physical and temporal boundaries, its light and temperature regime, its water
         composition, the turbulence and turnover rate, the ratio of benthic surface
         area to seawater volume, the sediment characteristics, and the water flow
         rate over the sediment surface.

      •  The results of microcosm or mesocosm experiments are measured by the
         ecological effects on the biota of the system.

      •  Microcosm studies have been designed for 30 days to more than a year in
         duration.
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                                           Section 403 Procedural and Monitoring Guidance
Analytical Method
      •  During microcosm studies, effluent to be tested should be added on a
         gradient. A typical experimental design might consist of six replicates in each
         of four treatment groups: a control, a single addition of a low concentration, a
         single addition of a high concentration, and repeated additions of low
         concentrations.

      •  Mesocosm systems can be classified by their degree of  planktonic/benthic
         coupling into single, well-mixed benthic coupled; single stratified; and totally
         benthic decoupled systems.

QA/QC Analysis

      •  The results of microcosm and mesocosm studies are limited by the size of
         the system.

      •  Scaling laws may be used to translate from microcosms to full-scale
         ecosystems.

Statistical Design Considerations

      •  A multivariate analysis of variance, followed by univariate analysis, and
         regression techniques are recommended for the analysis of all data.

      •  Efforts must be made to maximize the information gained and the statistical
         rigor of the analysis while minimizing the number of systems analyzed.

Use of Data

      •  Data can be used to estimate ecological  effects, chemical fate, transport
         mechanisms, bioaccumulation, and ecological risks.

      •  Data can also be used to assess impacts of individual or multiple discharges
         and to estimate recovery time.

      •  Mesocosms are most appropriate for use when maximum confidence in
         extrapolating back to a specific, large-scale environment is desired.

      •  Microcosms are most appropriate for use when simplification is the goal,
         either to isolate certain components to determine their importance  or to
         provide more easily analyzed ecosystems for test purposes.

      •  Microcosms and  rnesocosms may be most appropriate for use for  general
         permits issued for a whole category of discharges.

      •  There can be cost advantages to using microcosms and rnesocosms under
         certain circumstances.
                                                                            217
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                          5. LITERATURE CITED
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Boesch, D. F.  1977. Application of numerical classification in ecological investigations
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Borrego, JJ., F. Arrabal, A. de Vicente, L.F. Gomez, and P. Romero.  1983.  Study of
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gastroenteritis and water quality. Am. J. Epidemiol. 115: 606-61.

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Cailliet, G.M., M.S.  Love,  and A.W. Ebeling.  1986.  Fishes:  A field and laboratory
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CDC.  1979.  Viral hepatitis outbreaks-Georgia, Alabama.  Centers for Disease Control.
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Poll. Cont. Fed. 38:1000-1010.

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nutrients-eutrophication:  Saline water considerations In  Advances  in  water quality
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Austin, TX.

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76:1-44.
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                                          Section 403 Procedural and Monitoring Guidance
Couch, J.A., and J.W. Fournie. 1993.  Pathobiology of marine and estuarine organisms.
In Advances in fisheries science.  CRC Press, Boca Raton, FL.

Couch, J.A., and J.C. Harshbarger.  1985. Effects of carcinogenic agents on aquatic
animals: An environmental and experimental overview.  Environ. Carcinogenesis Revs.
3(1): 63-105.

Cuff, W., and N. Coleman.  1979.  Optimal survey design:  Lessons from a stratified
random sample of macrobenthos. J. Fish. Res. Board Can. 36: 351 -361.

Curtis, M.A.,  and  G.H.  Peterson.   1978.   Size-class  heterogeneity with spatial
distribution of subarctic marine benthos populations. Astarte 10:103-105.

Gushing, D.J.   1975.   Marine ecology and  fisheries.   Cambridge  University Press,
Cambridge, UK.

Darcy, G.  H. and E. J. Gutherz.  1984.   Abundance of demersal fishes on the west
Florida shelf, January 1978.  Bull. Mar. Sci. 34:81-105.

Davis, R.E. 1988. Modeling eddy transport of passive tracers. J. Mar. Res. 45: 635.

Dawe, C.J., J.C. Harshbarger,  R. Wellings,  and J.  D. Strandberg.  In press.   The
pathobiology  of spontaneous  and induced  neoplasms  in  fishes:  Comparative
characterization, nomenclature, and literature.  Academic Press, New York, NY.

Day, J.W., C.A.S. Hall,  W.M. Kemp, and A. Yanez-Arancibia. 1989. Estuarine ecology.
John Wiley and Sons, New York, NY.

Dayton, P. K.   1985.  The structure and regulation of some South American kelp
communities.  Ecol. Monagr. 55(4):447-468.

deBoer, J.  1988. Chlorobiphenyls in bound and non-bound lipids of fishes: Comparison
of different extraction methods. Chemosphere 17:1803-1810.

D'Elia, C.F.,  J.G.  Sanders,  and  D.G.  Capone. 1989.    Analytical  chemistry  for
environmental  sciences:    A question  of  confidence.    Environ.  Sci.   Technol.
23(7):768-774.
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Dodge, R. E., A. Logan, and  A. Antonius. 1982.  Quantitative reef assessment studies
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Donaghay, P.L.  1984.  Utility of mesocosms to assess marine pollution.  In Concepts in
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Eganhouse, R.P.  1990.   Sources  and magnitude  of  error associated with PCM
measurements. In Southern California Coastal Water Research Project annual report
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bottom sediment samplers. Appl. Environ. Microbiol. 44:1152-1158.

Engel, D., and G. Roesijadi. 1987.  Metallothioneins: A monitoring tool. \nPollution
physiology of estuarine organisms, ed. W. Vernberg, A. Calabrese, F. Thurberg, and
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Ferraro, S.P., F.A. Cole, W.A. DeBen, and R.C. Swartz. 1989.  Power-cost efficiency of
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Gilliom, R.J., and D.R. Helsel.   1986.  Estimation of distributional parameters  for
censored trace  level water quality data. 1, Estimation Techniques.  Water Res. Res.
22(2): 135-146.

Goldberg, E.D.,  V.T. Bowen, G.H. Farrington, J.H. Martin, P.L Parker, R.W. Risebrough,
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Grace, R.A. 1978. Marine  outfall systems planning, design, and construction.  Prentice
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Hansen, P.O.,  H.  Von   Westerhagen, and H.   Rosenthal.   1985.    Chlorinated
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Hardy, J.T.  1982. The sea surface microlayer:  Biology, chemistry, and anthropogenic
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Heath, A.  1987. Water pollution and fish physiology. CRC Press, Boca Raton, FL.

Hiatt, M.H.  1981.  Analysis of fish and sediment for volatile priority pollutants.  Anal.
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Hill K.D., T.M. Dauphinee, and D. J. Woods.  1986. Extension of the practical salinity
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Hinton, D., J. Couch, S. Teh, and L. Courtney.   1988.  Cytological changes during
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Pielou, E.G.  1984. The interpretation of ecological data. John Wiley and Sons, New
York, NY.

Plafkin, J.L., et. al.  1989.  Rapid bioassessment protocols for use in streams and rivers.
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Plumb,  R.H., Jr. 1981. Procedure for handling and chemical analysis of sediment and
water samples.  Technical report EPA/CE-81-1.  Prepared for the U.S.  Environmental
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Pomeroy, L.R.   1984. Significance  of microorganisms in  carbon and energy flow  in
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Pond, S., and G.L. Pickard.   1983.  Introductory dynamic oceanography.  3d ed.
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Pontasch,  K.W.,  B.R. Niederlehner, and  J.  Cairns, Jr.   1989.    Comparisons  of
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856-861.
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Prophet, E.B.,  B.  Mills, J.B. Harrington, and  L.H.  Sorbin.  1992.   AFIP laboratory
methods in histotechnology.  Armed Forces Institute of Pathology, American Registry of
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Rees, H.L.  1984.  A note on mesh selection and sampling efficiency in benthic studies.
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marine bottom samples.  Ecology 40: 307-309.

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study. IEEE Conference Proceedings, Oceans '86, pp. 885-888.

Rhoads, D.C., and J.D. Germano.  1982.  Interpreting long-term  changes in benthic
community structure: A new protocol.  Hydrobiologia 142: 291 -308.

Rhoads, D.C.,  and  J.D. Germano.   1986.   Characterization of organism-sediment
relations using sediment profile imaging: An  efficient method of remote ecological
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Ricker,  W.E.   1975.  Computation  and interpretation of biological  statistics of fish
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National Park Service initiates regional program.  Proc. 6th Int. Coral Reef Symp.  1:
399-403.

Rogers, C.S., M. Gilnack, and H.C. Fitz III. 1983.  Monitoring of coral  reefs with linear
transects: a study of storm damage. J. Exp. Mar. Biol. Ecol. 49: 179-187.

Romesburg, H.C.    1984.   Cluster  analysis  for  researchers.   Lifetime Learning
Publications, Belmont, CA.

Rothschild, B.J.  1986. Dynamics of marine fish populations. Harvard University Press,
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Spies,  R.B., D.W.  Rice, and J. Felton.  1988.  Effects of organic contaminants  on
reproduction of the starry flounder Platichthys stellatus in San Francisco Bay. Mar. Biol.
98: 181-189.

St.  John, E.W., J.R. Matches, and M.M. Wekell. 1982.  Use of iron milk medium  for
enumeration of Clostridium perfrigens. J. Assoc. Off. Anal. Chem. 65: 1129-1133.

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Swartz, R.C., W.A. DeBen, K.A. Serco, and J.O. Lamberson.  1982. Sediment toxicity
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Bull. 13(10) :359-364.

Swartz, R.C.,  D.W. Schultz, G.R.,  Ditsworth, W.A.  DeBen,  and F.A.  Cole.   1985.
Sediment toxicity, contaminsition, and macrobenthic communities near a large sewage
outfall. In Validation and predictability of laboratory methods for assessing the fate and
effects of contaminants in aquatic ecosystems, ed. T.P. Boyle, pp. 152-175. ASTM STP
865. American Society for Testing and Materials, Philadelphia, PA.

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Concepts in marine pollution measurements, ed. H.H. White, pp. 159-192. Maryland
Sea Grant College, College Park, MD.

Tetra Tech.  1985.  Bioaccumulation monitoring guidance:  Recommended analytical
detection limits. Vol. 3. Tetra Tech, Inc., Bellevue, WA.

Tetra Tech.  1986.  Bioaccumulation  monitoring guidance: Analytical methods for U.S.
EPA priority pollutants and 301 (h) pesticides in tissues from estuarine and  marine
organisms.  Vol. 4. Tetra Tech, Inc., Bellevue, WA.

Tetra Tech.   1987.  Bioaccumulation  monitoring  guidance:  Strategies for sample
replication and compositing.  Vol.5. Tetra Tech, Inc.

Thomann, R.V., and J.A. Mueller.  1987.  Principles of surface water quality modeling
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USEPA.  1982b.  Handbook for sampling and  sample preservation  of  water  and
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USEPA.  1982c.  Method for use of caged mussels to monitor for bioaccumulation and
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USEPA.   1983a.  Methods  for chemical analysis of  water and  wastes.  EPA
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USEPA.  1985a.  Bioaccumulation monitoring guidance: Selection of target species and
review of available bioaccumulation data.  Vol. 2.  EPA 403/9-86-006.  Office of Marine
and Estuarine Protection, Washington, DC.

USEPA.  1985b.  Interim guidance on quality assurance/quality control (QA/QC) for the
estuarine field and laboratory methods. U.S. Environmental Protection Agency, Office of
Marine and Estuarine  Protection, Washington, DC.

USEPA.  1985c.  Methods for measuring  the acute toxicity of effluents freshwater and
marine  organisms.   EPA 600/4-85-013.  U.S.  Environmental  Protection  Agency,
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USEPA.  1985d.  Recommended biological indices  for 301 (h) monitoring programs.
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USEPA.  1985e.   Test methods for Escherichia coli  and enterococci in water by the
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USEPA.  1986-1988.  Quality criteria for water.  EPA 440/5-86-001. U.S.  Environmental
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USEPA.  1986b. Bioaccumulation monitoring guidance: Analytical methods for USEPA
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USEPA.  1988c.  Methods  for aquatic toxicity identification evaluations:  Phase III
toxicity   confirmation   procedures.     Draft  phase   III   toxicity  series   report.
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244
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         APPENDIX A:
MONITORING METHODS REFERENCES
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                                                                     Appenctfx A
                        PHYSICAL CHARACTERISTICS

APHA. 1989. American Public Health Association, American Water Works Association,
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                                                                           A-3
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Appendix A
Hill K.D., T.M. Dauphinee, and D.J. Woods, 1986. Extension of the practical salinity
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                                      /

McCullough,  J.R. 1977.  Problems in measuring  currents  near the ocean surface.
Proceedings  of Oceans 77, Marine Tech. Soc. and Inst. of Electrical and Electronics
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Pickard,  G.L.,  and  W.J.  Emery.  1982.  Descriptive  physical  oceanography,  An
introduction. 4th (SI) enlarged ed. Pergamon Press, Inc., New York, NY.

Pond, S., and G.L. Pickard 1983. Introductory dynamic oceanography. 3d ed. Pergamon
Press, Inc., New York, NY.

Thomann, R.V., and  J.A.  Mueller. 1987.  Principles of surface  water quality modeling
and control. Harper and Row Publ., New York, NY.

USEPA.  1982.  Design of 301 (h) monitoring programs for  municipal wastewater
discharges to marine waters. EPA 430/9-82-010. U.S. Environmental Protection Agency,
Washington, DC.

USEPA. 1983. Methods for chemical analysis of water and wastes. EPA 600/4-79-020.
U.S. Environmental Protection Agency, Environmental Support  Laboratory, Cincinnati,
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USEPA.  1985.  Initial mixing  characteristics of municipal ocean  discharges.  Vol. I,
Procedures  and applications.    EPA-600/3-85-073a.  U.S.  Environmental  Protection
Agency, Narragansett, Rl.

USEPA.  1986.  Quality criteria  for water.  EPA  440/5-86-001.  U.S.  Environmental
Protection Agency, Office of Water Regulations and Standards. Washington, DC.
A-4
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                                                                     Appendix A
USEPA.  1987.  Quality assurance/quality control  (QA/QC)  for 301 (h) monitoring
program:  Guidance  on  field  and  laboratory methods.  EPA  430/9-86-004.  U.S.
Environmental  Protection  Agency,  Office of  Marine  and  Estuarine  Protection,
Washington, DC.

UNESCO.  1988.  The acquisition, calibration,  and analysis of CTD data. UNESCO
technical paper  in marine science  54.  United Nations  Educational, Scientific, and
Cultural Organization, France.

Wallace, J.W., and J.W. Cox.  1976. Design, fabrication and  system integration of a
satellite tracked,  free-drifting ocean data buoy. NASA Technical Memorandum X-72817.
January.

Wilson et al. 1986. Techniques of water-resources investigations of the United States
Geological Survey.  Fluorornetric procedures for dye tracing.  U.S. Department of the
Interior, U.S. Geological Survey, Washington, DC.

Wright,  S.J., et al. 1988.  Outfall plume dilution  in stratified  fluids.  Proc. Int. Symp.
Model-Prototype Correlation ofHydraul. Structures  148.
                             WATER CHEMISTRY

APHA. 1989. American Public Health Association, American Water Works Association,
Water Control Pollution Federation.  Standard methods for the examination of water and
wastewater. 17th ed. American Public Health Association, Washington, DC.

Armstrong, J.W., and  A.E. Copping. 1989.  Comparing the Regional Puget Sound
Marine Monitoring with the NOAA National Status and Trends Program. Coastal Zone
Proceedings 3: 2421-2435.

Becker,  D.S., and  J.W. Armstrong.  1988.  Development of regionally standardized
protocols for marine environmental studies. Mar. Poll. Bull. 19(7): 310-313.

D'Elia, C.F., et al. 1987. Nitrogen and phosphorus determinations in estuarine waters:
A comparison of methods  used in Chesapeake  Bay monitoring.  Final report to the
Chesapeake Bay Program, U.S. Environmental  Protection Agency, Region III.  U.S.
Government Printing Office, Washington, DC.
                                                                            A-5
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 Appendix A
 D'Elia,  C.F., N.L.  Kaumeyer, C.W. Keefe, K.V.  Wood, and C.F.  Zimmerman. 1988.
 Nutrient analytical services laboratory standard operating  procedures.  Chesapeake
 Biological Laboratory, University of Maryland.
 D'Elia,  C.F.,  J.G.  Sanders,  and  D.G. Capone. 1989.
 environmental  sciences:    A  question of  confidence.
 23(7):768-774.
Analytical  chemistry for
Environ.   Sci.  Technol.
 D'Elia, C.F.,  P.A. Steadier, and N. Corwin.  1977.  Determination of total nitrogen in
 aqueous samples using persulfate digestion.  Limnol. Oceanogr. 22: 760-764.

 Ferraro, S.P., F.A. Cole, W.A. DeBen, and R.C. Swartz.  1989.  Power-cost efficiency of
 eight macrobenthic sampling schemes in Puget Sound, WA, USA. Can. J. Fish. Aquat.
 Sc/. 46: 2157-2165.

 Gilliom,  R.J., and  D.R.  Helsel.  1986.   Estimation  of  distributional  parameters for
 censored trace level water quality data. 1, Estimation Techniques. Water Res. Res.
 22(2): 135-146.

 Gilbert, P.M., and T.C. Loder. 1977. Automated analysis of nutrient seawater: Manual
 of techniques. Woods Hole Oceanographic Institute Technical Report No. WH01-77-47.

 Goldberg, E.D., V.T. Bowen, G.H. Farrington, J.H. Martin, P.L. Parker, R.W. Risebrough,
 W. Robertson, E. Schneider and E. Gamble. 1978.  The mussel watch.  Environ.
 Conserv. 5:101-125.

 Hirsch, R.M.  1988.  Statistical methods and sampling design for estimating step trends
 in surface-water quality. Water Res. Bull. 24(3): 493-503.

 Ladd, J.M., S.P. Hayes, M. Martin, M.D. Stephenson, S.L. Coale, J. Linfield, and M. Brown.
 1984.  California state  mussel watch:  1981-1983.  Trace metals  and synthetic organic
 compounds in mussels from California's coast,  bays and estuaries.  Biennial report.  Water
 Quality Monitoring Report No. 83-6TS. Sacramento, CA.

 Porter, P.S., R.C. Ward, and H.F. Bell. 1988.  The detection limit.  Env. Sci. Tech. 22:
 856-861.

 Salley, B.A., J.G. Bradshaw, and B.J. Neilson. 1986.  Results of comparative studies of
 preservation  techniques for nutrient analysis on water samples.  Virginia Institute of
 Marine Science Report to the Chesapeake Bay Liaison Office. September.
A-6
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                                                                     Appendix A


Sokal, R.R. and F.J. Rohlf.  1981.  Biometry.  W.H. Freeman and Co., San Francisco,
CA.

Strickland, J.D.H., and T.R. Parsons. 1968. A practical handbook of seawater analysis.
Fisheries Research Board of Canada, Ottawa, Canada.

Tetra Tech.  1990.  National Estuary Program monitoring guidance document.  Draft
report.  Prepared  for U.S.  Environmental Protection Agency  by  Tetra  Tech, Inc.,
Lafayette, CA.

USEPA.  1979.   Handbook for  analytical quality control in water and wastewater
laboratories.  EPA 600/4-79-019.  Environmental Monitoring and Support Laboratory,
Cincinnati, OH.

USEPA.  1982.  Methods for organic chemical analysis of municipal and industrial
wastewater.  EPA 600/4-82-057.  U.S. Environmental Protection Agency, Environmental
Monitoring and Support Laboratory, Cincinnati, OH.

USEPA.  1983.  Methods for chemical analysis  of water and  wastes, 2d ed.  EPA
600/4-79-020.  U.S. Environmental Protection Agency, Environmental  Monitoring and
Support Laboratory, Cincinnati, OH.

USEPA. 1986. Test methods for evaluating solid waste.  EPA Publication SW-846,  3d
ed.  U.S. Environmental Protection Agency, Office of Solid Waste and Emergency
Response, Washington, DC.

USEPA. 1986-1991.  Recommended protocols for  measuring selected environmental
variables  in Puget Sound.  Looseleaf.  U.S. Environmental Protection Agency,  Region 10,
Puget Sound Estuary Program, Seattle, WA.

USEPA.  1987.   Quality assurance/quality control  (QA/QC) lor 301 (h)  monitoring
programs:   Guidance on  field  and laboratory methods.  EPA 430/9-86-004.  U.S.
Environmental  Protection  Agency,  Office  of  Marine  and   Estuarine   Protection,
Washington, DC.

USEPA.  1990.  Compendium of methods for marine and estuarine environmental
studies.   EPA 503/2-89/001.  U.S. Environmental Protection Agency, Office of Water,
Washington, DC.

Valderama, J.C. 1981. The simultaneous analysis of total nitrogen and total phosphorus
in natural waters.  Mar. Chern. 10:109-122.
                                                                          A-7
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Appendix A
Ward, B.C., and J.C. Loftis. 1986. Establishing statistical design criteria for water quality
monitoring systems, review and synthesis.  Water Res. Bull. 22(5): 759-767.
                            SEDIMENT CHEMISTRY

APHA. 1989. American Public Health Association, American Water Works Association,
Water Pollution Control Federation.  Standard methods for the examination of water and
wastewater. 17th ed. American Public Health Association, Washington, DC.

ASTM.  1990.  Standard guide for conducting 10-day static sediment toxicity tests with
marine and estuarine amphipods.  ASTM Guide E1367-90. American Society for Testing
and Materials, Philadelphia, PA.

ASTM. 1991. Standard guide for collection, storage, characterization, and manipulation
of sediments for toxicological testing. ASTM Designation E1391-90.  In Annual book of
ASTM standards. American Society for Testing and Materials, Philadelphia, PA.

DiToro, D.M., J.D. Mahony, D.J. Hansen, K.J. Scott, A.R. Carlson, and G.T.  Ankley.  In
press.  Acid volatile  sulfide  predicts the acute  toxicity of cadmium and nickel in
sediments.

DiToro, D.M., J.D. Mahony,  D.J. Hansen,  K.J. Scott, M.B. Hicks, S.M.  Mayr, and
M.S. Redmond.  In press.  Toxicity of cadmium in sediments:  The  role of acid
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EA and Battelle. 1990.  EA Engineering, Science, and Technology, Inc. and Battelle
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Estuarine Protection, Washington, DC.

Eganhouse, R.P.  1990.   Sources and magnitude of error associated  with  PCM
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Ferraro, S.P., H. Lee, R.J. Ozretich, and D.T. Specht. 1990. Predicting bioaccumulation
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A-8
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                                                                      Appendix A
Fredette, T.J., D.A. Nelson, T.  Miller-Way, J.A. Adair, V.A. Sotler, J.E. Clausner,  E.B.
Hands, and F.J. Anders. 1989. Selected tools and techniques for physical and biological
monitoring of aquatic dredged material disposal sites.  Final report.  U.S. Army Engineer
Waterways Experiment Station,  Vicksburg, MS.
Hiatt,  M.H. 1981. Analysis of fish and sediment for volatile priority pollutants.
Chem. 53:1541-1543.
Anal.
Karickhoff, S.W., D.S. Brown, and T.A. Scott. 1979. Sorption of hydrophobia pollutants
on natural sediments.  Wat. Res. 13: 241-248.

Knezovich,  J.P.,  and  F.L  Harrison.  1987. A new method  for  determining  the
concentration of volatile organic compounds in sediment interstitial water.  Bull. Environ.
Contam. Toxicol. 38: 837-940.

Lake, J.L., N.I.  Rubenstein, and  S.  Parvigano.  1987.  Predicting  bioaccumulation:
Development of a partitioning model  for use as  a screen tool in regulating ocean
disposal of wastes.   In Fate  and effects  of sediment-bound chemicals in aquatic
systems, ed.  K.L. Dickson, A.W. Maki, and W.A.  Brungo.   Sixth Pellston Workshop,
Florissano, CO.

Landrum, P.F., and J.A.  Bobbins. In press.  Bioavailability of sediment-associated
contaminants to benthic invertebrates.  In Sediments: Chemistry and toxicity of in-place
pollutants, ed. J.P. Giesy, R. Baudo, and H. Muntau.  Lewis Publishers.

McFarland, V.A., J.U.  Clarke, and A.B. Gibson.    1986.   Changing  concepts and
improved methods  for  evaluating the  importance  of PCBs as  dredged sediment
contaminants.  Miscellaneous Paper  D-86-5.   Department of the Army, Corps of
Engineers, Waterways Experiment Station, Vicksburg, MS.

Mclntyre, A.D., J.M. Elliot, and D.V. Ellis.  1984. Introduction: Design of sampling
programs.  IBP handbook no. 16.  In Methods  for the study of marine benthos, ed.
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Plumb,  R.H.,  Jr.  1981. Procedure for handling and chemical analysis of sediment and
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Fill Material, by Great Lakes Laboratory, State University College at Buffalo, Buffalo, NY.
U.S. Army Engineer Waterways Experiment Station, Vicksburg, MS.
                                                                            A-9
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Appendix A
 Swartz, B.C., W.A. DeBen, K.A. Serco, and J.O. Lamberson.  1982. Sediment toxicity
 and the distribution of amphipods in Commencement Bay, Washington, USA. Mar. Poll.
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 Swartz, R.C., D.W.  Schultz,  G.R.,  Ditsworth,  W.A. DeBen,  and  F.A. Cole. 1985.
 Sediment toxicity, contamination, and macrobenthic communities near a large sewage
 outfall.  In Validation and predictability of laboratory methods for assessing the fate and
 effects of contaminants in aquatic ecosystems, ed. T.P. Boyle, pp. 152-175. ASTM STP
 865. American Society for Testing and Materials, Philadelphia, PA.

 USEPA. 1986. Analytical methods for USEPA priority pollutants and 301 (h) pesticides in
 estuarine and marine sediments.   Prepared  for the Office of Marine and Estuarine
 Protection, Washington, DC.

 USEPA. 1986. Test methods  for evaluating solid wastes, physical/chemical methods.
 SW-846,3d ed. U.S. Environmental Protection Agency, Washington, DC.

 USEPA. 1986-1991.  Recommended protocols for measuring  selected environmental
 variables in Puget Sound.  Looseleaf.  U.S. Environmental Protection Agency, Region
 10, Puget Sound Estuary Program, Seattle, WA.

 USEPA. 1987. Bioaccumulation monitoring guidance: Strategies for sample replication
 and compositing.  Vol. 5.  EPA 430/9-87-003. U.S. Environmental Protection Agency,
 Office of Marine and Estuarine  Protection, Washington, DC.

 USEPA. 1987.   Quality assurance/quality  control (QA/QC)  for 301 (h)  monitoring
 programs: Guidance on field  and  laboratory methods.  EPA 430/9-86-004. Office of
 Marine and Estuarine Protection, Washington, DC.

 USEPA. 1989.  Sediment classification methods compendium.   Prepared for the U.S.
 Environmental Protection Agency, Office of Water Regulations and Standards.

 USEPA.  1990.  Statement   of  work  for   inorganics  analysis:  Multi-media,
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 USEPA/COE. 1977.  U.S. Environmental Protection  Agency and U.S. Army Corps of
 Engineers. Ecological evaluation of proposed discharge of dredged material into ocean
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 Station, Vicksburg, MS.
A-10
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                                                                     Appendix A
Washington State Department of Ecology.  1991.  Sediment management standards.
Washington Administrative Code (WAG) Chapter 173-204. Olympia, WA.
                            SEDIMENT GRAIN SIZE

ASTM. 1991. Standard guide for collection, storage, characterization, and manipulation
of sediments for toxicological testing. ASTM designation E1391-90. In  Annual book of
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Buller, AT., and J. McManus.  1979.  Sediment sampling and analysis.  In  Estuarine.
hydrography  and  sedimentation,  ed.  K.R. Dyer.   Cambridge  University  Press,
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Folk, R.L 1980. Petrology of sedimentary rocks.  Herpmill Publishing Co., Austin, TX.

Fredette, T.J., D.A. Nelson, T.  Miller-Way, J.A. Adair, V.A. Sotler, J.E. Clausner, E.B.
Hands,  and F.J.  Anders.   1989.  Selected tools  and  techniques for  physical and
biological monitoring of aquatic dredged material disposal sites. Final report.  U.S. Army
Engineer Waterways Experiment Station, Vicksburg, MS.

Mclntyre,  A.D., J.M.  Elliot, and D.V. Ellis.   1984. Introduction:  Design of sampling
programs.  IBP Handbook No. 16. In Methods for the study of marine benthos, ed. N.A.
Holme and A.D. Mclntyre, pp. 1-26.  Blackwell Scientific Publications, Oxford.

Plumb, R.H., Jr. 1981. Procedure for handling and chemical analysis  of sediment and
water samples.  Technical  report EPA/CE-81-1.  Prepared for the U.S. Environmental
Protection Agency/Corps of Engineers Technical Committee on Criteria  for Dredged and
Fill Material, by Great Lakes Laboratory, State University College at Buffalo, Buffalo, NY.
U.S. Army Engineer Waterways Experiment Station, Vicksburg, MS.
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1954.  Nomenclature based  on sand-silt-clay ratios. J.  Sed.  Petrol.
 USEPA.  1986-1991. Recommended protocols for measuring selected environmental
 variables in Puget Sound. Looseleaf.  U.S. Environmental Protection Agency,  Region
 10, Puget Sound Estuary Program, Seattle, WA.

 USEPA.  1987. Technical support document for ODES statistical power analysis.  EPA
 430/9-87-005. Office of Marine and Estuarine Protection, Washington, DC.
                                                                          A-11
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Appendix A
                      BENTHIC COMMUNITY STRUCTURE

Amjad, S.,  and J.S. Gray. 1983. Use of the nematode/copepod  ratio as an index of
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Anderberg, M.R. 1973.  Cluster analysis for applications.  Academic Press, New York,
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ASTM. 1991.  Standard guide for collection, storage, characterization, and manipulation
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ASTM standards. American Society for Testing and Materials, Philadelphia, PA.

Avent, R.M., M.E. King, and R.H. Gore.  1977. Topographic and faunal studies of shelf
edge  prominences off the central eastern Florida coast  USA.   Int. Rev. Gesamten
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Bazzaz, F.A.  1983. Characteristics of populations in relation to disturbance in  natural
and man-modified ecosystems.  In Disturbance and ecosystems, ed. H. A. Mooney and
M. Godron. Springer-Verlag, Berlin.

Becker,  D.S., and  J.W.  Armstrong.  1988.   Development of regionally standardized
protocols for marine environmental studies. Mar. Pollut. Bull. 19(7):310-313.

Bernstein, B.B., and R.W. Smith. 1986. Community approaches to monitoring.  IEEE
Conference Proceedings, Oceans '86, pp. 934-939.

Beukema,  J.J. 1988.   An evaluation  of the  ABC method (abundance-biomass
comparison) as applied to macrozoobenthic communities living on tidal flats in the Dutch
WaddenSea. Mar. Biol. 99: 425-433.  .

Bilyard, G.R. 1987. The value of benthic infauna in marine pollution monitoring studies.
Mar. Poll. Bull. 18: 581-585.

Boesch, D.F. 1977. Application of numerical classification in ecological investigations of
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Bohnsack,  J.A.  1979.   Photographic quantitative  sampling of  hard-bottom benthic
communities.  Bull.  Mar. Sci. 29:242-252.
A-12
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                                                                      Appendix A
Brown, B.E.  1988. Assessing environmental impacts on coral reefs.  Proc. 6th Int.
Coral Reef Symp. 1:71 -80.

Clifford, H.T., and W. Stephenson.  1975.  An introduction to numerical classification.
Academic Press, New York, NY.

Connell, J.H. 1978.  Diversity  in tropical  rain forests and coral reefs.  Science  199:
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Connell, J.H., and  M.J. Keough. 1985.  Disturbance and patch dynamics of subtidal
marine animals on hard substrata.  In The ecology of natural disturbance and patch
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Darcy,  G.H. and EJ. Gutherz. 1984. Abundance of demersal fishes on the west Florida
shelf, January 1978. Bull. Mar.  Sci.  34:81-105.

Dayton, P.K.  1985.   The  structure and regulation of  some  South American  kelp
communities. Ecol. Monagr. 55(4):447-468.

Dodge, R.E., A. Logan, and A. Antonius. 1982. Quantitative reef assessment studies in
Bermuda: A comparison of  methods  and  preliminary results.    Bull. Mar.  Sci.
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Downing, J.A. 1979.  Aggregation, transformation, and the design of benthos  sampling
programs. J. Fish Res. Board Cer. 36:1454-1463.

Dustan, P., and J.C. Halas.  1987.  Changes in the reef-coral community of  Carysfort
Reef, Key Largo, Florida: 1974 to 1982. Coral Reefs 6(2):91-106.

Eleftheriou, A., and N.A.  Holme. 1984.  Macrofauna techniques.  In Methods for the
study of marine benthos,  ed. N.A.  Holmes and A.D. Mclntyre, pp.  66-98.   Blackwell
Scientific Publications, Oxford.

Elliot, J.M. 1971.  Some methods for the statistical analysis of samples of benthic
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Ellis, D. 1985. Taxonomic sufficiency in pollution assessment.  Mar, Poll. Bull. 16: 459.
                                                           i
Elmgren, R., S. Hansson,  U. Larsson, B. Sundelin, and P.O. Boehm. 1983.  The "Tsesis"
oil spill: Acute and long-term impact on the benthos.  Mar. Poll. Bull. 15: 249-253.
                                                                           A-13
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Appendix A
Ferraro, S.P., F.A. Cole, W.A. DeBen, and B.C. Swartz. 1989. Power-cost efficiency of
eight macrobenthic sampling schemes in Puget Sound, Washington, USA. Can. J. Fish.
AquatSci. 46:2157-2165.

Fleishack,  P.C., AJ. DeFreitas, and  R.B. Jackson.   1985.   Two apparatuses for
sampling benthic fauna in surf zones. Est. Cstl. Shelf Sci. 21 -.287-293.

Fredette, T.J., D.A.  Nelson, T. Miller-Way,  J.A. Adair, V.A. Sotler, J.E. Clausner, E.B.
Hands,  and FJ. Anders. 1989.   Selected tools and techniques for  physical and
biological monitoring of aquatic dredged material disposal sites.  Final report.  U.S. Army
Engineer Waterways Experiment Station, Vicksburg, MS.

Gamble, J.C.  1984.  Diving.   In Methods  for the study of marine  benthos, ed.  N.A.
Holmes and A.D. Mclntyre, pp. 66-68. Blackwell Scientific Publications, Oxford.

Gauch,  H.G.,  Jr.   1982.  Multivariate analysis in community ecology.   Cambridge
University Press, New York, NY.

Gray, J.S., and  F.B. Mirza. 1979.   A possible method for  the detection of pollution
induced disturbance on marine benthic communities. Mar. Poll. Bull. 10:142-146.

Green, R.  1979.  Sampling design and statistical methods for environmental biologists.
John Wiley and Sons, New York, NY.

Grigg, R.W., and S.J. Dollar.   1990.  Natural and  anthropogenic disturbance on coral
reefs.  In Coral  reefs, ed. Z. Dubinsky, pp. 439-452.  Ecosystems of the world 25.
Elsevier, New York, NY.

Grizzle, R.E.  1984.   Pollution indicator species of macrobenthos in a coastal lagoon.
Mar. EcoL Prog. Ser. 18:191-200.

Hartley, J.P.  1982.  Methods for monitoring offshore macrobenthos.  Mar. Poll.  Bull.
13:150-154.

Holme, N.A. 1984.  Photography and television. IBP handbook no. 16. In Methods for
the study of marine benthos, ed. N. A. Holmes and A. D. Mclntyre, pp. 66-98. Blackwell
Scientific Publications, Oxford.

Holme, N.A., and A.D. Mclntyre, eds.  1984. Methods for the study of marine benthos.
Blackwell Scientific Publications, Oxford.
A-14
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                                                                     Appendix A
Hurlbert,  S.H. 1971.  The  nonconcept of species diversity: A critique and alternative
parameters. Ecology 52: 577-586.

Jackson,  J.B.C., J.D. Cubit, B.D. Keller, V. Batista, K. Burns, H.M. Caffey, R.L. Caldwell,
S.D. Garrity, C.D. Getter,  C. Gonzales, H.M. Guzman, K.W. Daufman, A.M. Knapp,
S.C. Levings,  M.J.  Marshall,  R.  Steger, R.C.  Thompson,  and E. Weil. 1989.
Ecological effects of a major oil spill  on Panamanian coastal  marine communities.
Science 243: 37-44.

Lambshead, P.J. 1984. The nematode/copepod ratio. Some anomalous results  from
the Firth of Clyde.  Mar. Poll. Bull. 15: 256-259.

Lambshead, P.J.,  and H.M. Platt. 1985.   Structural  patterns  of  marine benthic
assemblages and their relationship  with empirical statistical models.  Proc.  19th Eur.
Mar. Biol. Symp., pp. 371-380.

Livingston, R.J., R.S. Lloyd, and M.S.  Zimmerman.  1976.  Determination of sampling
strategy for benthic macrophytes in polluted and unpolluted coastal areas.  Bull.  Mar.
Sci. 26:569-575.

Loya, Y.  1978.  Plotless and transect methods.  In C<&al reefs: Research methods, ed.
D.  R.  Stoddart  and  R.   E. Johannes pp.  197-217.    UNESCO, Monographs on
Oceanographic Methodology, Paris.

Lunz, J.D., and D.R. Kendall. 1982.  Benthic resource analysis technique, a method for
quantifying the effects of benthic community changes on fish resources. In Conference
proceedings on marine pollution, Oceans 1982,  pp. 1Q21-1027.  National Oceanic and
Atmospheric Administration, Office of Marine Pollution Assessment,  Rockville, MD.

Mclntyre, A.D.,  J.M.  Elliot, and D.V.  Ellis. 1984.   Introduction: Design  of  sampling
programs.  IBP handbook no. 16.  In  Methods for the study of marine benthos, ed.
N.A. Holme and A.D. Mclntyre, pp.  1-26. Blackwell Scientific Publications, Oxford.

Moore, E.J. 1978. Underwater photogrammetry. Prog, in Und. Sci. 3:101-110.

NRC. 1990.  Managing troubled waters: The role of marine environmental monitoring.
National Academy Press, Washington, DC.
Ohlhorst, S.L., W.D. Liddell,  R.J. Taylor, and J.M. Taylor.  1988.
census techniques. Proc. 6th Int. Coral Reef Symp. 2:319-324.
Evaluation of reef
                                                                          A-15
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Appendix A
Palmer, M.A. 1988.  Epibenthic predators and marine meiofauna: separating predation,
disturbance, and hydrodynamic effects. Ecology 69:1251 -1259.

Pearson, T.H., and R. Rosenberg. 1978.  Macrobenthic succession in relation to organic
enrichment and pollution of the marine environment.  Oceangr. Mar. Biol. Ann.  Rev. 16:
229-311.

Phillips, N.W., D.A. Gettleson, and K.D. Spring. 1990. Benthic biological studies of the
southwest Florida shelf. Amer. Zoo/. 30(1):65-75.

Pielou, E.G.  1984.  The interpretation of ecological data.  John Wiley and Sons, New
York, NY.

Porter, J.W.  1972.  Patterns of species diversity in Caribbean  reef corals.  Ecology
53:745-748.

Rabalais, N.N.   1990.  Biological communities of the south Texas continental shelf.
Amer. Zoo/. 30(1):77-87.

Raffaelli, D. 1987.  The behavior of  the nematode/copepod ratio in organic  pollution
studies. Mar. Environ. Res. 23:135-152.

Raffaelli, D., and C.F. Mason. 1981. Pollution monitoring with meiofauna, using the ratio
of nematodes to copepods. Mar. Poll. Bull. 12:158-163.

Rees, H.L. 1984. A note on mesh selection and sampling efficiency in benthic studies.
Mar. Pollut. Bull. 15:225-229.

Reish, DJ. 1959. A discussion of the importance of screen size in washing quantitative
marine bottom samples. Ecology 40:307-309.

Reish, D.J. 1986.  Benthic invertebrates as indicators of marine  pollution: 35  years of
study. IEEE Conference Proceedings, Oceans '86, pp. 885-888.

Rhoads,  D.C., and  J.D. Germano. 1982.   Interpreting long-term changes  in benthic
community structure: A new protocol.  Hydrobiologia 142: 291-308.

Rhoads,  D.C.,  and  J.D.  Germano.  1986.   Characterization of organism-sediment
relations  using  sediment profile imaging:  An  efficient method  of remote  ecological
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A-16
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                                                                       Appendix A
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 SCCWRP. 1988.  Recovery of Santa Monica Bay after termination of sludge discharge.
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 Self, S.G., and R.H. Mauritsen. 1988. Power/sample size calculations for generalized
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 Sheills,  G.M.,  and   K.J.   Anderson.  1985.    Pollution   monitoring  using   the
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 Sneath, P.H.A. and R.R. Sokal.   1973.   Numerical taxonomy:  The principles and
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 Swartz, R.C., D.W. Schultz,  G.R. Ditsworth, W.A. DeBen, and  F.A. Cole.  1985.
 Sediment  toxicity, contamination, and macrobenthic communities near a large sewage
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Tomascik, T., and F. Sander.  1987.  Effects of eutrophication on reef-building corals. II.
 Structure of scleractinian coral communities on fringing reefs, Barbados, West Indies
 Mar. Biol. 94:77-94.
                                                                           A-17
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Appendix A
USEPA. 1985. Recommended biological indices for 301 (h) monitoring programs. EPA
430/9-86-002.  U.S. Environmental Protection Agency, Office of Marine and Estuarine
Protection, Washington, DC.

USEPA. 1986-1991.  Recommended protocols for measuring selected environmental
variables in Puget Sound.  Looseleaf.  U.S. Environmental Protection Agency, Region
10, Puget Sound Estuary Program, Seattle, WA.

USEPA. 1987.  Technical support document for ODES statistical power analysis. EPA
430/9-87-005.  U.S. Environmental Protection Agency, Office of Marine and Estuarine
Protection, Washington, DC.

USEPA. 1988.  ODES data brief: Use of numerical classification.  U.S. Environmental
Protection Agency, Office of Marine and Estuarine Protection, Washington, DC..

USEPA. 1989.  Sediment classification  methods compendium.  U.S. Environmental
Agency, Office of Water Regulations and Standards, Washington, DC.

Warwick, R.M.   1985.   A  new method for detecting pollution  effects  on  marine
macrobenthic communities.  Mar. Biol. 92:557-562.

Warwick, R.M. 1986. The level of taxonomic discrimination required to detect pollution
effects on marine benthic communities.  Mar. Poll. Bull. 19: 259-268.

Weinberg, S.  1981.  A comparison of coral reef survey methods.  Bijdragen tot de
Dierkunde 51 (2):199-218.

White, M.W.,  and J.W. Porter.   1985.   The establishment and monitoring  of two
permanent photograph  transects in  Looe  Key and Key  Largo  National  Marine
Sanctuaries (Florida Keys).  Proc. 5th Int. Coral Reef Congr.  6:531-537.

Witman, J.D.  1985.  Refuges, biological disturbance, and rocky subtidal community
structure in New England. Ecol. Mono. 55:421-445.

Word, J.Q. 1978.   The infaunal trophic index.  1978 Annual Report, Southern California
Coastal Water Research Project,  pp. 19-39.

Word, J.Q., B.L. Myers, and A.J. Mearns. 1977.  Animals that are indicators of marine
pollution. 1977 Annual Report, Southern California Coastal Water Research Project, pp.
199-207.
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                                                                      Appendix A
                     FISH AND SHELLFISH PATHOBIOLOGY

 Adams, S.M., ed.  1990.  Biological indicators of stress in  fish.  American Fisheries
 Society Special Symposium No. 8.

 Adams, S.M., L.R. Shugart, and G.R. Southworth. 1990. Application of bioindicators in
 assessing the health of fish populations experiencing contaminant stress. In Biomarkers
 of environmental contamination, ed. J. McCarthy and L.  Shugart.  CRC Press,  Boca
 Raton, FL.

 Anderson,  D. 1990.   Immunological indicators:  Effects  of  environmental stress  on
 immune protection and disease outbreaks.  Proceedings American Fisheries Society
 Symposium 8: 38-50, Washington, DC.

 Anderson, D. 1990.  Passive hemolytic plaque assay for detecting antibody-producing
 cells in fish. In Techniques in fish immunology, ed. J. Stolen, J. Fletcher, D. Anderson,
 B. Roberson, and W. van Muiswinkel. SOS Publications, Fair Haven, NJ.

 Anderson, D., B. Roberson, and O. Dixon. 1979.  Cellular immune response in Rainbow
 Trout Salmo Gairdneri, Richardson to Yersinia  Ruckeri O-antigen monitored by the
 passive haemolytic plaque assay test. J. Fish Dis. 2:169-178.

 Anderson, D., O. Dixon, and E. Lizzio. 1986.   Immunization and  culture of Rainbow
 Trout organ sections in vitro. Vet. Immunol. Immun. 12: 203-211.

 Anderson, D., O. Dixon, and W. van Muiswinkel.  1990.  Reduction in the numbers of
 antibody-producing cells in rainbow trout, Oncorhynchus mykiss, exposed to sublethal
 doses of phenol before bath immunization.   In  Aquatic toxicology, ed. J.  Nriagu. A
Wiley-lnterscience Publication, John Wiley and Sons, New York.

Anderson, R.S.  1987. Immunocompetence in invertebrates. In Pollutant studies in
 marine animals, ed. C. S. Giam and L. E. Ray. CRC Press, Boca Raton, FL.

Anderson,  R.S.   1990.   Eiffects  of pollutant exposure on bactericidal  activity  of
 Mercenaria  mercenaria  hemolymph.    In  Biological  markers  of  environmental
contaminants, ed. J.F. McCarthy and L.R. Shugart. American Chemical Society, Los
Angeles, CA.

Blaxhall, P., and K. Daisley. 1973.  Routine haematological methods for use with fish
blood. J. Fish Biol. 5: 771.
                                                                          A-19
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Appendix A
Bouck, G.R. 1984. Physiological responses of fish: Problems and progress toward use
in environmental monitoring. In Contaminant effects on fisheries, ed. V. W. Cairns, P. V.
Hodson, and J. O. Nriagu. John Wiley and Sons, New York, NY.

Brown, D.A., C.A. Bowden, K. Chatel, and T.R. Parsons. 1977.  The wildlife community
of lona Island  Jetty,  Vancouver, BC, and heavy metal pollution effects.   Environ.
Conserv. 4:213-216.

Brusick, D. 1980.  Principles of genetic toxicology.  Plenum Press, New York, NY.

Buckley,  L.J., T.A. Halavik,  G.C. Lawrence, S.J. Hamilton, and  P. Yevich.   1985.
Comparative  swimming  stamina,  biochemical  composition,  backbone  mechanical
properties, and  histopathology of juvenile striped bass from rivers and hatcheries of the
eastern United States. Trans. Amer. Fish. Soc. 114:114-124.

Cailliet, G.M., M.S. Love, and A.W. Ebeling.  1986.  Fishes:  A field and  laboratory
manual on their  structure,  identification,  and natural history.  Wadsworth Publishing
Company, Belmont, CA.

Couch, J.A.  1978.  Diseases, parasites, and toxic responses of commercial penaeid
shrimps of the Gulf of Mexico and  South Atlantic  coasts of  North America.  Fish.  Bull.
76:1-44.

Couch, J.A., and  J.C. Harshbarger.  1985.  Effects of carcinogenic agents on aquatic
animals: An environmental and experimental overview.  Environ. Carcinogenesis Revs.
3(1):63-105.

Cross, J., and J.E. Hose. 1988. Evidence for impaired reproduction in white croaker
(Genyonemus lineatus) from contaminated areas off southern California. Mar. Environ.
Res. 24:185-188.

Dawe, C.J., J.C. Harshbarger, R.  Wellings,  and J.D. Strandberg.  In  press.    The
pathobiology of spontaneous  and  induced  neoplasms in  fishes:   Comparative
characterization, nomenclature, and literature. Academic Press, New York,  NY.

Dixon. 1982. Mar. Biolog. Let. 3:155-161.

Dorigan, J.V., and F.L. Harrison.  1987. Physiological responses of marine  organisms to
environmental stresses.  U. S. Department of Energy, Washington, DC.

Ellis, A.  1977.  The leucocytes of fish: A review. J. Fish Biol. 11:453.
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                                                                      Appendix A
Elskus, A., and J. Stegeman. 1989. Induced cytochrome P-450 in Fundulus heteroclitus
associated  with  environmental  contamination  by  polychlorinated  biphenyls  and
polynuclear aromatic hydrocarbons. Mar. Environ. Res. 27: 31-50.

Engel, D. 1987.   Metal  regulation and molting in the blue crab, Callinectes Sapidus:
Copper, zinc and metallothionein. Biol. Bull. 172: 69-82.

Engel, D., and G. Roesijadi. 1987.  Metallothioneins: A monitoring tool.  In Pollution
physiology of estuarine organisms, ed. W. Vernberg, A. Calabrese, F. Thurberg, and
F. J. Vernberg, pp. 421-438.  University of South Carolina Press.

Engel,  D.   1988.   The  effect of  biological  variability on  monitoring  strategies:
Metallothioneins as an example. Water Res. Bull. 24(5): 981-987.

Ferguson, H.W.   1989.  Systemic pathology of fish: A  text and atlas of comparative
tissue responses in diseases of teleosts. Iowa State University Press, Ames, IA.

Fisher, W.S.  1988.  Disease processes in marine bivalve molluscs.  American Fisheries
Society Special Publication 18.

Gardner,  G.R.,   P.P.   Yevlch,  J.C.  Harshbarger,  and  A.R.   Malcolm.    1991.
Carcinogenicity of Black Rock Harbor sediment to  the  eastern  oyster and  trophic
transfer of Black  Rock Harbor carcinogens from the blue mussel to  the winter flounder.
Environ. Health Perspect. 90:53-66.

Garvey, J. 1990. Metallothionein:  A potential biomarker of exposure to environmental
toxins. In Biomarkers of environmental contamination, ed. J.  McCarthy and L. Shugart.
CRC Press,  Boca Raton, FL.

Giam, C.S.,  and  L.E. Ray.  1987.  Pollutant studies in  marine animals.  CRC Press,
Boca Raton, FL.

Haasch, M.,  P. Wejksnora, J. Stegeman, and J. Lech. 1989.  Cloned rainbow trout liver
P-I450 complementary DNA as a potential environmental monitor.  Toxicol. Appl. Pharm.
98: 362-368.

Hargis, W., M. Roberts, and D. Zwerner. 1984.  Effects of contaminated sediments and
sediment-exposed effluent water on estuarine fish: Acute toxicity.  Mar. Environ. Res.
14:337-354.

Hargis. W., and D. Zwerner. 1988.  Effects of certain contaminants  on  eyes of several
estuarine fishes.  Mar. Environ. Res. 24: 265-270.
                                                                           A-21
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Appendix A
Harrington, F.W., and J.O. Corliss.  1991.  Microscopic anatomy of invertebrates. Vol.
1-15 (some still in press).  Wiley-Liss, New York, NY.

Haux,  C.,  and L.  Forlin.   1988.   Biochemical methods for detecting effects  of
contaminants on fish.

Heath, A. 1987. Water pollution and fish physiology. CRC Press, Boca Raton, FL.

Hernberg, S. 1976.   Biochemical, subclinical, and  clinical responses to. lead and their
relation to different exposure levels as indicated by concentration of lead in blood.  In
Effects and dose-response relationships of toxic metals,  ed.  G.  Norberg.  Elsevier,
Amsterdam, 404.

Hinton, D.E., and J.A. Couch.  1984.  Pathobiological measures of marine pollution
effects. In  Concepts in  marine pollution  measurements, ed. H.H. White, pp.  7-32.
Maryland Sea Grant College, College Park,  MD.

Hinton, D.,  J. Couch,  S. Teh,  and L.  Courtney.  1988.  Cytological changes during
progression of neoplasia in selected fish  species.   In Aquatic life toxicology,  toxic
chemicals and aquatic life:  Research and management, ed. D. Malins, A. Jensen, and
M. Moore. Elsevier Science Publishers.

Holeton, G.  1972. Gas exchange in fish with and without hemoglobin. Respir. Physicol.
14:142.

Hose,  J.E., J.N. Cross, S.G. Smith, and D. Diehl. 1989.  Reproductive impairment in a
fish  inhabiting a contaminated  coastal  environment off southern California.  Environ.
Poll. 57:139-148.

Howard, D.W., and  C.S. Smith.  1983.   Histological techniques  for marine bivalve
molluscs.   NOAA Technical Memorandum.   NMFS-F/NEC-25.  U.S.  Department of
Commerce, National Oceanic and Atmospheric Administration, Woods Hole,  MA.

Hunn,  J. 1988. Field assessment of the effects of contaminants on fishes. Biological
Report 88, Fish and Wildlife Service, U.S. Department of the Interior, Columbia, MO.

Jeme, N., and A. Nordin. 1963.  Plaque formation  in agar by single antibody-producing
cells.  Science 140: 405.
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                                                                       Appendix A
Jimenez, B., A. Oikari, S. Adams, D. Hinton, and J. McCarthy. 1990.  Hepatic enzymes
as  bio-markers  of environmental,  physiological and  toxicological variables.    In
Biomarkers of environmental contamination, ed. J. McCarthy and  L.  Shugart, pp.
123-142. CRC Press, Boca Raton, FL.

Johnson, R., and H. Bergman.  1984.  Use of histopathology in aquatic toxicology:  A
critique.  In Contaminant effects on fisheries,  ed. V. Cairns, P. Hodson, J. Nriagu. John
Wiley and Sons, New York, NY.

Kieinow, K.,  M. MeLancon,  and J. Lech.  1987.   Biotransformation and induction:
Implications for toxicity, bioaccumulation and monitoring of environmental xenobiotics in
fish. Environ. Health Perspect. 71:105-119.

Klingerman, A.D. 1982.   Fishes as biological detectors of the  effects of genotoxic
agents. In Mutagenicity, new horizons in genetic toxicology, ed. J. Meddle. Academic
Press.

Klontz, G. 1985.  Diagnostic methods in fish  diseases:  Present status and needs.  In
Fish and shellfish pathology, ed. A. Ellis. Academic Press Inc.

Landolt, M.L., and  R.M. Kocan.   1983.   Fish  cell  cytogenetics: A  measure of the
genotoxic effects of environmental pollutants.  In Aquatic toxicology, ed. J. Nriagu.  John
Wiley and Sons, New York, NY.

Larson, A., C.  Haux, and M. Sjobeck.  1985.   Fish physiology  and metal pollution:
Results and experiences from laboratory and  field studies.  Ecotoxicol. Environ. Saf. 9:
250.

Lech, J., M. Vodicnik, and C.  Elcombe. 1982.  Induction of mono-oxygenase activity in
fish. In Aquatic toxicology, ed. L. Weber, pp. 107-148.  Raven Press.

Luna, L. 1968.  Manual of histologic staining methods of the Armed Forces Institute of
Pathology.  McGraw-Hill Book Company, The Blakiston Division, New York, NY.

Malins, D.C., and A. Jensen.  1988. Aquatic toxicology.  Elsevier Science Publishers,
Amsterdam.

Matthews, E., J. Warinner, and  B. Weeks. 1990.  Assays of immune function in fish
macrophages.  In Techniques in fish immunology, ed. J. Stolen, T. Fletcher, D. Anderson,
B. Roberson, and W. van Muiswinkel.  SOS Publications, Fair Haven, NJ.
                                                                           A-23
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Appendix A
May, E.B., M.J. Garreis, and M.M. Lipsky.  1985.  Histological markers of environmental
effect  4th Symposium on Coastal and Ocean Management.

McCarthy, J.F. 1990.  Concluding remarks:  Implementation of a biomarker-based
environmental  monitoring  program.  In Biomarkers of environmental contamination, ed.
J. McCarthy and L. Shugart. Lewis Publishers, Boca Raton, FL.

McCarthy,  J.F.,  and L.R. Shugart,  eds.   1990.   Biomarkers  of environmental
contamination. Lewis Publishers, Boca Raton, FL.

McLea, D., and M. Gordon. 1977.  Leucocrit:  A simple haematological technique for
measuring acute stress in salmonid fish, including stressful concentrations of pulpmill
effluent. J. Fish Res. Bd. Can. 34: 2164.

Meyers, T.R., and J.D. Hendricks.  1985.  Histopathology. \nFundamentalsofaquatic
toxicology: Methods and applications,  ed. G.M. Rand and S.R. Petrocelli,  pp. 283-331.
Hemisphere Publishing Co., New York.

Mix, M.C.  1986.  Cancerous diseases in aquatic animals  and their association with
environmental  pollutants: A critical literature review.  Mar. Environ. Res. 20 (1&2):1-141.

Myers, M.S.,  J.T.  Landahl, M.M.  Krahn,  and B.  B. McCain.   1991.  Relationships
between hepatic neoplasms and related lesions and  exposure to toxic  chemicals in
marine fish from the U.S. West Coast.  Environ. Health Perspect. 90:7-16.

Neff, J.M. 1985.   Use  of biochemical measurements to  detect pollutant-mediated
damage  to  fish.   American  Society  for  Testing and  Materials,  Special Technical
Publication 854:155-181.

Nielson, L.A., and D.L. Johnson, eds.  1984. Fisheries techniques. American Fisheries
Society, Bethesda, MD.

Overstreet, R.M.   1988.  Aquatic  pollution problems, southeastern  U.S.  coasts:
Histopathological indicators. Aquat. Toxicol. 11:213-239.

Passino, D.R.  1984.  Biochemical indicators  of stress in  fishes:  An overview.   In
Aquatic toxicology, ed. J. Nriagu. John Wiley and Sons, New York, NY.

Patton, J., and J. Couch. 1984. Can tissue anomalies that occur in marine fish implicate
specific pollutant chemicals?  In Concepts in marine pollution measurements, ed. H.
White.  Maryland Sea Grant College, University of Maryland.
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                                                                      Appendix A
Payne, J., L. Fancey, A. Rahimtula, and E. Porter. 1987. Review and perspective on the
use of mixed-function oxygenase enzymes in biological monitoring.   Comp. Biochem.
Physiol. C86: 233-245.

Peters, G., H. Delventhal, and H. Klinger.  1980.  Physiological and morphological effects
of social stress in the eel, Anguilla anguilla.  L. Art. Fisch. Wiss. 30:157.

Pickering, A. 1981.  Stress and fish. Academic Press.

Roberts, R. J. 1989. Fish pathology.  Balimore Tindall, London, England.

Rowley, A.  1990.   Collection, separation and identification of fish leucocytes.   In
Techniques in fish immunology, ed. J. Stolen, T. Fletcher, D. Anderson, B. Roberson,
and W. van Muiswinkel. SOS> Publications, Fair Haven, NJ.

Sanders, B.  1990.  Stress proteins. In Biomarkers of environmental contamination, ed.
J. McCarthy and L. Shugart. CRC Press,  Boca Raton, FL.

Schmid, W.  1982.  Chapter 36 in  Chemical mutagens: Principles and methods for their
detection,  ed. A. Hollaender.  Plenum Press.

Shugart, LR.  1990. Biological monitoring: Testing for genotoxicity.  \r\Biomarkersof
environmental contamination, ed. J. McCarthy and L. Shugart. CRC Press, Boca Raton,
FL.

Sindermann, C.J.  1983.  An examination of some relationships between pollution and
disease.  Rapp. P.-V. Reun. Cons. Int. Explor.  Mer. 182: 37-43.

Sindermann, C.J.  1990. Principal diseases of marine fish and shellfish.  Vol. 1 and 2.
Academic Press, San Diego, CA.

Snieszko,  S. 1974.  The effects of environmental  stress on  outbreaks of infectious
diseases of fish. J. Fish Biol. 6: 197-208.

Sorenson, E.M. 1991.  Metal poisoning in  fish.  CRC Press, Boca Raton, FL.

Sorenson, E.M.B., and N.K.R. Smith. 1981.  Hemosiderin granules: Cytotoxic response
to arsenic exposure in  channel catfish,  Ictalurns punctatus.  Bull. Environ.  Contam.
Toxicol. 27: 645-653.
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Appendix A
Sparks, A.K.  1985.  Synopsis of invertebrate pathology exclusive of insects.  Elsevier
Science Publishers, Amsterdam.

Stedman's Medical Dictionary. 1982. 24th ed. Williams & Wilkins, Baltimore, MD.

Stegeman, J.J., and J.J. Lech. 1991.  Cytochrome P-450 systems in aquatic species:
carcinogen metabolism and biomarkers for carcinogen and pollutant exposure.  Environ.
Health Perpect. 90:101-116.

Stegeman, J., K. Renton, B.  Woodin, Y. Zhang, and  R. Addison. 1990.  Experimental
and environmental induction of cytochrome P450E in fish from Bermuda waters.  J. Exp.
Mar. Biol. Ecol. 138: 49-67.

Stegeman, J., B. Woodin, A.  Goksoyr. 1988. Apparent cytochrome P-450 induction as
an indication of exposure to environmental chemicals  in the flounder Platichthys flesus.
Marine Ecol. Prog. Serv. 46: 55-60.

Sumner, B.E.H. 1988. Basic histochemistry. John Wiley and Sons, New York, NY.

Tabor's Cyclopedic Medical Dictionary.  1985.  F. A. Davis Company, Philadelphia, PA.

Thomas, P. 1990.  Molecular and biochemical responses of fish to stressors and their
potential use in environmental monitoring.  Proceedings American Fisheries  Society
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Turgeon, D.D., S.B. Bricker, and T.P. O'Connor.  In press. National status and trends
program:  Chemical  and biological monitoring of U.S. coastal waters.  In  Ecological
indicators, Vol. 1, ed. D.H. McKenzie and D.E. Hyatt.  Elsevier Applied Science, Essex,
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USEPA.  1985. Methods for measuring the acute toxicity of effluents to freshwater and
marine organisms.  3d. ed. EPA/600/4-85/013. U.S. Environmental Protection Agency,
Environmental Monitoring and Support Laboratory, Cincinnati, OH.

USEPA. 1986. Proceedings  and summary of the workshop on finfish as indicators of
toxic  contamination.   July 27-28,  1986,  Arlie, VA.   U.S. Environmental  Protection
Agency, Office of Marine and  Estuarine Protection, Washington, DC.

USEPA. 1987. Guidance for conducting fish liver histopathology studies during 301 (h)
monitoring. EPA 430/09-87-004.  U.S. Environmental Protection Agency, Washington,
DC.
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                                                                     Appendix A
USEPA.  1987.  Bioaccumulation monitoring guidance: Strategies for sample replication
and compositing.  Vol. 5.  EPA 430/9-87-003.  U.S. Environmental  Protection Agency,
Office of Marine and Estuarine Protection, Washington, DC.

USEPA.  1987.  Technical support document for ODES statistical power analysis. EPA
430/9-87-005.  U.S. Environmental Protection Agency, Office of Marine and Estuarine
Protection, Washington, DC.

USEPA.  1988.  Short-term methods for estimating the chronic toxicity of effluents and
receiving  waters  to  marine and  estuarine  organisms.  EPA/600/4-87/028.  U.S.
Environmental  Protection Agency, Environmental Monitoring and Support Laboratory,
Cincinnati, OH.
USEPA. 1989.  Rapid bioassessment protocols for use in streams and rivers.
444/4-89-001. U.S. Environmental Protection Agency, Washington, DC.
EPA
USEPA. 1990.  Three year assessment of reproductive success  in winter flounder,
Pseudopleuronectes americanus (Walbaum), in Long Island Sound, with comparisons to
Boston  Harbor:  1986-1988.   I. Reproductive cycle:  Vitellogenin.  II.  Comparative
reproductive success:  Biology, biochemistry, chemistry.  III.  Comparative embryo
development and mortality.  Final report.   U.S.  Environmental  Protection Agency,
Regions I, II. Long Island Sound Project.

Vogelbein, W., J. Fournie, P. VanVeld, and R. Huggett.  1990.  Hepatic neoplasms in
the mummichog Fundulus heteroclitus from a creosote-contaminated site.  Cancer Res.
50: 5978-5986.

Warinner,  J., E.  Mathews, and B.  Weeks. 1988.   Preliminary investigations  of the
chemiluminescent response in normal and pollutant-exposed fish.   Mar. Environ. Res.
24:281-284.

Wedemeyer G., R. Gould, and W. Yasutake. 1983.  Some potentials  and limits of the
leucocrit test as a fish health assessment method. J. Fish Biol. 23: 711.

Wedemeyer, G., and W. Yasutake. 1977.  Clinical methods for the assessment of the
effects of environmental stress on  fish health.  Technical  paper  89. U.S. Fish and
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Weeks, B., R. Huggett, J. Warinner, and E. Mathews. 1990.  Macrophage responses of
estuarine fish as  bioindicators of toxic contamination.  In Biomarkers of environmental
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                                                                         A-27
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Appendix A
Weeks, B., A.  Keisler, J. Warinner, and E.  Mathews. 1987.  Preliminary evaluation of
macrophage pinocytosis as a technique to  monitor fish health.  Mar.  Environ. Res.
22: 205-213.

Weeks, B.,  and J. Warinner. 1984.   Effects of toxic  chemicals  on macrophage
phagocytosis in two estuarine fishes.  Mar. Environ. Res. 14: 327-335.

Weeks, B., and J. Warinner. 1986.  Functional evaluation of macrophages in fish from a
polluted estuary.  Vet. Immunol. Immun. 12: 313-320.

Weeks, B., J. Warinner, P. Mason, and D. McGinnis. 1986. Influence of toxic chemicals
on the chemotactic response of fish macrophages.  J. Fish Biol.  28: 653-658.

Weis, J.S., P.  Weis, and E.J. Zimmerer. 1990. Potential utility of fin regeneration in
testing sublethal  effects of wastes.  In Oceanic processes in marine pollution.  Vol. 6,
Physical and chemical processes:   Transport and transportation, ed.  D. Baumgortner
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West, G. 1990. Methods of assessing ovarian development in fishes:  A review. Aust.
J. Mar. Freshwater Res. 41:199-222.

Wolke,  R., C.  George,  and V. Blazer.  1984.  Pigmented macrophage accumulations
(MMC;PMB): Possible monitor of fish health.  In Parasitology and pathology of marine
organisms in the world ocean, ed: W. Hargis, p. 93. NOAA Technical Report NMFS 25,
p. 93.

Wydoski, R., and G. Wedemeyer. 1976.  Physiological responses of fish: Problems and
progress toward use in  environmental monitoring.  In Aquatic toxicology, ed  V. Cairns,
P. Hodson, and J. Nriagu. John Wiley and Sons, New York, NY.

Yevich, PP.,  and  C.A.  Barszcz.    1980.   Preparation  of  aquatic animals  for
histopathological examination.  In  International mussel watch, Appendix  6-13,  pp.
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Yevich, P.P., and C.A. Barszcz. 1983. Histopathology as a monitor for marine pollution:
Results of histopathological examinations of the animals collected for the 1976 Mussel
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A-28
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                                                                      Appendix A
                             FISH POPULATIONS

Armstrong, N.E., P.M. Storrs, and H.F. Ludwig. 1970. Ecosystem-pollution interactions in
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Anderberg, M.R. 1973.  Cluster analysis for applications. Academic Press, New York,
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Bechtel, T.J., and B.J. Copeland. 1970. Fish species diversity indices as indicators of
pollution in Galveston Bay, Texas. Contrib.  in Mar. Sci. 15:103-132.

Boesch, D.F.  1977. Application of numerical classification in ecological investigations
of water pollution. EPA 600/3-77-033.  U.S. Environmental Protection Agency, Office of
Research and Development, Corvallis,  OR.

Bond, C.E. 1979. Biology of fishes. Sanders College Publishing, Philadelphia, PA.

Cailliet, G.M., M.S.  Love, and  A.W. Ebeling.  1986.  Fishes: A field and laboratory
manual on their structure,  identification,  and  natural history.  Wadsworth Publishing
Company, Belmont, CA.

Clifford, H.T., and W. Stephenson.  1975.  An introduction to numerical classification.
Academic Press, New York, NY.

Curtis, M.A., and G.H. Peterson. 1978. Size-class heterogeneity with spatial distribution
of subartic marine benthos populations. Astarte 10:103-105.

Gushing, D.J.  1975.  Marine  ecology and fisheries.   Cambridge University Press,
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Ferraro, S.P., F.A. Cole, W.A. DeBen, and  R.C. Swartz.  1989. Power-cost efficiency of
eight macrobenthic sampling schemes in Puget Sound, Washington, USA.  Can. J. Fish.
Aquat. Sci. 46: 2157-2165.

Fredette,  T.J., D.A. Nelson, T. Miller-Way, J.A. Adair, V.A. Sotler, J.E. Clausner,
E.B. Hands, and F.J. Anders.  1989.  Selected tools and techniques for physical and
biological monitoring of aquatic dredged material disposal sites.  Final report. U.S.
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                                                                           A-29
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Appendix A
Gauch, H.G.  1982.  Multivariate analysis in community ecology. Cambridge University
Press, Cambridge, UK.

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biologists. John Wiley and Sons, New York, NY.

Green, R.H.   1984.  Statistical and  nonstatistical  considerations for environmental
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Hurlbert,  S.H.  1971. The nonconcept of species diversity: A critique and alternative
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Hurlbert,  S.H.  1984.  Pseudoreplication and the design of ecological field  experiments.
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Margalef, R. 1969.  Diversity and  stability: A practical proposal and  a model of
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Nielson, L.A., and D.L. Johnson, eds. 1984. Fisheries techniques.  American Fisheries
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Pielou, E.G.  1966,  The measurement of diversity in  different types  of biological
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Ricker, W.E.   1975.  Computation and interpretation  of  biological statistics of fish
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Romesburg,  H.C.   1984.   Cluster analysis   for  researchers.   Lifetime Learning
Publications, Belmont, CA.

Rothschild, BJ. 1986. Dynamics of marine fish populations. Harvard University Press,
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Self, S.G., and  R.H.  Mauritsen.  1988.  Power/sample size calculations for generalized
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Sneath, P.H.A.,  and R.R. Sokal.  1973.  Numerical taxonomy: The principles   and
practices of numerical classification.  Freeman, San Francisco, CA.
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                                                                    Appendix A
Swartz,  R.C.,  D.W. Schultz, G.R. Ditsworth, W.A.  DeBen, and F.A.  Cole.   1985.
Sediment toxicity, contamination, and macrobenthic communities near a large sewage
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effects of contaminants in aquatic ecosystems, ed. T.T. Boyle.  American Society for
Testing and Materials (ASTM), Philadelphia, PA.

Tsai, Chu-Fa. 1968. Effects of chlorinated sewage effluents on fishes in Upper Patuxent
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USEPA.  1978.  Use  of  small otter trawls  in  coastal  biological surveys.   EPA
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USEPA.  1982.  Design of 301 (h)  monitoring  programs  for municipal wastewater
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USEPA.  1985.  Recommended biological Indices for 301 (h) monitoring programs. EPA
430/9-86-002.  U.S. Environmental Protection Agency, Office of Marine and Estuarine
Protection, Washington, DC.

USEPA.  1986-1991.  Recommended protocols for measuring selected environmental
variables in Puget Sound.  Looseleaf.  U.S. Environmental Protection Agency, Region
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USEPA.  1987.  Technical support document for ODES statistical power analysis. EPA
430/9-87-005.  U.S. Environmental Protection Agency, Office of Marine and Estuarine
Protection, Washington, DC.

USEPA.  1988.  ODES data brief: Use of numerical classification.  U.S. Environmental
Protection Agency, Office of Marine and Estuarine Protection, Washington,  DC.

USEPA.   1990.  Environmental Monitoring  and Assessment Program: Ecological
indicators.   EPA 600/3-90-060.   U.S. Environmental  Protection Agency, Office  of
Research and Development, Washington, DC.

Whipple, J.A., M.  Jung, R.B.  MacFarlane,  and R. Fischer. 1984.  Histopathological
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                                                                         A-31
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Appendix A
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Abaychi, J.K., and J.P. Riley. 1979. The determination of phytoplankton pigments by
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Ahlstrom,  E.  1969.   Recommended  procedures  for measuring the productivity of
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ASTM.  1979.  American Society for Testing and Materials. Water.  In Annual book of
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Azam, F., T. Fenchel, J.G. Field, J.S.  Gray, L.A. Meyer-Reil, and F. Thingstad.  1983.
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Beers, J.R. 1978.  Pump sampling. In Monographs on oceanographic methodology. 6.
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Beers, J.R., G.L. Stewart, and J.D.H. Strickland.  1967. A pumping system for sampling
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Boesch, D.F. 1977. Application of numerical classification in ecological investigations of
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Brown, L.M., B.T. Hargrave, and M.D. Mackinnon. 1981.  Analysis of chlorophyll a in
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Bumison, B.K. 1980.  Modified  dimethyl sulfoxide  (DMSO) extraction for chlorophyll
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Burrell, V.G., W.A. Van Engel, and S.G. Hummel. 1974. A new device for subsampling
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                                                                     Appendix A
Chesapeake Executive Council, 1988.  Living resources monitoring plan.  Chesapeake
Bay Program, Annapolis, MD.

Clifford, H.T., and W. Stephenson.  1975.  An introduction to numerical classification.
Academic Press, San Francisco, CA.

Cochran, W.G.  1963.  Sampling techniques. 2d ed. John Wiley and Sons, Inc., New
York, NY.
                       >       »
Cochran, W.G.  1977.  Sampling techniques. 3d ed. John Wiley and Sons, Inc.  New
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Copeland, B.J.  1966. Effects of industrial waste on the marine environment. J. Water
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Copeland,   B.J.,   and   D.E.   Wohlschlag.   1968.   Biological   responses   to
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Cooley, W.W.,  and  P.R. Lohnes. 1971.  Multivariate  data analysis. John Wiley and
Sons, Inc. New York, NY.

Grossman, J.S., R.L. Kaisler, and J. Cairns, Jr. 1974. The use of cluster analysis in the
assessment of spills of hazardous materials. Amer, Midland Natur. 92:94-114.

D'Elia,  C.F.,  K.L.  Webb,  D.V.  Shaw,  and  C.W. Keefe.    1986.  Methodological
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Garside, C.,  and J.P.  Riley.   1969.  A thin-layer chromatographic  method  for the
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                                                                          A-33
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 Appendix A
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 Gieskes, W.W.C.,  and G.W. Kraay.  1983.  Dominance of Cryptophyceae during the
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 Goeyens, L, E. Post,  F. Deharis, A. Vandenhoudt, and W. Baeyens.  1982. The use of
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 Gordon, D.C., and  W.H. Sutcliffe, Jr.  1974.  Filtration of seawater using silver filters for
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 Grasshoff, K., M. Ehrhardt, and K. Kremling.  1973. Methods of seawater analysis, 2d
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 Green, R.H.  1980.  Multivariate approaches in ecology:  The assessment of ecologic
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 Green, R.H.  1984.  Some guidelines for the design of biological monitoring programs in
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 Green, R.H., and G.L. Vascotto.  1978.  A method for the analysis of environmental
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 Heck, K.L., Jr., and R.J. Horwitz. 1984. Statistical analysis of sampling data to assess
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 Hollander,  M., and  D.A. Wolfe.  1973. Nonparametric statistical methods. John Wiley
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 Hurlbert, S.H. 1971.  The nonconcept of species diversity:  A critique and alternative
 parameters. Ecol. 52:577-586.
A-34
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                                                                      Appendix A
Inskeep, W.P., and P.R. Bloom. 1985.  Extinction coefficients of chlorophyll a and b in
N, N-dimetylformamide and 80% acetone.  Plant Physiol.  77:483-485.

Jacobs, F., and G.C. Grant.  1978.  Guidelines for zooplankton sampling in quantitative
baseline  and monitoring programs.  Rep. No. 600/3-7-78-026.   U.S. Environmental
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Jeffrey, S.W.,  and G.M. Hallegraeff.  1980.  Studies of phytoplankton  species and
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Jeffrey,  S.W., and G.F.  Humphrey.   1975.   New spectrophotometric equations for
determining  chlorophyll  a,  b,  cl  and  c2  in  higher plants,  algae and  natural
phytoplankton. Biochem.  Physicol. Pflanzen. 167:191-194.
Jeffrey,  S.W., M. Sielicki, and  F.T. Hazo.
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Lorenzen, C.J. 1966. A method for the continuous measurement of in vivo chlorophyll
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Lorenzen,  C.J.   1967.     Determination   of   chlorophyll   and   pheo-pigments:
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Magnien, R.E. 1986.  A comparison of estuarine water quality chemistry analysis on the
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Ecological Modeling and Analysis Division, Technical Report.  Baltimore, MD.

Mantoura,  R.F.C.,  and  C.A. Llewellyn.   1983.   The rapid  determination of algal
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                                                                           A-35
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 Appendix A
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 Odum,  H.T., R.P. Cuzan  du Rest,  R.J.  Beyers,  and C.  Allbaugh. 1963.  Diurnal
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 Parsons, T.R., Y. Maita, and C.M. Lalli.  1984.  A manual of chemical and biological
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                                                                      Appendix A
Saila, S.B., D. Chen, V.J.  Pigoga, and S.D. Pratt.  1984.  Comparative evaluation of
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                                                                          A-37
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Appendix A
Speziale,  B.J.,  S.P. Schreiner,  P.A.  Giammatteo,  and  J.E.  Schindler.    1984.
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UNESCO.  1978.   Monographs on oceanography methodology.   6.   Phytoplankton
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A-38
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                                                                     Appendix A
USEPA.  1979.  Handbook for analytical  quality  control in  water and  wastewater
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USEPA.  1985.  Interim guidance on quality assurance/quality control (QA/QC) for the
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USEPA.  1987.  Technical support document for ODES statistical power analysis. EPA
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USEPA.  1989.  Chesapeake Bay basin monitoring program atlas (Vols.  1 and 2). U.S.
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USEPA.  1989.  Compendium of methods  for marine and  estuarine environmental
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Wiebe, P.M., and W.R.  Holland. 1968. Plankton patchiness:   Effects on repeated  net
tows.  Limno. Oceanogr. 13:315-321.

Williams, W.T. 1971. Principles of clustering. An. Rev. Ecolo. Syst.  2:303-326.

Winer, B.J.  1971.  Statistical principles in experimental  design.   McGraw-Hill Book
Company, New York,  NY.

Wood, A.M.  1979. Chlorophyll a:b in marine planktonic algae.  J. Phycol. 15: 330-332.
                                                                          A-39
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Appendix A


Wood, L.W. 1985.,  Chloroform-methanol extraction  of chlorophyll a.  Can. J. Fish.
Aquat Sci. 42: 38-43.

Yentsch,  C.S.,  and D.W.  Menzel.   1963.   A method for the determination  of
phytoplankton  chlorophyll  and  phaeophytin   by  fluorescence.   Deep-Sea  Res.
10:221-231.

Zar, J.H. 1974. Biostatistical analysis.  Prentice-Hall, Inc., Englewood Cliffs, NJ.
                     HABITAT IDENTIFICATION METHODS

Abraham,  B.J.,  and  P.L.  Dillon.   1986.   Species profiles:    Life histories  and
environmental requirements of coastal fishes and invertebrates.  (Mid Atlantic)—Soft
shell clam.  U.S. Fish and Wildlife Service, FWS/OBS-82/11.68.  U.S. Army Corps of
Engineers, TR EL-82-4.

Adamus,  P.R.   1988.  The  FHWA/Adamus  (WET) method for wetland  functional
assessment.  In The ecology and management of wetlands, ed. D.D. Hook et al., pp.
128-133. Croom Helm Publishers.

Adamus, P.R., L.T. Stockwell,  EJ. Clairain, Jr., M.E. Morrow,  L.P. Rozas, and  R.D.
Smith.  1987.  Wetland  evaluation  technique (WET).  Vol. I.  Literature review and
evaluation  rationale.  U.S. Army Corps of Engineers, Waterways Experiment Station,
Vicksburg, MS.

Adamus,  P.R., E.J.  Clairain,  Jr.,  D.R. Smith, and  R.E.  Young.   1987.   Wetland
evaluation  technique (WET).   Vol.  II.  Technical report Y-87.   U.S.  Army Corps of
Engineers, Waterways Experiment Station, Vicksburg, MS.

Aggus,  L.R., and W.M.  Bivin.  1982.  Habitat suitability index models:  Regression
models based on harvest of cool  and coldwater fishes in reservoirs.  U.S. Fish and
Wildlife Service, Biological Services Program, Washington, DC.

Anon.  1985. Proposed policy and procedures for fish habitat management. Department
of Fisheries and Oceans, Ottawa, Ontario, Canada.

Beauchamp, R.B.,  ed. 1974.   Marine environment planning guide for the Hampton
Roads/Norfolk naval operating area. Spec. Pub. No. 250. Naval Oceanographic Office,
Washington, DC.
A-40
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                                                                       Appendix A
Bain, M.B., and J.L. Bain.  1982.  Habitat suitability index model:  Coastal stocks of
striped bass.  Rep. Natl. Coastal Ecosystems Team, U.S. Fish. Wildlife Service, Rep.
No. FWS/OBS 82/10.1, Washington, DC.

Chesapeake Executive Council. 1988. Habitat requirements for Chesapeake Bay living
resources. Chesapeake Bay Program, Annapolis, MD.

Colwell, M.A., and L.W. Oring.  1988.  Habitat use by breeding and migrating shorebirds
in south central Saskatchewan. Wilson Bulletin 100(4):554-566.

Cowardin, L.M., V. Carter, F,,C. Golet, and E.T. LaRoe.  1979. Classification of wetland
and deepwater habitats of the United States.  U.S. Fish and Wildlife Service, Office of
Biological Services, Washington, DC.

Dee, N.,  J. Baker,  N. Drobner, K. Duke,  I. Whitman, and  D.  Fahrigner.   1973.
Environmental  evaluation system for water resources planning,   Water  Res. Res.
9(3):523-534.

Department of Fisheries and Oceans.  1984.  A  fishery officer's guide for fish habitat
management and protection. Ottawa, Ontario, Canada.

Diaz, R.J., and C.P. Onuf,  1985.  Habitat suitability index models: Juvenile Atlantic
croaker.  Revised.  Biological reports of the U.S. Fish and Wildlife Service, Washington,
DC.

Fay, C.W., R.J. Neves, and G.B. Pardue.  1983.  Species profiles: Life histories and
environmental requirements of coastal fishes and invertebrates (mid-Atlantic) - Striped
bass. WWS/OBS 82/11.8. U.S. Fish and Wildlife Service, Washington, DC.

Hardy, J.D., Jr. 1978.  Development of fishes of the mid-Atlantic Bight: An atlas of the
egg, larval and juvenile  stages.  Vol. III.  U.S.  Department of the Interior, Fish  and
Wildlife Service, Biological Service Program.  FWS/OBS-78/12.

Heinen, J.I., and R.A. Mead.   1982.  The application of remote sensing to site-  and
species-specific wildlife  habitat analysis.  Technical Reports of Virginia Polytechnical
Institute, RR-82-2, NFAP-292.

Johnson, G.D.  1978.  Development of fishes of the mid-Atlantic Bight. An atlas of egg,
larval  and juvenile  stages,.   Volume  IV.  Carrangidae through Ephippidae.   U.S.
Department of  the  Interior, Fish and  Wildlife Service,  Biological  Services  Program.
FWS/OBS-78/12.
                                                                           A-41
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Appendix A
Johnson, H.B., B.F. Holland, Jr., and S.G. Keefe. 1977. Anadromous fisheries research
program, northern coastal area. N.C. Div. Mar. Fish. Rep. No. AFCS-11.

Jones, P.W., F.D. Martin, and J.D. Hardy, Jr.  1978.  Development of fishes of the
mid-Atlantic Bight An atlas of egg, larval and juvenile stages. Vol. I.  U.S. Department
of the Interior, Fish and Wildlife Service, Biological Service Program. FWS/OBS-78/12.

Klein, R.( and J.C. O'Dell.  1987.  Physical habitat requirement for fish and other living
resources inhabiting class I and II waters.  Internal document. Maryland Department of
Natural Resources, Tidewater Administration.

Lonard, R.I., E.J. Clairain, Jr., R.T. Huffman, J.W. Hardy, L.D. Brown,  P.E. Ballard, and
J.W. Watts.  1981. Analysis of methodologies for assessing wetlands value.  U.S. Water
Resources Council,  Washington, DC, and U.S. Army Corps of Engineers, Vicksburg,
MS.

Lonard, R.I., and E.J. Clairain,  Jr.  1986.   Identification of methodologies for  the
assessment of wetland functions  and values,  In  Proceedings: National wetlands
assessment symposium, pp.  66-72.  Association of State Wetland Managers,  Inc.,
Portland, ME, June 17-20.

Marble, A.D., and M. Gross. 1984. A method for assessing wetland characteristics and
values.  Landscape Plan. 2:1-17.

Reppert, R.T.,  W. Siglero,  E.  Stakhiv, L. Messman, and C. Meyers.   1979.   Wetland
values:   Concepts  and methods  for wetlands evaluations.  IWR Research Report
79-R-1, U.S. Army Engineer Institute for Water Resources, Fort Belvoir, VA.

SCS Engineers.  1979. Analysis of selected functional characteristics of wetlands.
Contract No. DACW73-78-R-0017, Reston, VA.

Segar, D.A. 1987. The Aquatic Habitat Institute: A new concept  in estuarine  pollution
management.  Proceedings of the Tenth National Conference Estuarine and Coastal
Management:  Tools of the Trade.  New Orleans, Louisiana,  12-15 October 1986.  Vol.
2, p. 501.

Solomon,  R.D., B.K. Colbert,  W.J.  Hanses, S.E. Richardson, L.W. Ganter, and  E.G.
Vlachos.   1977.    Water Resources Assessment Methodology  (WRAM)-lmpact
assessment and alternative evaluation. Technical report Y-77-1 /Environmental. Effects
Laboratory, U.S. Army Engineer Waterways Experiment Station, CE, Vicksburg, MS.
A-42
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                                                                     Appendix A
State of Maryland  Department of Natural Resources.   Undated.   Environmental
evaluation of coastal wetlands. Draft. Tidal Wetlands Study, pp. 181-208.

Toole,  C.L., R.A.  Barnhart, and C.P.  Onuf.  1987.  Habitat suitability index models:
Juvenile English  sole.   Biological reports  of. the  U.S.  Fish  and  Wildlife Service,
Washington, DC.

U.S. Army Construction Engineering  Research Lab.  1987. Environmental gradient
analysis, ordination and  classification in  environmental impact assessments.  Final
report.  Champaign, IL.

U.S. Army Engineer Division, Lower  Mississippi Valley.  1980.  A habitat evaluation
system for water resources planning. U.S. Army Corps of Engineers, Lower Mississippi
Valley Division, Vicksburg, MS.

USEPA.  1982.  Research on fish and wildlife  habitat.  U.S. Environmental Protection
Agency, Office of Research and Development, Washington, DC.

USEPA.  1984.  Final ocean discharge criteria evaluation Navarin Basin OCS lease sale
83. U.S. Environmental Protection Agency, Region 10, Seattle, WA.

USEPA.  1988.  Use of geographic information systems for wetlands protection.  U.S.
Environmental Protection Agency, Office of Wetlands  Protection, Washington, DC.

USFWS.  1978.  Classification,  inventory and analysis of fish and wildlife habitat:
Proceedings of a national symposium, Phoenix, Arizona, January  24-27, 1977.  U.S.
Fish and Wildlife Service, Office of Biological Services, Washington, DC.

USFWS.  1980.  Habitat evaluation  procedures   (HEP)  manual (102ESM).   U.S.
Department of the Interior, Fish and Wildlife Service, Washington, DC.

USFWS. 1982. Standards for the development of habitat suitability index models. 103
ESM. U.S. Fish and Wildlife Service.
                             BIOACCUMULATION

Boehm, P.D. 1984. The Status and Trends Program: Recommendations for design and
implementation of the chemical measurement segment. Workshop report.  NOAA,
Rockville, MD.
                                                                          A-43
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Appendix A
deBoer, J. 1988.  Chlorobiphenyls in bound and non-bound lipids of fishes: Comparison
of different extraction methods. Chemosphere 17:1803-1810.

DiToro, D.M., J.D. Mahony, D.J. Hanson, KJ. Scott, A.R. Carlson, and G.T. Ankley. In
press.  Acid volatile  sulfide  predicts the acute  toxicity of cadimum and nickel in
sediments.

Ferraro, S.P., H. Lee, R.J. Ozretich, and D.T. Specht. 1990. Predicting bioaccumulation
potential: A test  of  a fugacity-based model.   Arch.  Environ. Contam.  Toxicol. 19:
386-394.

Fowler, S.W. 1982.  Biological transfer and transport processes.  In Pollutant transfer
and transport in the sea, vol. 2, ed. G. Kullenberg. CRC Press, Boca Raton, FL.

Gardner, W.S., W.A.  Frez, E.A.  Cichocki, and C.C. Parrish. 1986.  Micromethod for
lipids in aquatic invertebrates.  Limnol. Oceanogr. 30:1099-1105.

Gardner, W.S., T.F.  Nalepa, W.A.  Frez, E.A. Cichocki, and P.F. Landrum.  1985.
Seasonal patterns in lipid content of Lake Michigan macroinvertebrates. Can. J. Fish.
Aquat Sci. 42:1827-1832.

Goldberg, E.D., V.T. Bowen, G.H. Farrington, J.H. Martin, P.L. Parker, R.W. Risebrough,
W.  Robertson,  E. Schneider and E. Gamble.  1978.   The mussel watch. Environ.
Conserv. 5:101-125.

Hansen, P.O., H. Von Westerhagen, and H. Rosenthal. 1985.  Chlorinated hydrocarbons
and hatching success in Baltic herring spring spawners. Mar.  Environ. Res. 15: 59-76.

Hiatt, M.H. 1981. Analysis of fish and sediment for volatile priority pollutants. Anal.
Chem. 53:1541-1543.

Karickhoff, S.W., D.S. Brown,  and T.A. Scott. 1979. Sorption of hydrophobic  pollutants
on natural sediments.  Wat. Res. 13: 241-248.

Knezovich, J.P.  and  F.L. Harrison. 1987.   A  new method for determining  the
concentration of volatile organic compounds in sediment interstitial water. Bull. Environ.
Contam. Toxicol. 38: 837-940.
A-44
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                                                                       Appendix A
Ladd, J.M., S.P. Hayes, M. Martin, M.D. Stephenson, S.L. Coale, J. Linfield, and
M.  Brown. 1984.  California  state  mussel  watch: 1981-1983.  Trace metals and
synthetic organic compounds in  mussels from  California's  coast,  bays, and
estuaries. Biennial  Report.    Water Quality  Monitoring Report No. 83-6TS.
Sacramento, CA.

Lake, J.L., N.I.  Rubinstein, and S.  Parvignano.  1987.   Predicting bioaccumulation:
Development of a  partitioning model for use as a screen  tool in  regulating  ocean
disposal of wastes.  In  Fate  and  effects  of sediment-bound  chemicals  in aquatic
systems,  ed  K.L.  Dickson, A.W. Maki,  and W.A.  Brungs. Sixth Pellston  Workshop,
Florissant, CO.

Landrum, P.P.,  and J.A. Robbins.  In press. Bioavailability  of sediment-associated
contaminants to benthic invertebrates. In Sediments: Chemistry and toxicity of in-place
pollutants, ed. J.P. Giesy, R. Baudo, and H. Muntau. Lewis Publishers.

Niimi, A.J. 1983.  Biological and toxicological effects of environmental contaminants in
fish and their eggs.  Can. J.  Fish. Aquat. Sci. 40: 306-312.

Pearson, T.H., and R. Rosenberg. 1978. Macrobenthic succession in relation to organic
enrichment and pollution of the marine environment. Oceanogr. Mar. Biol. Ann. Rev. 16:
229-311.

Phillips, D.J.H. 1980.  Quantitative aquatic biological indicators. Applied Science Publ.
Ltd., London,  England.

Phillips, D.J.H., and D.S. Segar. 1986. Use of bio-indicators in monitoring conservative
contaminants: Programme design imperatives.  Mar. Poll. Bull. 17:10-17.

Rubinstein, N.I., J.L. Lake, R.J. Pruell, H. Lee, B. Taplin, J. Heltshe, R. Bowen, and S.
Parvignano.   1987.    Predicting  bioaccumulation  of  sediment-associated  organic
contaminants: Development of a regulatory tool for dredged material evaluation. Internal
report. U.S. Environmental Protection Agency,  Narragansett, Rl.

Spies, R.B.,  D.W. Rice,  and J. Felton. 1988.   Effects  of organic  contaminants on
reproduction of the starry flounder Platichthys stellatus in San Francisco Bay. Mar. Biol.
98:  181-189.

Tetra Tech. 1985. Bioaccumulation  monitoring guidance: Estimating the potential for
bioaccumulation of priority pollutants and 301 (h) pesticides discharged Into marine and
estuarine waters.  Vol. 1. Tetra Tech,  Inc., Bellevue, WA.
                                                                           A-45
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Appendix A
Tetra Tech. 1985.  Bioaccumulation monitoring guidance: Selection of target species
and review of available bioaccumulation data. Vol. 2.  Tetra Tech, Inc., Bellevue, WA.
Tetra Tech.  1985.   Bioaccumulation monitoring guidance:
detection limits. Vol. 3. Tetra Tech, Inc., Bellevue, WA.
Recommended analytical
Tetra Tech. 1986.  Bioaccumulation monitoring guidance:  Analytical methods for U.S.
EPA priority pollutants and 301(h) pesticides in tissues  from  estuarine and marine
organisms. Vol. 4.  Tetra Tech, Inc., Bellevue, WA.
Tetra Tech. 1987.   Bioaccumulation monitoring guidance:
replication and compositing.  Vol.5. Tetra Tech, Inc.
  Strategies  for sample
USEPA. 1982.  Method for use of caged mussels to monitor for bioaccumulation and
selected biological responses of toxic substances in municipal wastewater discharges to
marine waters. Draft.  U.S. Environmental Protection Agency, Environmental Monitoring
Support Laboratory, Cincinnati, OH.

USEPA.  1985.  Bioaccumulation monitoring guidance:  Selection of target species and
review of available bioaccumulation data. Vol. 2.  EPA 403/9-86-006. Office of Marine
and Estuarine Protection, Washington, DC.

USEPA.  1986.  Bioaccumulation monitoring guidance:  Analytical methods for USEPA
priority pollutants and 301 (h) pesticides in tissues from estuarine and marine organisms.
Vol. 4. EPA 503/6-90-002. Office of Marine and Estuarine Protection, Washington, DC.

USEPA.  1987.  Quality assurance/quality  control (QA/QC) for  301 (h)  monitoring
programs:  Guidance on field and laboratory methods. EPA 430/9-86-004.  Office of
Marine and Estuarine  Protection, Washington, DC.

USEPA. 1989.  Assessing human  health risks from  chemically contaminated fish and
shellfish: A guidance  manual.  U.S. Environmental Protection Agency, Office of Marine
and Estuarine Protection, Washington, DC.

USEPA.  1989.  Guidance  manual:    Bedded sediment   bioaccumulation  tests.
EPA/600/X-89/302.  ERLN-N111. U.S. Environmental  Protection Agency, Environmental
Research Laboratory - Newport, OR.

USEPA. 1990.  Assessment and control of bioconcentratable contaminants in surface
waters. Draft report. U.S. Environmental Protection Agency, OWEP, Washington, DC.
A-46
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                                                                     Appendix A
USEPA.  1990.   Computerized  Risk and Bioaccumulation  System  (Version  1.0).
ERLN-N137.  U.S.  Environmental  Protection  Agency,  Environmental  Research
Laboratory, Narragansett, Rl.

USEPA.  1991.  Technical support document  for water quality-based toxics control.
EPA/505/2-90-001. U.S. Environmental Protection Agency, Washington, DC.

Young,   D.R.,  A.J.  Mearns,  and  R.W.  Gosset.  1990.    Bioaccumulation  and
biomagnification of DDT and PCB residues in a benthic and a pelagic  food web of
Southern California.
                                 PATHOGENS

Andrews, W.H., and M.W.  Presnell. 1972. Rapid  recovery of Escherichia coli from
estuarine water, Appl. Microbiol. 23:521.

APHA. 1989. American  Public Health Association, American Water Works Association,
American Water Pollution Control Federation. Standard methods for the examination of
water and wastewater. 17th ed. American Public Health Association, Washington, DC.

Bisson,  J.W., and V.J. Cabelli. 1979. Membrane filtration enumeration method for
Clostridium perfringens.  Appl. Environ. Microbiol. 37:55-66.

Bitton, G., B.N. Feldberg,  and S.R.  Farrah.  1979. Concentration of enteroviruses from
seawater and tap water by organic flocculation using non-fat dry mile and casein water.
Air Soil Pollut. 10:187.

Booz-Allen & Hamilton.   1983.  A  background document on  pathogenic organisms
commonly found  in municipal sludge.  Prepared  for U.S. Environmental Protection
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Bordner, R.H.,  J.A Winter, and P.V Scarpino, eds. 1978.  Microbiological methods for
monitoring the environment,  water and waste. EPA/600/8-78-017.  U.S. Environmental
Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, OH.

Borrego, JJ., F. Arrabal, A. de Vicente, L.F. Gomez, and P. Romero.  1983. Study of
microbial inactivation  in  the  marine environment.  J.  Water Pollut.  Control Fed.
55:297-302.
                                                                          A-47
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Appendix A
Brezenski,  F.T., and J.A. Winter. 1979. Use of the delayed incubation membrane filter
test for determining coliform/bacteria in sea water. Water Res. 3:583.

Buras, N.  1974.  Recovery  of  viruses from waste-water and effluent by the  direct
inoculation  method.  Water Res. 8:19.

Cabelli, VJ. 1983. Health effects criteria for marine waters.  EPA/600/1-80-0431, U.S.
Environmental Protection Agency, Cincinnati, OH.

Cabelli, V.J., A.P.  DuFour,  M.A.  Levin,  L.J.  McCabe, and  P.W. Haberman.  1979.
Relationship! of microbial indicators to health effects at marine bathing beaches.  Am.J.
Public Health 69:690-696.

Cabelli, VJ., A.P. DuFour, L.J.  McCabe, and  M.A.  Levin. 1982. Swimming-associated
gastroenteritis and water quality. Am. J. Epidemiol. 115:606-61.

Cabelli, V.J., A.P. DuFour, L.J.  McCabe, and  M.A.  Levin. 1983. A marine recreational
water quality criterion consistent with indicator concepts and risk analysis.  J.  Water
Pollut. Control Fed. 55:1306-1314.

CDC. 1979. Viral  hepatitis outbreaks-Georgia,  Alabama. Centers for Disease Control.
Morbid. Mortal. Weekly Rep. 28:581.

Chang, S.L., G. Berg, K.A. Busch, R.E. Stevenson, N.A.  Clarke, and P.W. Kabler.  1958.
Application of the Most Probable  Number method for estimating  concentrations  of
animal viruses by tissue  culture technique. Virology 6:27.

Clark, H.F., E.E. Geldreich, H.L. Jeter, and P.W Kabler. 1951. The membrane filter in
sanitary bacteriology. Pub. Health Rep. 66:951.

Clark, J.A.  1969.  The detection of various bacteria indicative of water pollution by a
presence-absence (P-A) procedure. Can. J. Microbiol. 15:771.

Clark, J.A.  1980. The influence  of increasing numbers of nonindicator organisms upon
the detection of indicator organisms by the membrane filter and presence-absence tests.
Can. J. Microbiol. 26:827.

Clark, J.A., and O.T. Vlassoff. 1973.  Relationships among pollution  indicator bacteria
isolated from raw water and distribution systems by the presence-absence (P-A) test.
Hea/tf7Lab.Sc/.10:163.
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                                                                      Appendix A
Oliver, D.0.1967. Detection of enteric viruses by concentration with polyethylene glycol.
In Transmission of viruses by the  water route, ed. G. Berg.  Interscience Publ., New
York, NY.

Cooney, M.K. 1973. Relative efficiency of cell cultures for detection of viruses. Health
Lab. Sci. 4:295.

Cowles, P.B. 1939. A modified fermentation tube. J. Bacteriol. 38:677.

Dalla Vallee,  J.M.  1941. Notes on the most probable  number index as  used in
bacteriology. Pub. Health Rep. 56:229.

Dobberkau, H.J., R. Walter, and S. Rudiger. 1981. Methods for virus concentration from
water. In Viruses and wastewater treatment, ed. M. Goddard and M. Butler. Pergamon
Press, New York, NY.

DuFour,  A.P., E.R. Strickland,  and VJ.  Cabelli.  1981.  Membrane filter method for
enumerating Escherichia coli. Appl. Environ. Microbiol. 41:1152-1158.

Dutka, B.D. 1981.  Membrane filtration applications, techniques and problems. Marcel
Dekker, Inc., New York, NY.

Emerson, D.J., and V.J. Cabelli. 1982. Extraction of Clostridium perfingens spores from
bottom sediment samplers. Appl. Environ. Microbiol. 44:1152-1158.

Ericksen,  T.H.,  C.  Thomas, and  A.  Dufour. 1983.  Comparison of two  selective
membrane filter methods for enumerating fecal streptococci in freshwater  samples.
Abs. Annual Meeting, American Soc. Microbiology, p. 279.

Evans, T.M., C.E. Warvick, RJ. Seidler, and M.W. LeChevallier.  1981. Failure of the
most-probable number techniques to detect conforms in drinking water and raw water
supplies. Appl. Environ. Microbiol. 41:130.

Farrah, S.R. 1982. Isolatin of viruses associated with sludge particles.  In Methods in
environmental virology, ed. C.P. Gerba and S.M. Goyal. Marcel Dekker, Inc., New York,
NY.

Feingold, A.O. 1973. Hepatitis from eating steamed clams.   J. Am.  Med. Assoc.
225:526-527.
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Appendix A
Ferraro, S.P., F.A. Cole, W.A. DeBen, and R.C. Swartz. 1989. Power-cost efficiency of
eight macrobenthic sampling schemes in Puget Sound, Washington, USA. Can. J. Fish.
AquatSci. 46:2157-2165.

Fifield, C.W., and C.P. Schaufus.  1958.  Improved membrane  filter medium for the
detection of coliform-organisms. J. Amer. Water Works Assoc. 50:193.

Gameson, A.L.N. 1983. Investigation of sewage discharges  to some British coastal
waters.  Water Resources Centre Technical Report, TR 193.  Bucks, United Kingdom.
Geldreich, E.E., P.W. Kabler, H.L. Jeter, and H.F.  Clark. 1955.  A delayed incubation
membrane filter test for coliform bacteria in water. Amer. J. Pub. Health 45:1462.

Geldreich, E.E., H.L. Jeter, and J.A. Winter. 1967. Technical considerations in applying
the membrane filter procedure. Health Lab. Sci. 4:113.

Gerba, C.P., and S.M. Goyal. 1982. Methods in environmental virology. Marcel Dekker,
Inc., New York, NY.

Gerhards, P., ed. 1981. Manual of methods for general bacteriology. American Soc.
Microbiology, Washington, DC.

Greenberg, A.E., and D.A. Hunt, eds. 1985. Laboratory procedures for the  examination
of seawater and shellfish.  5th ed. American Public Health Association, Washington, DC.

Halvorson, H.O., and N.R. Ziegler. 1933-35.  Application of  statistics to  problems  in
bacteriology.  J. Bacteriol. 24:101; 26:4331, 559, 29:609.

Hardy, J.T. 1982. The  sea surface microlayer: Biology, chemistry, and anthropogenic
enrichment. Prog. Oceanogr. 11:307-328.

Homma, A., M.D. Sobsey, C.Wallis, and J.L. Melnick. 1973.  Virus concentration from
sewage. Water Res. 7:945.

Hsiung, G.D. 1973. Diagnostic virology. Revised ed. Yale Univ. Press, New Haven, CT.

Inhorn, S.L., ed. 1977. Quality assurance practices for health laboratories. American
Public Health Assoc., Washington, DC.
 A-so
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                                                                      Appendix A
Jacobs, N.J., W.L. Zeigler, F:.C. Reed, T.A. Stukel, and E.W. Rice. 1986. Comparison of
membrane filter, multiple-fermentation-tube,  and presence-absence techniques  for
detecting total coliforms in small community water systems.  Appl. Environ. Microbiol.
51:1007.

Kaplan, J.E., R.A. Goodman,  LB. Schonberger, E.G. Lippy, and G.W. Gary. 1982.
Gastroenteritis due to Norwalk virus: An outbreak  association with municipal water
system. J. Infect. Dis. 146:190-197.

Kabler, P.W. 1954. Water examinations by membrane filter and MPN procedures. Amer.
J. Pub. Health 44:379.

Kelly, S., and W.W.  Sanderson. 1962.  Comparison  of various tissue cultures for the
isolation of enteroviruses. Amer. J. Pub. Health 52:455.

Lee, L.H., C.A. Phillips, M.A. South, J.L  Melnick, and M.D. Yow.  1965. Enteric virus
isolations in different cell cultures.  Bull. World Health Org. 32:657.

Lennette, E.H., A. Balows, W.J. Hausler, Jr., and H.J. Shadomy, eds. 1985.  Manual of
clinical microbiology.  4th ed. American Soc. for Microbiology, Washington, DC.

Levin,  M.A.,  J.R.  Fischer,  and  V.J.  Cabelli. 1975. Membrane  filter  technique  for
enumeration of enterococci in marine waters. Appl. Microbiol. 30:66.

Lin, S. 1973. Evaluation of coliform test for chlorinated secondary effluents. J. Water
Pollut. Control Fed. 45:498.

Lin, S.D. 1976. Evaluation of Millipore  HA and HC membrane filters for the enumeration
of indicator bacteria. Appl. Environ. Microbiol. 32:300.

Lund, E., and C.E. Hedstrom. 1969. A study on sampling and isolation methods for the
detection of virus in sewage.  Water Res.  3:823.

Lydholm, B.,  and A.L. Nielsen. 1979. Methods for  detection of virus in wastewater
applied to samples from small scale treatment systems.  Water Res. 14:169.

McCarthy, J.A.,  H.A.  Thomas, and J.E.  Delaney.   1958.  Evaluation of reliability of
coliform density test. Amer. J. Pub. Health 48:12.

McCarthy, J.A., and J.E. Deianey.  1958. Membrane filter media studies. Water Sewage
Works 105:292.
                                                                           A-51
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Appendix A
McCarthy, J.A., J.E. Delaney, and R.J. Grasso.  1961.  Measuring coliforms in water.
Water Sewage Works 108:238.

McCarthy, J.A., and J.E. Delaney. 1965. Methods for measurig the coliform content of
water. Sec. III.  Delayed holding procedures for coliform bacteria. PHS Res. Grant WP
00202 NIH Rep.

McCrady, M.H. 1915. The numerical interpretation of fermentation tube results. J. Infect.
Dis. 12:183.

Morris, R., and W.M. Waite.1980. Evaluation of procedures for recovery of viruses from
water - II detection systems. Water Res. 14:795.

NOAA.  1988. National Marine Pollution  Program  federal plan  for  ocean pollution
research, development and monitoring.  U.S.  Department of  Commerce, National
Oceanic and Atmospheric Administration, Washington, DC.

Ohara, HM H. Naruto, W. Watanabe, and I. Ebisawa. 1983. An outbreak of Hepatitis A
caused by consumption of raw oysters.  J. Hyg. Camb. 91:163-165.

Olson, B.H. 1978. Enhanced accuracy of coliform testing in seawater by a modification
of the most-probable number method. Appl. Microbiol. 36:438.

OTA. 1987.  Wastes in marine environments.  U.S. Congress, Office of Technology
Assessment.  OTA 0-334.

Panezai, A.K., T.J. Macklin, and H.G. Coles. 1965. Coli-aerogenes and Escherichia coll
counts on water samples by means of transported membranes. Proc. Soc. Water Treat.
Exam. 14:179.

Payment, P., C.P. Gerba, C. Wallis and J.L Melnick. 1976. Methods for concentrating
viruses from large  volumes of estuarine water on plated membranes.  Water Res.
10:893.

Pederson, D.C. 1980. Density levels of pathogenic organisms in municipal wastewater
sludges: A literature review. Prepared by Camp Dresser and McKee,  Inc.  for U.S.
Environmental Protection Agency, Office of Research and Development, Cincinnati, OH.

Ramia, S., and S.A. Sattar. 1979. Second-step concentration of viruses in drinking and
surface waters using polyethylene glycol hydroextraction. Can. J. Microbiol. 25:587.
 A-52
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                                                                       Appendix A


 Rao, V.C., U. Chandorkar, N.U. Rao, P. Kumaran, and S.B. Lakhe. 1972.  A simple
 method for concentrating and detecting viruses in wastewater. Water Res. 6:1565.

 Rehm, R., S. Duletsky,  J. Pierce, and R. Sommer. 1983. Contaminants of concern in
 sewage  sludge.  Draft  prepared for Office  of Program Policy and Evaluation, U.S.
 Environmental Protection Agency, Washington, DC.

 Rovozzo, G.C., and  C.N. Burke. 1973. A  manual  of basic  virological techniques.
 Prentice-Hall, Englewood Cliffs, NJ.

 Schmidt, N.J., H.H. Ho, J.L. Riggs, and E.H. Lennette. 1978. Comparative sensitivity of
 various cell culture systems for isolation of viruses from wastewater and fecal samples.
 Appl. Environ. Microbiol. 36:480.

 Seidler, R.J., T.M. Evans,  J.R. Kaufman, C.E. Warvick, and M.W. LeChevallier. 1981.
 Limitations of standard coliform enumeration techniques. J.  Amer. Water Works Assoc.
 73:538.

 Selna, M.W., and R.P. Miele. 1977. Virus sampling in wastewater-field experiences. J.
 Environ. Eng. Div., Proc. Amer. Soc. Civil Eng. 103:693.

 Shuval, H.I., S.  Cymbalista, B. Fattal, and N.  Goldblum. 1967. Concentration of enter?:
 viruses in water by hydro-extraction and two-phase separation.  Transmission of viruses
 by the water route, ed. G. Beirg. Interscience Publ., New York, NY.

 Shuval, H.I., B. Fattal, S.  Cymbalista, and N. Goldblum. 1969. The phase-separation
 method for the concentration  and detection of viruses in water. Water Res. 3:225.

 Slanetz, L.W., and C.H. Bartley. 1957. Numbers of enterococci water, sewage and feces
 determined by the membrane filter technique with an improved medium. J. Bacteriol.
 74:591.

 Sobsey, M.D. 1976. Methods for detecting enteric viruses in water and wastewater. In
 Viruses in water, ed. G. Berg, H.L. Bodily, E.H. Lennette, J.L. Melnick and T.G. Metcalf,
 American  Public Health Assoc., Washington, DC.

 Sobsey, M.D. 1976. Field monitoring techniques and data analysis.  In Virus aspects of
 applying municipal waste  to land, ed. L.B. Baldwin, J.M. Davidson, and J.F. Gerber.
 Univ. Florida, Gainesville, FL.

 Sobsey, M.D. 1982.  Quality of currently available methodology for monitoring viruses in
the environment. Environ. Internal 7:39.
                                                                           A-53
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Appendix A


Sobsey M.D., C.P. Gerba,  C.  Wallis,  and  J.L. Melnick.  1977.   Concentration  of
enteroviruses from large volumes of turbid estuary water. Can. J. Microbiol. 23:770.

St. John, E.W., J.R. Matches, and M.M. Wekell. 1982.  Use of iron milk medium for
enumeration of Clostridium perfrigens. J. Assoc. Off. Anal. Chem. 65:1129-1133.

Strandridge, J.H., and J.J Delfino. 1981.  A-1  Medium: Alternative techniques for fecal
coliform organism enumeration in chlorinated wastewaters.  Appl. Environ. Microbiol.
42:918.

Thomas, H.A., Jr. 1942. Bacterial densities from fermentation tube test. J. Amer. Water
Works Assoc. 34:572.

Taylor, R.H.,  R.H.  Bordner,   and  P.V.  Scarpino.  1973.  Delayed   incubation
membrane-filter test for fecal conforms. Appl. Microbiol. 25:363.

Thomas, H.A., and R.L. Woodward. 1956. Use of molecular filter membranes for water
potability control. J. Amer. Water Works Assoc. 48:1391.

USEPA.  1978.  Microbiological methods  for monitoring the environment.  EPA
600/8-78-017.   U.S. Environmental Protection Agency,  Office of Research and
Development, Environmental Monitoring and Support Laboratory.

USEPA. 1985.  Test methods  for Escherichia coli and enterococci in water by the
membrane  filter procedure.  U.S. Environmental Protection Agency, Environmental
Monitoring and Support Laboratory, Cincinnati, OH.

USEPA. 1986. Ambient water quality criteria for bacteria - 1986.  EPA 440/5-84-002.
U.S. Environmental Protection Agency, Washington, DC.

USEPA.  1986-1991. Recommended protocols for measuring selected environmental
 variables in  Puget Sound.  Looseleaf. U.S. Environmental  Protection Agency, Region
 10, Puget Sound Estuary Program, Seattle, WA.

 USEPA. 1988.   Water quality  standards  criteria  summaries:  A  compilation   of
 state/federal criteria - Bacterial.  U.S. Environmental Protection Agency, Office of Water
 Regulations and Standards, Washington, DC.

 Wallis, C., and J.L. Melnick. 1967. Virus concentration on aluminum and calcium salts.
 Amer. J. EpidemioL 85-459.
 A-54
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                                                                      Appendix A
 Wallis, C., and J.L. Melnick.  1967. Concentration of viruses on aluminum hydroxide
 precipitates.  In Transmission  of viruses by the water route, ed. G. Berg. Interscience
 Publ., New York, NY.

 Wellings, P.M., A.L. Lewis, and C.W. Mountain. 1976. Viral concentration techniques for
 field sample  analysis.   In  Virus aspects of applying  municipal waste to land, ed.
 L.B.Baldwin, J.M. Davidson, and J.F. Gerber. Univ. Florida, Gainesville, FL.

 Weiss, J.E., and C.A. Hunter.  1939. Simplified bacteriological examination of water. J.
 Amer. Water Works Assoc. 31:689.
                       EFFLUENT CHARACTERIZATION

Adelman,  I.R., L.L. Smith Jr., and G.D. Siesennop.  1976.  Acute toxicity of sodium
chloride, pentachlorophenol,  guthion, and hexavalent chromium to fathead minnows
(Pimephales promelas) and  goldfish (Carassius auratus).   J. Fish. Res. Board Can.
33:203-208.

APHA.  1989.  American Public Health Association, American Water Work Association,
Water Pollution Control Federation. Standard methods for the examination of water and
wastewater. 17thed. American Public Health Association,  Washington, DC.
Bergman, H.,  R.  Kimerle, and  A.W.  Maki,  eds.    1985.
assessment of effluents.  Pergamon Press, Inc. Elmsford, NY.
Environmental hazard
Bowman, M.C., W.L. Oiler, T. Cairns, A.B. Gosnell, and K.H. Oliver.  1981.  Stressed
bioassay systems for rapid screening of pesticide residues.   Part I:   Evaluation of
bioassay systems. Arch. Environ. Contam. Toxicol. 10:9-24.

Dowden, B.F., and  H.J. Bennett.  1965.  Toxicity of selected chemicals to  certain
animals. J. Water Pollut. Control Fed. 37(9):1308-1316.

Kimerle, R., W. Adams, and D. Grothe.   1985,  Tiered  assessment of effluents.  In
Environmental hazard assessment of effluents, eds. H. Bergman, R.  Kimerle, and
A. Maki.

Kimerle, R., A. Werner, and W.  Adams.  1983. Aquatic  hazard evaluation, principles
applied to the development of water quality criteria.  In Aquatic toxicology and  hazard
assessment (7th symposium), eds. R. Cardwell and R. Purdy.
                                                                          A-55
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Appendix A
Magnuson, V.R., O.K. Harriss, M.S.  Sun, O.K. Taylor,  and  G.E.  Glass.   1979.
Relationships of activities of metal-ligand species of aquatic toxicity. ACS symposium
series, no. 93.  Chemical modeling in aqueous systems, ed. E.A. Henne, pp. 635-656.

OSHA.  1976.   OSHA safety and health standards, general industry.  OSHA 2206
(revised).  29 CFR 1910.  Occupational Safety and Health Administration, Washington,
DC.

Patrick, R., J.  Cairns, Jr., and A. Scheier.  1968. The relative sensitivity of diatoms,
snails,  and  fish to  twenty  common  constituents of  industrial   wastes.   Prog.
Poirier, S.H., M.L. Knuth, C.D. Anderson-Buchou,  L.T. Brooke, A.R.  Lima,  and
PJ.   Shubat.       1986.      Comparative   toxicity   of   methanol    and
N.N-dimethylformamide  to freshwater fish  and  invertebrates.   Bull.   Environ.
Contam.  Toxicol. 37(4) :6 1 5-621 .

Randall, T.L., and P.V. Knopp.  1980. Detoxification of specific organic substances by
wet oxidation. J. Water Pollut. Control Fed. 52(8): 217-2130.

Schimmel, S.C., G.E. Morrison, and M.A. Heber. 1989.  Marine complex effluent toxicity
program: Test sensitivity,  repeatability, and  relevance to receiving water toxicity.  Env.
Tox. and Chem. 8:739-746.

Stumm, W., and J.J. Morgan.  1981. Aquatic chemistry - An introduction emphasizing
chemical equilibria in natural waters. John Wiley and Sons, Inc., New York, NY.

U.S. Department of Health, Education and Welfare. 1977.  Carcinogens - working with
carcinogens.  Publication no.  77-206.   Public Health Service, Center for Disease
Control, National Institute of Occupational Safety and Health.

US EPA.   1982.   Handbook for  sampling  and  sample  preservation  of  water and
wastewater. EPA 600/4-82-01 9.  U.S. Environmental Protection Agency, Environmental
Monitoring and Support Laboratory, Cincinnati, OH.

USEPA.  1982. Test methods - Technical additions to methods for chemical analysis of
water and wastes.  EPA 600/4-82-055. U.S. Environmental Protection Agency, Office of
Research and Development, Cincinnati, OH. December.

USEPA.   1982.   Water  quality assessment:  A  screening  procedure  toxic and
conventional pollutants.   Parts 1 and  2.  EPA  600/6-82-004.   U.S. Environmental
Protection Agency, Office  of Research and Development, Athens,  GA.
 A-56
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                                                                     Appendix A
USEPA.   1983.   The   treatability  manual.   Vol.  IV.   EPA 600/2-82-001.   U.S.
Environmental  Protection Agency,  Office of  Research and  Development.   GPO,
Washington, DC.

USEPA.  1984.  Effluent and ambient toxicity testing and instream community response
on the Ottawa River, Lima,  Ohio.  EPA 600/2-84-080.  Permits Division, Washington,
DC,  U.S.  Environmental  Protection Agency,  Office of Research and Development,
Duluth, MN.

USEPA.  1984.  CETIS:  Complex Effluent Toxicity Information System. Data encoding
guidelines and procedures. EPA 600/8-84-029. U.S. Environmental Protection Agency,
Office of Research and Development, Duluth, MN.

USEPA.  1984.  CETIS: Complex Effluent Toxicity Information System.  CETIS retrieval
system user's manual.  EPA 600/8-84-030.  U.S.  Environmental.Protection Agency,
Office of Research and Development, Duluth, MN.

USEPA.   1984.  Development of water quality based  permit  limitations for toxic
pollutants; National policy. U.S. Environmental Protection Agency.  Fed. Regist, March
9,1984,49(48).

USEPA.  1984.  Technical guidance manual for performing wasteload allocations, Book
III estuaries.  U.S. Environmental Protection Agency, Office of Water Regulations and
Standards, Washington, DC.

USEPA.  1985.  Methods for measuring the acute toxicity of effluents freshwater and
marine  organisms.   EPA  600/4-85-013.  U.S. Environmental  Protection  Agency,
Environmental Monitoring and Support Laboratory, Cincinnati, OH.

USEPA.  1985.  Short-term methods for estimating the chronic toxicity of effluents and
receiving  waters to freshwater  organisms.  EPA 600/4-85-014.  U.S. Environmental
Protection Agency, Cincinnati, OH.

USEPA.  1986-1988.  Quality criteria for water.  EPA 440/5-86-001. U.S. Environmental
Protection Agency, Office of Water Regulations and Standards, Washington, DC.

USEPA.  1988.  Methods for aquatic toxicity identification evaluations:  Phase I toxicity
characterization procedures.   Draft EPA research series report.  EPA 600/3-88-034.
U.S. Environmental  Protection Agency, Environmental  Research Laboratory,  Duluth,
MN.
                                                                          A-57
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Appendix A
USEPA.  1988. Methods for aquatic toxicity identification evaluations: Phase II toxicity
identification procedures. Draft EPA research series report.  EPA 600/3-88-035.  U.S.
Environmental Protection Agency, Environmental Research Laboratory, Duluth, MM.

USEPA.  1988. Methods for aquatic toxicity identification evaluations:  Phase III toxicity
confirmation procedures.  Draft phase III toxicity series report. EPA/600/3-88-036. U.S.
Environmental Protection Agency, Environmental Research Laboratory, Duluth, MM.

USEPA.  1988. Short-term methods for estimating the chronic toxicity of effluents and
receiving  waters  to  marine and estuarine organisms.   EPA 600/4-87-02.   U.S.
Environmental Protection Agency, Office of Research and Development, Cincinnati, OH.

USEPA.   1988.   Draft  generalized methodology  for conducting  industrial  toxicity
reduction evaluations (TREs).  Draft EPA Research Series Report. U.S. Environmental
Protection Agency, Water Engineering Research Laboratory, Cincinnati, OH.

USEPA.   1989.   Toxicity reduction  evaluation  protocol for  municipal wastewater
treatment plants.   EPA  600/2-88-062.   U.S.  Environmental Protection Agency, Water
Engineering Research Laboratory, Cincinnati, OH.

USEPA.  1989. Short-term methods for estimating the chronic toxicity of effluents and
receiving  waters  to freshwater  organisms.   EPA 600/4-89-001.  U.S. Environmental
Protection Agency, Water Engineering Research Laboratory, Cincinnati, OH.

USEPA.  1989. Biomonitoring for control of toxicity in effluent discharges  to the marine
environment.   EPA 625/8-89-015.  U.S. Environmental Protection  Agency, Office of
Research and Development, Center for  Environmental Research Information.

USEPA.  1990. Permit writer's guide for marine and estuarine discharges. Draft.  U.S.
Environmental  Protection Agency, Office  of   Water Enforcement and Permits,
Washington, DC.

USEPA.  1990. Assessment and control of bioconcentratable contaminants in surface
waters.  Draft.  U.S. Environmental Protection Agency, Office of Water Enforcement and
Permits, Washington, DC.
USEPA.  1991.  Technical support document for water quality-based toxics control.
EPA  505/2-90-001.    U.S.  Environmental  Protection  Agency,  Office  of  Water
Enforcement and Permits, Office of Water Regulations and Standards, Washington, DC.
Walters,  C.I., and C.W. Jameson.
Butterworth Publ., Woburn, MA.
1984.  Health and safety for  toxicity testing.
A-58
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                                                                    Appendix A
                      MESOCOSMS AND MICROCOSMS

Davey, E.W., K.T. Perez, A.E. Soper, N.F. Lackie, G.E. Morrison, R.L Johnson, and J.
F. Heltsche.  In press.  Significance of the surface micro-layer to the environmental fate
of di(2-ethylhexyl) phthalate predicted from marine microcosms.   U.S. Environmental
Protection Agency, Environmental Research Laboratory,  Ecosystems Effects Branch,
Narragansett, Rl.

Donaghay, P.L.  1984.  Utility of mesocosms to assess marine pollution. In Concepts in
marine pollution measurements,  ed. H.H. White, pp. 589-620.  Maryland Sea Grant
College, College Park, MD.

Dwyer,  R.L., and K.T.  Perez.   1983.   An  experimental examination of ecosystem
linearization. The American Naturalist 121 (3):305-323.

Grassle, J.P., and J.F. Grassle. 1984. The utility of studying the effects of pollutants on
single species populations in  benthos  of mesocosms and coastal ecosystems.  In
Concepts in marine pollution measurements, ed. H.H. White, pp. 621-642.   Maryland
Sea Grant College, College Park, MD.

Grice, G.W.  1984. Use of enclosures in studying stress on plankton communities.  In
Concepts in marine pollution measurements, ed. H.H. White, pp. 563-575.   Maryland
Sea Grant College, College Park, MD.

Leffler, J.W. 1984. The use of self-selected, generic aquatic microcosms for pollution
effects assessment. In  Concepts in marine pollution measurements, ed.  H. White, pp.
139-158. Maryland Sea Grant College, College Park, MD.

Oviatt, C.A. 1984.  Ecology as  an experimental science and management tool.  In
Concepts in marine pollution measurements, ed. H.H. White, pp.  539-548  Maryland
Sea Grant College, College Park, MD.

Perez, K.T., E.W. Davey, N.F. Lackie,  G.E. Morrison, P.G. Murphy, A.E. Soper, and
D.L. Winslow. 1984.  Environmental assessment of phthalate ester, di(2-ethylhexyl)
phthalate (DEHP), derived from a marine microcosm. Special Technical Publication
802. American  Society for Testing and Materials (ASTM),  Philadelphia, PA.

Perez, K.T., and G.E. Morrison. 1985.   Environmental assessments from simple test
systems and a  microcosm:  Comparisons of monetary costs.  In  Multispecies toxicity
testing, ed. J. Cairns, pp. 89-95.
                                                                         A-59
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Appendix A
Perez, K.T.,  E.W. Davey, G.E. Morrison, J.A. Cardin, N.F. Lackie, A.E. Soper,
R.J. Blasco, C. Bearce, R.L. Johnson, and S. Marino. 1989. Influence of organic
matter and industrial contaminants  in sewage effluent on marine ecosystems.
ERLN   Publication.  U.S.  Environmental  Protection  Agency,  Environmental
Research Laboratory, Ecosystems Effects Branch, Narragansett, Rl.

Perez, K.T.,  E.W. Davey, J. Heltsche, J.A. Cardin, N.F.  Lackie, R.L. Johnson,
R.J. Blasco, A.E. Soper, and E. Read. 1990. Recovery of Narragansett Bay, Rl:
A feasibility study.  ERLN Contribution No. 1148.   U.S. Environmental Protection
Agency,   Environmental Research  Laboratory,   Ecosystems  Effects  Branch,
Narragansett, Rl.

Perez, K.T.,  G.E.  Morrison, E.W.  Davey, N.F. Lackie, A.E. Soper, R.J.  Blasco,
D.L. Winslow, R.L. Johnson, P.G.  Murphy, J.F. Heltsche.  In press. Influence of
size on  the  fate and  ecological  effects of the pesticide  kepone in  a  physical
simulation model.   U.S.  Environmental  Protection  Agency,  Environmental
Research Laboratory, Ecosystems Effects Branch, Narragansett, Rl.

Pilson, M.E.Q. 1984.  Should we know the  fates of pollutants.  In Concepts in marine
pollution measurements, ed. H.H. White, pp. 575-588.  Maryland Sea Grant College,
College Park,  MD.

Pontasch, K.W., B.R.  Niederlehner,  and  J.  Cairns,  Jr. 1989.   Comparisons  of
single-species microcosm and field responses to a complex effluent.  Environ. Tox. and
Chem. 8:521 -532.

Pritchard, P.H., and A.W. Bourquin.  1984.  A perspective on the role of microcosms in
environmental  fates and  effects assessments.   In Concepts in  marine  pollution
measurements, ed. H.H. White, pp.  117-138.  Maryland Sea  Grant College, College
Park, MD.

Santschi, P.H., U. Nyffeler, R.  Anderson, and S. Schiff. 1984.  The enclosure as a tool
for the assessment of transport and effects of pollutants in lakes. In Concepts in marine
pollution measurements, ed. H.H. White, pp. 549-562.  Maryland Sea Grant College,
College Park,  MD.

Taub, F.B. 1984.  Introduction to  laboratory  microcosms.   In Concepts in marine
pollution measurements, ed. H.H. White, pp. 113-116.  Maryland Sea Grant College,
College Park,  MD.
A-60
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                                                                    Appendix A
Taub, F.B. 1984.  Measurement of pollution in standardized aquatic microcosms.  In
Concepts in  marine pollution measurements, ed. H.H. White, pp. 159-192.  Maryland
Sea Grant College, College Park, MD.
USEPA. 1983.  Project summary:  Experimental marine microcosm test protocol and
support document.   EPA-600/S3-83-055.  U.S. Environmental Protection Agency,
Environmental Research Laboratory, Narragansett, Rl.
USEPA.  1987.
36352-36360.
Site-specific  aquatic  microcosm test.   Fed. Regist.  52(187):
USEPA. 1990.  Experimental marine microcosm test protocol and support document.
Revised.  U.S. Environmental Protection Agency, Environmental Research Laboratory,
Ecosystems Effects Branch Narragansett, Rl.
                                                                         A-61
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              APPENDIX B:

       OCEAN DISCHARGE CRITERIA
PUBLISHED AT FR Vol. 45, No, 194, 65942-65954
            OCTOBERS, 1980
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 J35942
                                                                                      Appendix B

Federal Register / Vol. 45, No. 194  /  Friday,  October 3, 1980 / Rules and Regulations	
ENVIRONMENTAL PROTECTION
AGENCY

40 CFR Part 125

[FRL 1609-1]

Ocean Discharge Criteria

AGENCY: Environmental Protection
Agency.

ACTION: Final rule.

SUMMARY: EPA is promulgating final
guidelines under section 403(c) of the
Clean Water Act. These guidelines will
be applied in issuing and revising
National Pollutant Discharge
Elimination System Permits for
discharges into the territorial seas, the
contiguous zone and the oceans.

DATES: These guidelines become
effective on November 3,1980.

FOR FURTHER INFORMATION CONTACT
Kenneth Farber,  Office of Water
Regulations  and Standards (WH-586),
Environmental Protection Agency, 401 M
 Street, SW. Washington, D.C. 20460,
 202-472-5746.
 SUPPLEMENTARY INFORMATION:

 I. Background

   EPA is today promulgating revised
 guidelines for determining  the
. degradation of the territorial seas, the
 contiguous zone and the oceans.
 Pursuant to section 403(a) of the Clean
 Water Act, no National Pollutant
 Discharge Elimination System
 ("NPDES") permit for discharges into
 these marine waters may be issued
 when these guidelines are  in effect
 except in compliance with the
 guidelines.
   These guidelines are issued pursuant
 to section 403(c)(l) which provides that:
   The Administrator shall, within one
 hundred and eighty days after enactment of
 this Act (and from time to time thereafter),
 promulgate guidelines for determining the
 degradation of the waters of the territorial
 seas, the contiguous zone, and the ocean,
 which shall include:
   (A) the effect of disposal of pollutants on
 human health or welfare, including but not
 limited to plankton, fish, shellfish, wildlife,
 shorelines, and beaches.
    B) the effect of disposal of pollutants on
 • ..irine life, including the transfer,
 concentration, and dispersal of pollutants or
 their byproducts through biological, physical,
 and chemical processes: changes in marine
 ecosystem diversity, productivity, and
 stability; and species and community
 population changes;
   (C) the effect of disposal of pollutants on
 esthetic, recreation, and economic values:
   (D) the persistence and permanence of the
 effects of disposal of pollutants;
                            (E) the effect of the disposal at varying
                          rates, of particular volumes and
                          concentrations of pollutants;
                            (F) other possible locations and methods of
                          disposal or recycling -of pollutants including
                          land-based alternatives; and
                            (G) the effect on alternate uses of the
                          oceans, such as mineral exploitation and
                          scientific study.
                            On October 15,1973, EPA
                          promulgated combined regulations
                          implementing section 102{a) of the
                          Marine Protection. Research, and
                          Sanctuaries Act and section 403(c) of
                          the Clean Water Act. The primary focus
                          of these regulations was on the ocean
                          disposal of waste material, including
                          sewage sludges, liquid and solid
                          industrial wastes and dredged materials,
                          by dumping from moving vessels.
                            In practice, these regulations proved
                          unworkable in many respects as section
                          403 ocean discharge criteria. At the
                          same time, operating experience
                          demonstrated that the ocean dumping
                          regulations themselves required
                          revision. EPA therefore determined that
                          both the ocean dumping regulations and
                          the ocean discharged criteria should be
                          revised and published as separate
                          regulations. All reference  to section
                          403(c) guidelines was deleted from the
                          revised ocean dumping regulations
                          which were promulgated on January 11,
                          1977 (42 FR 2468). However, the Agency
                          encountered substantial difficulty in
                          developing revised ocean discharge
                          guidelines, and, until recently, there
                          have been no published national
                          guidelines in place. Since withdrawal of
                          the original guidelines, permit writers
                          have been implementing section 403 on
                          a case-by-case basis.
                             On June 21,1979, the Pacific Legal
                          Foundation filed suit in United States
                          District Court for the Eastern District of
                           California, seeking, among other things,
                           that EPA promulgate revised section
                          403(c) guidelines, Pacific Legal
                          Foundation v. Costle, Civ. No. S-79-429-
                           PCW. The National Wildlife Federation
                           intervened in that lawsuit. On October1
                           31.1979, the Court ordered EPA both to
                           promulgate these guidelines and to
                           publish interim guidelines stating
                           Agency policy in reviewing, issuing, or
                           denying NPDES permits under  section
                           403, pending promulgation of the final
                           guidelines. The interim guidelines were
                           published in the Federal Register on
                           November 15i 1979, 44 FR 65751, and
                           they will be superseded by the final
                           guidelines published today.
                             The Agency then published proposed
                           ocean discharge criteria in the  Federal
                           Register (45 FR 9548) on February 12,
                           1980, held an oral hearing on the
                           proposal on March 21,1980, and
                           provided a comment period for
submission of written comments which
was to end on March 28,1980. At the
request of various interested groups, the
comment period was extended for 30
days. Based on the extended comment
period and on the large volume of
comments received, the Agency moved
the court to extend the final
promulgation date by 120 days beyond
the July 30 deadline. The court extended
the final deadline until September 30.
1980.
II. Development of the 403 Guidelines

1. Synopsis of the Guidelines
  Section 403 is intended to prevent
unreasonable degradation of the marine
environment and to authorize imposition
of effluent limitations, including a
prohibition of discharge, if necessary, to
ensure this goal. These guidelines were
developed to satisfy this intent. They
provide flexibility to permit writers to
tailor application requirements, effluent
limitations, and reporting requirements
to-the specific circumstances of each
discharger's situation, while ensuring
consistency and certainty by imposing
minimum requirements, in situations
where the long-term impact of a
discharge is not fully understood.
  Under these guidelines, no NPDES
permit may be issued which authorizes
a discharge of pollutants that will cause
unreasonable degradation of the marine
environment. Prior to permit issuance,
the director, defined as either the
Regional Administrator or the State
Director where there is an approved
State program, or an authorized
representative, is required to evaluate
whether a proposed discharge will cause
such degradation. In making this
determination, the director is to consider
the factors specified in § 125.122 (a) and
tb).
   In cases where sufficient information
is available for the director to make a
reasonable determination whether
unreasonable degradation of the marine
environment will occur, the director is
governed by § 125.123 (a) and (b) of the
regulations. Discharges which will cause
unreasonable degradation will be
prohibited; other discharges may be
permitted under conditions necessary to
ensure that such degradation will not
occur.
   In those cases where the director is
unable to determine whether
unreasonable degradation will occur,
 § 125.123(c) governs. No discharge in
 this situation is allowed unless the
 director can reasonably determine that:
 (1) the discharge will not cause
irreparable harm to the marine
 environment while further evaluation is
 undertaken; (2) there are no reasonable
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   AopendixB

             Federal Register  /  Vol. 45,  No.  194 / Friday October 3, 1980 /  Rules and Regulations
                                                                       65943
 alternatives to the discharge; and (3) the
 discharge will comply with certain
 mandatory permit conditions, including
 a bioassay-based discharge limitation
 and monitoring requirements. These
 permit conditions will assist in
 determining whether and to what extent
 further limitations are necessary to
 ensure that the discharge does not cause
 unreasonable degradation. If further
 limitations are necessary, § 125.123(d)(4]
 provides that the permit must be then
 modified to include these additional
 limitations or else revoked.
  These guidelines encourage the use of
 available information in addition to any
 supplied by the permit applicant. Thus,
 the director may make determination
 based on information such as that
 contained in any relevant environmental
 impact statement section 301(h) or other
 variance applications, existing technical
 and environmental field studies, or EPA
 industrial and municipal waste surveys.
 2, Relationship Between the Statute and
 the Guidelines
  (a) Section 403(c)(l}—Section 403(c)(l)
 specifies seven factors which are to be
 included in guidelines for determining
 the degradation of marine waters. These
 factors form the basis for the
 determinations which must be made
 pursuant to these guidelines.
  Most of the statutory factors,
 including 403(c)(l](A). (B), (C). (D), (E).
 and a portion of (G), involve
 consideration of the biological ef