Puget Sound Estuary Program
SAMPLING AND ANALYSIS DESIGN
FOR DEVELOPMENT OF
EVERETT HARBOR ACTION PROGRAM

DRAFT REPORT

PREPARED BY:
TETRA TECH, INC.

FOR:
U.S. ENVIRONMENTAL PROTECTION AGENCY
REGION X - OFFICE OF PUGET SOUND
SEATTLE, WASHINGTON

JULY, 1986



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Draft Report
TC 3991-03
SAMPLING AND ANALYSIS DESIGN
FOR DEVELOPMENT OF EVERETT HARBOR
ACTION PROGRAM
by

Tetra Tech, Inc.
for

U.S. Environmental Protection Agency
Region X - Office of Puget Sound
Seattle, Washington
July, 1986
Tetra Tech, Inc.
11820 Northup Way, Suite 100
Bellevue, Washington  98005

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                           CONTENTS


                                                                 Page

LIST OF FIGURES                                                  iii

LIST OF TABLES                                                    iv

INTRODUCTION                                                       1

GENERAL APPROACH                                                   3

    STUDY TYPES AND INTEGRATION                                    3
    SPATIAL ANALYSIS                                               4
    REFERENCE AREA                                                 6
    CRUISE PROCEDURES                                              7
    STATION LOCATION METHODS                                       7

SEDIMENT QUALITY SURVEY                                            9

    DATA GAPS                                                      9
    GENERAL STUDY DESIGN                                          11
    STATION LOCATIONS                                             13
    SAMPLING METHODS, PROCESSING, AND ANALYSES                    17

SEDIMENT BIOASSAYS AND BENTHIC MACROINVERTEBRATE COMMUNITIES      20

    DATA GAPS                                                     20
    GENERAL STUDY DESIGN                                          21
    STATION LOCATIONS                                             25
    SAMPLING METHODS AND SAMPLE PROCESSING                        26
    LABORATORY PROCEDURES                                         27

BIOACCUMULATION AND PATHOLOGY                                     29

    DATA GAPS                                                     30
    GENERAL STUDY DESIGN                                          31
    STATION LOCATIONS                                             36
    SAMPLING METHODS                                              37
    SAMPLE PROCESSING                                             38

DATA MANAGEMENT                                                   40

SUMMARY                                                           41

REFERENCES                                                        42

APPENDIX:  MAPS
                              ii

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                                  FIGURES

        NOTE:  Figure found immediately following page indicated
Number                                                                  Page
   1    Project area:  Everett Harbor and the Lower Snohomish River       1
   2    General approach to development of Everett Harbor Action Plan     1
   3    Components of recommended study design                            3
   4    Sample processing scheme for pathology and bioaccumulation
        component                                                        38

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                                  TABLES


        NOTE:  Table found immediately following  page  indicated

Number                                                                  Page

   1    Determination of minimum detection levels for  elevated
        incidence of disease given 10 sample sizes and three  back-
        ground levels of disease                                        34

   2    Summary of basic study design                                   41

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                              INTRODUCTION
     Under the  Puget Sound Estuary Program, the U.S.   Environmental 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  and microbial contamination in Port  Gardner
and the  lower  Snohomish  River.   The resulting Everett Harbor Action Plan
will:   1) identify existing problems  of toxic chemical  contamination, microbial
contamination, and associated biological effects  within the study area
boundaries (Figure 1); 2) identify ongoing and historical sources of  toxic
chemical  pollution; and 3) identify appropriate remedial  actions and agency
responsibilities  for implementing  the foregoing  actions.  Recommendations
for short-term corrective  actions will be  presented  in  the Interim Work
Plan.   The sampling and analysis plan presented  here  represents the  study
design for a field investigation to supplement  the existing database.
Results of the  field studies will  be used to develop the  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 problem
areas  and rank them in terms of priority for action.  Thus, study areas
defined in Figure 1 will  be used in the data  evaluation  stage,  but  all
areas  will not  be sampled as part  of this project.   Elements of the decision-
making approach and summaries of available data are provided in the  Initial
Data Summaries and Problem Identification report (Tetra Tech 1985b).  The
conceptual basis of the Everett  Harbor Action Program and the sampling
and analysis specifications  described herein are  similar to those used
previously for  the Commencement Bay Superfund Project (Tetra Tech  1985a)
and the Elliott Bay Toxics 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  (Tetra Tech  1986b) provides  details

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                                                  EVERETT HARBOR AREAS
  MUKILTEO
              ^^^^^••^^ NAUTICAL MILES
             KILOMETERS
            2           CONTOURS IN FEET
     EAST WATERWAY

     SOUTH PORT GARDNER

(T)  OFFSHORE PORT GARDNER

(7)  SNOHOMISH RIVER DELTA

(?)  SNOHOMISH RIVER

(£)  PORT GARDNER DISPOSAL SITE

@  EBEY SLOUGH

(?)  STEAMBOAT SLOUGH

(9)  UNION SLOUGH
Figure 1.   Everett Harbor project area.

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    DATA COLLECTION

    DATA EVALUATION

     PROBLEM-AREA
       EVALUATION

   POLLUTION SOURCE
      EVALUATION

    REMEDIAL  ACTION
         PLAN
   DATA
   GAPS
FIELD STUDY
  DESIGN
	I
Figure 2.   General  approach to development of Everett Harbor
           Action Plan.

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of sample handling, analytical chemistry, and other  Quality Assurance/Quality
Control (QA/QC) procedures.

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                            GENERAL APPROACH


     The  general objectives of the study  design are to:

     0    Identify problem  areas related  to  chemical contamination

     •    Define the spatial  extent and chemical/biological characteristics
          of  problem areas

     •    Prioritize problem areas for  remedial action.

The approach used to  develop the study design  involved:   1)  evaluation
of existing data for each study component (e.g., 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 sediments,  and biological
effects;  4)  identification of data gaps  in terms of spatial  and temporal
coverage  of existing data; and 5) selection  of study components,  conceptual
approach, specific variables to be measured,  and  station locations for
this study plan.  Sampling stations for  each recommended study component
were  positioned to fill  gaps in the  spatial coverage of previous studies,
to ensure adequate characterization of  areas near major pollutant sources,
and  to confirm selected  problem areas identified in previous studies.
All maps  referred to in the text are in Appendix A.

STUDY TYPES AND INTEGRATION

     Components of  the 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 identification
process.  The  rationale for the choice of  indicators, their interrelationships,

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                                  SURVEY OF CONTAMINATION AND
                                     BIOLOGICAL EFFECTS IN
                                   EVERETT HARBOR STUDY AREA


SEDIMENT QUALITY
• Surface Sediment
Chemistry
• Grain-Size
• Subtidal and Intertidal







SEDIMENT BIOASSAYS
• Amphipod Survival
• Subtidal and Intertidal









BENTHIC INFAUNA
• Community Structure
• Subtidal









BIG-ACCUMULATION
• Fish Muscle
• Crab Muscle
• Subtidal









FISH PATHOLOGY
• External
• Liver
• Subtidal

Figure  3.   Components  of recommended study design.

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and action-level criteria  that define  problem  areas are described in the
Initial  Data Summaries and Problem Identification  report (Tetra Tech 1985b).

     Concurrent  sampling of related variables  can  help ensure timely completion
of the project,  efficient  to use of  cruise resources, and collection  of
appropriate data.  The general timing of the field work is outlined below:

     •    August             — Bioaccumulation
                             — Pathology

     •    September/October   — Sediment Quality
                             — Benthic Infauna
                             — Bioassays

Timing of individual  surveys is justified below in  the  discussion of detailed
study designs.   Briefly, the comprehensive benthic survey  is scheduled
after the major recruitment period (spring-summer)  for  many infaunal species.
The timing of the  bioaccumulation and pathology  study ensures that English
sole will have  resided  in shallow-water sampling  locations  for several
months before sampling.

SPATIAL ANALYSIS

     Discrimination of spatial  patterns  in  contaminant distributions and
biological responses is a major objective of this project.   To facilitate
spatial  analysis, the project area has been divided  into nine smaller areas
based on geographic  features, bathymetry, and  locations of  major  pollutant
sources  (Figure 1).  The East Waterway was defined as a  distinct area.
The Snohomish River  and Estuary includes five  areas.   The remaining portion
of Port Gardner  includes three areas:  the deep, offshore areas; the southern
shoreline; and the Port Gardner Disposal Site.  Area  boundaries  and  major
features are as  follows:

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1.   East Waterway—All  of the East Waterway  north and east of
     a line  from the Snohomish  River mouth black can  buoy  "3A"
     to the southern-most  boundary of the  former  Weyerhaeuser
     pulp mill dock.

2.   South Port Gardnei—Shoreline areas (<30 ft  deep) from Elliot
     Point (Mukilteo) to the southernmost boundary of the  former
     Weyerhaeuser pulp mill  dock

3.   Offshore Port Gardner—All deep-water (>30  ft) areas  of
     Port Gardner exclusive of the  Port  Gardner Disposal  Site
     (see No.  6 below).

4.   Snohomish River  Delta—The area west of a line between the
     downstream boundary of Ebey and Smith Islands out  to the
     30-ft depth contour.

5.   Snohomish River—The main  navigable river channel downstream
     from the  Interstate-5 (1-5) bridge to the mouth  of  the river
     (marked by the black can bouy "3A").

6.   Port Gardner Disposal  Site—The designated disposal  site
     in South  Port Gardner.

7.   Ebey Slough—The  channel  adjacent to the  northern boundary
     of Ebey Island, west of 1-5 to a line downstream between
     Priest  Point and the western tip of Ebey Island.

8.   Steamboat Slough—The channel between Ebey and Smith Islands,
     west of 1-5 to a line between the western tip of Ebey  Island
     and the northwestern tip of Smith Island.
                                                        <
9.   Union  Slough—The portion of  the slough  west and north of
     1-5.

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During the  data analysis phase,  some of the above areas may be subdivided
based on  observed distributions of  contamination and effects.

     Several approaches to  spatial analysis will be used:  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  among areas for input  to
the  priority ranking procedure;  and 4) comparisons of individual  stations
and area  averages with data from reference  sites.   Each approach  will  be
important  for  assessing the  distribution of contamination and biological
effects in  the  project area.   If major sites .of contamination and  effects
are  found  beyond the influence of all  known sources,  other causes will
be investigated (e.g., historical contamination, unidentified local  source,
undefined transport process).

REFERENCE 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 chemical
contamination.   The upper portion of Port Susan will be used as a reference
area for comparison with the Everett   Harbor study area.   The reference
area is located approximately 20 km northwest of the study area.  The physical
characteristics of Port Susan appear to be similar to those of inner Everett
Harbor.  Port Susan has  muddy to  sandy sediments at shallow depths,  is
a  partially enclosed embayment,  and is influenced by freshwater from a
major river, the Stillaguamish.  Data on  the biota (i.e., fishes, benthos)
of  upper Port  Susan are not available at present.  Limited data on sediment
chemistry indicate that contaminant levels for total aromatic hydrocarbons,
polychlorinated biphenyls (PCBs), chlorinated  butadienes, hexachlorobutadienes,
and most  metals  are  low (Mai ins 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.

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CRUISE PROCEDURES

     Standard methods of operation will  be developed as part of the cruise
plan.  They incorporate  QA/QC procedures consistent with the Quality Assurance
Plan for this  project.  Special precautions will be taken to prevent contami-
nation of samples during collection and initial  processing aboard the survey
vessel.   Cleaning of samples, working areas, and instruments before collection
of each sample for  chemical analysis 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 (Tetra Tech 1986b).

STATION LOCATION METHODS

     Because  of the many sources and documented spatial heterogeneity of
contamination  in the  Everett Harbor study area, precise   positioning  of
sampling stations is  essential.   The intent  of  the navigational  control
effort is to accurately determine and document the locations of all  sampling
stations and transects.  In the Snohomish  River  and inner portion of Everett
Harbor 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  Snohomish River
and the East  Waterway will  be photographically recorded  (i.e., corners
of buildings and piers, spires, towers,  smoke stacks,  and  other  readily
distinguishable,  permanent objects).  The shipboard  photos will be compared
to aerial photographs  and USGS quadrangle  maps for the area.   Objects  that
can be  recognized clearly  on the  aerial  photograph or map, and hence can
be accurately  located,  will be selected and numbered as  reference  points.
The series of  shipboard  photos, with the reference points  identified and
numbered, will  constitute 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  along-channel

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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
the project.  In the outer  portions of the study  area, Loran C wi11  be
used as  the primary positioning tool,  with line-of-site  and photographic
confirmation.

     The boat will  be  anchored  at  stations whenever possible, and 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 database.
                                     8

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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 chemical  contamination
of the Everett Harbor  study area is based on analyses  of  sediment quality.
Station  locations  for the accepted data sets obtained from  previous studies
are shown in Maps 3 and  4.   Only  in  a few areas  are the data sufficient
to indicate whether problems may exist, and even in those areas the sampling
has not been oriented  toward developing relationships between high  levels
of toxic chemicals and sources.  Compared with other areas of Puget Sound,
the total suite of  chemicals measured in previous studies in Everett  Harbor
has been limited  [i.e. PCBs, polynuclear aromatic hydrocarbons (PAH), and
metals only].   Specific data gaps  for each of the areas are  discussed below.

East Waterway

     East Waterway has been  sampled extensively (Map  4).   A large number
of grab samples and deep cores have adequately characterized the horizontal
and vertical  distributions  of the  selected indicator chemicals used in
the analysis below (i.e., PAH, PCBs, and selected metals). However, information
regarding the concentrations and distributions of organic compounds other
than PAH and PCBs is needed.  Of particular interest  are those substances
that may be associated specifically with the pulp mills,  such  as the phenolic
and chlorinated phenolic compounds.  Results of recent studies in Commencement
Bay (Tetra Tech 1985a) and other studies suggest that  the following substances
of concern may accumulate in sediments as a result of  pulp-mill discharges:

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     •    Monochloro- and dichlorodehydroabietic acid

     t    Dehydroabietic acid

     •    Isopimaric acid

     •    3,4,5- and  4,5,6-trichloro- and  tetrachloroguaiacol

     •    2,4-dichloro-, 2,4,6-trichloro-, 2,3,4,6-tetrachloro-, and
          pentachlorophenol

     t    4-methyl phenol

     •    Chloroform.
     Historical  sampling in East Waterway  also has been aimed primarily
at determining  the spatial  extent of contamination  in  that area and  has
not addressed  the identification  of the sources of that  contamination.
Additional  data are needed to better  define the impact of specific sources
on the receiving  environment in  East  Waterway.

South Port  Gardner and Offshore  Port  Gardner

     Much of the deeper portions of South  Port Gardner have been sampled
adequately  for  selected indicator chemicals (e.g.  PAH,  PCBs, and selected
metals), but data regarding the distributions  of  other toxic substances
are lacking.  In  addition, the South  Port Gardner shoreline,  the deepwater
area  near  the  effluent discharge  points for the Scott pulp mill  and the
City of Everett CSO, and the area near Mukilteo  where  high  levels of  PAH
have  been  observed are characterized poorly at present.  All  of these areas
are close to known sources, including CSOs,  storm drains,  and industrial
effluents.
                                     10

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     Although Offshore Port Gardner  has been sampled  infrequently  (Map
3), the available data and the distance of this area from  sources indicate
that high  levels of toxic substances  probably are not a problem.  However,
the slope area bordering the Snohomish  River  Delta may be  a  depositional
zone for material transported by the river, and hence may accumulate  higher
concentrations of toxic substances than those measured in shallower waters.

Snohomish River, the Sloughs and Delta

     Insufficient data are available for  all river, slough,  and delta areas
(no analyses  of  priority pollutants in  sediments  are available from Ebey,
Steamboat,  and  Union Sloughs).  The  few samples collected from the lower
Snohomish River  and the delta have  contained  elevated concentrations of
some selected  indicator substances (i.e., PAH, PCBs, and selected metals).
In addition, pulp mills and other industries, CSOs, storm drains, and landfill
leachate discharge into these areas.   The impact of these sources  on the
receiving environment has not been evaluated.

Port Gardner  Disposal Site

     A limited  number of samples has  been collected near the disposal  site
southwest of  East Waterway.  Of these,  only one had a data  set acceptable
for evaluating selected organic indicator chemicals (i.e., PAH and  PCBs).
This limited database is insufficient to  characterize adequately the disposal-
site area.

GENERAL STUDY DESIGN

     The objectives of the sediment quality survey are to:

     t   Determine  the kind  and extent of chemical  contamination
         in  intertidal and subtidal sediments
                                      11

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

     •    Relate environmental contamination to  specific sources

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

Relationships among physical and chemical  properties of sediments, toxicity
measured in the sediment  bioassays, and benthic infaunal community character-
istics  will  be examined  using the results  of this survey and the related
benthos/bioassay studies described later.

     The sediment quality survey consists of the sampling of surface sediments
(i.e., 0-2 cm) at  57  stations throughout the Everett Harbor study  area
and at 3 stations in the reference area. The variables to be measured include
the following:

     Target Chemicals

     •    Bulk sediment  concentrations of priority pollutants, hazardous
          substances,  and miscellaneous compounds

     Ancillary Parameters

     a    Total organic  carbon and nitrogen
     •    Water-soluble  sulfides
     •    Total  solids
     a    Grain-size distribution.

     The chemical  analyses  for  toxic chemicals will include EPA priority
pollutants and hazardous substance list compounds.   Volatile EPA  priority
pollutant compounds will  only be measured  at stations close to potential
sources.  Miscellaneous compounds to be  analysed  at  stations  near  pulp
                                      12

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mill discharges will include  the chemicals of  concern listed  previously
(see Data Gaps  and East Waterway above).

     Ancillary  variables will  be analyzed in every surficial  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
will be  useful for determining the origin  of organic material.  Data on
sulfide content will indicate the potential toxicity of bottom  sediments
because of anoxic conditions.   Grain-size distributions will  allow stratifi-
cation of station comparisons  based on the  physical characteristics of
samples,  and will thereby help distinguish effects of pollutants  on benthic
infaunal  communities from the effects of physical influences  of  the habitat
itself.

     In the the sediment quality survey, only  the upper 2 cm  of  each sediment
sample will  be  collected and analyzed (Tetra Tech  1986d).   At  undisturbed
sites, the surface sediments  are expected  to represent  the most recent
contaminant profiles.  Because the surface sediment layer also  is the  most
biologically  active zone  of the sediments, contaminant concentrations in
surface sediments are of most interest for  relating contamination to  biological
uptake,  bioaccumulation,  and  other associated effects.  Future  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.

STATION LOCATIONS

     Fifty-six subtidal  and four  intertidal sites  will  be  established
as part of the  overall  sampling program  (Map  11).  The majority of the
stations  will  be located  at depths of 30 ft  where possible.   The 30-ft
depth is  shallow enough to be within the potential  influence  of  most contam-
inant  sources, yet deep enough to be beyond the influence of sunface waves.
Station locations reflect the following objectives of the  sampling plan:
to fill data gaps, to confirm suspected  toxic  "hot-spots" near major sources,
and to verify questionable data.
                                      13

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     The majority of subtidal  stations are  located in shallow nearshore
areas for the following  reasons:

     •    An evaluation of recent studies  in  the  Port Gardner/Everett
          project area showed a major data gap for the  shallow, nearshore
          environment

     •    Most pollutant sources discharge  directly  to nearshore areas

     •    The nearshore environment has the highest  incidence of human
          contact with toxicants entering Port Gardner, and the greatest
          probable effects

     •    Previous  studies in Elliott Bay showed that a major source
          of contaminants  (Denny Way  CSO) had 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.

     The  breakdown of  stations  by area within  the Port Gardner/Everett
project boundaries is listed below:

                                            Subtidal       Intertidal

1.   East Waterway                             15               0
2.   South Port  Gardner                         11               4
3.   Offshore Port Gardner                       7
4.   Snohomish River Delta                       3
                                      14

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5.   Snohomish River                             8              0
6.   Port Gardner  Disposal Site                  0
7.   Ebey Slough                                 3              0
8.   Steamboat Slough                            6              0
9.   Union Slough                                0              0
     Reference area                              3              0
                             TOTAL             56               4

East Waterway

     Fifteen  subtidal  stations are located in East Waterway.  Five stations
are located near active CSOs, storm drains, or industrial  outfalls.   Seven
stations  arelocated  at  varying distances from these sources to evaluate
contamination  gradients.  Three stations are located near a potential historical
source  of toxic contaminants, the former Weyerhaeuser pulp mill.  The main
rationale for station  placement is to quantify the degree  of contamination
and the magnitude of  biological effects in relation to potential sources
of contaminants.

South Port Gardner

     Eleven  subtidal  and  four intertidal  stations are located in this area.
Subtidal stations occur  near the Mukilteo sewage treatment plant, the Mukilteo
fuel  facility, Japanese Gulch, Powder Mill  Gulch, and Pigeon Creek #1 and
#2.  The South Port Gardner shoreline is an area with multiple storm drains,
CSOs,  and industrial  discharges of which little is known.  The proposed
station locations represents the  major  known point sources for this area
and will  enable a comparison of the  potential impacts of these sources.
Intertidal sites are  located shoreward from several of these subtidal stations
to determine contaminant levels in sediments at public access areas and
beaches.
                                      15

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Offshore Port Gardner

     Seven  subtidal  stations  are located  in  this area.   All of them are
clustered around the Scott deepwater outfall to evaluate potential  contamination
and biological  effects  related to that discharge.

Snohomish River Delta

     Three  subtidal  stations  are located  on  the river delta  to provide
information on levels of contamination in sediments  at stations  removed
from  known sources.   One station is  located near the mouth of Steamboat
and Ebey Sloughs downstream  from  the Tulalip landfill  and the  Weyerhaeuser
lagoon  discharge.   The other two stations are  located 1  and 2 km west of
East Waterway and the mouth  of the Snohomish River.

Snohomish River

     Eight subtidal  stations are  located within the Snohomish River downstream
of the 1-5 bridge.  One station  is  positioned  near a  major CSO discharge
into the Everett Marina Basin.  Three stations are located off  the historical
discharge from the Weyerhaeuser kraft mill.  One station is  located mid-channel
off the Everett Wastewater  Treatment Plant outfall. One station is located
near CSOs 016  and 017 below the 1-5 bridge.  Finally, two  stations are
located at mid-channel  near  the mouth of the river.  These latter two stations
are located away from known sources  and will be used  to evaluate the integrated
potential effects from  upstream  sources of contaminants.

Port Gardner Disposal Site

     The disposal site will be  evaluated as part  of the  Puget  Sound Dredged
Disposal Analysis (PSDDA)  program.   If an initial  evaluation indicates
that  the site is  suitable for  future disposal   operations, then further
sampling and analysis of the site may be specified as  part  of PSDDA.  Therefore,
sampling of the  disposal  site will not be conducted as  part of the Everett
Harbor Action Program.
                                      16

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Ebey Slough

     Three subtital stations are located  in the main channel  of Ebey  Slough.
All  of these  stations are positioned to  evaluate levels of  contamination
and  biological  effects potentially associated with leachate  from the  Tulalip
landfill.

Steamboat Slough

     Six subtidal  stations are  located in  the main  channel  of  Steamboat
Slough. One  station is  located near the Tulalip landfill,  whereas the
remaining stations are  located near the discharge from the  Weyerhauser
kraft mill lagoon.

Union Slough

     No sampling  is  proposed  in  Union  Slough.  Because there are no  known
sources of pollutants in this area, problems are not expected.

SAMPLING METHODS,  PROCESSING, AND ANALYSES

     Sediment  samples  will be  collected  with a modified  O.l-m^ van Veen
grab according to protocols recommended  under the Puget Sound  Estuary  Program
(Tetra  Tech  1986d).  Each  station will be located using  the navigation
techniques discussed earlier.  Before each sample is taken,  vessel  position
will  be visually rechecked (range alignments)  and necessary adjustments
will be made.  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.

     If several attempts to sample a station are  unsuccessful,  another
nearby station meeting similar sampling needs will be selected  and documented.
Standardized data (e.g.,  collection date, time, station  location, depth,
and  replicate number) will be recorded  at each station.
                                      17

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     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,
aliquots will  be taken from a composite sample.  The upper 2  cm of sediment
away from the edge of the  grab will be removed carefully with a stainless
steel  spatula,  transferred to a clean stainless steel  bowl, and homogenized
by stirring  with a glass rod.  Aliquots will be collected  as  follows:

     •    500 cm3 will be transferred to a precleaned glass  jar with
          teflon cap liners (for  organic chemical analysis)

     •    125 cm3 will be transferred to a precleaned glass  jar (for
          metals analysis)

     •    100 cm3 will be transferred to a Whirl-pak bag (for grain-size
          analysis)

     •    1,500 cm3 will  be transferred to precleaned glass jars (for
          bioassays)

     •    500 cm3 will be transferred to a precleaned glass  jar with
          teflon cap liner (for archival storage).

     Precleaned  (solvent-rinsed and muffle-furnaced) stainless steel bowls
will be brought aboard with replicate solvent-washed spatulas to  provide
spares in the event of loss.   Bowls  will be of adequate size  for compositing
samples.  Between samples,  the  bowls 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.
Spatulas  and stirring rods  also will  be rinsed with site  water, rinsed
with solvent, and wrapped in aluminum foil.
                                     18

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     Intertidal 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 to the
above.

     In  the laboratory,  analytical chemistry methods will  follow procedures
of the Puget Sound Estuary  Program and the U.S.  EPA 301 (h) program  (Tetra
Tech 1986a) or comparable methods  (see  Quality  Assurance Project Plan,
Tetra Tech  1986b).
                                     19

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       SEDIMENT BIOASSAYS AND BENTHIC MACROINVERTEBRATE  COMMUNITIES
     The present study design  includes analysis of sediment toxicity (bioassays)
and benthic macroinvertebrate  communities at 27 of the subtidal sites chosen
for sediment chemistry (Maps 11  and  12).  Sediment bioassays are also planned
for the four intertidal  chemistry stations.  Station locations for acceptable
data sets  from previous  studies  are shown in Maps 5-8 to allow comparisons
with proposed sampling stations  shown in Map 12.  The detailed study  design
for these components is presented  following a summary of data gaps.

DATA GAPS

Benthic Infauna

     Major  data gaps  exist  for characterizing benthic infaunal  community
structure throughout most  of the Everett Harbor study area  (Maps 5  and  6).
The main exception is the East  Waterway where two recent projects  sponsored
by the U.S. Navy have provided adequate spatial  coverage.   The following
sections present  the major  data gaps  for benthic community structure in
the remaining areas:

     •    No benthic infauna data  for Ebey, Steamboat, and Union Sloughs
          are available

     •    Limited data on  benthic  infauna are available for the Snohomish
          River.  Only two stations  within the river have been  sampled
          for determination  of species composition and abundance of
          benthic infauna

     •    South Port Gardner lacks adequate data on benthic infauna
                                       20

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     t    Benthic infauna of the Port  Gardner  Disposal Site have not
          been  characterized adequately.

Sediment  Bioassa.ys

     Almost all  of  the available  information  on  sediment toxicity in the
Everett Harbor  study area  is for East Waterway.   Only three stations outside
this area have  acceptable  data (Maps 7 and 8).  The  major gaps  in the existing
sediment  toxicity data  are the following:

     •    For the East Waterway, the amphipod  data are adequate, but
          the oyster data  are for frozen  sediments

     •    The information  for Offshore Port Gardner  is extremely limited,
          being based on  three  stations  near the  mouth of  the  East
          Waterway

     •    Acceptable  sediment-bioassay  data are  not available for
          remaining areas.

GENERAL STUDY DESIGN

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

     •    Determine the abundance and distribution of benthic macroinvertebrates

     •    Determine the  toxicity  of sediments to a representative
          sensitive species

     t    Relate sediment contamination to  biological effects (i.e.,
          community structure of benthic  infauna  and bioassay  responses)
                                      21

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     •    Rank areas and  contaminants with  respect to environmental
          impacts.

     For most areas,  amphipod bioassays  are recommended.   If sediments
with salinities  less than 15 ppt are  encounted, other tests may  be  needed.
The amphipod  sediment bioassay  measures  short-term (i.e.  10-day)  response
to bioavailable contaminants  and provides an  index to estimate  effects
on sensitive  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  infaunal communities and
bioassay  data in selected nearshore areas  will  fill  major  data  gaps for
the project area.

Benthic Infauna

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

     •    Total  abundance

     •    Abundances of higher taxa,  e.g.,

               Polychaeta

               Mollusca

               Crustacea

               Amphipoda

     •    Species abundances

     •    Species richness
                                      22

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     t    Species dominance

     •    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.

     Total abundance (of all species) and abundances of higher taxa  (e.g.,
Echinodermata, Amphipoda,  Polychaeta, Gastropoda) will be  determined to
assess gross  disturbances of benthic communities.  Comparisons  of  community
characteristics among  study areas based on  analyses of higher taxa will
provide input  to site  ranking for the Decision Criteria. Statistical comparisons
of data from each area  or individual station with reference conditions
will  establish a quantitative basis  for  describing the presence, magnitude,
and spatial  extent of  biological responses to  contamination.

     Species  richness and dominance  and  the abundances of indicator species
can be used  directly to analyze community properties in relation to site
characteristics.   Using  numerical  clustering techniques,  the entire data
set on species abundances can be reduced  to an  interpretabl e  form, whereby
groups of  stations are  identified  on the  basis  of similarities in their
species composition and relative species  abundances (Boesch 1977).  Multiple
regression,  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, organic carbon  content, and chemical concentrations.   Discrimination
among  the  potential  causes of observed  alterations in infaunal communities
will address the importance of conventional  physical-chemical parameters
relative  to  contamination levels.

     Because  of the high degree of spatial variability in benthic  community
characteristics, it is necessary to analyze a  sufficient number  of  replicate
samples.   In  previous studies of Puget Sound and elsewhere,  a minimum of
four  to  six  replicate 0.1-m2  van Veen grabs has been recommended  (Holme
                                      23

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and Mclntyre 1971; Lie 1968).   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 assemb-
lages.

     A power  analysis  of  replicate infaunal  samples from outer  Elliott
Bay and the Seahurst area was performed using total  abundance, total  number
of taxa,  echinoderm abundance,  and  amphipod abundance  (data from METRO
Seahurst  Baseline Study).   The power analyses indicate that five replicate
O.l-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 for characterizing species richness as  well as for performing
numerical  classification analyses.  Although 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 further does not increase statistical  sensitivity sufficiently
to justify higher costs.

Sediment  Bioassays

     The amphipod  sediment  bioassay will be conducted according to Swartz
et al. (1985), as modified by the Puget Sound Estuary Program (Tetra  Tech
and E.V.S. Consultants 1986).

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

     0    Acute (i.e., 10-day) mortality of  amphipods  (Rhepoxynius
          abronius)

     •    Sublethal effects on R. abronius  (moribund, emergence)
                                      24

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     •    Temperature

     •    pH of sediment

     0    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 Port Gardner  and the Snohomish River system are shown on Map  12.
Sediment chemistry  (Map 11) will be evaluated at all stations sampled  for
benthic infauna and/or sediment  bioassays.  In  the project  area, sediment
for toxicity bioassays  will be  collected at both intertidal  and subtidal
sites, whereas benthic infauna will be characterized only at subtidal  sites.
Sampling and analysis of  benthic infauna at intertidal  sites is not recoirmended
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 project 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.  Fourth, knowledge
about individual species  responses is limited, interpretation of the results
would be difficult.

     Benthic samples  will  not be collected at the subtidal  biological  station
at the Scott deepwater outfall because reference data will  not  be available
for benthic infaunal  communities  at that depth.  The three benthic stations
in the upper Snohomish River will  be sampled,  but will  not  be analyzed
unless adequate reference conditions can be found.
                                      25

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     The rationale for placement of biological  stations is related to that
of the sediment  quality studies discussed earlier.   In  particular, most
of the  biological stations  are positioned  near contaminant sources within
the nearshore  areas to  allow  analysis  of  gradients in  sediment toxicity
and benthic community structure.

SAMPLING METHODS AND SAMPLE PROCESSING

          Benthic  infauna will be sampled and analyzed according to protocols
recommended under the  Puget Sound Estuary Program (Tetra  Tech 1986g).
A 0.1-m2 modified  van Veen grab will  be used  to collect sediment for chemical
and benthic infaunal analyses and for toxicity bioassays.  At each station,
replicate  samples will  be taken for analysis of benthic infauna, sediment
chemistry, and bioassays.  To avoid disturbance and loss of benthic organisms,
samples  for benthic infauna analyses  will  not be obtained by subsampling
grab samples used  for other analyses.  Eight  to  ten sequential  grabs will
be made, with  alternate  grabs for benthos and chemistry/bioassays.  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.

     Because  the  high cost  of  chemical analyses and bioassays limits  the
number of these measurements that  can  be made for in  replicate samples
in 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.  After a benthic sample
is collected,  the  sample will  be washed on  a  1.0-mm screen.   Mesh of this
size has been used in  most  previous  studies  of  benthic infauna in Puget
Sound.   Samples  will be  transferred  to  a container and  preserved with  10
percent buffered formalin.

     Sediment samples obtained for bioassays will be placed in clean jars
following the  homogenizing procedure.  Samples will be  placed immediately
                                      26

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in the dark  on ice, transported to the laboratory, refrigerated  (4° C),
and then  assayed within 14 days of collection.

LABORATORY  PROCEDURES

Benthic Infauna

     After sitting  at  least 24  h  in  fixative, infaunal  samples will be
washed on a 0.5-mm  screen,  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.  All
benthic  organisms will be  identified to species,  if possible, or  to the
lowest taxon  practical.

     Benthic  samples  will  be analysed sequentially according  to salinity
of the habitat in  the Snohomish River estuary  and  sloughs.   Samples from
the most  saline areas will  be  analyzed before samples from  less  saline
environments.   Benthic community structure will  not  be analyzed for  samples
taken upstream from  stations where the  results indicate a freshwater benthic
community.

     QA/QC procedures will  follow the Quality Assurance Project  Plan  (Tetra
Tech 1986b).   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 on 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,
temperature and salinity will  be changed gradually at rates  no  greater
than 1°  C  and 2 ppt per day until  the bioassay  conditions of 15° C and
                                      27

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25 ppt are  attained.  Five replicate assays will  be  performed in 1-1  glass
beakers containing 2 cm (weighted) of test sediment.

     Prior to initiation  of the bioassay,  sediment samples will  be  mixed
within their  storage containers,  and  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  0.5-mm screen.  Numbers
of surviving amphipods will  be counted  as those capable of discernible
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
the  study area.   Both organic and inorganic contaminants will  be used in
separate  series of control  experiments.

     Sediments from the Snohomish River  estuary may  be subjected to wide
ranges in salinity  depending on  the  river discharge  and  tidal pumping.
Interstitial  salinities  between 15  and  24 ppt will be adjusted upward to
25 ppt.   The amphipod  bioassay will  not be conducted on  samples having
an interstitial salinity less than 15 ppt.
                                     28

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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, whereas
pathological  analyses  may indicate  sublethal responses of organisms to
chronic exposure  to toxic  chemicals.

     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 the  Everett Harbor study
area, increasing  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.,
Malins et al.  1984; Tetra  Tech 1985a,b).  It is therefore likely that  this
species  is being influenced by contamination of bottom  sediments.  Finally,
because selected bottomfish species are captured and consumed by some recrea-
tional  fishermen (McCallum 1985), English sole  are indicative of a food-
web pathway through which  contaminants can move from  sediments to humans.
According to  a recent angler survey in areas of the East Waterway and lower
Snohomish River,  however,  English sole is not eaten  by a large number of
recreational anglers  in  Everett (McCallum 1985).

     Dungeness crab   (Cancer magister) was chosen as  the representative
invertebrate species  primarily because it has shown a  tendency to accumulate
contaminants  in  tissues that are  consumed  by humans.  This species is
sufficiently abundant throughout the  study area to allow statistical comparisons
among all study sites.  Moreover, Dungeness crabs are an  important recreational
and commercial  fisheries  resource in the Everett area (McCallum 1985; Shapiro
and Associates  1985).
                                      29

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DATA GAPS

Bioaccumulation

     Data on bioaccumulation of toxic chemicals  in edible tissues of indigenous
marine organisms  from the  study area are  available  only from one study
by Washington Department of  Ecology (Cunningham 1982).   Concentrations
of priority pollutants were analyzed in dorsal muscle of rock sole  (Lepidopsetta
bilineata) and English sole from five stations in three areas:   East Waterway,
lower Snohomish River, and a reference site  on the  southwest side of  Gedney
Island  (Maps 9 and 10).   Data gaps exist  for all other study areas.  Edible
muscle tissue of Dungeness crab has not been  analyzed for toxic  contamination
in the project area.  Therefore, the lack  of data for target bioaccumulation
variables represents a major data gap.

Pathology

     All historical fish pathology data were collected  from the  East Waterway,
the Snohomish River, the Mukilteo-Everett  shoreline, and the area immediately
adjacent to the  mouth of  the Snohomish River.   Major data  gaps for fish
pathology include the following areas:

     •    Parts of South Port Gardner

     •    Offshore Port Gardner

     •    The Snohomish River Delta

     •    Parts of the Snohomish River

     •    The Port Gardner Disposal Site

     0    Ebey Slough
                                       30

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     •    Steamboat Slough

     •    Union Slough.

GENERAL STUDY  DESIGN

     The primary objectives of this component are to:

     •    Determine levels  of tissue contamination and frequencies
          of pathological disorders  in  representative  fish species
          in the Everett Harbor study  area

     t    Compare the  level  of tissue contamination and prevalence
          of disorders among areas

     •    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 local  "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  concentrations in fish and crab muscle

     •    Pesticides and PCBs

     t    Mercury

     •    Total extractable lipid material
                                      31

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     Pathological  abnormalities  in  fishes and invertebrates

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

     •    Internal  abnormalities for English sole

     •    Selected  liver lesions for English sole, primarily:

               - Neoplasms
               - Preneoplasms
               - Megalocytic hepatosis
               - Nuclear pleomorphism
               - Hepatocellular  regeneration
               - Excessive melanin  macrophage centers

     Ancillary parameters

     •    Individual  English sole

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

     •    Individual  Dungeness crabs

               - Weight
               - Width
               - Sex

     •    Species composition (numerical) of trawl samples.

Chemical  analysis of  edible portions of  target species will allow estimation
of potential  human  health hazard.   PCBs and  pesticides  will  be analyzed
because  these  substances accummulate  in muscle tissue of marine organisms
                                      32

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and are  a  potential threat  to public health.   Metals other than mercury
will  not  be analyzed in fish muscle samples  because previous  data  (e.g.,
Cunningham  1982)  indicate that no problem exists.

     Analyses of contaminants  in  fish  livers is not recommended for this
study because:

     •   Compounds such as PAH that have been implicated  as one  potential
         cause of liver lesions  are  detected infrequently in liver
         samples using standard techniques

     t   For PCBs, there may be little relationship between contaminant
         concentration in the liver and the prevalence of liver lesions
          (Tetra  Tech 1985a)

     •   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 1985a).  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

     •   Because of the  small mass of liver  tissue (typically less
         than 2 g), compositing of individual  samples would be necessary
         for chemical analyses

     •   Detection limits are generally high even when samples are
         composited because of the high  lipid content of livers.

     The liver  is singled out  for histopathological  analyses because it
is the organ most heavily afflicted with pathological  disorders  (e.g.,
Mai ins 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 pleomorphism, hepatocellular  regeneration, and  excessive  melanin
                                      33

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macrophage  centers.  These disorders are wel1-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  determined, morphologically  similar  lesions
have been induced in laboratory  mammals and fishes  by  exposure to toxic
chemicals  (Maiins et al.  1984).

     Pathological and contaminant analyses  will  be biased toward larger
English  sole (i.e., larger than 230 mm total  length,  or at least  3 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
greatest  threat to public health if their edible tissue is contaminated.
Second,  prevalence of several pathological disorders in  English sole livers
increases with age (Malins et  al.  1982; McCain  et al.  1982;  Tetra  Tech
1985a).   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 sufficient samples sizes  can
be obtained,  an upper size limit (e.g., 300 mm) will  also be set for  fish
used in  the  pathology study.

     Ancillary data  (weight,  length,  sex,  and  age) 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 1).  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?"
                                      34

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     TABLE 1 .   DETERMINATION OF MINIMUM DETECTION LEVELS  FOR ELEVATED
      INCIDENCE OF  DISEASE GIVEN 10 SAMPLE SIZES AND THREE BACKGROUND
                            LEVELS OF  DISEASE*
Sampl e
Size
20

40

60

80

100

120

140

160

180

200


0 Percent
tf> 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

Background Level
s of Disease
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 chi-
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).

-------
Results showed  that for all three background  levels, discriminatory improvement
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 comparisonwise 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 tissue in English sole,  five fish
from each study site will be used.   This  sample size is a  balance between
analytical costs  and even  representation  across all stations.   Gahler et
al.  (1982) 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 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  (e.g., 100-150  percent  of the
grand  mean among  stations),  but  may be insufficient for discriminating
smaller differences.

     For bioaccumulation in Dungeness  crabs, a composite of muscle tissue
samples from 5-10 crabs will be analyzed from  each transect.   The  number
of individual  samples in the composite  will be the same for all  transects.
Results will allow risk estimates to be made for human consumption  of contam-
inated  crabs.   To provide an estimate of variance for QA/QC purposes, three
composite samples will be analyzed  from the East Waterway.  However, because
estimates of variance will  not be available for all sites,  statistical
comparisons between transects will  not be possible.
                                      35

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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  season.  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.  Late August  has been selected
as the optimal sampling period.

STATION LOCATIONS

     It is recommended that  11 transects be sampled in the Everett Harbor
study area to evaluate  bioaccumulation and  fish  pathology (Map 12).   In
addition, a  single transect  will  be located  in the Port Susan reference
area.  Most of the transects in the  Everett Harbor study area are  located
near known or suspected  sources of contamination.   In  the East Waterway
and the Snohomish River, transects will  run  parallel  to the longitudinal
axis of  each water body.   In other parts of  the  project area, transects
will  be positioned along the 30-ft isobath to  coincide  with the sampling
depth  for sediment chemistry, sediment bioassays,  and  benthic infauna.
Trawl locations are described below with respect  to the specific  areas
defined earlier (see above, Figure 1).
                                     36

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     East Waterway—One transect is  recommended for East Waterway.  Although
historical data exist for this area,  new data will allow interstudy comparisons
to be made  and  interannual variation to be evaluated.

     South Port Gardner—Four trawl transects are recommended for South
Port Gardner.   One is located directly offshore  from the Mukilteo  sewage
treatment  plant.   A second  transect is located off the fuel storage tanks
at Mukilteo.  A third transect is  located offshore from  Powder Mill  Gulch.
Finally, a  fourth transect is located off the former Weyerhauser pulp mill.

     Snohomish River Delta—Two transects are recommended  for the Snohomish
River Delta.   One is located off Mission Beach near  the Tulalip  sewage
outfall.   The  other transect  is  located approximately 1.5 km northwest
of the mouth of the Snohomish River  and East Waterway.

     Snohomish  River—Two transects  are recommended for  the Snohomish River.
One transect is located near Baker Island, downstream  from the discharge
point of Everett's secondary  treatment sewage ponds.   The second transect
is located  near the mouth of the river, off an adjacent  industrial area.

     Ebey, Steamboat,  and Union Sloughs—Two transects are  recommended
for the slough  system.   One transect in Steamboat Slough is located  off
the Tulalip  landfill.  A  second  transect in Steamboat  Slough is located
off the discharge point of Weyerhauser's kraft-mill lagoon  system.

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.
                                      37

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     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).
Generally, a 5-min  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 Port Gardner and the Snohomish River may be 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.

     Dungeness  crabs will be sampled with the trawls and crab pots. Crabs
captured when trawling will be pooled with those from crab pots if required
to obtain the desired number.

SAMPLE PROCESSING

     The recommended  sample processing scheme  is illustrated in Figure 4.

     After  each  trawl  sample  is  brought aboard, the catch will  be sorted
into three categories:  1) English  sole,  2)  Dungeness crabs,  and  3) mis-
cellaneous fishes and invertebrates.  All  organisms will be examined  for
grossly visible  external abnormalities while  being processed.

     Five to ten  Dungeness crabs will  be randomly  selected from those caught
at each site, measured (nearest mm, carapace  width), weighed (nearest  gm,
wet weight), sexed,  frozen whole, and stored  for  later contaminant analysis
of edible tissue.  Invertebrates  other than Dungeness  crabs and  fishes
other  than English  sole  will  be  identified to  species and released.   If
                                      38

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       CRAB-POT SAMPLE
                                       TRAWL SAMPLE
        CANCRID CRABS
                    ENGLISH SOLE
                                 COLLECT
                                 ANCILLARY
                                 DATA
                                MISCELLANEOUS FISHES
                                AND  INVERTEBRATES
                               SELECT 5 LARGE
                               FISH AFTER
                               LIVER REMOVAL
    DETERMINE CONTAMINANT
    LEVELS IN EDIBLE  TISSUE
    IN  5 CRABS AND 5  FISH
                                   I
FILLET
                                                    SELECT 60 FISH
                                                    LARGER THAN
                                                    230 MM TL
                        ±
                                                         IDENTIFY, COUNT
                                                         AND RELEASE
                  FIX a 1-CM3
                  SUBSAMPLEin 10%
                  FORMALIN
I
                                  REMOVE AND
                                  STORE OTOLITHS
                                                      EXAMINE FOR
                                                      PATHOLOGICAL
                                                      DISORDERS
                                                          DETERMINE AGES
Figure 4.   Sample  processing scheme  for  pathology  and  bioaccumulation component.

-------
sufficient  numbers of  crabs are caught in  the  nets, the crabs in the pots
will  be released.

     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 gross
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  selected randomly, wrapped in aluminum,  and stored
on ice.   The whole fish  will  be returned  to the laboratory where fillets
of dorsal muscle will be removed with stainless steel scalpels.  The fillets
will  be stored frozen in glass jars for later chemical analysis.

     From each of the 60  livers, a 1-cm^ 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.
                                      39

-------
                             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,  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.   Quality assurance tests  for each data set will be conducted
by the appropriate scientist.   These outputs  will  be reviewed to ensure
that accurate  data have been entered into the data files.
                                      40

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                                SUMMARY
     A  summary of spatial coverage  and  sampling effort for the Everett
Harbor  study  design  is provided in Table 2.  The  study design has been
developed to  provide  a  comprehensive assessment of contamination and its
effects within the study area.  The objective has been to  gain as much
information  as  possible within the constraints of preliminary estimates
of program cost and available funding  resources.
                                     41

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                             TABLE 2.   SUMMARY  OF  BASIC  STUDY DESIGN
Project Sediment Quality Bioassays3
Subarea Subtidal Intertidal Subtidal Intertidal
East
Waterway
South Port
Gardner
Offshore Port
Gardner
Snohomish
River Delta
Snohomish
River
Ebey Slough
Steamboat
Slough
Port Susanc
Total No.
Stations
Total No.
Samples
15
11
7
3
8
3
6
3
56
56
0 6
4 6
1
0 2
0 5
0 3
0 2
0 3
4 28
4 28
0
4
	
0
0
0
0
0
4
4
Benthic Fish
Infauna3 Bioaccumulation^ Pathologyb
Subtidal Subtidal Subtidal
6
6
0
2
5
3
2
3
27
135d
1
4
0
2
2
1
2
1
13
80e
1
4
0
2
2
0
2
1
12
720f

a Sediment  chemistry  will  be measured  at  all  stations sampled for benthic infauna and sediment
bioassays.
b Subtidal bioaccumulation  and pathology samples will be taken from the same trawls.
c Reference  area.
& Five replicate 0.1-m2 van Veen  samples per  station for benthic infauna.
e Edible muscle tissue from  five  English  sole at each station and a single composite of 5-10
Oungeness crabs at  all  stations except East Waterway, where 3 composites will be analyzed.
f Sixty English sole  livers for histopathological analyses at each station.

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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.

Boesch,  D.F.  1977.  Application of numerical  classification in ecological
investigations of  water pollution.  EPA-600/3-77-033.  U.S. EPA Environmental
Research Laboratory, Corvallis, OR.  125 pp.

Cunningham, D. 10  November 1982.  Memo:   Assessment of  toxic pollutants
in English sole and rock  sole in Everett Harbor and  Port Gardner.  Washington
Department of Ecology, Olympia, WA.  28 pp.

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.

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.

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

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  Ocean and Atmospheric Administration,
Boulder, CO.   295 pp.

Malins,  D.C., B.B. McCain,  D.W. Brown et al.  1982.  Chemical contaminants
and abnormalities  in fish  and invertebrates from Puget Sound.  NOAA Technical
Memorandum OMPA-19. National Oceanic and  Atmospheric Administration, Boulder,
CO.  168 pp.

Malins,  D.C., B.B. McCain,  D.W. Brown  et  al. 1984.  Chemical pollutants
in sediments and diseases  of bottom-dwelling fish in  Puget Sound, Washington.
Environ. Sci. Technol. 18:705-713.

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.

McCallum, M.   1985. Recreational and  subsistence catch  and consumption
of seafood from three urban industrial bays of Puget Sound:  Port Gardner,
Elliott Bay,  and Sinclair Inlet.  Washington State Division of  Health,
Epidemiology Section, Department of  Social  and  Health Services, Olympia,
Washington.  59 pp.

                                      42

-------
Mearns,  A.J.,  and J.M. Allen.   1978.  Use of small  otter  trawls in coastal
biological surveys.  EPA-600/3-78-083.  U.S. Environmental Protection Agency,
Con/all is,  OR.   33 pp.

Roff, D.   1982.  Reproductive  strategies in flatfish:  a first synthesis.
Can. 0.  Fish. Aquat. Sci. 39:1686-1698.

Romberg,  G.P., S.P- Pavlou, and  E.A. Crecelius.  1984.  Presence, distribution,
and fate  of toxicants in Puget Sound and Lake  Washington.   Metro Toxicant
Program  Report  No.  6A.  Toxicant  Pretreatment Planning Study Technical
Report Cl.   Municipality of Metropolitan Seattle, Seattle,  WA.  231 pp.

Shapiro  and Associates,  Inc.   1985.  Draft Environmental  Impact Statement
Supplement.   Everett Harbor and Snohomish River Navigation Project.  Prepared
for U.S. Army  Corps of Engineers  by Shapiro and  Associates, Seattle, WA.
51 pp. +  appendices.

Swartz,  R.C.,  W.A. DeBen  J.K.P. Jones, J.O.  Lamberson, and F.A.  Cole.
1985.  Phoxocephalid amphipod  bioassay for marine  sediment toxicity.
pp. 284-307.   In:   Aquatic  Toxicology and Hazard  Assessment, Proc.  of the
Seventh Annual   Symposium.   ASTM-STP 854.  American  Society for Testing
and Materials, Philadelphia, PA.

Tetra Tech. 1985a.  Commencement Bay nearshore/tideflats  remedial  investi-
gation.   Tetra Tech, Inc., Bellevue, WA.

Tetra Tech.  1985b.  Everett Harbor  action plan:   initial data summaries
and problem identification.   Prepared for U.S.  Environmental  Protection
Agency,  Region  X, Office  of Puget Sound.  Tetra Tech, Inc., Bellevue, WA.
81 pp. +  appendix.

Tetra Tech.  1985c.  Sampling and analysis design for development of Elliott
Bay toxics  action plan.  Prepared for U.S. Environmental Protection Agency,
Region X,  Office of Puget Sound. Tetra Tech, Inc.,  Bellevue, WA.  69 pp.  +
appendix.

Tetra Tech.  1986a.  Analytical methods for U.S.  EPA priority pollutants
and 301(h)  pesticides  in  marine and estuarine sediments.  Prepared for
Office of  Marine and Estuarine Protection, U.S.  Environmental Protection
Agency, Washington, DC.  Tetra Tech,  Inc., Bellevue,  WA.

Tetra Tech 1986b.   Quality assurance project plan  for field investigations
to support  development  of  the Everett Harbor action plan.  Prepared for
U.S.  Environmental  Protection  Agency,  Region  X,  Office of Puget Sound.
Tetra Tech, Inc., Bellevue, WA.

Tetra Tech. 1986c.   Recommended protocols  for conducting fish pathology
studies in  Puget Sound.   Draft  Report.  Prepared  for U.S. Environmental
Protection Agency,  Region  X, Office of Puget  Sound.  Tetra Tech,  Inc.,
Bellevue, WA.
                                     43

-------
Tetra Tech.  1986d.  Recommended  protocols for measuring conventional  sediment
variables in Puget Sound.   Prepared  for U.S. Environmental Protection Agency,
Region X, Office of Puget Sound.  Tetra Tech, Inc., Bellevue, WA.  46 pp.

Tetra Tech.  1986e.  Recommended  protocols for  measuring  metals in Puget
Sound sediment and tissue samples.   Draft Report.  Prepared for U.S.  Environ-
mental Protection Agency, Region X, Office  of  Puget Sound.   Tetra Tech,
Inc., Bellevue, WA.

Tetra Tech.  1986f.   Recommended  protocols  for measuring organic compounds
in Puget Sound sediment and  tissue samples.   Draft Report.   Prepared for
U.S.  Environmental  Protection  Agency, Region  X,  Office of  Puget Sound.
Tetra Tech, Inc., Bellevue,  WA.   55 pp.

Tetra Tech.  1986g.  Recommended  protocols for sampling and analyzing  subtidal
benthic macroinvertebrate assemblages in Puget  Sound.  Draft Report.  Prepared
for  U.S.  Environmental  Protection Agency, Region X, Office of Puget Sound.
Tetra Tech, Inc., Bellevue,  WA.   37 pp.

Tetra Tech and E.V.S. Consultants.   1986.  Recommended protocols for conducting
laboratory bioassays on  Puget Sound sediments.  Final  Report.  Prepared
for  U.S.  Environmental  Protection Agency, Region X, Office of Puget Sound.
Tetra Tech, Inc., Bellevue,  WA.   55 pp.
                                       44

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                                                                                     EVERETT
•
-------
                                                        EVERETT
                                EAST WATERWAY
    SURFACE RUNOFF
    CSO
 • INDUSTRIAL DISCHARGE - EXISTING
 D INDUSTRIAL DISCHARGE  HISTORICAL
    SCOTT
    EVERETT
(WG) WESTERN GEAR
 0         250
     Contaminant sources and selected industry
     locations in East Waterway  of Everett Harbor.

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                  1                  2
                                      NAUTICAL MILES
                      KILOMETERS
                    2               CONTOURS IN FEET
Sediment Chemistry:  Sampling stations for selected data
sets in  Everett Harbor.
                                                      MAPS
 •   SAMPLING STATION

	 INTERTIDAL AREAS

-------
                                                    EP19-31 ;
                                                  • B6-E8 /
                                                    EP19-30
 30
                                            B6-E14
                               EAST WATERWAY  EP19-29
                                     B8-29  •
                                        B6-E16     EP20-23

                                   EP19-28 •
                                                       EVERETT
                               EP19.26   B6-E18
  SAMPLING STATION
          250
                    500
0          250

CONTOURS IN FEET
                      YARDS

                         METERS
                      500
  Sediment Chemistry:  Sampling stations for selected data
  sets in East Waterway of Everett Harbor.
MAP 4

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                1                   2
                                    NAUTICAL MILES
                   KILOMETERS
                  2               CONTOURS IN FEET
Benthic Infauna: Sampling  stations for selected data
sets in  Everett  Harbor.
                                                     MAPS
 •   SUBTIDAL SAMPLING STATION

	 INTERTIDAL AREAS

-------
                                        U5-E2

                             EAST WATERWAY
• SAMPLING STATION


60
0         250

CONTOURS IN FEET
    Benthic Infauna: Sampling stations for selected data
    sets in  East Waterway of  Everett Harbor.             MAP 6

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                   1                  2
                                       NAUTICAL MILES
                      KILOMETERS
                     2               CONTOURS IN FEET
Sediment Bioassay: Sampling stations for selected data
sets in Everett  Harbor.
                                                       MAP 7
 0   OYSTER LARVAE BIOASSAY
 4   AMPHIPOD BIOASSAY
	  SAMPLES WERE COMPOSITED
	 INTERTIDAL AREAS

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 30
      .-•    o *B9'5
U8-E15'4>.  CH8-22
                                             U8-E14
                              EAST- WATERWAY
                                          U8-E16
 0  OYSTER LARVAE BIOASSAY
 •  AMPHIPOD BIOASSAY
	 SAMPLES WERE COMPOSITED
         250
                    500
                      YARDS
                        METERS
0         250
CONTOURS IN FEET
EVERETT
  Sediment Bioassays: Sampling stations for selected data
  sets in East Waterway of Everett Harbor.               MAP 8

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 EVERETT
FISH TRAWL/PATHOLOGY

BIOACCUMULATION

INTEFITIDAL AREAS

(TRAWL LOCATIONS ARE APPROXIMATE)
Fish Pathology and Fish  Bioaccumulation: Sampling
stations for selected data sets in Everett Harbor.
                                                     MAP 9

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                                                  EVERETT
      FISH TRAWL/PATHOLOGY
  •   BIOACCUMULATION


TRAWL LOCATIONS ARE APPROXIMATE
         250
                   500
0         250

CONTOURS IN FEET
                     YARDS

                       METERS
                     500
   Fish  Pathology and Fish Bioaccumulation: Sampling
   stations for selected data sets in  East Waterway
   of Everett Harbor.                                   MAP 10

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                                                     NOTE: 3 STATIONS WILL BE SAMPLED
                                                         FOR PP + PM IN THE PORT SUSAN
                                                         REFERENCE AREA.
Sampling locations for recommended chemical
studies in Everett Harbor.
                                              MAP 11
o  PP
•  PP-V
®  PP + PM
*  PP-V+PM
A  V + PM
PP - Priority Pollutants
 V » Volatile Compounds
PM * Pulpmill Compounds

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NOTE: 3 BIOASSAY/BENTHOS STATIONS
    AND 1 TRAWL TRANSECTWILL BE
    SAMPLED IN THE PORT SUSAN
    REFERENCE AREA.
                                    NAUTICAL MILES

                                 CONTOURS IN FEET
      ®   INTERT1DAL BIOASSAY
      *   SUBTIDAL BIOASSAY
      •   SUBTIDAL BIOASSAY/BENTHOS
      •4 SUBTIDAL PATHOLOGY/BIOACCUMULATION
Sampling locations for recommended biological
studies in Everett Harbor.

-------