Final Report
TC 3991-01
SAMPLING AND ANALYSIS DESIGN
FOR DEVELOPMENT OF ELL I OH BAY
TOXICS ACTION PLAN
by

Tetra Tech",' In'c.
for

U.S. Environmental Protection Agency
Region X - Office of Puget Sound
Seattle, Washington
July, 1985
Tetra Tech, Inc.
11820 Northup Way, Suite 100
Bellevue, Washington  98005

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                           CONTENTS


                                                                 Page

LIST OF FIGURES                                                   iv

LIST OF TABLES                                                     v

INTRODUCTION                                                       1

GENERAL APPROACH                                                   2

    STUDY TYPES AND INTEGRATION                                    2
    REFERENCE AREAS                                                6
    CRUISE PROCEDURES                                              7
    STATION LOCATION METHODS                                       8
    OTHER STUDIES                                                  9

CONTAMINANT SOURCES                                               11

    DATA GAPS                                                     11
    GENERAL STUDY DESIGN                                          13
    STATION LOCATIONS                                             16
    SAMPLING METHODS, PROCESSING, AND ANALYSES                    21

SEDIMENT QUALITY SURVEY                                           23

    DATA GAPS                                                     23
    GENERAL STUDY DESIGN                                          25
    STATION LOCATIONS                                             28
    SAMPLING METHODS, PROCESSING, AND ANALYSES                    31

SEDIMENT BIOASSAYS AND BENTHIC MACROINVERTEBRATE COMMUNITIES      35

    DATA GAPS                                                     35
    GENERAL STUDY DESIGN                                          37
    STATION LOCATIONS                                             41
    SAMPLING METHODS AND SAMPLE PROCESSING                        42
    LABORATORY PROCEDURES                                         44

BIOACCUMULATION AND PATHOLOGY                                     48

    DATA GAPS                                                     48
    GENERAL STUDY DESIGN                                          50
    STATION LOCATIONS                                             55
    SAMPLING METHODS                                              56
    SAMPLE PROCESSING                                             57

OPTIONAL TIERED STUDY DESIGN                                      59


                              11

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DATA MANAGEMENT                                                   61

SUMMARY                                                           62

REFERENCES                                                        67

APPENDIX MAPS:

    MAP 1 - CONTAMINANT SOURCES AND SELECTED INDUSTRY
            LOCATIONS IN ELIOTT BAY AND THE LOWER
            DUWAMISH RIVER

    MAP 2 - SEDIMENT CHEMISTRY:  SAMPLING STATIONS FOR
            SELECTED DATA SETS IN ELLIOTT BAY AND THE
            LOWER DUWAMISH RIVER

    MAP 3 - BENTHIC INFAUNA:  SUBTIDAL AND INTERTIDAL
            SAMPLING STATIONS FOR SELECTED DATA SETS
            IN ELLIOTT BAY AND THE LOWER DUWAMISH RIVER

    MAP 4 - SEDIMENT BIOASSAYS:  SAMPLING STATIONS FOR
            SELECTED DATA SETS IN ELLIOTT BAY AND THE
            LOWER DUWAMISH RIVER

    MAP 5 - BIOACCUMULATION AND FISH PATHOLOGY:  SUBTIDAL
            AND INTERTIDAL SAMPLING STATIONS FOR SELECTED
            DATA SETS IN ELIOTT BAY AND THE LOWER DUWAMISH
            RIVER

    MAP 6 - SAMPLING LOCATIONS FOR FIELD STUDIES IN ELIOTT
            BAY AND THE LOWER DUWAMISH RIVER
                              111

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                                  FIGURES
                                                                   Following
Number                                                                  Page
   1    Project area:  Elliott Bay and the Lower Duwamish River           1
   2    General approach to development of Elliott Bay Toxics
        Action Plan                                                       1
   3    Components of recommended study design                            2
   4    Hypothetical spatial patterns of contamination and effects
        in Elliott Bay/Duwamish River subareas                            5
   5    Sample processing scheme for pollutant source study               22
   6    Elliott Bay Power Analyses                                        41
   7    Elliott Bay Power Analyses                                        41
   8    Flow chart depicting decision criteria for conducting  sediment
        bioassays                                                         46
   9    Temporal trends  in  selected  liver disorders of English sole
        from Elliott Bay and the lower Duwamish River                     49
  10    Sample processing scheme for pathology and bioaccumulation
        study  in subtidal areas                                           57
                                      1v

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                                  TABLES


                                                                   Following
Number                                                                  Page

   1    Summary of source sampling sites                                 16

   2    Determination of minimum detection levels for elevated
        incidence of disease given 10 sample sizes and three back-
        ground levels of disease                                         53

   3    Summary of basic study design                                    62

   4    Source sampling sites for Elliott Bay Toxics Action Plan         62

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                               INTRODUCTION
     Under the  Puget Sound Water Quality Management  Program, the U.S.  Environ-
mental Protection  Agency  (EPA) and -the Washington Department of Ecology
in cooperation with other local, state, and  federal agencies are developing
an action plan  to solve  problems of  toxic  contamination in E.lliott  Bay
and  the  lower Duwamish  River.  The  resulting Elliott  Bay  Toxics Action
Plan  will:  1) identify existing problems of toxic  contamination and associated
biological  effects within the study area boundaries (Figure 1); 2) identify
ongoing and historical sources of toxic pollution; and 3)  identify appropriate
remedial  actions and agency responsibilities  for implementing those actions.
This sampling  and  analysis  plan represents  the study  design for a  field
investigation to support  development of a final action plan (Figure 2).
Recent historical  data and results from the studies described herein  will
be integrated into a decision-making process to identify  toxic problem
areas and rank  them  in  terms of priority for  action.  Further sampling
of some  of the project study areas defined  in  Figure 1 (i.e., Areas 10-12)
will  not  be conducted as part of this study.   Prioritization of these  areas
will  be  accomplished using available data.  Elements of the decision-making
approach  and summaries of  historical  data  are described in the  Initial
Data Summaries  and Problem Identification report (Tetra Tech 1985b).  Recommen-
dations for short-term  corrective  actions  are presented itt the  Interim
Action Plan (Tetra Tech 1985c).

     Each major section of this  sampling  and analysis plan  describes a
study design  component, including study objectives, kinds of  samples, variables
to be measured, sampling methods, sample processing, and laboratory analyses.
The Quality Assurance Project Plan  provides details of  sample handling,
analytical  chemistry, and  other Quality Assurance/Quality Control (QA/QC)
procedures.  Optional modifications of the comprehensive study design  based
on tiered-analysis are  described  in  a separate  section after the basic
study plan is  presented.

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                                  • COMBINED SEWER OVERFLOW (UAJOR)

                                  • OOMMNCD tCWIN OVCWLOW (UNO*)

                                            KWCH OVEtViaWSTDW OMAN

                                 
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    DATA COLLECTION
DATA EVALUATION
k
r
DATA
GAPS
   HAZARD EVALUATION
FIELD STUDY
  DESIGN
                                                  	I
    POLLUTION  SOURCE
       EVALUATION
    REMEDIAL ACTION
         PLAN
Figure 2.   General  approach to development of Elliott Bay
           Toxics Action Plan.

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                             GENERAL APPROACH
     Implementation of the  recommended  study  design  will  fill major data
gaps and provide input  to  the decision-making process  to achieve  the following
objectives:

     t    Prioritize  toxic problem areas based on sediment contamination,
          bioaccumulation,  sediment toxicity to amphipods,  benthic
          community  structure, and fish liver pathology

     •    Prioritize toxic  pollutant  sources  based on contamination
          of sediments within storm-drain and combined  sewer overflow
          (CSO)  systems.

The approach used to  design  the  recommended studies  involved:   1) evaluation
of existing data  for  each study  component (e.g.,  pollutant sources,  sediment
chemistry, benthic infauna);  2)  mapping of station locations  with acceptable
historical data;  3)  preliminary  assessment of problem areas based on  spatial
and  temporal distribution  of  pollutant sources,  contamination of various
media, and biological  effects;  4)  identification  of  data gaps  in terms
of  spatial  and  temporal  coverage  of  existing  data;  and 5) selection of
recommended study components, conceptual  approach,  specific  variables to
be measured, and station locations.  Sampling stations  for each recommended
study component were  positioned  to fill  gaps in spatial  coverage of  previous
studies,  to ensure  adequate characterization  of  known pollutant  sources,
and to confirm problem areas  identified in previous  studies.

STUDY TYPES AND INTEGRATION

     Components  of the recommended study  design  are  shown  in Figure 3.
Each study component will provide data for a specific environmental  indicator
(e.g., sediment chemistry, benthic infauna) relevant to  the problem  identifi-
                                    2

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                                                            ELLIOTT BAY TOXICS
                                                            ACTION PLAN SURVEY
                                  SOURCE  CHARACTERIZATION

                                    • CSOt
                                    • Stem Drains
                                                             CONTAMINATION AND
                                                             BIOLOGICAL EFFECTS
   SEDIMENT QUALITY
    • Suffice Sedlnnt
     Meal stry
    • Grain-Size
    • Subtldal and
     Intertldal
SEDIMENT BIOASSAVS

 •  Anphlped Survival
 •  Subtldal and Intertldal
BENTHIC INFAUNA

  • Community Structure
  • Subtldal
BIOACCUMULATION

 •  Fish Muscle
Figure  3.   Components  of recommended  study design.

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cation  process.  The rationale for the  choice of  indicators, their inter-
relationships, and action-level criteria  that define  problem areas  are
described  in  the Initial  Data Summaries and  Problem Identification report
(Tetra Tech 1985b).  Sampling and analysis  of  pollutant  sources are  also
recommended.   These new data  in conjunction with existing data will be
used to evaluate  the relative importance of  different sources of pollution
in terms of a  mass-loading  indicator for individual  contaminants (or chemical
classes).  When possible, source data will  also  be used to correlate environ-
mental contamination and effects with specific  sources.

     Scheduling  the  individual  studies  can  help  ensure timely completion
of the project, efficiency of cruise resources,  and collection of appropriate
data.  The general timing of the field work  is  outlined below:

     •    August/September    --   Sediment  Quality
                                  Benthic Infauna
                                  Bioassays

     t    September           --   Bioaccumulation
                                  Pathology

     •    September/October   --   Pollutant Sources

Timing of individual surveys is justified  later in the discussion of detailed
study designs.   In general, the comprehensive benthic  survey coincides
with a time of relative stability of benthic infaunal assemblages and ensures
that English sole will have resided  in shallow-water  sampling locations
for several  months before sampling.

     Discrimination  of  spatial  patterns  in  contaminant distributions and
biological  responses is a major objective  of this project.   To facilitate
spatial  analysis, Elliott Bay and  the  Duwamish  River  Estuary have been
divided into 12 areas based on geographic  features  and  locations of major
storm  drains  and CSOs (Figure 1).  The nearshore area of Elliott Bay (i.e.,
less than 150  ft  deep) and  the lower Duwamish River includes nine  areas.

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The remainder  of Elliott  Bay  includes three deep-water areas:  the inner
bay, the outer bay, and the Fourmile  Rock Disposal  Site.  Area  boundaries
and major features are as follows:

     1.    Magnolia -- West Point, south to Smith  Cove; residential
          and public park.

     2.    Seattle Waterfront North  -- Terminal 90/91  to Pier 70; Interbay
          CSO at T90/91, Denny Way  CSO, Myrtle Edwards public fishing
          pier.

     3.    Seattle Waterfront South  -- Pier 70  to  Terminal 37; main
          Seattle waterfront, ferry terminals, King Street and Connecticut
          Street CSOs, Seattle Aquarium, public fishing pier.

     4.    North Harbor Island -- Southern end of  Elliott  Bay from
          T37 west to Harbor Avenue/West Seattle;  northern Harbor
          Island, Longfellow Creek.

     5.    East  Waterway  --  Mouth  to Spokane  Street bridge; Hanford
          and Lander CSOs.

     6.    West  Waterway — Mouth to  Spokane Street bridge; S.W.  Lander
          and S.W. Florida CSOs.

     7.    Kellogg Island -- Spokane Street bridge to  Kellogg Island/Slip
          1; Hanford-1 CSO.

     8.    Upper Duwamish Estuary -- Kellogg  Island/Slip  1 to head
          of navigation; Michigan Street CSO, Georgetown  flume.

     9.    Duwamish Head/Alki  Beach  -- Eastern  shoreline of Duwamish
          Head,  north of Fairmount Avenue S.W., to Alki Point;  primarily
          residential, public park.

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     10.   Four-mile Rock  Disposal Site -- The designated disposal site
          and surrounding  areas that showed elevated  contaminant concen-
          trations  in  the Toxicant Pretreatment Planning Study (Romberg
          et  al.  1984).

     11.   Inner Elliott Bay — All areas east of a north/south transect
          from Ouwamish Head to T90/91 not included  in other subareas.

     12.   Outer Elliott  Bay -- All areas west of a line from Duwamish
          Head to T90/91  boundary not included in other subareas.

During the  data analysis  phase, some  of the above areas may be further
divided based on  actual distributions of contamination and effects.

     Spatial analysis of the  data will  be  conducted in several ways:  1)
assessment of contamination/response at individual  stations for detection
of "hot spots"  (i.e.,  relatively localized areas of contamination and biological
effects);  2)  gradient  analysis; 3)  comparisons  of averages for areas as
input  to  the priority ranking procedure; and 4) comparisons of individual
stations  and  area averages with data from external  reference sites.   Each
approach  will  be  important for assessing the distribution  of contamination
in the project area.   This information  can also  be used  to determine the
extent of contamination  associated with individual  sources.   If major  sites
of contamination  are found beyond the influence of all  known sources, then
other causes  must be investigated (e.g., historical  contamination,  unidentified
local source, or  undefined transport process).

     Sampling locations have  been positioned to  characterize gradients
of contamination  at different scales.   First,  gradients  may exist  at the
level  of  an  individual  waterway or  project  area.  Examples  of possible
contamination patterns in the East and West Waterways are shown in Figure 4.
Concentrations  of many  toxic  contaminants in  sediments  may  reach  their
peak near  the mouth of the lower Duwamish River and  decline  upstream (Dexter
et al. 1981; Harper-Owes  1983).  Gradients at the scale  of a waterway or
area can  be  longitudinal  or transverse.  Similar  gradients  in contaminant
                                    5

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                                         GRADIENTS
                        WATERWAY LEVEL
  POINT SOURCE RELATED
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       waterway.  Arrows indicate direction of in-
       creasing contamination or effects.  Gradients
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          Figure  4.   Hypothetical spatial patterns  of contamination
                      and  effects In  Elliott  Bay/Duwamish  River  subareas.

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concentrations  may also exist at  a  finer  scale around  individual point
sources.   Although other patterns  of contamination are  possible (e.g.,
random or  uniform distribution), available data  suggest that some gradients
in contaminant concentrations  probably exist, at least in the heavily contami-
nated areas.

     Gradient analysis  provides  the framework to  test hypotheses about
spatial  patterns regardless  of  the actual pattern.  Several  gradients may
co-exist,  obscuring individual  source-related contamination, patterns.
Moreover,  a pattern of localized hot spots may  be superimposed on a more
general spatial pattern in many areas, especially in the lower Duwamish
River where large-scale contamination may be the  result of upstream sources.

REFERENCE  AREAS

     Three potential  reference areas  are  suggested for the  Elliott  Bay
studies:   upper  Port Susan,  Blakely Harbor  (Bainbridge Island), and the
Seahurst  area.   The ideal  reference  area exhibits physical characteristics
(e.g., sediment  type, water  depth,  wave exposure, freshwater influence)
that  are  similar to those  of  the  study area, but  is  without significant
human influence.

     Two of the suggested  reference areas may be  appropriate  for  inner
Elliott Bay sites.  The physical  characteristics of  Port Susan appear  to
be most similar  to  those of inner  Elliott Bay.  Port Susan has muddy sediments
at shallow depths,  is a  partially enclosed  embayment,  and is influenced
by freshwater from a major river, the Stillaguamish.   Blakely Harbor  is
also a partially enclosed  embayment and has muddy  sediments at shallow
depths.   However, it lacks a  major freshwater influence  and  the exposure
is different.  The  third  referenc*  area, Seahurst,  has habitats similar
to those  found  in outer Elliott Bay, although exposure and  some physical
characteristics  differ  (e.g.,  Seahurst  is  not  an  embayment).  This area
has  similar depth range, with coarse sediments occurring  at shallow depths
and finer sediments  at deep-water  sites.

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     Data on sediment  chemistry and the  biota  (i.e., fishes, benthos)  of
Blakely Harbor are not presently  available, precluding  further evaluation
of this  potential  reference area.   Such data will become available soon,
since Blakely Harbor was sampled  in Junp, 1985.  A more  informed evaluation
of this  potential  reference area may  be made at that time.  At present,
however,  there is no a priori  reason to dismiss  Blakely  Harbor as a control
area,  since there are no major industrial discharges  into its waters.
Data on the  biota (i.e., fishes,  benthos) of upper  Port Susan are also
not  available  at present.  Limited  data on sediment chemistry indicate
that contaminant  levels for  total aromatic hydrocarbons,  polychlorinated
biphenyls,  chlorinated butadienes,  hexachlorobutadienes, and most metals
are low (Malins et  al. 1982).   Sediment  concentrations of  mercury are elevated
slightly, however.  Additional data on sediment  chemistry (e.g., pesticides,
acids, bases, volatile  solids)  are not available.  A  large data set for
the  Seahurst  area  is presently available (Dinnell et  al.  1984; Word  et
al. 1984).   Preliminary evaluation  of the benthic infauna and bioassay
results  indicates  that the Seahurst area is  an adequate  reference site
for deep-water stations in  outer  Elliott Bay.

     It  is apparent  that  an optimal control  area  does not exist for  the
lower Duwamish River and nearshore Elliott Bay.  Nevertheless, stations
at 30-ft  depth in upper Port Susan  and/or Blakely Harbor  should yield reasonable
control data.  Other sites  in Puget Sound were also considered as potential
reference areas  for Elliott Bay  (i.e., Skagit Bay,  Padilla Bay, and  the
Nisqually Delta).  However,  none  offered an  appropriate combination  of
depth, sediment  type, exposure, and proximity to the  study area.  Confir-
mation of the suitability of  Port  Susan and Blakely  Harbor as reference
areas  will  be made during  this study.  Exact locations  of the sampling
stations  will be  determined  during  the cruise  by qualitative analysis  of
sediment  characteristics and benthic infauna.

CRUISE PROCEDURES

     Special precautions  will  be  taken to prevent contamination of samples
during collection and  initial  processing onboard  the vessel.  Cleaning
                                   7

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of samples, working areas, and instruments before collection  of  each sample
for chemical analyses  is essential.  Work areas of the ship  will  be arranged
to avoid contamination of samples by engine exhaust, oil, and other interfering
substances.  Details of  QA/QC procedures are provided in the Quality Assurance
Project Plan.

STATION LOCATION METHODS

     Because of  the many sources and documented spatial  heterogeneity  of
contamination in the Elliott Bay and Duwamish River system, precise  positioning
of sampling  stations is essential.  The intent of the navigational  control
effort is to  clearly  determine  and  precisely  document where  all  of the
samples were collected.   In the  Duwamish River and waterways,  this  is
complicated because  routine electronic navigation equipment  (e.g., microwave
units or Loran  C) will not function accurately.  At the same time, horizontal
distances to  fixed shore objects  are short, and there are many fixed  points
available for  referencing station  locations.  During  the cruises, the available
visual reference points in the Duwamish River and the East and  West Waterways
will  be photographically  recorded  (i.e., corners of buildings and piers,
spires, towers, smoke  stacks, and other readily distinguishable, permanent
objects).  The water-surface photos will be compared to aerial  photographs
and USGS quadrangle maps for the area.  Objects which can be clearly recognized
on the  aerial  photograph or map, and hence can be accurately located,  will
be selected and numbered  as allowable reference points.   The series  of
surface photos,  with the  reference  points  identified and  numbered,  will
provide the primary  station location tool in the waterways.

     In practice,  stations  will be located by establishing  ranges  between
two reference points, if  possible,  and/or by  establishing long-channel
and  cross-channel  distances to  shore objects.  All station  locations  will
be documented  by  photographs  taken  at the time  samples are  collected and
by written descriptions of  relationships to the reference points.   Station
positioning methods  will  be accurate enough  to define locations within
approximately  a 10-ft  radius.  Standardized procedures will  be used throughout
                                    8

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the  project.  In Elliott Bay  and in the  reference  area,  Loran  C  will  be
used  for primary positioning, with line-of-site and photographic  confirmation.

     The boat will be anchored at the stations whenever possible ar,j  sampling
positions determined by reference to the shore-object fixes.   Station  locations
will  be  verified  just before  each sample is taken.  The  plotted  station
locations will  be converted to state plane coordinates for  entry  into the
data base.

OTHER STUDIES

     This  study  has  been designed to recognize and incorporate information
from other simultaneous studies, including:

     t    EPA pollutant loading  study

     •    EPA contaminant transport study

     t    National Marine Fisheries Service  (NMFS) pathology, bioaccumu-
          lation, and sediment contamination study

     •    National  Oceanographic and Atmospheric Administration (NOAA)
          Status  and Trends Program

     0    NOAA urban anglers study

     0    Corps of Engineers (COE) Duwamish  Capping project

     0    COE Duwamish Turning Basin Studies

     0    WDOE  Harbor Island Superfund Project

     0    METRO Alki Sewage Treatment Plan Facilities planning  studies

     0    METRO combined sewer overflow (CSO) studies
                                     9

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     •    METRO Duwamish  head baseline studies

     •    METRO sewage  effluent monitoring studies

     0    Port of Seattle dredging studies.

     Data from these studies  are expected to add valuable information for
the final  evaluation  of problem areas, and will  be incorporated into  the
project data base.  To  the extent possible, expertise and information will
be exchanged  freely  among investigators.
                                      10

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                            CONTAMINANT SOURCES
     This study  is designed to provide additional data on sources of toxic
contaminants to  the project area.  Other ongoing studies  by  METRO, NOAA,
and EPA  will provide  substantial data  on  mass loading  and transport of
toxic contaminants.  The  sampling design recommended  below  will support
and enhance  these  other studies.  In turn, results of the other investigations
may be used  in  the data analysis and interpretation phase of this study.

DATA GAPS

     Thirty-four  combined sewer overflows (CSOs) and approximately 60 city
storm drains have  been  identified  in  the project area  (Map 1).  Only  the
larger METRO CSOs  (i.e.,  Denny Way,  Hanford Street, Lander Street,  and
Michigan  Street)  have  been sampled  for analyses of toxic contaminants.
They were analyzed for  priority pollutants as part of the Toxicant Pretreatment
Planning  Study  (TPPS).   Relatively  little is known about the quality of
discharges  from  the remaining CSOs  and drains.  Sediment samples from 12
storm drains have  been  analyzed for  metals and PCBs.   Additionally,  the
wastewater collection system has been sampled at  various locations to  charac-
terize inflows to the  West Point wastewater treatment  plant.  However,
storm  events and  overflow were not  monitored.  Deficiencies in pollutant
source data  are  detailed below:

     t    Information  on  the local groundwater  system  is  unavailable,
          particularly  with respect to contamination beneath industrial
          sites.   The  extent of migration of pollutant  plumes into
          Elliott  Bay and the Duwamish River is unknown.  Sweet Edwards
          and Associates is currently Investigating groundwater conditions
          in the  area and will  be proposing  a  monitoring scheme to
          identify and  characterize aquifers in the area.
                                     11

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•    Industrial storm drains along the waterfront may be  a significant
     source of pollutants.  Individual industrial  sites are served
     by private  storm  drains that discharge directly  in the
     waterways.   Little  1s  known about  the  flow, composition,
     or even  location of most of these discharges.  METRO conducted
     a survey of 34 industrial sites  in  the  Harbor  Island/Ouwamish
     River area to identify problem areas  and found several Instances
     of illegal dumping of industrial wastes via storm drains.

•    Many  Port of  Seattle facilities  drain directly to the waterways.
     Although data on discharge quality  are  unavailable,  these
     storm  drains serve only the  port  property and  therefore
     would not have a large contributing area.  Annual  discharge
     is expected to be relatively small.

•    Flow  and water chemistry information on the city  storm drains
     is not  available.   METRO sampling  of sediments  in  the 12
     storm  drains in the  study area  was  intended  to  identify
     hot spots, and did not attempt to characterize the discharge.
     Monitoring of both  wet- and dry-season discharge is required
     to estimate pollutant  loadings.   Sampling  locations  could
     be selected on the basis of  size  and  characteristics of
     the area served and METRO'S sampling results.

•    The city of Seattle  CSOs have not been  sampled.  Total discharge
     from  these sources has been estimated  at approximately 240
     million gal/yr.   Because many city CSOs  serve  as emergency
     overflows and  would overflow only in the event of  an equipment
     failure, sampling   could  be confined  to the few major  CSOs.
     Monitoring would  be conducted during  the  overflow  event
     in both  wet and dry  seasons.

0    Three abandoned landfills are located within  the  study  area--
     Interbay,  West Seattle, and South Park.   Both South Park
     and West Seattle have  been recommended  for further  study
                                12

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          by the King County Health Department.  At this time,  little
          is known about the impact of these sites on groundwater.

     •    Four  of  METRO'S 17 CSOs  have been analyzed for priority
          pollutants.  These four (I.e., Denny Way,  Lander Street,
          Hanford Street, and Michigan Street) account for 50-85 percent
          of the total METRO CSO discharge.   However, no  overflow
          samples were taken during  the  dry season, and three  of the
          four  samples at each site were  taken within  a 4-day period.
          The  available dry-season samples were  taken from within
          the collection  system over a  1-yr  period  (12/80-11/81),
          but did not coincide with  overflows.   Further monitoring
          of the major METRO CSOs is required to adequately estimate
          loadings to Elliott Bay and the Duwamish River.

     •    Several seeps have been identified along the Duwamish  River,
          but there  is no  information  on volume  or composition of
          flow.

GENERAL STUDY DESIGN

     Because of the  number and complexity of pollutant sources in  the lower
Duwamish River  system, an accurate model  of contaminant  inputs and outputs
based  on mass  balances would be difficult  and  expensive  to  construct.
Prioritization  of sources can be accomplished for a large number  of sources
in a cost-effective way  by  analyzing  sediments  within drainage systems.
Thus, the primary indicators used to rank sources will be based on  contaminant
concentrations  in sediments collected from within storm  drains  or CSO conduits.
The multiple of contaminant concentration and  annual flow  (estimated from
basin area and  land use)  will serve as a  ranking index to prioritize sources.
Analyses of priority pollutants and hazardous substances in source  sediments
are proposed for 55 storm drains and CSOs.  These constitute a  major known
source of contaminants to the bay/river system.
                                     13

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     A secondary objective  of  the  source study will be to verify the sediment-
ranking technique by  sampling  stormwater discharges at  two  selected  sites
and correlating  pollutant concentrations in  the discharge with corresponding
concentrations  in  drain sediments.  Contaminant concentrations  in both
the bulk stormwater sample  and the suspended  solids fraction will be analyzed
in a composite of at  least  four grab samples taken during  a storm event.
Data  on  the four  major CSO discharges are  already available and additional
data will be gathered as part  of METRO NPDES monitoring.   These  data can
be used to further evaluate the relationship between contamination  of  source
discharges and sediment composition within the drain system.

     METRO'S TPPS study identified a list  of frequently  detected  contaminants
in the major CSOs and collection system,  including:
               Inorganic Substances
Organic Compounds
               aluminum
               antimony
               arsenic
               beryl Hum
               cadmium
               chromium
               copper
               lead
               manganese
               mercury
               nickel
               selenium
               silver
               tnallium
               zinc
               cyanide
phenol
1,4 dichlorobenzene
naphthalene
phenanthrene
dimethyl phthalate
diethyl phthalate
di-n-butyl phthalate
butyl-benzyl phthalate
di-octyl phthalate
PCB-1254, 1260
methylene chloride
chloroform
trichloroethylene
tetrachloroethylene
benzene
ethyl benzene
toluene
                                      14

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Because  these  contaminants  represent the major analytical  groups  (i.e.,
volatiles,  acid extractables, bases, neutrals, etc.), full-scan analyses
for priority  pollutants will be conducted  on several selected source samples.
Additional target chemicals (e.g., carbazoles) will be specified  for analysis,
in consultation  with U.S.  EPA, after the analytical laboratory has been
selected  and  the results  of comprehensive  analyses  of  selected samples
are available.  A reduced  list of target chemicals will be developed  in
consultation  with U.S. EPA after the results of the initial  comprehensive
analyses  are  available.

     In  summary,  the variables  to be measured during the pollutant source
study  are:

     Target Chemicals

     t    Sediments  in drain and CSO systems
     •    Bulk  water sample  (two storm drains)
     •    Particulate fraction  (two storm drains)

     Conventional  Pollutants

     t    Total  suspended  solids (two storm drains)
     •    Particulate organic carbon

For stormwater analyses,  the particulate fraction should also be analyzed
because:   1)  Information on  the partitioning  of pollutants between  water
and particulate phases  indicates their biological  availability,  2) contaminants
bound to  suspended  matter represent  the major form  of contamination  in
the water  column for  most priority  pollutants,  and  3)  the  composition of
suspended matter  can be compared with sediment contamination data to  infer
transport  pathways  between water  and  bottom sediments.   Since volatile
organic compounds  are  not  expected  to associate  strongly with  suspended
matter, volatile  contaminants need not be measured  in the  particulate fraction.
                                     15

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     Measurements of participate organic carbon allow contaminant concentrations
to be normalized to  the  organic carbon content  of a  sample.   During data
analysis  and interpretation, this  normalization procedure is  useful  for
linking sediment contamination to pollutant sources.   Measurement of total
suspended  solids allows  calculation of the fraction of contaminants associated
with suspended  particulate matter  in the discharges from  the  two storm
drains.

STATION LOCATIONS

     Forty-one  stations  have been selected for sampling (Table  1).   Seven
sites are  METRO  CSOs, the  remainder  are city CSOs and storm drains  (SO).
Storm drain locations were selected on the basis of size and characteristics
of the contributing  area,  as well as results from METRO storm drain sediment
analyses.  CSO  locations were based on overflow volume—any CSO with an
estimated discharge greater than 30 million gal/yr was  selected.  A breakdown
of source sampling locations by subarea  is listed below:

Magnolia:

     1.   32nd  Avenue W.  SO  -- 48-in storm drain that drains a large
          part of Magnolia between Thorndyke Avenue W. and 37th Avenue W.

     2.   Magnolia CSO (W006)l — Located  at 32nd Avenue W. with average
          annual flows estimated between 40 and  100 million gal/yr.

Seattle Waterfront North:

     1.   Interbay  CSO/SD (068)  --  Located  at Pier 90.   Annual CSO
          flows  estimated  at 60 million  gal.'yr.   Also provides  storm
          drainage for industrial  area in  Interbay.
^Numbers in parentheses are NPDES  permit  number.
                                      16

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            TABLE  1.   SUMMARY OF SOURCE SAMPLING SITES
Project Subarea
Magnolia
Seattle Waterfront North
Seattle Waterfront South
North Harbor Island
West Waterway
East Waterway
Duwamish Head/Alki Beach
Kellogg Island
Upper Duwamish Estuary
No. CSO
1
1
3
0
1
2
0
0
1
No. CSO/ SO
0
1
0
2
6
2
0
1
3
No. SO
1
1
•0
1
1
2
1
2
8
TOTALS                      Q                15               17

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     2.    Denny Way  CSO (W027) -- Annual  discharge  ranges between
          80 and 600 million gal/yr.

     3.    Pier 91 SO -- Drains industrial area north of Pier 90/91.

Seattle  Waterfront South:

     1.    King  Street CSO  (W028)  -- Annual discharge ranges between
          10 and 60 million gal/yr.

     2.    Connecticut Street CSO  (W029) -- Annual  discharge  ranges
          between 30 and 100 million gal/yr.

     3.    Vine Street CSO (069) -- Annual discharge averages 35 million
          gal/yr.

North Harbor Island:

     1.    llth  Avenue S.W.  CSO/SD  (077) -- 30-in CSO/storm drain that
          drains northeast corner of Harbor Island.

     2.    Longfellow Creek  -- Historically carried flow from  3 city
          CSOs  (120 million  gal/yr).  The CSOs  are now controlled
          and  can  handle up  to a 10-yr  storm without  overflowing.
          Longfellow Creek currently discharges only non-contact cooling
          water from  Seattle Steel  (formerly Bethlehem Steel) and
          storm drainage from  areas  along  Delridge  Way  during  large
          storm events.

     3.    S.W. Fairmont SO -- Drains  180 ac in West Seattle.

Duwamish Head/Alki Beach:

     1.    56th Avenue S.W. storm drain -- 72-in storm drain that  drains
          most of central portion of Alki/West Seattle.
                                     17

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West Waterway:
     1.    S.W.  Florida CSO/SD (106)  --  36-in CSO/storm  drain that
          drains the northwest corner of Harbor Island.

     2.    S.W.  Lander CSO/SO (105)  -- 48-in CSO/storm drain  that  drains
          central area of Harbor Island  around the old lead  smelter.
          Was  a significant source  of lead and other metals.   Sediments
          have  since been removed and parking  lots in the area  paved.
          Sampling will show effectiveness of  cleanup.

     3.    S.W.  Lander SO --  21-in  private  line.   Source of  oil to
          West  Waterway.

     4.    S.W.  Florida CSO/SD (098)  --  54-in CSO/storm  drain that
          drains area along S.W. Florida  Street and  26th Avenue S.W.
          Known source of  PCBs,  PAHs, and metals.   In  March, 1985,
          Wyckoff Company was  found  guilty  of illegally discharging
          wastes into  both  the Florida drainage  system and Elliott
          Bay  and was fined one million dollars.  U.S. EPA is  currently
          installing monitoring wells to evaluate groundwater  conditions
          beneath the site.  PCBs  believed  to originate upstream of
          Wyckoff at Purdy Scrap yard.

     5.    Spokane Street  CSO/SD  (102) -- 27-in  CSO/storm drain  that
          drains southwest corner of  Harbor Island.   Reports  of milky
          white plume in vicinity of  discharge.

     6.    S.W. Hind Street  CSO/SD (099) -- 96-in CSO/storm drain  with
          annual CSO discharge estimated at 40 million gal/yr.  Drains
          area along 26th  Avenue  S.W. and Delridge Way.   Longfellow
          Creek discharges  through  this drain  except during  large
          storms.
                                     18

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     7.    Chelan CSO (W036) -- Annual  discharge ranges between 1 and
          50 million gal/yr.

     8.    SW  16th CSO/SD (104) --  Drains 12 ac  in  southwest corner
          of Harbor Island.

East Waterway:

     1.    Hanford CSO (W032) --  Annual overflow ranges between 0 and
          700 million gal/yr.

     2.    Lander CSO (W030) -- Annual  overflow ranges between 20 and
          300 million gal/yr.

     3.    S.W.   Hanford  CSO/SD (162) --  42-in CSO/storm  drain  that
          drains  entire southeast  corner of Harbor Island.

     4.    S.W.  Florida SO (36  in)  -- Located on east side of Harbor
          Island.  Drains part of  east side of  island  around  fuel
          storage tanks.

     5.    S.  Hinds Street CSO/SD (107) -- 54-in CSO/storm drain that
          drains  area along  Alaska Way and South Spokane Street.

     6.    S.W.  Lander SO (15  in)  -- Drains 8 ac on east side of Harbor
          Island.

Kellogg  Island:

     1.    Diagonal  Way CSO/SD  (111)  -- !44-1n  CSO/storm drain that
          receives overflow  from METRO'S Hanford regulator  and drains
          Rainier Avenue area.

     2.    S.W.  Dakota Street  SD  -- 30-in storm drain that discharges
          to an  open ditch.   Drains  area around Seattle Steel.
                                     19

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     3.    S.W.  Idaho Street SO  --  Drains 390  ac  along W. Marginal
          Way  S.

Upper Duwamish Estuary:

     1.    S.W. Michigan Street SO -- 72-in  storm drain  that drains
          area along  West Marginal Way and  Highlands Park.

     2.    Fox  Street CSO/SD (116) -- 30-in CSO/storm drain that drains
          industrial  area along  East Marginal Way.   Source of  heavy
          metals.

     3.    Michigan Street CSO (W039) --  Annual overflows range between
          90 and 200  million gal/yr.

     4.    2nd  Avenue  South SO -- 30-in storm drain that drains industrial
          area along  West Marginal Way.

     5.    Georgetown flume  --  66-in  pipe that  discharges to Slip 4.
          Known source of PCBs.

     6.    1-5   storm  drain --  60-in storm drain  that discharges to
          Slip 4.  Elevated metals.

     7.    Boeing SO  --  60-in  storm  drain that  discharges to Slip 4.
          Drains Boeing Field.   Elevated  metals.

     8.    Boeing CSO/SD (117)  -- 30-in CSO/storm drain that discharges
          to Slip 4.  Drains Boeing.   Elevated metals.

     9.    Isaacson CSO/SD  (156) --  Drains 290-ac industrial area at
          Boeing Field.
                                     20

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     10.   Slip 6 SD  --  Drains 120 ac  of industrial  land  in southern
          end  of  Boeing  Field.

     11.   S.W.  Graham SD -- Drains 170 ac along  West Marginal Way S.

     12.   2nd  Avenue S.  SD -- Drains  240 ac along West Marginal Way
          S. near South  Park.

SAMPLING METHODS, PROCESSING, AND ANALYSES

Sediment

     Samples  of  the 0-2 cm  sediment layer  will  be collected from within
storm-drain and CSO  systems using a  stainless steel  "cookie-cutter"  and
spatulas.   Samples  of  the upper 2  cm  wi 11  be taken  because it is assumed
that the surface  layer represents the most recent  sediments  deposited within
a drain  system.   Access  to a drainage system will  be gained at the "end
of the pipe" or through  a manhole.  The preferred  approach will be to sample
from  a  sedimentation basin whenever  possible.  Processing and analyses
of sediment samples will follow procedures in Tetra  Tech  (1985a) or comparable
methods.

Storm Drain Discharges

     Composite samples are  necessary  to estimate mean concentrations of
contaminants and  sediment in the discharge from a  storm drain throughout
a storm  event.  Samples will be collected by an automatic sampler, composi-
ted in proportion to  flow,  and the flow rate measured  throughout the  sample
collection period.  Beginning and ending times must  be noted, and rainfall
data for the same time  period recorded.  If flow-proportional  sampling
is not feasible,  automatic  sampling at fixed time  intervals  is an alternative.
Sample collection at  30-min  intervals for the duration  of the storm  event,
or a  minimum  of 24  h,  is  recommended.  Because  contaminant  concentrations
are highest during the beginning of the  storm event,  sampling  of the initial
                                     21

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flow is critical.   A grab sample of the  initial  flow should also be collected
and analyzed  for  the volatile organic priority  pollutants.

     Processing  and analyses of stormwater  samples are summarized in Figure 5.
Analytical techniques will follow procedures in Tetra Tech (1985a) or comparable
methods.
                                       22

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                                                  SOURCE WATER SAMPLE
       SUBSAMPLE
        80 nl
ARCHIVE
 45 nl
 PURGABLE
 ORGANIC
SUBSTANCES
  35 ml
                                          i
                                     FILTER 1  1 HATER
                                 0.45 u NUCLEOPORE  FILTER
                                                    FILTER  1-4 1 WATER
                                                0.45 u GLASS-FIBER FILTER
                                         ANALYZE
                                   PARTICIPATE FRACTION
       i
  ANALYZE 0.5-2 g
PARTICULATE FRACTION
                             TOTAL SUSPENDED
                                  HATTER
                                                                   BASE NEUTRALS
                                                                 ACID EXTRACTABLES
                                                                  PCBs  PESTICIDES
                                                                                            BASE/NEUTRALS
                                                                                          ACID EXTRACTABLES
                                                                                           PCBs PESTICIDES
        Figure 5.    Sample  processing  scheme  for  pollutant  source  study.

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                         SEDIMENT QUALITY SURVEY
     The detailed  study design for the sediment quality survey  Is  presented
In this section,  following a brief summary of data gaps.

DATA GAPS

     Most of the  available information on toxic contamination of the Elliott
Bay and lower Duwamish River  is related to sediment quality.  Station  locations
for  the  acceptable data  sets derived  from previous studies  are shown  in
Map 2.   The data  are sufficient for defining broad areas of  contamination
(e.g.,  Seattle waterfront, lower Duwamish River) but are not  suitable  for
clear delineation of problem areas in relation to sources.   The major gaps
in the existing sediment contamination data are the following:

Magnolia:

     •    There is  very  limited information frcm this area, with virtually
          no recent chemical data from the shallower sediments.   While
          extreme contamination is not expected because of the distance
          from known  major  sources, the  area is a site of frequent
          human contact and shellfish  harvesting.   For this  reason,
          the  present  lack of data should be remedied.

Seattle Waterfront:

     t    The  sampling density has been so minimal along the waterfront
          that  few general characteristics of the area can be determined.
          In light of  the potential  for major  unidentified  sources
          (both historical and ongoing),  the scarcity of information
          on sediment  contamination  along the  waterfront represents
          one  of  the largest data gaps.
                                     23

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     t    Virtually no intertidal  data are available from  this portion
          of Elliott Bay, in part  because  there is  little  intertidal
          sediment in this  heavily developed area.   Nonetheless, some
          information would be useful  to establish relationships with
          sources (e.g.,  Denny Way CSO) and to make comparisons with
          concentraions in other intertidal sediments.

Duwamish River, Waterways, and North Harbor Island:

     0    Data on intertidal  sediment chemistry in the East  and West
          Waterways and  the  remainder of the  lower Duwamish  River
          are  not  available.

     •    There  is  a  lack of subtidal  sediment contamination data
          near several sources (especially drains and CSO/drain combi-
          nations) in the East  and West Waterways.  Available data
          are  limited in their ability to clearly define  the  complex
          gradients that may be present  in the system, and  to delineate
          relationships between sediment concentrations  and  many of
          the  identified sources.

     •    In  comparison to the lower  river, the upriver sections south
          of Kellogg  Island have  received  such  limited  sampling in
          recent  studies that the  full extent of identified "hot  spots"
          is not  known, nor is there  assurance that  additional  areas
          of high  concentrations of toxic  contaminants do not exist.

Duwamish Head/Alki Beach:

     •    Data for shallow waters (less  than 100 ft), including areas
          near several drains and  CSOs, are missing.   Because of the
          primarily residential  nature  of  the  watershed, this data
          gap  is  not considered major.
                                     24

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Four-mile Rock Disposal Site:

     t    Only limited  samples have been obtained  from  the designated
          disposal  site and the surrounding  sediments.  The  data are
          presently  too  limited to  determine with  any accuracy the
          maximum  elevations in sediments or the  extent  to which
          contaminated  dredged-material extends  beyond  the designated
          disposal  site.

Deep-water Elliott  Bay:

     •    Given the  lack of immediate sources and the  relatively low
          levels observed consistently throughout this  area, the  available
          data seem sufficient to determine the status of most deepwater
          sediments.

GENERAL STUDY  DESIGN

     The objectives of the  sediment quality survey are to:

     •    Determine  the kind  and extent  of toxic  contamination  in
          intertidal  and  subtidal sediments

     •    Characterize the  physical properties of sediments  related
          to contaminant availability, transport pathways, and engineering
          aspects of remedial action  (e.g., dredging)

     0    Relate  the kinds  and  magnitudes  of toxic contamination  to
          biological  effects.

Relationships among physical and chemical  properties of sediments, toxicity
measured  in the sediment bioassays, and benthic infaunal  communities will
be  examined, using results of this survey and the related benthic  biological
studies described  later.   Data  on  sediment  concentrations  of pollutants
can also  be used to relate specific sources to  environmental contamination.
                                      25

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     The sediment  quality survey consists of the  sampling of surface sediments
(0-2 cm) throughout  the nearshore region of Elliott  Bay,  in the East  and
West Waterways,  in other  areas of the lower Duwamish River, and at the
Four-mile Rock  subarea.  The variables to be measured  include  the following:

     Target Chemicals

     t  Bulk sediment  concentrations

     Ancillary Parameters

     t  Total  organic  carbon and nitrogen
     •  Total  sulfide
     •  Percent solids
     •  Grain-size analysis
     •  Oi1 and grease

     For the  first 10 samples to be analyzed, the list of  target chemicals
will  be comprehensive [complete scan of priority pollutant, hazardous substance
list compounds, and miscellaneous substances specified by Tetra Tech (1985b)].
Based on existing  data and  the results of this  first phase of  analyses,
a  list  of  important  contaminants  to  be analyzed in the remaining samples
will be finalized  and  submitted to EPA for review and approval.   Results
of the  METRO TPPS  and  the Commencement Bay Superfund Project showed  that
the following 25 organic compounds were either undetected  or were detected
infrequently  in  sediment samples  from Puget  Sound:  nitrophenols (4);
halogenated ethers (5);  hexachlorocyclopentadiene;  aniline; chloro- and
nitroanilines (4); benzidines  (2); endosulfans (3); endrins  (2); heptachlors
(2); and toxaphene.

     Ancillary parameters will be analyzed for every surface sediment sample.
Concentrations  of organic  contaminants can  then be normalized  to  total
organic carbon values of each sediment sample to account for varying  ratios
of organic  to inorganic substances  among samples.  Nitrogen content data
                                     26

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will be  useful for interpreting the origin  of organic material.   Data on
sulfide content will indicate the potential  toxicity of  bottom sediments
due to  conventional  pollutants and processes  (e.g., deoxygenation of surficial
sediments, high BOD, and  release of  toxic forms of sulfur).   Grain-size
analyses are necessary  to  establish the physical characteristics of the
samples and to distinguish effects of pollutants from physical influences
of the  habitat on benthic  infauna-1 communities.  Procedures of Buchanan
and Kain (1971) will be followed for grain-size analyses.

     In the sediment quality survey, only the top 2-cm layer of each sediment
sample  is to be collected and analyzed.   At undisturbed  sites,  the  surface
sediments are expected  to  represent the most recent contaminant profiles.
In the  study area,  the  surface sediment layer is the most biologically
active  zone of the  sediments.  Hence, contaminant  concentrations in surface
sediments are of most  interest from the standpoint  of relating contamination
to biological uptake,  bioaccumulation, and effects.

     Because  the  Duwamish River is a dynamic system with extensive sediment
transport, resuspension, and deposition, the sediments  at any specific
location may be a mixture of materials  from local  sources as well as other
areas (e.g., upstream  influences).  Existing data are  not  adequate to describe
vertical  profiles of contamination  in  the river sediments.  Moreover, a
detailed study of  sediment cores is  beyond the scope of  this study.   The
study  of the 0-2 cm  layer  represents  the  best approach for both Elliott
Bay and the Duwamish  River because:  (1)  analysis of  a more extensive surface
layer  (e.g., 0-10 cm) would preclude  comparison of  the  results of this
study with the results of previous  studies, most of which have analyzed
the 0-2  cm layer;  (2) sampling will be conducted during the dry season,
i.e., the time of year  when bottom conditions  are expected to be most stable;
and (3) sampling of  the 0-2 cm surface layer is appropriate for  characterizing
the most recent conditions.  In any case, it  is recognized  that collection
and analysis of sediment  cores may be  necessary to  define the depth of
problem sediments  before  an area is subjected to sediment remedial action.
                                     27

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     If  sediments contaminated  by an ongoing source  were recently covered
by a layer of  clean  sediments transported from upstream,  then analysis
of  the 0-2 cm  layer  could miss the  contamination.   However, this is not
expected to be ? widespread occurrence, especially during the dry  season.
Also,  in the East and  West Waterways  and in the Kellogg Island segment,
analyses of benthic  infauna may detect subsurface peaks  in  contamination
because many infauna  are expected to'burrow into the deeper sediments (greater
than 0-2 cm).   To evaluate  the alternative approach of  sampling an extensive
surface  layer, the 0-10  cm  layer will be sampled and analyzed at two selected
locations in  the Duwamish River.   Comparisons will  be  made between  the
results  from the 0-2  cm  layer and those from the 0-10 cm layer.

STATION LOCATIONS

     Ninety-two subtidal stations and 13 intertidal stations will be sampled
for analyses of sediment chemistry, bioassay, and benthic  infauna  (Map  6),
including two reference-area stations.  Station locations reflect the following
objectives of the sampling  plan:  fill data gaps, confirm suspected toxic
"hot  spots", and analyze  gradients  near major sources.  The majority of
proposed stations occur along the  30-ft contour in  Elliott Bay, and  in
the Duwamish waterways.

     An  evaluation  of recent  studies in  Elliott  Bay showed a major data
gap for the nearshore  environment.  Most pollutant sources discharge directly
to nearshore areas.  The  nearshore  environment has  the highest incidence
of human contact with  toxicants entering Elliott Bay,  and  the greatest
probable effects.  Previous  studies at the Denny Way CSO showed that this
major source of contaminants has the  greatest  impact  at  depths less than
100 ft  (Armstrong et al.  1978; Romberg et al. 1984).  For many fish and
invertebrate  species, the  nearshore environment is prime  habitat for foraging,
reproduction,  and juvenile development (nursery grounds).  Feasible remedial
actions are most effective in the  nearshore environment.   These  reasons
provided the main rationale for placing the majority of subtidal stations
within nearshore areas.
                                     28

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     The breakdown  of  stations by subarea is listed  below:

                                  Subtidal    Intertidal

Magnolia                              4          0
Seattle Waterfront  North               6          2
Seattle Waterfront  South              12          0
North Harbor Island                   8          2
East Waterway                        17          3
West Waterway                        15          1
Kellogg Island                         8          3
Upper Duwamish  Estuary                16          1
Duwamish Head/Alki                     4          0
Reference Area                         2          1

Magnolia—Four  stations are located  along the 30-ft  contour to establish
presence or absence of contaminants (potentially  from  Fourmile Rock Disposal
Site material)  in the  nearshore environment.  Two of the  stations  are  also
near  CSOs.  METRO will  be  analyzing  toxic  contaminants  in clams at two
stations and in  sediments  at one station along  Magnolia Beach.  These results
can be  used in conjunction  with results from the  present  study to perform
an initial  assessment of the impact  of toxic chemicals  in the  Magnolia
area.

Seattle Waterfront  North—Four stations are  located  along the 30-ft contour.
Two other stations  are placed within the east and  west  slips at  Terminal
90/91.  The rationale  for station placement  at T90/91 is to assess contamination
potentially originating  from the CSO and  storm drains  at the terminal.
The remaining  stations  were established  to determine toxicant levels and
effects near a  public  fishing pier  and to  determine longshore gradients
away  from  the  Denny Way  CSO.   Because  the  subtidal areas near Denny Way
CSO has been extensively sampled in  previous  studies, it  is not  proposed
for sampling during  this  survey.  Intertidal  sites at the public fishing
pier and the Denny  Way CSO are recommended to  determine contaminant  levels
in intertidal sediments.
                                     29

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Seattle Waterfront South—The main Seattle  waterfront has been poorly sampled
in all  previous  studies.  To fill in this data gap, 12 stations are proposed
at the  30-ft  contour  along  the waterfr'- it  (or  at shallower depths  within
the slips).

North Harbor  Island—Eight stations are positioned along the 30-ft contour
north of Harbor  Island  and in southeast  Elliott Bay.   The rationale  for
placement  of  these stations is to determine the areal extent of the effects
of materials  originating in the Duwamish waterways  and to determine gradients
away from  known sources  along northern Harbor Island (e.g., shipyards and
fuel-pier facilities).  Two intertidal sites are  also recommended in  this
area.

East Waterway—Five  stations are  placed along  the center of the channel
to assess longitudinal  gradients in the waterway.  One station is positioned
at the  head of  each of the two major slips.  At each of  eight CSOs or storm
drains, an  additional  station is located  near the source.   One intertidal
site is located  at the head of the East Waterway.

West Waterway--The rationale for  station  placement  in  the West  Waterway
is similar  to that in the  East Waterway, except that sites near  10 drains
or CSOs will be  sampled in the West  Waterway.  One  station is located at
Terminal  5  and  one station is  located in a slip north of SW Florida Street.
Five stations  are placed along the center of the channel.  Three intertidal
sites are proposed  in  this waterway.

Kellogg Island—Three stations  are placed  along  the center channel, with
four additional stations  at major  drains or CSOs  in  this segment.   One
station is  located in Slip 1.  An  additional   three intertidal  sites are
proposed in  this segment of the river.

Upper  Duwamish Estuary—Seventeen stations are proposed  for  this segment
of the river (16 subtidal; 1  intertidal).  Two  sampling   strategies  were
combined.  First, to provide a broad-scale  survey  to characterize  the general
                                     30

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distributions of contaminants, and confirm that  the upper estuary is less
contaminated  than other areas, five  stations are  placed  along the center
channel  of the river.  Second, specific areas  near potential sources were
designated  for sampling (e.g., 1n Slips  2-5, at the Mi-higan Street  CSO,
and at  six other drains or CSOs).  One intertidal  station was also selected
for sampling  in a public-access area of concern.

Duwamish Head/Alki Beach—Because of the quantity of available information
in this area  and indications that it is largely uncontaminated, tlie density
of stations  is low  relative to other areas.  Four  stations are placed along
the 30-ft contour from Alki Point  to  the eastern side  of Duwamish  Head.
One of  these stations  is  near a 72-in  storm drain at Alki Beach.  These
sites will  be sampled  to ensure data comparability with  past studies and
to provide  data from a relatively clean area for comparative impact analyses.

Reference Area—Two  stations are located at a reference site.  Both stations
will  be placed at  a  depth of 30 ft.   Only fine-grained habitats will be
sampled because data  on benthic infauna in other habitat types  are available.

SAMPLING METHODS, PROCESSING, AND ANALYSES

     Subtidal sediment samples for chemical  analyses and  bioassays will
be collected with  a  chain-rigged 0.1-m2 van Veen grab.   Each station will
be located using navigation techniques discussed earlier.  Before each
sample is taken, vessel position will be visually rechecked (range alignments)
and necessary adjustments will be made.  The benthic grab will be deployed
upon  arrival  on station,  as directed  by the field  supervisor.  Following
deployment, and as  the grab is recovered onboard  the  sampling vessel, it
will  be placed in  a  sampling  tray with the grab remaining in the closed
position.   The hinged  lids of the van Veen sampler will  be opened for
observation  of the sample.  Following judgment of the  penetration  depth
and subsequent sample  acceptability by the  field  supervisor, qualitative
observations  of sediment color,  odor, texture, and the presence of recognizable,
living organisms will  be recorded on the  log sheets.
                                     31

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     Care will be taken  to  ensure recovery  of an  intact surface sediment
layer,  with four major criteria  for rejection of a sample:

     0     Overfilling, with  sediment touching  the  top of the close''
          cover

     •     Water  leaking  from sides  or  bottom, or visible  scour of
          the  surface near the  edges of the van Veen  sampler  when
          opened.

     •     Turbid water overlying  the sediments

     •     Insufficient sampler penetration.

     If, through visual  check of the substrate surface contained  in the
grab, it is determined that the  grab has  either  misfired,  been disturbed,
or lost  a  significant portion  of the substrate, the field supervisor will
direct  discarding the sample  and resetting of  the  gear.   In response  to
variability of  substrates in  the study area, the field supervisor may use
a series of grabs at the  same  station  to obtain  an acceptable depth  of
grab penetration.   In medium  to  coarse sand,  a minimum of 4-5 cm is an
acceptable penetration depth.   In fine sand and  sandy silt, a penetration
depth of 7-10  cm  is the minimum  acceptable depth, and in  silt, a penetration
depth of at  least 10 on is the minimum acceptable depth.  If two attempts
to sample a station are unsuccessful,  another nearby station meeting similar
sampling needs will be selected  and documented.   Standardized data  (i.e.,
collection  date, time, station location, depth, and replicate number) will
be recorded along with the qualitative features discussed above.

     Once onboard, the sample will be held in a vertical position by  blocks
and the overlying water carefully drained off  by an aspirator  hooked  to
the  ship's  hose.  The subsamples  for volatile organic  analyses will be
taken  first by  placing 40-cm3  glass vials (duplicates) at the undisturbed
sediment surface and filling them using a stainless steel spatula.   No
air  space will  remain in the  vials. For the remainder of the subsamples,
                                     32

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allquots  will  be  taken  from a composite sample.  The upper  2  on  of sediment
away from the  edge of  the grab will be carefully removed with  a glass plate,
transferred  to a clean glass beaker,  and homogenized by stirring with a
glass rod. Aliquots will be collected as follows:

     •    500  cm3 win  be  transferred  to precleaned glass  jars with
          teflon cap liners (for organic chemical analyses)

     •    125  cm3 wiii  be  transferred  to precleaned glass  jars (for
          metals analyses)

     •    100  cm3 W1'il  be  transferred to freon-rinsed glass  jars (for
          oil  and  grease analyses)

     •    100  cm3  will be transferred to Whirl-pak bags (for grain-size
          analyses)

     t    1,500 cm3 W1'il  be transferred to precleaned glass  jars (for
          bioassays)

     t    500  cm3 win  be  transferred  to precleaned glass  jars with
          teflon cap liners (for archival).

     Precleaned  (solvent-rinsed and muffle-furnaced)  beakers will be brought
onboard together with  replicate (solvent-washed) spatulas to provide  spares
for  loss  or breakage.   Beakers should be of adequate size for  compositing
of samples.  Between samples, the beakers will  be washed with  site  water
to remove all  residual  particulates, washed with pesticide-grade methanol
and pesticide-grade dichloromethane, and then covered with aluminum  foil.
Also  between  stations, the  spatulas  and glass rods will  be  rinsed with
site water, rinsed with  solvent, and wrapped in aluminum foil.

     The  van Veen sampler will  be emptied over the side and rinsed of all
residual  particulate matter.  Between stations, the sampler will be  stored
closed on the  sampling tray.
                                     33

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     Intertldal  samples will  be  collected  from shore using  a stainless
steel "cookie-cutter"  and spatula.  Otherwise intertidal  and subtidal sediment
samples will  be  processed and analyzed in similar fashion.

     In the  laboratory, analytical chemistry methods  will follow procedures
of Tetra Tech (1985b)  or  comparable  methods (see the Quality Assurance
Project Plan).

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       SEDIMENT BIOASSAYS AND BENTHIC  MACROINVERTEBRATE COMMUNITIES
     The study  design includes  analysis  of sediment toxicity (bioassays)
and benthic  macroinvertebrate communities at the majority of subtidal sites
chosen  for  sediment  chemistry (Map 6).  Sediment bioassays are also planned
                                                               •
for the remaining  subtidal chemistry stations,  which are  located in the
lower Duwamish River  south of Kellogg Island,  and at intertidal sites throughout
the project  area.   Station locations  for acceptable data sets from previous
studies are  shown  in  Maps  3 and 4 to  allow comparisons with proposed sampling
stations shown in  Map 6.

DATA GAPS

     The available  information  on  sediment toxicity  and benthic infauna
for deep-water (greater than 100 ft deep) areas of Elliott  Bay is adequate
for  characterization of  toxic effects at those sites, although the number
of replicate deep-water samples is small.  However, major  data gaps exist
for  the shallow  nearshore environment and much of the lower Duwamish  River
system.  The sections below address data gaps for subtidal  benthic  infauna
and sediment bioassays.

Benthic Infauna

     •    Along  Magnolia Bluff, the Discovery Park area, and the eastern
          side of Duwamish  Head, benthic Infaunal communities in shallow
          waters (less than 100 ft deep) have not been characterized.

     •    Except  for the  Denny Way  CSO area,  the Seattle waterfront
          has not  been sampled adequately for benthic infauna.
                                     35

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     t    Limited data are available for the Duwamish  Estuary.  Except
          for several sites near Kellogg  Island, the benthic  infauna
          have not been adequately characterized.

     •    Qualitative estimates of infaunal abundance and community
          structure  are  available  for the  Duwamish Head/Alki  Beach
          area, but the data are insufficient for detailed quantitative
          analyses because of limited replication of samples.

     t    Reference data  for  benthic  infaunal  communities in shallow
          waters with fine sediments are unavailable.

Sediment Bioassays

     •    Spatial coverage  of the  subtidal shoreline  along Elliott
          Bay from West Point to  Alki Point is mostly limited to intensive
          sampling at the Denny Way  CSO and Pier 56.

     t    Spatial  coverage of the entire  Duwamish River  system is
          limited to repeated sampling of  several stations around
          Harbor Island  plus  one  site (14th Street  bridge) between
          Kellogg Island and the head of navigation.

     •    There  is limited  characterization of  sediment toxicity in
          the area  between the Fourmile  Rock  Disposal Site  and the
          beach to the north.

     •    Data  from the  Duwamish River estuary are based on bioassays
          using marine organisms that may be  inadequate to characterize
          toxicity of sediments from potentially brackish environments.
          Appropriate procedures  to  test sediments  having reduced
          interstitial  salinity with  these organisms have  not yet
          been developed.
                                    36

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GENERAL STUDY DESIGN

     The benthic  ecology study consists of an assessment  of benthic infaunal
communities and sediment toxicity  as determined  through bioassays.   The
main objectives of this  study are to:

     •    Determine  the abundance  and  distribution  of biota  in the
          sediments

     •    Relate  sediment contamination to biological  effects  (i.e.,
          community structure of benthic  infauna and  toxic  responses
          to sediments)

     t    Rank areas and  contaminants  with respect  to environmental
          impacts.

The  amphipod sediment bioassay measures  short-term response  to bioavailable
contaminants and provides  an  index to  estimate effects on indigenous  organisms
by  integrating  physical,  chemical,  and  biological aspects  of environmental
contamination.  The benthic infaunal assessment  indicates  the ultimate,
long-term effects of sediment contamination  at the community level.   In
addition, benthic infauna  data and  bioassays  in selected nearshore areas
of  Elliott  Bay  and  the Duwamish  River  will fill a major data gap relative
to prioritizing problem areas.

Benthic  Infauna

     The variables recommended for  the benthic  infaunal  study are the following:

     a    Total abundance

     •    Abundances of higher taxa, e.g.,
               Polychaeta
               Mollusca
               Amphipoda
                                      37

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     •    Species abundances

     t    Species richne'3

     t    Species composition/similarity.

Using these  parameters, spatial  patterns  in biological responses  to pollution
can be defined and the  relative degree of  response at each site  can be
estimated.   Comparisons of  community characteristics among  project areas
based on  analyses of higher taxa will provide  input to site ranking for
the Decision Criteria.  Statistical comparisons  of data from each area,
study area segment, or individual station with  reference  conditions will
establish a quantitative  basis for describing  the presence,  magnitude,
and spatial  extent of biological responses to contamination.

     At 20 selected stations, species richness, dominance, and  the abundances
of indicator species will be used to  analyze community properties and define
conditions  in problem areas more precisely.  Based on the initial analyses
of data on higher taxa, these 20 stations will be selected (in  consultation
with  U.S.  EPA) to represent conditions  in and near the worst problem  areas.
Using  numerical clustering  techniques, the entire data set on species abundances
can be reduced to an interpretable form, whereby groups of stations are
identified on the basis of similarities  in their  species  composition and
relative species abundances (Boesch 1977).   Multiple regression, rank
correlation, discriminant analysis, and other multivariate  techniques may
be used  to  relate station-group membership (defined by infaunal community
characteristics) to site characteristics, such  as  grain size  composition,
depth, conventional  pollutant concentrations, organic carbon content, and
priority  pollutant concentrations.  Discrimination among the potential
causes of observed alterations in  infaunal  communities will  address the
importance of conventional physical-chemical  parameters relative  to con-
tamination levels.
                                     38

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     Because of the  high degree of  spatial  variability  in  benthic community
 characteristics, it is  necessary to analyze  a  sufficient number of replicate
 samples.   Based  on  previous studies  in Puget  Sound and elsewhere,  a  minimum
of four  to  six  replicate O.l-m2  van  "sen grabs has been recommended  (Holme
and Mclntyre  1971; Lie  1968;  Malins  et al . 1982).  A total of  five  0.1-m2
replicate samples  is usually  adequate for most  impact assessment  work.
Lie's analysis  of species-area  curves showed 75-85 percent of the total
species  at  a site could  be  found in  five replicates  (Lie 1968).  Fewer
replicate  samples were  adequate to  characterize the composition  of the
dominant  species assemblages.

     Although most  previous studies  of benthic infauna in  Pug.et--Sound have   .    [J
used>-a^0. 1 -m2  van Veen sampler, a  smaller sampler of^sjmi'liar design (0.06-m2         ^
van VeenjWi-lJ^be used  in this study.  The^sma'lTer sampler has  several   {f^f^ff^
advantages.   First^more precise  estimates  of organism abundances  can be
obtained  with  the smaller samplers-Michael et al. 1981).  Statistical  tests
of differences between study's ites  and  reference sites based  on  the 0.06-m2
samples will be more powerful than those based on 0.1-m2 samples.   (Examples
of statistical p,ower analyses are provided below).  Finally,  smaller samples   wsf
require  le-s's  effort for sorting and identification of organisms^ thereby
reduc-ing  laboratory processing costs.
 educ-i
  ^
     To establish  the comparability  of  results from the 0.1-m2  and 0.06-m2
samplers,  a  limited  number  of 0.1-m2 samples will be analyzed  during this
study.  Using  a 0.1-m2 van  Veen  grab,  five additional replicates will be ^  *~
                                                                                -
collected  at  each of two  stations sampled  concurrently with  the 0.06-mZ
sampler.   One  station  will be located  in  the Duwamish River,  and the other
will be located in Elliott  Bay.   The results will be used  to  compare  the
two sampling devices in terms of statistical precision of infaunal  variable
estimates, representativeness of samples {i.e., characterization of  species
composition),  and statistical  power to discriminate among station  means,
                                                                            f*
     A power analysis of replicate  infaunal samples  from  outer Elliott   J
Bay and the Seahurst area was performed using  total abundance, total  number
of taxa,  echinoderm  abundance, and amphipod  abundance  (data from METRO
                                     39
                                                                                 ,.\
                                                                                   /

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Seahurst  Baseline Study).  For these analyses, power (1-beta)  was  set equal
to 0.8 and the significance level  (alpha) of  the  test was  set  equal to
0.05.  The  resultant  power relationships are presented  in  Figures 6 and 7.
The mean value for each variable over all sites is marked by a  solid horizontal
line.  Because  these  analyses were based on data from 0.1-n? van Veen samples,
they are  conservative indicators of results expected  for  0.06-m2  (i.e.,  tests
based on  smaller  samples will exhibit greater power).

     Total number of  taxa  proved to be the most powerful variable for detecting
differences  among stations (e.g.,  using an ANOVA design).   For  example,
differences of  about  20-25 species among stations could  be statistically
detected with five replicates at each of 7-16 stations (Figure  6).  Differences
equal  to  the  mean value  could be detected using only two replicate samples.
That is,  use of two replicates would  detect close to a 100 percent  reduction
in total  number  of  species at a  study site  relative to  a reference  site
with a number  of species  equal to the grand mean for the data used  in these
power  analyses.   Reference site  values are  expected  to  range from  about
30 to about 80 taxa.

     For total  abundance  and  amphipod abundance, five replicates are adequate
to detect statistically a difference of about 250  individuals  and about
15  individuals,  respectively.  Reference  site  values are expected to  range
from about  200  to about  700 for  total abundance and  from about 5 to  35
for  amphipod abundance.

     Echinoderms are  relatively rare  and patchy  in their  distribution.
Because of  the variability in the echinoderm abundance data, no significant
differences of  a magnitude equal  to the grand  mean  could be  detected  using
as many as  15 0.1-m?  replicate  samples.

     In conclusion, the  power  analyses indicate  that five replicate 0.1-m2
samples are adequate for  statistically detecting differences in total number
of  taxa  (and  possibly total abundance  and  amphipod  abundance) among sites.
At this level  of replication, species-level  identifications  are  desirable
at  selected stations for characterizing  species  richness  as well as for
                                     40

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performing numerical  classification analyses.  However,  use of five replicates
probably has limited  value for detecting differences  in  individual  species
abundances (or abundances  of rare  higher taxa) among  sites.  Increasing
the number of replicates so as to  increase  statistical sensitivity  would
not be  warranted, since  the power  relationships  in Figures  6  and 7 begin
to level off at about 5 replicates.  Thus, for present purposes,  five replicates
were  accepted as sufficient to  characterize benthic communities  in the
project area and  at reference sites.

Sediment Bioassays

     The  variables  to be measured during  the sediment  toxicity bioassays
are:

     •    Acute mortality of  amphipods (Rhepoxynius  abronius)

     0    Sublethal effects on ]?.  abronius (moribund, emergence)

     •    Temperature

     •    pH of sediment

     •    Salinity  (interstitial and overlying seawater)

     •    Dissolved oxygen.

Measurement of physical and chemical variables during the  bioassays  provides
QA/QC and ancillary data for  interpretation of results.

STATION LOCATIONS

     The  locations  of proposed  sampling  stations for benthic  infauna and
bioassays in Elliott  Bay and  the Duwamish River system  are shown on Map 6.
In  the  project area,  sediment for  toxicity bioassays  will be  collected
at all intertidal and  subtidal sites designated  for  sediment chemistry
                                     41

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          ELLIOTT BflY POWER flhJflLYSIS
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Figure  6.  Elliott Bay  Power Analyses.

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      • l
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Figure 7.   Elliott  Bay Power Analyses.

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(13 intertidal  and 92 subtidal sites).   Benthic  infauna will be characterized
at 76 subtidal  sites in Elliott Bay and  the Duwamish  River.   Sampling  and
analysis  of benthic infauna at intertidal  sites is not recommended  for
several  reasons.  First,  an intertidal  infaunal survey  would require  a
major effort because of  the diversity of substrate  types within the study
area.  Second,  comparisons among study  areas  would require  a substantial
sampling  effort  at a reference site'to  characterize "background" conditions
for a variety of  substrate types.  Third, relatively few  impact assessment
studies  have included  intertidal Infauna in  the past.  Because knowledge
about individual  species responses is  limited,  interpretation of the results
would be  difficult.  In  addition, characterization of  subtidal benthic
infauna  upriver of the Kellogg  Island  area is not recommended.  Benthic
communities are exposed to frequent physical perturbations, due  to  a combination
of dredging, resuspension  of sediments  by  vessel   traffic, and natural
disturbance.  Discrimination of effects  of  toxic contaminants from those
of physical disturbance would be difficult.

     The rationale  for  placement  of  benthic  ecology stations is related
to that  of the  sediment quality studies  discussed  earlier.   In particular,
most of  the benthic ecology stations  are positioned within the nearshore
areas to allow  analysis  of longshore gradients  in response to different
contamination levels.

     Benthic infauna  will  be  analyzed and bioassays  conducted at the same
two reference sites used for sediment  chemistry analyses.   Because  of  the
natural  variability of biological communities and  the  variation  in background
sediment contamination among reference  sites, use  of more than one reference
site is  recommended.  The  sediment  bioassay  will also be conducted  at  the
intertidal  reference station chosen for  chemistry  analyses.

SAMPLING METHODS  AND SAMPLE  PROCESSING

     The 0.06-m2  modified van Veen grab  will be used for the benthic infauna
survey.   A 0.1-m2 modified van Veen  grab will  be  used  for collection of
sediment for chemical analyses and  bioassay.  At each station, replicate
                                     42

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samples  will  be taken for analysis of benthic  infauna, sediment chemistry,
and bioassays.   To  avoid disturbance and loss  of benthic organisms, samples
for benthic analyses will not be subsampled.   Eight to ten sequential grabs
will be made,  with  alternate grabs for benthos (0.06-n£)  and chemistry/bioassays
(0.1-m2)t  respectively.   Aliquots of  the  upper 2 cm from  all replicate
chemistry/bioassay  grabs  will  be composited to form  a single  sample  per
station.   Chemical and  physical properties of the sediments and bioassay
responses will  thus be measured from  a single composite  of all replicate
samples collected  at a given station.

     Since  the  high  cost of  chemical  analyses and bioassays limits these
measurements in replicate  samples to the QA/QC program,  analyses of replicate
grabs  is  not  desirable.   It is preferable to perform bioassays and chemical
analyses on composite  sediment samples  to characterize  average  toxicity
at  a  site, not the variability associated with that site.  The  alternation
of separate replicate  grabs for benthic infauna and  composite grabs  for
the chemistry/bioassay  provides the best assurance that measurements made
on the composite sample correspond  to  the habitat  conditions experienced
by the benthic community.

     After  a  benthic  sample  is  collected,  the sample will  be washed on
a 1.0-rim screen.  Mesh of  this size ensures that representative  population
samples  of most species are obtained.  Samples will be transferred to a
container and  preserved with 10 percent buffered formalin.

     Sediment  samples obtained for bioassays will  be  placed  in  clean poly-
ethylene bags  following the homogenizing procedure.  The bag will be  sealed
following  expulsion  of  air.   Samples  will  be immediately  stored in the
dark  on  ice,  transported to the  laboratory, refrigerated  (4° C), and then
assayed within 5 days  of  collection.
                                     43

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LABORATORY PROCEDURES

Benthic Infauna

     After sitting  at least  24  h  in fixative,  infaunal samples will be
washed, transferred  to  glass jars,  and  covered with  70 percent  ethanol.
Using  a  dissecting microscope, organisms will  be  removed from the sediment
and sorted in  to major  taxonomic categories (e.g.,  Polychaeta, Oligochaeta,
Pelecypoda, Gastropoda, Amphipoda, Isopoda).   Specimens  from a given sample
and taxonomic  group  will be placed in separate vials.   At twenty  selected
stations, all  benthic  organisms will be identified to  species, if  possible,
or to lowest practical  taxon.

     The  present  study design  includes sampling  benthic macroinvertebrate
communities  at the majority of the  subtidal sites  chosen for  sediment
chemistry.   Benthic  samples will  be  analyzed sequentially according to
salinity  of the habitat in the Duwamish River estuary.   Samples  from the
most  saline  areas  will  be  analyzed  before  samples  from  the less saline
environments.   Benthic  community structure will  not be  analyzed for samples
taken upstream from  stations where the results indicate a freshwater benthic
community.

     Details  of  procedures for identification and enumeration of  specimens
are given by Holme and  Mclntyre (1971) and Swartz (1978).  QA/QC procedures
will follow the Quality Assurance Plan for this project.   A reference  collection
of species identified  during the study will  be compiled and archived at
U.S. EPA  Region X.

Sediment  Bioassays

     Rhepoxynius  abronius will  be collected  from West Beach and Whidbey
Island, transported  to the  laboratory, and incubated  in their native  sediments
for  a  week prior to  use in the assay.  During this period  of acclimation,
                                     44

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temperature and salinity  will be gradually changed  at  rates no greater
than  10  c  and 2 ppt per  day until the  bioassay conditions of 15° C and
25 ppt are  attained.

     The amphipod  bioassay  originally designed  by Swartz  et  al.  (1979,
1984)  has been used and modified by several investigators  (Chapman et al. 1984;
Ott et  al.  in preparation;  Pierson et al.  1983).  For  the purposes of this
study, the  bioassays will  be conducted following the  protocol of  Swartz
et al.  (1984).  Five  replicate assays  will be performed  in 1-1 beakers
containing  2 cm (weighted) of test  sediment.  Power  analyses of sediment
toxicity bioassay data  have been conducted by Rick  Swartz of EPA Environmental
Research Lab in Newport, Oregon.  The results show  that  the common procedure
of using five replicate trials with 20 amphipods  per trial gives an appropriate
level  of statistical  sensitivity  (i.e.,  a minimum  detectable difference
of about 15 percent mortality).

     Prior  to  initiation of the  bioassay,  sediment  samples will be mixed
within their storage containers as described by Swartz  et al.  (1985).   Pore
water will  be included in the final assay of bulk  sediments.  Once sediment
samples  are placed  in the beakers, 750 ml  of filtered 25 ppt seawater (1 ug,
nominal  filter diameter) will be layered onto the sediments and the resultant
suspended particulate  matter allowed to settle.   Twenty amphipods  will
then be  added to each replicate beaker and the water  overlying the sediments
agitated by gentle bubbling with scrubbed (oil-free),  water-saturated  air.
Bioassays will  be conducted under continuous illumination.

     Following 10-day exposure  to the  test sediments, bioassays will be
terminated  by sieving  beaker contents through  a  1.0-mm screen.  Numbers
of surviving  amphipods will be counted as those  capable  of discernable
movement (i.e., pleopod streaming) under a light microscope.   At this  time,
moribund animals will be identified  in a separate assay of burial response
(Swartz  et  al.  1984).

     Appropriate positive (clean sediments) and negative (spiked sediments)
controls will be performed in addition to assays of  sediment samples  from
                                     45

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the  study area.  Both  organic and inorganic  contaminants will be used in
separate series of control experiments.

     Of the  105 subtidal and  in  ertidal stations proposed in the Elliott
Bay area, 64 are in the Duwamish River system.   Sediments from these stations
may be subjected to  wide ranges in salinity depending on the river discharge
and tidal  pumping.   Bioassay results have demonstrated  that the marine
amphipod R. abronius is  sensitive to salinities  <2Q ppt (Swartz et al.  1985).
Thus, it was decided that, conservatively, a standard  bioassay salinity
of 25 ppt in both overlying and interstitial  seawater would eliminate effects
due to salinity.  This  effectively  limits the use of amphipod bioassays
to  field  sediments  meeting  this restriction.  As interstitial salinity
was not a parameter  commonly measured  in earlier sampling programs conducted
in this estuary, there  is  no information on the  upstream extent of sediments
appropriate to amphipod assays (i.e., with an interstitial salinity _25 ppt).
However,  saltwater  intrusions have  been observed as far upstream as 16 km
(10 mi) (Santos and  Stoner 1972), which greatly exceeds  the 9.6 km  (6  mi)
upstream limit of proposed Elliott Bay sample collection area.

     Recently, the salinity limitation of the  amphipod assay was addressed
at a bioassay meeting sponsored by the Seattle district  of the Army  Corps
of Engineers (COE).  Under the current dredged  material disposal guidelines
(EPA interim criteria),  there is no  alternative bioassay  to the amphipod
test for brackish water  sediments.  Since much of  the COE's proposed dredging
programs involve  sediments  collected within the Duwamish River system,
it is imperative to  COE  that modifications of or alternatives for the amphipod
bioassay be established  to encompass low salinity  sediments.

     Based  on  the outcome of  the COE meeting,  the  following  flow-chart
is proposed for  bioassays  with Elliott Bay area sediments (Figure  8).
The  interstitial salinity  of all sediments from the Duwamish River system
will be determined by centrifuging a  small subsample.   Standard amphipod
bioassays  will be  conducted on all sediments with an interstitial salinity
^25 ppt and on all Elliott Bay sediments.  The remaining Duwamish  River
                                     46

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      COLLECT ALL
 BIOASSAY AND CHEMISTRY
        SAMPLES
  ELLIOTT BAY AREA ?
YES
          NO
    DUWAMISH RIVER
       SEDIMENTS
            CONDUCT STANDARD
           AMPHIPOD BIOASSAY
 INTERSTITIAL SALINITY
       >. 25 ppt ?
           I
          NO
YES
  SALINITY ADJUSTMENT
    TEST  DEVELOPED  ?
          NO
YES
      CONDUCT
 STANDARD AMPHIPOD
   BIOASSAY WITH
SALINITY ADJUSTMENT
     DO NOT ASSAY
       SEDIMENT
Figure 8.   Flow chart depicting decision criteria for conduct-
           ing sediment bioassays.

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sediments  either will  not be assayed or will  be assayed with  the  standard
bloassay test  preceded with a salinity adjustment, if the latter  procedure
proves successful.
                                     47

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                      BIOACCUMULATION AND PATHOLOGY
     Because of the potential  relationship between bioaccumulation of toxic
substances  and  prevalence of  pathological conditions, these aspects  of
the study design are discussed  together in this section.  Tissue concentrations
of target chemicals provide a measure of contamination of biota,  while
pathological  analyses indicate sublethal responses of organisms to chronic
toxic exposures.

     English  sole (Parophrys  vetulus) was selected as the target fish species
for bioaccumulation and pathology analyses for  several reasons.   First,
this species  is  abundant and  widespread throughout Elliott Bay and the
lower Duwamish River, enhancing the probability that adequate sample  sizes
can  be obtained  at all study  sites.   Second,  English sole  live in close
contact with bottom sediments,  prey mainly on  small benthic infauna,  and
exhibit high  levels of tissue contamination and disease in  urbanized areas
of Puget Sound (e.g., Mai ins  et al.  1984).   It  is therefore likely that
this species  is  being influenced  by  contamination of bottom sediments.
Finally, because English sole  is captured and  consumed by at least some
recreational  fishermen, this  species  is part of a food-web  pathway through
which contaminants can move from sediments to  humans.  English sole also
serves as  a  conservative indicator of potential  health effects because
it is expected  to have higher concentrations  of contaminants in  edible
portions than  are other recreatonally caught  species (see discussion in
Tetra Tech  1985b).

DATA GAPS

     Station locations for  the  accepted  data sets compiled  from previous
studies of  bioaccumulation and  pathology are shown in Map 5.
                                     48

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Bioaccumulation

     Recent data  on bioaccumulation of toxic  substances in English sole
from specific  locations  in the project  area are limited  to two reports
(Mai ins  et  al.  1980; Romberg et al. 1984).   Because of the limited nature
of previous  surveys,  the lack of comprehensive data on  toxic contaminant
concentrations  in biota  of Elliott Bay  represents a major data gap.  In
particular,  data on metals, chlorinated  compounds,  and PAH in edible muscle
tissue of English sole  throughout  the  nearshore environment-of Elliott
Bay and  the  lower  Ouwamish River are needed.  Recent data are available
only at  Denny Way  CSO, Alki Point, and West Point (Romberg et al. 1984).

Pathology

     Although several  investigations  of pathological conditions in fishes
have been conducted  in the  project  area (e.g., Mai ins et  al. 1980,  1982,
1984; McCain et al.  1982;  see Map 5), the data can not be used to establish
existing  conditions  or to delineate specific problem areas.  First, analysis
of data  from  multiyear  studies suggests that the prevalence of certain
liver disorders in English  sole may be increasing  over  time (McCain et al.  1983;
Figure 9).   Second, past  studies have generally  pooled samples taken from
different stations (Map 5)  and from different seasons without controlling
for  differences  in  sample size among  sites  and among times.  Small sample
sizes at  individual  trawl sites probably motivated this  approach.  Third,
previous  studies have failed to correct data for differences  in age composition
of fishes among sites.  Because the prevalence of  several  liver disorders
is related  to fish age, comparison of samples  with  different age distributions
can greatly  bias the  results (Tetra Tech, 1985a).

     In  addition  to  the above limitations, past studies  do  not characterize
the pathology of English Sole at the following sites:

     •    Directly inshore  of Fourmile Rock Disposal Site

     •    Public fishing pier near Myrtle Edwards Park
                                     49

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                -• DENNY WAY CSO
             • — —O WATERFRONT
     30 -
     20 -
     10 -
          D—	D 1«   AYE. BRIDGE
                                                NEOPLASMS
                                                 PRENEOPLASMS
     60 -n
     40 -
     20 —
              1979   1980
 1981   1982   1983

YEAR
                     MEGALOCYTIC
                       HEPATOSIS
         NOTE:  SUHtfR SAMPLES
              ALL POINTS BASED ON 10 OK MORE FISH
                                    SOUKCE:  OATA FHOM McCAIN ET AL. 1M3
Figure  9.   Temporal trends in  selected  liver  disorders of
            English Sole from  Elliott  Bay and  the Lower
            Duwamish River.

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     •    Southern  waterfront outside of East Waterway

     •    Michigan  Street  CSO in Duwamish River

     0    West Seattle  between  Alki Point and Duwamish Head.

GENERAL STUDY DESIGN

     The  primary objectives  of this  study are to:   1)  determine  levels
of tissue contamination and  frequencies of  pathological disorders in  rep-
resentative fish  in  areas of Elliott Bay  and  the lower  Duwamish River;
2) compare the level  of tissue  contamination and prevalence of disorders
among areas; and 3) relate contamination and  disease of organisms to sediment
contamination.  Emphasis is placed  on  obtaining data suitable for statistical
analysis.  Results  of this study will allow ranking of areas based on degree
of tissue contamination and  disease,  identification of disease "hot spots,"
and  evaluation  of risk to public  health  from  consumption of contaminated
organisms.

The  variables to be measured  during  the bioaccumulation and pathology study
are:

Chemical  analyses in English sole  muscle tissue:

     t    Pesticides and PCBs

     0    Mercury

     0    Total extractable lipid  material

Pathology:

     0    External  abnormalities for  all biota  (e.g.,  lesions, epidermal
          papillomas,  fin erosion, parasites)
                                      50

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     •    Internal  abnormalities for English  sole

     t    Selected  liver lesions for English  sole, primarily:

               - Neoplasms
               - Preneoplasms
               - Megalocytic hepatosis
               - Nuclear pleomorphisms
               - Hepatocellular regeneration
               - Melanin macrophage centers

Ancillary  parameters:

     t    Individual English sole

               - Length of all English sole
               - Weight, sex,  and age of those  fish  subsampled for histo-
                pathological analysis

     •    Species  composition (numerical)  of  trawl samples.

Chemical  analysis  of edible portions of target species  will  allow estimation
of potential  human  health hazard.  These analyses  will  focus on PCBs because
this  group  of compounds was identified  as a problem  during the initial
data review  (Tetra  Tech 1985b).  Although  pesticides  are not expected  to
accumulate  in large  amounts in fish from the project area, they will also
be analyzed  because the data are easily obtained along with the PCB  data.
Most  metals do not accumulate to abnormally high  levels in fish muscle
tissue (Tetra Tech 1985a,b).  However,  mercury is  of  potential  concern
and  therefore will be analyzed  in  this  program.  More  extensive analyses
of toxic  chemicals  in  fish tissue are not  being proposed  here because  other
studies by  U.S.  EPA  and NOAA will  be conducted  in  the  project area soon.
                                     51

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     Analyses of contaminants  in  fish livers  is not recommended  for this
study because:  1) Compounds such as PAH that have been implicated as one
potential  caus'  of liver lesions are  not  detected in liver samples  using
standard  techniques; 2) For PCBs, there  is little relationship  between
contaminant  concentration  in the liver and  the  prevalence of liver lesions
(Tetra Tech 1985a); 3) The total mass  of a contaminant  in  the liver is
generally  less  than the total mass  of that contaminant in edible muscle
tissue (Tetra Tech 1985b).  Even  for human subpopulations  that  consume
fish  livers, the health hazard  associated  with  liver ingestion is generally
less than that associated with  muscle (fillet) ingestion;  4)  Because of
the  small  mass  of liver tissue (typically less  than 2 g), compositing of
individual  samples  is necessary  for chemical  analyses.  Thus, statistical
estimates of  population variability cannot  be obtained for full-scan chemical
analyses; and 5) Detection limits  are generally high even  when  samples
are  composited  because of the  high lipid  content  of livers.  Liver samples
will be archived in the event  that  financial  resources  for contaminant
analyses  become  available in the future.

     The liver  is  singled  out for histopathological  analyses because it
is the organ  most heavily afflicted  with  pathological disorders  (Malins
et  al. 1980, 1982).  To enhance  study efficiency, pathological  analysis
of livers will be restricted  to six  types of idiopathic  lesions.   These
include  hepatic neoplasms, preneoplastic  nodules, megalocytic hepatosis,
nuclear pleomorphisms, hepatocellular  regeneration, and melanin macrophage
centers.  These disorders are well-defined lesions that are likely  to be
prevalent enough  in the  study  area to ensure adequate  statistical  power
of  the data analyses.  Although the causes of these  lesions in field-caught
specimens have not been definitely  determined,  morphologically  similar
lesions  have been induced in  laboratory mammals and fishes by exposure
to toxic  chemicals  (Malins et  al. 1984).

     Pathological  and contaminant analyses will be biased toward larger
English sole (i.e., larger than 230 mm total length,  or greater than 2 years
old)  for two reasons.  First, larger fish are the  ones most likely to be
retained and consumed  by  recreational fishermen and  therefore  pose the
                                     52

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greatest  threat to public  health if their  edible tissue is contaminated.
Second,  prevalence of several pathological  disorders in English sole livers
increases  with  age (Tetra Tech 1985a;  Malins et  al. 1982; McCain et al. 1982).
Biasing  samples  toward larger (i.e., older)  fish will ensure that the study
focuses on  that portion of the English  sole  population most likely to  show
signs of stress  (i.e., lesions).  If adequate  sample sizes  can be obtained
with  reasonable effort (e.g., two trawls  per station), an upper size limit
(e.g., 300 mm)  will be set for English sole used in the  histopathological
study.

     Ancillary  data  (weight, length, sex) will be  collected for those English
sole subsampled  for histopathological  analysis.  Weight-length relationships
for  each  sex can serve as  "condition" indices  (e.g., for comparisons among
sites).  Length  of all remaining English sole will  also be measured.  Species
composition of each catch  will be  determined and these data will be used
to characterize and compare  fish assemblages.

Sample Sizes

     To determine the desirable  sample  sizes for  pathological  analyses
of English sole livers,  2x2  contingency analysis was  conducted  on three
sets of data (Table  2).  The question asked was:  "Given  a certain background
level  of disease (i.e., 0, 5, and 10 percent),  at what point does an  increase
in  sample size  lead  to  a  negligible  improvement (i.e.,<2.0 percent) in
the ability  to  statistically discriminate an elevated  level of disease?"
Results showed  that for  all  three  background levels,  improvement in dis-
criminatory  ability dropped  below  2.0 percent when sample size  exceeded
60.   Based  on  experience  in  Commencement Bay, a minimum sample size of
60 English sole per  station  will allow reasonable discriminatory  ability
after comparison- wise error  rates  are adjusted  to  compensate  for multiple
comparisons  with the  reference  area.  Sixty fish will  therefore be  used
for pathological analysis at each trawl  station.

     For contaminant  analysis  of edible  tissues, a minimum  of five  individual
fish  from each  study site  will be used.  This  sample size is  a balance
                                     53

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      TABLE  2 .  DETERMINATION OF MINIMUM DETECTION LEVELS FOR ELEVATED
       INCIDENCE OF DISEASE GIVEN 10 SAMPLE SIZES AND THREE BACKGROUND
                            LEVELS OF DISEASE*
Sample
Size
20

40

60

80

100

120

140

160

180

200
Background Levels of Disease
0 Percent
*b Dc
20.0

10.0

6.7

5.0

4.0

3.3

2.9

2.5

2.2

2.0

10.0

3.3

1.7

1.0

0.7

0.4

0.4

0.3

0.2

5 Percent
% D
30.0

20.0

16.7

15.0

13.0

12.5

12.1

11.3

10.6

10.5

10.0

3.3

1.7

2.0

0.5

0.4

0.8

0.7

0.1

10 Percent
* D
40 .-0

27.5

23.3

21.3

20.0

19.2

18.6

18.1

17.2

17.0

12.5

4.2

2.0

1.3

0.8

0.6

0.5

0.9

0.2

a Comparisons were made  using a 2x2  contingency  formulation and the chl-
square criterion.


b Minimum level of disease that Is significantly different (P<0.05) from
background levels.


c Difference In minimum  detection levels  between two consecutive sample
sizes (I.e..  Improvement of discriminatory ability).

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between analytical  costs and even  representation  across  all stations.
Gahler et al.  (1983)  and Tetra Tech  (1985a)  used the  same  sample size  to
compare contaminant  levels in muscles of English  sole between Hylebos  and
City  Waterways in Commencement Bay and  Discovery Bay reference sites.
Tissue  levels of PCBs were relatively high  in  the waterways and could be
discriminated from  those at the reference site (P<0.05,  Mann-Whitney U-test).
However, levels of DDT were only slightly elevated in the waterways  and
could be discriminated from background levels only at City Waterway.  These
results suggest that  a sample size of five  may  be  adequate for discriminating
large differences between contaminated and reference  sites, but may  be
insufficient for discriminating smaller differences.

Sampling Times

     To maximize sample sizes and thereby enhance  the  ability to discriminate
spatial patterns of contamination and disease, all  sampling will be conducted
during  a single week during early  September.   Sampling efficiency can be
maximized by sampling  between July  and  September.   Because larger fish
migrate into the nearshore zone to feed during this period, catch rates
of fish larger than 230 mm reach an annual  peak, and  fewer  trawl samples
should be needed to obtain required sample  sizes.

     A second  reason to sample English sole between July and  September
is that fish are rapidly replenishing lipid  reserves  following winter fasting
and  subsequent spawning  (review in Roff 1982).   Tissue concentrations of
lipophilic contaminants  (e.g., chlorinated hydrocarbons)  may therefore
reach an annual  peak  (i.e., worst-case scenario) during this  period.  Finally,
because most recreational fishing presumably occurs during spring and summer,
determination of contaminant levels  in  edible tissue during this period
is probably the most meaningful method of assessing risk to  public health
from consumption of contaminated organisms.
                                     54

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STATION LOCATIONS

     Eleven  trawl stations  are  proposed for the project area (Map 6),  with
one additional  transect near Pt.  Pully  for characterizing reference conditions.
Within Elliott Bay and  the  lower  Duwamish River, trawl locations were selected
to fill data gaps, and  to sample more  intensively major areas of contamination
or  recreational  fishing that have'been sampled  previously  by Malins  et
al. (1984) and McCain et al.  (1982).  As much as possible,  trawl  locations
were  chosen  to  correspond with  areas to be sampled for sediment-chemistry,
benthos, and  bioassays.  In the East and West  Waterways  and  the Duwamish
River,  transects  will run parallel to the longitudinal axis of  each water
body and will be positioned at mid-channel.  Transects along  the Elliott
Bay shoreline  and  in  the  reference area will be positioned along the 30-ft
isobath to coincide  with the sampling depth  for  sediment  chemistry and
benthic infauna.

     Trawl locations  are described below with respect to the 10 areas delineated
for sediment  sampling.

Magnolia  Bluff—One  trawl  station to fill the data gap in the area directly
inshore from  the Fourmile Rock Disposal Site.

Seattle Waterfront  North—One  trawl  station to fill the data gap off the
public fishing area.

Seattle Waterfront  South—One  trawl  station  to  sample  near the Pier  70
public fishing area and one station  to sample the central waterfront area.

North Harbor Island—Two trawl  stations to sample the areas off northern
Harbor Island and Longfellow  Creek.

East Waterway--One trawl station  to  sample East Waterway.

West Waterway--One trawl station  to  sample West Waterway.
                                     55

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Kellogg  Island—One trawl  station to sample  the area off the Diagonal  Way
CSO/SD.

Upper Duwamish  Estuary--0ne trawl station to  fill the data gap off the Michigan
Street CSO.

Duwamish  Head/Alki Beach—One trawl station to  fill the data gap just east
of Duwamish  Head  and to provide data from an  area expected to be relatively
uncontaminated.

Reference Area—Point Pully, one trawl station.

     The relative  proximity of  trawl sites  along the shoreline from the
lower Duwamish  Estuary to the Denny Way CSO will  allow  testing of  the null
hypothesis  that the prevalences of pathological conditions  in English sole
are equal among sites (i.e., that pathological conditions  are not site-specific
indicators).   Moreover, if  the hypothesis  is rejected, the selected trawl
locations  will  allow analysis of  gradients in tissue  contamination  and
pathological  conditions from the  lower Duwamish River north to the Seattle
Waterfront and west to  the Duwamish Head area.   Also,  the results  from
trawls at  the  Magnolia  site and  the  reference site will  be compared to
assess effects  of dredged-material dumping practices on nearshore biota.

SAMPLING METHODS

     English sole  will  be sampled using a 7.6-m  (headrope)  otter trawl
having a body mesh size  of 3.2 cm (stretched) and a  cod-end liner mesh
size  of  0.8 cm.  As this  net has  been  used  by other researchers in Puget
Sound (e.g., University of Washington, National Marine  Fisheries Services,
Tetra Tech), data collected in the present study  will be directly comparable
with results of most past studies.   Mearns and Allen  (1978) describe  the
sampling device and its operation.

     Trawls will be made at a  constant  vessel speed of approximately 1.3
m/sec  (2.5 knots) and each  transect will extend  approximately 400 m (0.25 mi).
                                     56

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Generally,  a 5-mln haul  will  cover the  required distance,  but this may
vary depending  upon  strength and direction of currents.   Transects based
on distance rather  than time are recommended  to  ensure that sampling effort
is standardized.  A minimum of one haul will  be  made  at each site.  Additional
hauls may be necessary to obtain required  sample  sizes.

     Because trawling in Elliott  Bay  and the  Duwamish waterways is often
complicated  by  snags and capture of bottom  debris, the trawl  will  include
a polypropylene (i.e., floatable) retrieval  line attached to' a float  at
one end and  to  the cod end (by shackle)  at  the other end.  This line  allows
the net  to  be pulled in  a  reverse direction,  and generally frees it from
snags and bottom debris without tearing  it. Two  complete  trawl assemblies
will be onboard, including otter boards, bridles, and nets.

SAMPLE PROCESSING

     The recommended sample processing scheme  is  illustrated in Figure 10.

     After  each trawl sample  is  brought  onboard, the catch will be sorted
into two categories:  1)  English  sole,  and  2)  miscellaneous fishes and
invertebrates.   All organisms will be examined for grossly visible external
abnormalities while  being processed.

     All English sole will be measured  (nearest mm, total length).  Sixty
fish larger  than 230 mm will be selected randomly,  and weighed  (nearest gm,
wet weight).  The body cavity of each individual will then be opened and
the sex will be  determined.  These fish  will  then be examined for  grossly
visible  internal abnormalities, and  the liver and otoliths (sagitta)  of
each specimen removed.   If  60 fish  cannot be obtained  from the  initial
trawl  sample,  additional hauls will be made  until the required sample size
is obtained.  Otoliths will be stored for  later age determination.

     After livers and  otoliths have been removed, five of the 60 fish larger
than 230 mm  will be  randomly selected and  stored  on  ice.   The whole  fish
will  be  returned to  the  laboratory where fillets of dorsal muscle will
                                     57

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                                                                     TRAWL SAMPLE
                                                     JL
                                                 ENGLISH SOLE
                            COLLECT
                            ANCILLARY
                            DATA
                                                       MISCELLANEOUS FISHES
                                                       AND  INVERTEBRATES
                          SELECT 5 LARGE
                          FISH AFTER
                          LIVER REMOVAL
DETERMINE CONTAMINANT
LEVELS  IN EDIBLE
TISSUE  OF 5 FISH
                              I
                                               SELECT 60 FISH
                                               LARGER THAN
                                               230 MM TL
FILLET
                        ±
                                                         IDENTIFY. COUNT
                                                         AND RELEASE
                    REfOVE AND
                    SPLIT LIVER
REMOVE AND
STORE OTOLITHS
                                    FIX a 1-CM3
                                    SUBSAMPLEin 10X
                                    FORMALIN
                                          I
                            FREEZE REMAINDER
                            OF LIVER (ARCHIVE)
     I
DETERMINE AGES
                                     EXAMINE FOR
                                     PATHOLOGICAL
                                     DISORDERS
FigurelO.   Sample processing  scheme for  pathology and  bioaccumulation study  in
              subtidal  areas.

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be removed with stainless steel  scapulas.  The  fillets will  be divided
and stored frozen  in  glass  jars  for volatile, organic, and metals  analysis.

     From  each  of the 60  livers, a l-ai)3 subsample will be excised, placed
in 10 percent buffered formalin,  and retained for histopathological analysis.
If a liver contains  grossly visible abnormalities, the subsample will  be
taken at the border between  the  normal and abnormal  tissue and will  include
both types of tissue.   If  no abnormalities are visible, the subsample will
be taken from the  center  of  the  liver  at its broadest point.
                                     58

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                       OPTIONAL TIERED STUDY DESIGN
     The  study design described in earlier sections specifies additional
analysis of samples determined to meet  stated program needs after consideration
of the deficiencies in  available  information.  The advantage of this approach
is that a complete and  synoptic data set will  be available  to define the
extent of problems and  to  establish  quantitative relationships among contami-
nation, bioassay responses, and  community  structure of benthic  infauna.
Existing data (approximately 1979 to present) will be used to further delineate
the boundaries of identified problem areas.   Moreover, existing data will
be used to delineate problem areas in deep-water portions of Elliott Bay.

     EPA  has  requested an analysis  of  elements of the study design in which
total cost could be reduced.  The following  discussion is  a review of  an
optional  tiered  approach  to laboratory processing and analyses that could
result  in program cost savings at the  expense of a reduced pool of information
for  decision-making.   Tiered approaches imply that processing and detailed
analyses of some samples would be contingent  upon initial  results derived
from analysis of selected  samples.

     Tiering  options may  be applied  to sediment chemistry,  benthic infauna,
and bioassays.  Because of the gaps  in bioaccumulation and  pathology data,
and  the  relatively  small  numbers  of  samples specified  for these efforts
in the comprehensive study  design,  a tiered  approach to  bioaccumulation
and  pathology studies will  not be used.   Because sediment bioassays must
be conducted on  fre'ih  (not frozen) sediment,  it is impossible  to delay
bioassay  analysis  of  samples while other analyses are  being conducted.
Also, elimination of bioassay samples  is not desirable because:  1) bioassays
are cost-effective in terms of amount of information gained  for the expense;
2) consistency of bioassay  results depends strongly on consistent techniques;
and  3)  quantitative relationships  between bioassays and sediment chemistry
are possible only if both  tests are  conducted on the same sample.
                                     59

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    The following  tiered approach may  be implemented during  this  study:

    o     If  survival of  amphipods in the sediment toxicity bioassay
          is  not significantly different  from control  survival, then
          benthic  infauna  and sediment chemistry may not be analyzed.
          In  this case, the station  would  not be considered a problem
          area,  and it would not be prioritized for remedial  action
     o    If  survival of  amphipods in the  sediment toxicity bioassay
          is  very low (e.g., less than  50 percent)  and significantly
          different th?n control survival,  then  only sediment chemistry
          may be analyzed.

     Regardless of  the  choice  of  tiering options,  all  samples specified
in the comprehensive study design should be  collected.   Adoption of a tiering
approach  will  require archival of some samples  until the results from other
samples  are available.
                                     60

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                       DATA MANAGEMENT
     All  data  for  the project. Including field observations, will be entered
onto pre-formatted data log sheets.  The completed sheets  will be entered
into the  project Data Management System  (DMS) in National Oceanographic
Data Center (NODC) formats.

     Upon entry  of a data set, or segment thereof, the  scientist who generated
the data will be provided with hard copies of  the computer data file, together
with basic data  quality  analyses (e.g.,  means, standard deviations, and
ranges)  of each  parameter.   Appropriate tests  for  each  data  set will  be
provided.   These outputs  will  be reviewed to ensure that  accurate data
have been entered  into the data files.
                                     61

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                                 SUMMARY
                      ELLIOTT BAY TOXICS ACTION PLAN
                       SAMPLING AND ANALYSIS DESIGN
     The objectives  of this sampling and analysis design  are to:  1) determine
the relative priority  ranking of  known sources  of  toxic contaminants to
Elliott  Bay and the  lower Duwamish River, and 2) identify  and rank  problem
areas in the receiving  environment  based on chemical  contamination and
biological  effects.  The information gathered during  this  survey  will be
used with other  available  data to develop the 1986 Elliott Bay Toxics Action
Plan.

     The focus  of  this sampling design  is  the nearshore area of  Elliott
Bay and the lower Duwamish  River.  An evaluation of recent studies in  Elliott
Bay  showed  a major data  gap for  the nearshore environment.  This is the
area of initial contamination from  shoreline sources, the area most frequently
used  by  humans, and the area of  highest biological productivity.  Moreover,
feasible remedial actions  are most  effective in the nearshore  environment.

     A summary  of  spatial coverage  and sampling effort for  each component
of the study design  is provided in  Tables 3 and  4.  Most portions of this
survey  will be implemented during September-October 1985.   Some stormwater
sampling may  be postponed to  later in  the  year depending on weather conditions.

SOURCE STUDIES

     Because  of the number and complexity of pollutant sources in the lower
Duwamish River system, an  accurate  model of contaminant  inputs and  outputs
based on mass  balances would be difficult  and expensive  to construct.
Prioritization of sources  can be  accomplished for a large number of  sources
in  a cost-effective  way  by analyzing sediments within drainage systems.
Thus, the primary indicators used to  rank sources will be based on contaminant
                                      62

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                                             TABLE 3.      SUMMARY  OF BASIC  STUDY DESIGN*
Pollutant Sources Sediment Quality and Bloassaysd Benthlc Inf aimed
Project Subarea CSO/SO Sediments* Sub tidal Intertldal Subtldal
Magnolia 2
Seattle Waterfront-North 3
Seattle Waterfront-South 3
North Harbor Island 3
West Waterway 8
East Waterway 6
Kellogg Island 3
Upper OuwMlih Estuary 12
OuMMlsh Head/AlM 1
Reference
Total Number of Stations 55
Total Number of Samples' 55
4 0
6 2
12 0
8 2
17 3
IS 1
8 3
16 1
4 0
2 1
92 13
941 13
4
6
12
8
17
IS
8
0
4
2
76
390f
Bloaccumulatlon*
Subtldal
1
1
2
2
1
1
1
I
1
1
12
600
Fish Pathology*
Subtldal
1
1
2
2
1
1
1
1
1
1
12
720"
1 Numbers of stations may change with Implementation of tiered study-design options.
b Discharge will also be sampled at five selected drains.
Includes 14 sites to be selected during the study.
c Does not Include QA/QC samples for chemical analyses.
Total numbers of stations and samples







* Stations for bent hk Infaunal community structure and sediment bloassays coincide with those
for sediment quality. At two stations, additional samples will be taken with a 0.1 m* sampler
for a grab Intercomparlson study.
• Subtldal bloaccuaulatlon and pathology samples to be taken from the same trawls.
' Five replicate 0.06-m2 van Veen samples per station for benthlc Infauna. Five replicate
0.1-mZ van Veen samples at two stations.
V Edible muscle tissue fro* five English sole at each station.

" Sixty English sole livers for hlstopathologlcal analyses at each station.

1 At two  stations In the Ouwamlsh River,  samples of the 0-10 cm sediment layer will also be
analyied for chemical variables.

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        TABLE 4.    SOURCE SAMPLING SITES FOR ELLIOTT BAY
                       TOXICS ACTION PLAN
Magnolia

Magnolia CSO
32nd Ave W SO

Seattle Waterfront-North

Pier 91 SO
Interbay CSO/SD
Denny Way CSO

Seattle Waterfront-South

Vine St. CSO (072)
King St. CSO
Connecticut CSO

North Harbor Island

llth Ave SW CSO/SD (077)
Longfellow Creek
SW Fair-mount SO

West Waterway

SW Florida CSO/SD (098)
SW Florida CSO/SD (106)
SW Lander SD (21")
SW Lander CSO/SD (105)
SW Hinds CSO/SO(099)
Chelan CSO
SW 16th CSO/SD (104)
SW Spokane CSO/SD (102)
East Waterway
              (36")
SW Florida SD
Lander CSO
Hanford CSO
SW Hanford CSO/SD (162)
S Hinds CSO/SD (107)
SW Lander SD (15'}
                                        Kellogg Island

                                        SW Dakota SD
                                        SW Idaho SD
                                        Diagonal Way CSO/SD (111)

                                        Upper Duwamish Estuary

                                        Brandon CSO (W041)
                                        SW Graham SD
                                        SW Michigan SD
                                        Michigan CSO
                                        S Fox CSO/SD (116)
                                        2nd Ave S CSO
                                        Georgetown flume
                                        1-5 SD
                                        Slip 4 SD
                                        Slip 4 CSO/SD (117)
                                        Isaacson CSO/SD (156)
                                        Slip 6 SD

                                        Alki Beach/Duwatnish Head

                                        56th Ave SW SD

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concentrations in sediments collected from within storm drains or CSO conduits.
The multiple of contaminant concentration and annual  flow (estimated  from
basin area and land  use) will serve as a ranking index to  prioritize sources.
Analyses of priority pollutants and hazardous substances in source sediments
are  proposed for 55  storm drains and CSOs.  These  constitute  a major known
source of contaminants  to the bay/river system.

     A secondary objective of the source study will  be to  verify the sediment-
ranking technique by sampling stormwater discharges  at two selected  sites
and correlating pollutant concentrations in the discharge  with  corresponding
concentrations  in drain sediments.   Contaminant  concentrations  in  both
the bulk stormwater  sample and the suspended solids  fraction will be analyzed
in a composite of four grab samples  taken during a  storm event.   Data on
the  four major CSO  discharges are  already available and additional data
will be gathered  as part of METRO  NPOES monitoring.  These data  can be
used  to further evaluate  the relationship between  contamination of source
discharges and sediment composition within the drain  system.

     The  results of this survey  will be  used to  prioritize sources for
implementing source  control.  The  information  will  also be  valuable for
determining  locations where further characterization of actual discharges
may be needed during future surveys.

BENTHIC STUDIES

     Each of the analyses for benthic studies will be conducted using samples
taken at the same stations during the same sampling  period.  At each  site,
chemical  analyses  and amp hi pod bioassays will be conducted  on subsamples
of the same homogenized composite sample consisting  of several  grab samples.
Benthic infauna analyses  will be  conducted  on separate replicate grabs
taken at the same time as the other benthic samples.  The total  numbers
of  stations quoted  in the  sections  below Include two stations located in
a reference area (e.g.. Port Susan,  Blakely Harbor, or Samish  Bay).   The
final reference stations will be selected during the cruise after qualitative
analyses of sediment characteristics and benthic  infauna are collected
                                     63

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from several  candidate  sites.  The objective will be to choose a shallow,
clean habitat  with  fine-grained sediments.

Benthic Infauna

     Subtidal  infauna will  be sampled at 76 stations during late summer
using  a  0.06 m2 van  Veen  grab (five  replicates per  station) and a 1-mm
screen.  In Elliott Bay,  samples will be collected  along  the 30-ft  contour
(or the shallowest  depth  possible where bulkheads prevent sampling at 30 ft).
In the Duwamish River, most samples will be collected  from 30-ft deep areas,
but  other depths  may be sampled depending on bathymetry  in  the vicinity
of pollutant  sources.  However,  the  upper Duwamish Estuary will  not  be
sampled  because of problems  with  interpreting the  data  (dredging and normal
vessel impacts versus  toxic impacts).   At most  stations,  benthic  infauna
will  be identified to  higher  level  taxa (e.g., Gastropoda, Amphipoda,
Asteroidea).  Full  species-level  taxonomy is  planned  for 20  stations  to
be selected after initial analyses of data for higher  level  taxa.

     Using a 0.1-m2 van  Veen  grab, five additional  replicates will be collected
at  each  of two  stations sampled concurrently with the  0.06-m2 sampler.
One station will be located in  the Duwamish River, and the other  will  be
located  in Elliott  Bay.   The  results will be used to compare  the two sampling
devices  in terms of statistical precision of infaunal variable estimates,
representativeness  of samples (i.e., characterization  of species composition),
and statistical power to discriminate among station means.

Amphipod Sediment Bioassays

     Sediment samples  will  be assayed for toxicity  at a total  of 105 sites
using  the  standard  acute lethal  amphipod bioassay  (Rhepoxynius abronius).
Problems of interpreting the  meaning of bioassay response (i.e., contaminant
concentrations vs.  response), as  encountered  in previous surveys, can be
minimized by  conducting bioassays  on the same  samples  used  for  sediment
chemical  analyses.   Both sediment  chemistry and bioassays will  be  conducted
                                      64

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at 16 sites  in the Upper Duwamish Estuary and at 13 intertidal  sites where
benthic  infaunal analyses will not be done.

Sediment Chemistry

     Bulk sediment  samples of the  0-2  cm layer will  be collected  from a
total of 105 sites.  For  the first  10 samples  to be analyzed,  the list
of target chemicals will be comprehensive [i.e., priority pollutants, hazardous
substances,  and miscellaneous chemicals specified by Tetra  Tech (1985b)].
Based on  existing data and  the  results of the 10 comprehensive  analyses,
a reduced list  of target chemicals will be developed in consultation with
U.S.  EPA.   At  two stations  in  the Duwamish  River, the 0-10  cm  layer of
sediment will also be  sampled and  analyzed.   Comparison  of the results
from  the  0-10  cm samples  with  those from  the  0-2 cm samples will  allow
an evaluation of the latter approach for  the Duwamish River system.

Tiered Approach

     All bioassay  samples will  be  analyzed within 96 h after collection
to avoid freezing of samples.  Initially,  benthic infauna  and  sediment
chemistry  samples will be archived. Based on  the results of the  bioassay
tests, it may not be necessary to analyze the benthic infauna and chemistry
samples  at  selected stations.  For example, extremely high mortality  (i.e.,
greater  than 50 percent of the test  population)  in the amphipod  bioassay
may be considered sufficient for classification of a site as a "high priority
problan area."  In such cases, further benthic analyses might not be conducted.
Final decisions  regarding  elimination of sample analyses will be made by
U.S.  EPA.

BIOACCUMULATION

     Bioaccumulation will be assessed by measuring concentrations of selected
priority pollutants  (PCBs,  pesticides, mercury) in edible muscle tissue
of English  sole.   Five replicate  samples will be collected with an otter
trawl at each of 12 sites, including one  reference area  station.  Fish
                                     65

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samples  used for bioaccumulation analyses will be selected  from  those  used
for pathological  studies.

FISH PATHOLOGY

     Pathological  conditions (especially neoplasms, preneoplasms,  megalocytic
hepatosis, and nuclear  pleomorphisms) will be assessed in livers  of  60  English
sole at each of 12 sites.   External  abnormalities will be noted as appropriate. -
Existing data on  fish pathology  in the study  area have major 1'imitations
because  of small sample sizes, tendency of investigators  to pool  samples
across widely-separated trawl  sites  and across  seasons (with  different
sample  sizes within-site and within-season), and  failure to correct for
age-related effects  among  sites.  Because of suspected increases in disease
prevalence over  time in the study area, historical data  may not  reflect
existing conditions.
                                     66

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                                REFERENCES
Armstrong, J.W.,  R.H.  Thorn,  K.K. Chew,  et al.  1978.   The Impact  of the
Denny Way combined sewer overflow on the adjacent flora and fauna in Elliott
Bay,  Puget  Sound, Washington.   Municipality of  Metropolitan Seattle,
Seattle, WA.  102 pp.  MET0056F

Boesch, D.F.   1977.   Application of numerical classification 1n-ecological
Investigations  of water pollution.   EPA-600/3-77-033.   U.S.  EPA Environ-
mental Research Laboratory, Corvallis,  OR.   125 pp.  BOES001F

Buchanan, J.B.,  and  J.M. Ka1n.   1971.   Measurement  of the  physical  and
chemical  environment,   pp. 30-58.   In:  Methods  for the  Study of  Marine
Benthos.   N.A. Holme  and A.D. Mclntyre  (eds).   IBP  Handbook  No. 16.
Blackwell Scientific Publications,  Oxford,  UK.  HOLM001F

Chapman,  P.M., and  R.M.  Kocan.   1984.    Survey  of biological  effects  of
toxicants upon  Puget Sound biota.   Ill:  Tests in  Everett  Harbor,  Samish,
and Bellingham Bays.   NOAA  Technical Memorandum NOS OMS 2.  National  Oceanic
and Atmospheric Administration, Rockvilie,  MD.  CHAP008F

Chapman, P.M., M.A. Farrel, and R.O. Brinkhurst.   1982.  Effects of species
interactions  on the survival  and  respiration of  Limnodrllus  hoffmeisteri
and Tubifex tubifex (Oligochaeta, Tubificidae)  exposed to various pollutants
and environmental  factors.   Water Res.  16:1405-1408.  CHAP005F

Dexter, R.N.,  D.E.  Andersen,  and E.A. Quinlan.  1981.   A  summary of know-
ledge  of  Puget Sound  related to  chemical  contaminants.  NOAA Technical
Memorandum OMPA-13.   National  Oceanic and  Atmospheric  Administration,
Boulder, CO.  435 pp.  DEXT001F

Dlnnell,  P.A.,  F.S.  Ott,  and  Q.J. Stober.   1984.   Renton  sewage treatment
plant  project.  Seahurst  baseline  study.   Volume  X.   Section  12.   Marine
toxicology.   University of  Washington  Fisheries Research  Institute, Seattle,
WA.  192 pp.   UWFR019F

Gahler, A.R.,  R.L. Arp, and J.M.  Cummins.   1982.   Chemical  contaminants
in edible  non-salmonid fish and crabs from Commencement  Bay,  Washington.
U..S. EPA Environmental  Services Division, Seattle, WA.  117 pp.  GAHL001F

Harper-Owes  Company.   1983.   Water  quality assessment  of  the Duwamlsh
Estuary,  Washington.   Municipality  of  Metropolitan  Seattle,  Seattle,  WA.
MET0026F

Holme,  N.A.,  and A.D.  Mclntyre.   1971.   Methods  for the study  of marine
benthos.  IBP Handbook  No. 16.   Blackwell  Scientific  Publications,  Oxford,
UK.  334 pp.   HOLM001F


                                    67

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Lie,  U.   1968.   A quantitative  study  of benthic  infauna  in Puget  Sound,
Washington,  U.S.A.  in  1963-1964.   Fisk.  Skr.,  Ser.  Hav.  14:229-556.
LIE 001F

Long,  E.R.   1984.   Sediment bioassays:   a  summary of  their use in  Puget
Sound.  NOAA Ocean Assessments Division, Seattle,  WA.   30 pp.   LONG004F

Mai ins,  D.C.   1984.    Chemical  pollutants  in  sediments  and diseases  of
bottom-dwelling fish  in Puget  Sound,  Washington.   Environ.  Sci. Technol.
18:705-713.  MALI009F

Mai ins,  D.C.,  B.B. McCain,  and D.W. Brown.   1982.    Chemical  contaminants
and abnormalities  in fish and invertebrates from  Puget Sound.   NOAA Techni-
cal Memorandum OMPA-19.  National  Oceanic and Atmospheric  Administration,
Boulder, CO.  168 pp.  MALI003F

Malins,  D.C.,  B.B. McCain,  and D.W. Brown.   1980.    Chemical  contaminants
and biological  abnormalities in  central   and  southern Puget  Sound.   NOAA
Technical Memorandum OMPA-2.   National  Oceanic and Atmospheric  Administra-
tion,  Boulder, CO.  295 pp.   MALI002F

Malins, D.C.,  B.B. McCain,  D.W.  Brown,  S.L. Chan, M.S. Myers,  J.T. Landahl.
1984.   Chemical pollutants in sediments  and diseases of bottom-dwelling fish
in Puget Sound, Washington.   Environ. Sci. Technol. 18:705-713.

McCain, B.B., D.C. Malins.  and  S.-L.  Chan.  1983.  A  multiyear  (1979-1983)
comparison of  disease  prevalence in English  sole and Rock sole  from  eight
selected sites in  Puget  Sound.  NOAA Northwest  and Alaska Fisheries Center,
Seattle, WA.  38 pp.  MCCA002F

McCain, B.B., M.S. Myers, and U.  Varanasi.  1982.  Pathology  of two species
of flatfish from urban estuaries  in Puget  Sound.   NOAA Northwest  and Alaska
Fisheries Center,  Seattle, WA.  100 pp.   MCCA001F

Michael, A.D., R.A. McGrath,  and C.D. Long.   1981.   Benthic grab compara-
bility  study.   Final  Report.   U.S. Department of  the Interior, Bureau  of
Land Management.   Prepared by Taxon, Inc., Salem,  MA.   32 pp.  + Appendices.

Ott, F.S., P.O. Plesha,  R.D.  Bates, C.  Smith, and B.B. McCain.   (In prepa-
ration).  An evaluation  of  an amphipod  bioassay using  sediments  from  Puget
Sound.  36 pp.  OTT 001D

Pierson, K.B.,  B.D. Ross,  and C.L.  Melby.   1983.   Biological  testing  of
solid  phase  and  suspended  phase dredge  material from  Commencement  Bay,
Tacoma, WA.  U.S.  Army  Corps of Engineers, Seattle, WA.  59  pp.   PIER001D

Roff,   D.   1982.    Reproductive  strategies 1n  flatfish:  a first  synthesis.
Can. J. Fish. Aquat. Sci. 39:1686-1698.   ROFF001F
                                    68

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                                      COUWNCD S£W€fl OVENFLOW (UAJOR)
                                      COMOMCD SEWER QVEflFlOW (MINOR*
                                  •   COMBINED SEWER OVfRFLOWSTOflM DRAIN
                                  •*!   STOAW DRAIN (f 10 34')
                                      STORM DRAIN (25* 10 *•}
                                  •«C   STORU DRAW | > 4f'}
                                  0   TPWTMiNT PtAMT
                                  ®   OTHM POTENTIAU SOURCES
Contamin;     ources and sel«xted industry locations
in Elliott Ba     >d the lower Duwamish River
                                                     MAPI

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                                    COMBINED SEWER OVlftRXW (MAXWl
                                    COMBINED SCWE* OVERFLOW {MINOR)
                                    COMBINED SCWEA OvtRFLOWSTORM DAMN
                                    STORM DRAIN (I* w 24')
                                    STORM DRAW («• 10 «T)
                                    STORM DRAM (> «']
                                           M_*NT OUTFALL
                                (?)  OTMCB POTENTIAL SOURCES
Sediment Chemistry: Sampling stations for selected
data sets in Elliott Bay and the lower Duwamish River
                                                   MAP 2

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                                     COMBINED SEWER OVERFLOW (MAJOR)
                                     COMtMEO «WER WIRfLD* (MHOfl)
                                     COMBINED SOrtR OVERFLOW'STORM DRAIN
                                     KOMI
                                     STORM DRAM («• 10 41'}
                                 -«   STORM DRAM (> 4T)
                                 O   TRCATMEWT PLANT OUTFALI
                                     OTHER POTFNT.AL SOURCfS
Benthic Infauna: Subtidal and intertidal sampling
stations for selected data sets in Elliott Bay and the
lower Duwamish  River
                                               MAPS
• SUBTItMl

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                                 *  COMBINED SEWER OVERFLOW (MAJOR:
                                     COMBINED SEWER OVERFLOW (MINOR)
                                  »  COMBINED SEWER OVERFLOwrSTORM DRAIN
                                 ^.  STORM DRAIN (i- w 2«-j
                                 4$  STORM DRAIN («• ic 40-}
                                 •«$  STORM OfUUN (>•«•}
                                  O  TRCATweNT PUUT OUTFALL
                                     OTMffl POTENTIAL SOURCES
Sediment Bioassays: Sampling stations for selected
data sets in Elliott Bay and the lower Duwamish River
                                                   MAP 4

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                               •*  COMBINED SEWER OVERF

                               *  CQUWNED SEWER OVEWUJW (UMOR)

                               *  COMBINED SEWER OVERFLOW/STORM DRAW

                                   STORM DRAIN (•• to 24-|

                                   STOAM DRAIN (»• ID 4«-|
Bioaccumulation and Fish Pathology: Subtidal and
intertidal sampling stations for selected data sets in
Elliott Bay and the lower Duwamish River      MAp 5
FISH TRAWUMTHOLOGY

BIOCCUMULATION

O»TA WERE POOLED

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                               *  COUMMCO SCWCR OVERaOW (UUOA)
                               •  COM0MCD SeWCM WW*UJW
                                               	   DRAM
                              4$  STORM DRAIN (•' to 24')
                                  STORM OAAM (»' ID *•')
                                  STORM OftUN (>4C*)
                                            OUTWU.
Sampling locations for field studies in
Elliott Bay and the lower Duwamish River
                                                  MAP 6
     NTBTOM. BOWS*/CHO«TWr
•    SU8T1QM. BlOASSW/CHEMlSTW
•    SUBTIDW. BKHSSAY/CHEUISTBY/IN
Tb

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