FIELD VERIFICATION PROGRAM
               (AQUATIC DISPOSAL)


              TECHNICAL REPORT D-85-3


LABORATORY  EVALUATION OF ADENYLATE
ENERGY  CHARGE  AS A  TEST FOR STRESS
   MYTILUS EDULIS AND  NEPHTYS  INCISA
   TREATED  WITH  DREDGED MATERIAL

                       by
          Gerald E. Zaroogian, Carol Pesch
             Paul Schauer, Diane Black

          Environmental  Research Laboratory
         US Environmental Protection Agency
          Narragansett, Rhode  Island 02882
                  February 1985

                   Final Report

          Approved For Public Release; Distribution Unlimited
       prepared for  DEPARTMENT OF THE ARMY
           US  Army Corps of Engineers
           Washington, DC  20314-1000
       and US Environmental Protection Agency
             Washington, DC  20460
         Monitored  by  Environmental Laboratory
   US Army Engineer Waterways Experiment Station
    PO Box 631,  Vicksburg, Mississippi  39180-0631

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   Destroy this report  when  no longer  needed.  Do not return
                      it to the originator.
The findings  in this report are not  to be  construed  as  an official
     Department of the Army position unless so designated
                by other authorized documents.
       The contents of this report are not to be used for
       advertising,  publication, or promotional purposes.
        Citation of trade names  does not constitute an
        official  endorsement  or  approval of the  use of
                  such  commercial  products.
      The  D-series of reports includes publications of the
         Environmental  Effects  of Dredging Programs:
            Dredging Operations Technical Support
           Long-Term  Effects of Dredging Operations
       Interagency Field  Verification of Methodologies  for
       Evaluating  Dredged Material  Disposal  Alternatives
                  (Field  Verification Program)

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 SUBJECT:  Transmittal of Field Verification Program Technical Report Entitled
          "Laboratory Evaluation of Adenylate Energy Charge as a Test for
          Stress in Mytilus edulis and Nephtys incisa Treated with Dredged
          Material"
TO:  All Report Recipients
1.  This is one in a series of scientific reports documenting the findings of
studies conducted under the Interagency Field Verification of Testing and
Predictive Methodologies for Dredged Material Disposal Alternatives (referred
to as the Field Verification Program or FVP).  This program is a comprehensive
evaluation of environmental effects of dredged material disposal under condi-
tions of upland and aquatic disposal and wetland creation.

2.  The FVP originated out of the mutual need of both the Corps of Engineers
(Corps) and the Environmental Protection Agency (EPA) to continually improve
the technical basis for carrying out their shared regulatory missions.  The
program is an expansion of studies proposed by EPA to the US Army Engineer
Division, New England (NED), in support of its regulatory and dredging mis-
sions related to dredged material disposal into Long Island Sound.  Discus-
sions among the Corps' Waterways Experiment Station (WES), NED, and the EPA
Environmental Research Laboratory (ERLN) in Narragansett, RI, made it clear
that a dredging project at Black Rock Harbor in Bridgeport, CT, presented a
unique opportunity for simultaneous evaluation of aquatic disposal, upland
disposal, and wetland creation using the same dredged material.  Evaluations
were to be based on technology existing within the two agencies or developed
during the six-year life of the program.

3.  The program is generic in nature and will provide techniques and inter-
pretive approaches applicable to evaluation of many dredging and disposal
operations.  Consequently, while the studies will provide detailed site-
specific information on disposal of material dredged from Black Rock Harbor,
they will also have great national significance for the Corps and EPA.

4.  The FVP is designed to meet both Agencies' needs to document the effects
of disposal under various conditions, provide verification of the predictive
accuracy of evaluative techniques now in use, and provide a basis for deter-
mining the degree to which biological response is correlated with bioaccumula-
tion of key contaminants in the species under study.  The latter is an
important aid in interpreting potential biological consequences of bioaccumu-
lation.  The program also meets EPA mission needs by providing an opportunity
to document the application of a generic predictive hazard-assessment research
strategy applicable to all wastes disposed in the aquatic environment.  There-
fore, the ERLN initiated exposure-assessment studies at the aquatic disposal
site.  The Corps-sponsored studies on environmental consequences of aquatic
disposal will provide the effects assessment necessary to complement the EPA-
sponsored exposure assessment, thereby allowing ERLN to develop and apply a
hazard-assessment strategy.  While not part of the Corps-funded FVP, the EPA
exposure assessment studies will complement the Corps' work, and together the
Corps and the EPA studies will satisfy the needs of both agencies.

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SUBJECT:  Transmlttal of Field Verification Program Technical Report Entitled
          "Laboratory Evaluation of Adenylate  Energy Charge as a Test for
          Stress in Mytilus edulis and Nephtys incisa Treated with Dredged
          Material"

5.  In recognition of the potential national significance,  the Office,  Chief
of Engineers, approved and funded the studies  in January 1982.   The  work is
managed through the Environmental Laboratory's Environmental  Effects of
Dredging Programs at WES.  Studies of the effects of upland disposal and
wetland creation are being conducted by WES and studies of  aquatic  disposal
are being carried out by the ERLN, applying techniques worked out  at the
laboratory for evaluating sublethal effects of contaminants on aquatic  organ-
isms.  These studies are funded by the Corps while salary,  support  facilities,
etc., are provided by EPA.  The EPA funding to support the  exposure-assessment
studies followed in 1983; the exposure-assessment studies are managed and
conducted by ERLN.

6.  The Corps and EPA are pleased at the opportunity to conduct  cooperative
research and believe that the value in practical implementation and improve-
ment of environmental regulations of dredged material disposal will be con-
siderable.  The studies conducted under this program are scientific in nature
and will be published in the scientific litefature as appropriate and in a
series of Corps technical reports.  The EPA will publish findings of the
exposure-assessment studies in the scientific literature and in EPA report
series.  The FVP will provide the scientific basis upon which regulatory
recommendations will be made and upon which changes  in regulatory implementa-
tion, and perhaps regulations themselves, will be based.  However, the docu-
ments produced by the program do not in themselves constitute regulatory
guidance from either agency.  Regulatory guidance will be provided under
separate authority after appropriate technical and administrative assessment
of the overall findings of the entire program.
      Choromokos, Jr., Ph.D.,  P.E.
Director, Research and Development
U. S. Army Corps of  Engineers
Bernard D. Goldstein, M.D.
Assistant Administrator for
Research and Development
U. S. Environmental Protection
Agency

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        Unclassified	
SECURITY" CLASSIFICATION OF THIS PACE (When Date Entered)
REPORT DOCUMENTATION PAGE
t. REPORT NUMBER 2. OOVT ACCESSION NO.
Technical Report D-85-3
4. TITLE (mid Subtlll*)
LABORATORY EVALUATION OF ADENYLATE ENERGY CHARGE
AS A TEST FOR STRESS IN MYTILUS EDULIS AND NEPHTYS
INCISA TREATED WITH DREDGED MATERIAL
7. AUTHORS
Gerald E. Zaroogian, Carol Pesch, Paul Schauer,
Diane Black
9. PERFORMING ORGANIZATION NAME AND ADDRESS
US Environmental Protection Agency
Environmental Research Laboratory
Narragansett, Rhode Island 02882
It. CONTROLLING OFFICE NAME AND ADDRESS
DEPARTMENT OF THE ARMY, US Army Corps of Engineer^
Washington, DC 20314-1000 and US Environmental
Protection Agency, Washington, DC 20460
14. MONITORING AGENCY NAME A ADORESSf/f different tram Controlling Olllce)
US Army Engineer Waterways Experiment Station
Environmental Laboratory
PO Box 631, Vicksburg, Mississippi 39180-0631
READ INSTRUCTIONS
BEFORE COMPLETING FORM
3. RECIPIENT'S CATALOG NUMBER
5. TYPE OF REPORT 4 PERIOD COVERED
Final report
6. PERFORMING ORG. REPORT NUMBER
8. CONTRACT OR GRANT NUMBERfi)
10. PROGRAM ELEMENT, PROJECT. TASK
AREA & WORK UNIT NUMBERS
Field Verification Program
(Aquatic Disposal)
12. REPORT DATE
February 1985
13. NUMBER OF PAGES
56
IS. SECURITY CLASS, (ol thlt report)
Unclassified
ISa. DECLASSIFIC ATI ON/ DOWNGRADING
SCHEDULE
16. DISTRIBUTION STATEMENT (ol thlt Reparl)
  Approved for public release; distribution unlimited.
17. DISTRIBUTION STATEMENT (ol the ebetrtct entered In Block 20. II dlllerent from Report)


  Available from National Technical Information  Service,  5285  Port Royal Road,
  Springfield, Virginia  22161.
 IB. SUPPLEMENTARY NOTES
19. KEY WORDS (Continue on revetee tide II neceeimry end Identity by block number)
 Adenylate energy charge  (WES)             Dredged material   (WES)
 Aquatic biology   (LC)                       Dredging (Biology)   (LC)
 Biological assay   (LC)                      Marine ecology—Research
 Dredging—Environmental aspects—Evaluation   (LC)
                                                         (LC)
20. ABSTRACT (Continue ea renree *Mfc H neteemeair ead I dent I IT by block number;
       Changes in adenine nucleotide metabolism were evaluated as indices of
  stress in the marine bivalve Mytilus edulis  and the polychaete Nephtys incisa
  when treated with  highly contaminated dredged material under laboratory condi-
  tions.  Anesthetization of N.  incisa is necessary to maximize the adenosine
  triphosphate (ATP) concentration  and the adenylate energy  charge (AEC) by
  eliminating all swimming activity and facilitating handling upon their
  removal from sediment.  Extraction of adenine nucleotides  from N_. incisa
                              	                         (Continued)
DO,
     JAN 71
COITION OF * NOV 65 IS OBSOLETE
                                                          Unclassified
                                             SECURITY CLASSIFICATION OF THIS PA!»E (When D*t» Entered)

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  	Unclassified	
SECURITY CLASSIFICATION OF THIS PAGEfWlMl Dmtm Enlfttd)
  20.  ABSTRACT (Continued).

  required the addition of ethylenediaminetetraacetic acid to perchloric acid to
  obtain consistently high recovery efficiencies of 96 ± 0.3 percent for ATP.
  Perchloric acid alone gave consistent recovery efficiencies of 92 ± 0.5 percent
  of ATP from adductor muscle tissue of M. edulis.

       The AEC for M. edulis treated with 50 percent reference REF/50 percent
  Black Rock Harbor (BRH) dredged material was significantly different from all
  other treatments in test I (17 Nov 83).  In test II (19 Mar 84) no significant
  differences occurred among treatments.  At the end of treatment, M. edulis
  from the 50 percent REF/50 percent BRH had significantly lower AEC values than
  those AEC values obtained at the start of treatment.  Reproducibility between
  tests was determined by comparison of the same treatment between tests for all
  treatments.

       The AEC for M. edulis treated with 50 percent REF/50 percent BRH differed
  significantly between tests.  No other significant differences occurred among
  the other treatments between tests with M. edulis.  Although a significant
  difference occurred with the treatment 50 percent REF/50 percent BRH between
  tests, the data support the fact that the reproducibility of AEC for the same
  treatment between tests is excellent when variations in experimental conditions
  are considered.

       The AEC for IJ. incisa treated with BRH/REF was significantly different
  from all other treatments within a test for test I (2 Sept 83) and test II
  (20 Sept 83).   No other significant differences occurred among the other
  treatments in either test.  Tests for reproducibility indicated that the AEC
  for Jfl. incisa treated with REF/BRH differed significantly between tests.  The
  biological significance of this difference is questionable.  No other signifi-
  cant differences occurred among the other treatments.

       Reproducibility within and between tests is exceptionally good for both
  M.  edulis and II. incisa.  Both M. edulis and N^. incisa are excellent species
  with which AEC can be used to accurately assess their metabolic state and
  health condition when exposed to sublethal environmental perturbations.

       This investigation is the first phase in developing field-verified
  bioassessment evaluations for the Corps of Engineers and the US Environmental
  Protection Agency regulatory program for dredged material disposal.  This
  report is not intended for regulatory purposes; appropriate assessment
  methodologies that are field verified will be available at the conclusion of
  this program.
                                                   Unclassified
                                        SECURITY CLASSIFICATION OF THIS PAGEfHTim DM* Entered;

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                                 PREFACE






     This report describes work performed by the U.S. Environmental




Protection Agency (EPA) Environmental Research Laboratory, Narragansett,




R.I. (ERLN), as part of the Interagency Field Verification of Testing and




Predictive Methodologies for Dredged Material Disposal Alternatives




Program (Field Verification Program (FVP)).   The FVP is sponsored by the




Office, Chief of Engineers (OCE), and is assigned to the U.S. Army Engineer




Waterways Experiment Station (WES), under the purview of the Environmental




Laboratory's (EL) Environmental Effects of Dredging Programs (EEDP).  The OCE




Technical Monitors for FVP were Drs. William L. Klesch and Robert J. Pierce.




The objective of this program is to verify existing predictive techniques




for evaluating the environmental consequence of dredged material disposal




under aquatic, wetland, and upland conditions.  The aquatic portion of




the FVP study is being conducted by ERLN, with the wetland and upland



portions conducted by WES.




     The principal ERLN investigators for this aquatic study were




Dr. Gerald Zaroogian, biochemist; Dr. Paul Schauer; Ms. Carol Pesch,




research aquatic biologist; and Ms. Dianne Black, research aquatic




biologist.  Sample preparation and nucleotide analyses were conducted




under the supervision of Dr. Zaroogian assisted by Ms. Mary Johnson.




Laboratory exposures of N._ incisa were performed under the supervision




of Dr. Schauer and assisted by Dr. Gerald Pesch, Mr. John Sewall, and




Mr. Michael Balboni.  Ms. Dianne Black supervised the laboratory




exposures of M. edulis with assistance from Ms. Melissa Hughes and

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Mr. Greg Tracey.  Data management and data analysis were conducted by

Mr. Jeffery Rosen and Mr. James Heltsche.

     The EPA Technical Director for the FVP was Dr. John H. Gentile;

the Technical Coordinator was Mr. Walter Galloway; and the Project

Manager was Mr. Allan Beck.

     The study was conducted under the direct WES management of Drs.

Thomas M. Dillon and Richard K. Peddicord, Contaminant Mobility and Regu-

latory Criteria Group (CMCG); Ecosystem Research and Simulation Division

(ERSD); EL.  Dr. C. Richard Lee was Chief, CMCG.  Mr. Donald L.

Robey was Chief, ERSD.  Dr. John Harrison was Chief, EL.  The FVP

coordinator was Mr. Robert L. Lazor, and the EEDP Manager was

Mr. Charles C. Calhoun, Jr.

     Commander and Director of WES during preparation of the report was

COL Tilford C. Creel, CE.  Technical Director was Mr. F. R. Brown.


     This report should be cited as follows:

     Zaroogian, G. E., et al.  1985.  "Laboratory Evaluation of
     Adenylate Energy Charge as a Test for Stress in Mytilus edulis
     and Nephtys incisa Treated with Dredged Material," Technical
     Report D-85-3, prepared by US Environmental Protection Agency,
     Narragansett, R. I., for the US Army Engineer Waterways Experi-
     ment Station, Vicksburg, Miss.

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                                 CONTENTS


                                                                     Page

PREFACE	    1

LIST OF TABLES	    4

LIST OF FIGURES	    5

PART I:  INTRODUCTION	    6

      Background	    6
      Ob j ectives	    8

PART II:  MATERIALS AND METHODS	    9

       Overview	    9
       Sediment Collection and Preservation	    9
       Mytilus edulis	   12
       Nephtys incisa	   22
       Statistical Analyses	   29

PART III:  RESULTS	   30

        Mytilus edulis	  30
        Nephtys incisa	  33

PART IV:  DISCUSSION	  41

PART  V:  CONCLUSIONS	  49

REFERENCES	  50

APPENDIX A:  EFFECTS OF HANDLING AND ACCLIMATION PROCEDURES ON AEC..  Al
                                     3

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                              LIST OF TABLES

No.

 1       Measured concentrations (dry weight) of  suspended
         particles for tests with M. edulis	   14

 2       Measured concentrations (dry weight) of  suspended
         particles for tests with N_._ incisa	   26

 3       Sample size determinations for the detection of fixed
         magnitudes of differences in AEC for M.  edulis  after
         being treated with BRH sediment under laboratory
         conditions for 26 and 28 days	   30

 4       The response of adenine nucleotides in adductor muscle
         tissue of M^ edulis after treatments with BRH sediment
         for 26 and 28 days under laboratory conditions	   32

 5       Adenylate energy charge and adenine nucleotide
         concentrations in N^ incisa with and without
         anesthetization with magnesium chloride	   34

 6       Comparison of perchloric and trichloroacetic acids
         for extraction of adenine nucleotides from
         N^_ incisa for AEC determination	   36

 7       Sample size determination for the detection of  fixed
         magnitudes of differences in AEC for N.  incisa  after
         being treated for 10 days with BRH sedTment
         under laboratory conditions	   38

 8       The response of adenine nucleotides in N. incisa after
         treatment with BRH sediment for 10 days  under
         laboratory conditions (test I and II)	   40

 Al      The response of adenine nucleotides in N^_ incisa after
         treatment with BRH sediment for 10 days  under
         laboratory conditions (test III)	   A2

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                             LIST OF FIGURES

No.                                                                  Page

 1       Central Long Island Sound disposal site and South
         reference site	   11

 2       Black Rock Harbor, Connecticut,  source of
         dredged material	   11

 3       Schematic of the dosing system used to treat M.  edulis
         with various concentrations of BRH and REF sediment
         suspensions	   15

 4       Sediment dosing system with chilled water bath and
         argon gas supply	   15

 5       Summary of the procedure for the extraction of adenine
         nucleotides from the adductor muscle of M. edulis	   17

 6       Summary of the procedures for analysis of ATP, ADP, and
         AMP in adductor muscle tissue of M. edulis and whole
         N^ incisa	77.777777	   21

 7       Suspended sediment dilution system, distribution chamber,
         and exposure chamber used for acute toxicity tests
         with N^ incisa	   24

 8       Summary of the procedure for the extraction of adenine
         nucleotides from the marine polychaete N. incisa	  28

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        EVALUATION OF ADENYLATE ENERGY CHARGE AS A TEST FOR STRESS




    IN MYTILUS EDULIS AND NEPHTYS INCISA TREATED WITH DREDGED MATERIAL








                         PART I:  INTRODUCTION






                                Background






         1.  Historically, toxiclty studies involving aquatic organisms




have measured classical parameters such as growth, reproduction, and




mortality.  In most situations, these measures suffer from their lack of




sensitivity.  A biochemical marker or indicator of stress such as the




adenylate energy charge (AEG) (Atkinson 1971) can be used to gain infor-




mation on the physiological condition of an organism prior to the occur-




rence of irreversible changes.  Since most biochemical systems react to




specific stressors, a more generalized Indicator such as AEC is advanta-




geous in natural areas which may be influenced by the Interaction of




pollutants and environmental factors simultaneously.




        2.  Adenylate energy charge is an indication of the amount of




energy available to an organism from the adenylate pool.  It is calcu-




lated from measured concentrations of three adenine nucleotides,




adenosine triphosphate (ATP), adenosine diphosphate (ADP) , and adenosine




monophosphate (AMP), which are integral to the energy metabolism of all




organisms (Atkinson 1971).  The AEC, defined as (ATP + 1/2 ADP)/ (ATP +




ADP + AMP), has a maximum value of 1.0 when all adenylate is in the form




of ATP and a minimum value of 0 when all adenylate is in the form of AMP




(Atkinson and Walton 1967).  The energy charge has been considered impor-




tant in the control of key catabolic and anabolic pathways (Atkinson 1971).

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Values of energy charge correlate with physiological condition: energy




charges between 0.8 and 0.9 are typical of organisms which are actively




growing and reproducing, usually under optimal environmental conditions




(Atkinson 1971; Chapman et al. 1971; Ball and Atkinson 1975; Wiebe and




Bancroft 1975; Chapman and Atkinson 1977; Karl and Holm-Hansen 1978;




Rainer et al. 1979; Ivanovici 1980; Karl 1980; Giesy et al. 1981;




Mendelssohn and McKee 1981; Romano and Daumas 1981; Skjoldal 1981;




Dickson et al. 1982; Vetter and Hodson 1982; Zaroogian et al. 1982;




Geisy et al. 1983; Hoya et al. 1983).  Values in the range of 0.5 to 0.7




have been observed in organisms which are stressed (Ball and Atkinson




1975; Behm and Bryant 1975; Wiebe and Bancroft 1975; Wijsman 1976; Karl




and Holm-Hansen 1978; Rainer et al. 1979; Christensen and Devol 1980;




Ivanovici 1980; Karl 1980; Giesy et al. 1981; Mendelssohn and McKee




1981; Romano and Daumas 1981; Vetter and Hodson 1982; Zaroogian et al.




1982) and whose growth and reproduction rates are reduced (Chapman et




al. 1971).  Values below 0.5 have been associated with irreversible loss




of viability under detrimental conditions (Ridge 1972; Montague and




Dawes 1974; Ball and Atkinson 1975; Wijsman 1976; Karl and Holm-Hansen




1978; Skjoldal and Bakke 1978; Christensen and Devol 1980; Giesy et al.




1983; Vetter and Hodson 1982).  If these relationships apply generally,




a knowledge of the energy charge of key species with known responses to




particular environmental conditions would provide a convenient measure




to assess the extent to which these species are stressed.

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                                  Objectives






     3.  The objective of this study was to evaluate the applicability of AEG




as a measure of stress in M. edulis and jfl. incisa treated with highly contami-




nated dredged material under laboratory conditions and to determine the degree




of variability and reproducibility inherent in the test.  This objective is




referred to as the Field Verification Program (FVP) and is the subject of




this report.

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                     PART II:  MATERIALS AND METHODS






                                Overview






        4.  The types of tests conducted for AEC included both suspended




particulate and solid phase exposures to Black Rock Harbor (BRH) sedi-




ments.  Suspensions of either reference (REF) or BRH sediment were




used in various combinations with a solid phase ranging from 100 per-




cent REF to 100 percent BRH sediment where appropriate.  Tests combining



the solid and particulate phase were representative of the type of




condition at the disposal site; however, the concentrations of suspended




material used in the tests did not necessarily simulate actual field




concentrations.  Concentrations were chosen to produce a dose response




in the endpoint measurements.




        5.  The tests described below generally follow methods prescribed




in Standard Practice for Conducting Acute Toxicity Tests with Fishes,




Macroinvertebrates, and Amphibians (ASTM 1980).  Although the ASTM test




methods were not specifically designed for sediment tests, they provide




guidelines for experimental designs, water quality parameters, statis-




tical analyses, and animal care, handling, and acclimation.






                   Sediment Collection and Preservation






        6.  Reference sediment for these studies was collected from the




FVP South reference site (40°7.95'N  and  72°52.7'W), which is  approximately




700 m south of the southern perimeter of the Central Long Island  Sound




(CLIS) disposal site  (Figure  1).  Reference  sediment was  collected with a




Smith-Mclntrye grab sampler  (0.1 m2) in  August and  December  1982  and  May

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1983 (collections I, II, and III, respectively).  Sediment from each


collection was returned to the laboratory, press sieved (wet) through a


2-mm mesh stainless steel screen, homogenized in a tub with a paddle, and


stored in polypropylene (collection I) or glass (collections II and III)


containers at 4°C until used in experiments.  Each container of material


was coded with collection number, date, and jar number.*


        7.  Black Rock Harbor sediment was collected from 25 locations


within the highly industrialized Black Rock Harbor (Bridgeport,  Conn.)  study

                 2
area with a 0.1-m  gravity box corer to a depth of 1.21 m (Figure 2).  The


sediment was homogenized,  distributed to barrels,  and stored at 4°C.  The


contents of each barrel were homogenized in a tub with a paddle, wet sieved


through a 1-mm sieve,  distributed to glass jars, and stored at 4°C until used


in experiments.   Samples of sediment were taken at various points in the


collection, mixing,  and distribution procedure for moisture content and


chemical analysis.
  See Lake et al.  (1984) for complete details.
                                     10

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                                          SOUTH REFERENCE
                                               • SITE
Figure 1.  Central Long Island Sound disposal site
           and South reference site
    Figure 2.  Black Rock Harbor, Connecticut
               source of dredged material
                        11

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                             Mytilus edulis


 Collection  and  Holding

        8.   Mytilus edulis were  collected with a scallop dredge from an

 uncontaminated  site near Dutch  Island in the West Passage of Narragansett

 Bay, R.I.  (71°24.0'W and 41°29.4'N) from depths ranging between 5 m and

 10 m.   Collection information for each test is listed below:

            Collection        Seawater                         Testing
  Test        Date       Temperature °C   Salinity(ppt)     Initiated

   I        10 Nov 83          13               31           17 Nov 83
  II        9 Mar 84            5               29           19 Mar 84


 The mussels were sorted to obtain a size range of 50 to 55 mm shell

 length  and held in a laboratory flow-through system with unfiltered

 seawater at ambient temperature.  Acclimation, if necessary, was

 conducted in running unfiltered seawater at a rate of 1°C per day to

 15°C as this was the temperature selected for testing.


 Exposure Methods

        9.   Since 50 mg/£, of  suspended REF sediment did not adversely

 affect  M. edulis during 28 days exposure, this concentration of

 particulate was selected as  the no-observable-effeet-concentration.

 Therefore, 50 mg/jj, was used  as the suspended solids concentration in

 all tests.  Two experiments  were designed to examine the effects of BRH

dredged material on M.  edulis (Lake et al.  1984).   The experimental

 design  included three concentrations of particulate exposure in the

 following ratios:  100 percent REF/0 percent BRH, 50 percent REF/50

 percent BRH, and 0 percent REF/100 percent BRH.  The measured
                                     12

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concentrations are given in Table 1.   The exposure  conditions were




chosen on the basis of previous  experiments  and were  expected to be




sublethal for the 28-day exposure period.  Survival of  M.  edulis was



100 percent in all treatments except  for the 100 percent BRH and the




50 percent REF/50 percent BRH treatments in test I  (Table  1).




       10.  Figure 3 illustrates the  M. edulis exposure system  and




Figure 4 illustrates the sediment dosing system.  In  the conduct of




tests with M^ edulis, the REF and BRH mixing and distribution chambers




(Figure 3) were maintained at 50 mg/jj, and treatment combinations were




obtained by proportionally siphoning  suspended sediment from the




appropriate distribution chambers to  produce a combined flow of 300 mfc/min




in each exposure chamber.  On day 0 and 28, M. edulis were sampled for




AEC.  Test I was terminated on day 26 instead of day 28 since the




reduction in feeding, especially in the 100 percent BRH treatment,  which




was observed to be quite significant  on day 26, could have indicated




a rapidly deteriorating health condition, which in turn could have




resulted in death prior to 28 days.  Therefore, it was  thought prudent




to terminate at 26 days.




       11.  Spectrophotometrie measurements of  the amount of suspended




participates entering the exposure chambers were made daily using the




relationship between absorbance and dry weight  of  suspended particulates.




The latter was determined by collecting triplicate samples of suspended




sediment directly  from the diluter or  by  preparing serial dilutions




from the highest concentration.  The dry  weight of these samples was




measured using the methods reported in Lake et  al. (1984).  Linear
                                    13

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                                          Table 1
                Measured Concentrations (Dry Weight) of Suspended Particles
for Tests with M. edulis












Concentration (mg/i) Concentration (mg/£)
Added to Chamber
Treatment

100% BRH
100% REF
50%REF/50%BRH

100% BRH
100% REF
50%REF/50%BRH
X

62.
56.
59.

56.
49.
52.
+

8 ±
2 ±
4 ±

2 ±
4 ±
9 ±
SD

9
8
5

8
6
5


.9
.2
.5

.6
.1
.7
In Chamber
x ±
Test I
30.
11.
24.
Test II
29.
14.
23.
SD
17
2 ±
6 ±
5 ±
Concentration Range
In Chamber
Nov 83
17.5
5.1
15.4

73
24
63

.4 -
.3 -
.3 -
(mg/*)

5.0
2.0
9.4
Death
%*

0
3
13
19 Mar 84
1 ±
1 ±
5 ±
11.4
6.4
10.1
48
28
45
.6 -
.9 -
.4 -
5.0
6.4
6.4
0
0
0
*Based on a total of 40 mussels per treatment.

-------
reference (lurry
     L
               mixing 	
                chamber
                            seowoler
                                                                BRH slurry
                                                                   L
                  magnetic/
                   tlirrer
                          distribution
                           chamber —
                                 lo drain
                           /
                                                                      seowoler
                                                    u
                                                     overflow lo water both, Ihen lo drain
                                                      eipoiure chamber
                                     lo drain
                                               magnetic tlirrer
Figure 3.   Schematic of the dosing system used to  treat  M.  edulis with
             various concentrations of  BRH and REF sediment suspensions
                        SEPARATORY
                          FUNNEL
                                               DELIVERY
                                               MANIFOLD
                                                         DOSING
                                                         VALVE
                                                    TO EXPOSURE
                                                      SYSTEM
                                                  SLURRY
                                                  RESERVOIR
         Figure 4.
  Sediment  dosing system with chilled  water bath
  and  argon gas  supply
                                           15

-------
 regression  analysis of  the data established the relationship between




 absorbance  at 660 nm and dry weight.  Analysis of variance and multiple




 comparison  tests were performed on the suspended particulate data




 collected daily during  the experiment.




        12.  Forty M^ edulis were continuously fed laboratory-cultured




 Isochrysis  galbana at a rate of 94 mg (dry weight) per mussel per day.




 Conditions  and techniques of algal culture were modified after Guillard




 (1975).  Guillard's "f/2" nutrient media was used, except that all




 trace metals but iron were eliminated and the concentration of the




 vitamins thiamin and B12 were doubled.






 Adenylate Extraction




        13.  The adductor muscle was rapidly dissected out, blotted dry,




 placed  on a labelled polythene strip (Gladwrap®), and freeze clamped with




 aluminum blocks cooled to -196°C with liquid nitrogen (Ivanovici 1980;




 Bergmeyer 1965).  The time between sampling and dissection never exceeded




 10 minutes.  Tissue samples were removed and freeze clamped in less




 than 30 sec and the labelled samples were stored in liquid nitrogen




 until homogenization.




        14.  Adenine nucleotides were extracted from tissues with a




method similar to that of Ivanovici (1980) (Figure 5).  The freeze-




 clamped tissue was quickly transferred from its wrapping to a tared




 stainless steel homogenizing tube previously cooled in liquid nitrogen




 and placed in a polyurethane insulator and weighed.  Tissue samples




 (approx. 0.2 g) were ground to a fine powder at -196°C.  Perchloric




acid (PGA) (1 ml, 6% v/v) was added to the ground tissue and allowed
                                     16

-------
           Sample:   Adductor Muscle Tissue (> 0.2 g wet wt)
                    Freeze Clamp in polythene film
                           Store in liquid N2
                     Weigh, homogenize in liquid N2
                         add 1 ml 6% PGA (v/v)
                     Add more 6% PGA (tissueracid,  1:10)
                     Stand for approximately 15 min @ 0°C
                     Centrifuge 20 min, 6000 g @ 5°C
 Supernatant:
 adjust to pH 6.5-7.0
 with solid K2C03
 Centrifuge 20 min, 6000 g @ 5°C
                    Discard pellet
 Discard pellet
   1
Neutralized supernatant,
Analyze ATP, ADP, AMP
or freeze and store @ -20°C
Figure 5.  Summary of the procedure for the extraction of adenine
           nucleotides from the adductor muscle of M. edulis
                                    17

-------
 to  freeze,  ground  to a powder, and mixed with the tissue sample.

 This mixture was kept on ice and allowed to thaw, after which additional

 ice-cold PGA was added (the final ratio of tissue to PCA was 1:10,

 w/v) and then centrifuged at 5°C and 6000 g for 20 min after thorough

 mixing.  The supernatant was decanted into a (polyethylene) centrifuge

 tube containing 5  pi of Universal indicator and adjusted to pH 6.5-7.0

 with solid  1^003.  These tubes were left on ice for approximately

 15 min to allow C02 evolution and then centrifuged as above.  The

 supernatant was decanted from the KC104 precipitate into clean (poly-

 ethylene) centrifuge tubes and assayed or stored at -20°C.  Generally

 20 samples  were prepared each day.  Recovery efficiency of the extrac-

 tion was determined by spiking tissue samples with ATP, ADP, and

 AMP and recovery was calculated by the following equation:



                        [Sample + Standard] - [Sample]             (1)

            % recovery =   	——   x 100%

                             [Standard]
where
            Sample + Standard = concentration of adenylates in
                                sample spiked with adenylates

            Sample            « concentration of adenylates in
                                sample

            Standard          = concentration of adenylate
                                standard
       15.  Extraction efficiences of adenine nucleotides from adductor

muscle tissue of M._ edulis by PCA were consistently greater than 92 +

0.5 percent.  It is important to note that the extraction efficiencies
                                    18

-------
refer to the extraction procedure and do not necessarily  reflect  intra-

cellular extraction efficiencies.


Adenylate Assay

       16.  Upon thawing, frozen samples were centrifuged as above to

remove any KC104 as precipitate before assaying for adenylates.

       17.  The concentrations of ATP, ADP, and AMP were  determined

spectrophotometrically (340 nm) with hexokinase (Lamprecht and Trautschold

1974) , pyruvate kinase, and myokinase (Adam 1963) ,  respectively (Figure

6).  All enzymes, chemicals, and reagents (analytical grade) were obtained

from Boehringer Mannheim, Indianapolis, Indiana.

       18.  The principle of the ATP assay is as follows:  glucose is

phosphorylated by ATP to glucose-6-phosphate (G6P) with hexokinase (HK)

(reaction 1).  Glucose-6-phosphate then reacts with nicotinamide-adenine

dinucleotide phosphate (NADP) to form 6-phosphoglucono-6-lactone and

reduced nicotinamide-adenine dinucleotide phosphate (NADPH).  This

reaction is catalyzed by glucose-6-phosphate dehydrogenese  (G6P-DH)

(reaction 2).

                                      HK
1 .  ATP + glucose
                                               > G6P + ADP
2.  G6P + NADP
                               G6P - DH        6-phosphoglucono-fi-lactone
                                          >
                                               + NADPH 4- H+
                                     19

-------
Thus for every micro-mole of ATP, one micro-mole of NADPH is formed and




causes an increase in absorbancy at 340 nm.




       19.  "The principle of the ADP and AMP assays is as follows:




pyruvate kinase (PK) catalyzes the phosphorylation of one micro-mole of




ADP by phosphoenolpyruvate (PEP) to form one micro-mole of ATP and




pyruvate (reaction 2).  Pyruvate in turn is converted to lactate by



lactate dehydrogenase (LDH).  Thus, one micro-mole of ADP results in




the formation of one micro-mole of nicotinamide-adenine dinucleotide




(NAD) (reaction 3).  The decrease in absorbancy at 340 nm caused by the




formation of NAD from NADH is, therefore, proportional to the amount of




ADP present in the sample.  After this absorbance change has been




measured in a sample, myokinase (MK) is added.  This enzyme catalyzes




the formation of 2 micro-moles of ADP from one micro-mole each of AMP




and ATP (reaction 1).  In turn, 2 micro-moles of NAD are formed (reac-




tions 2 and 3).




                                     MK
                 1.   AMP + ATP
—>  2 ADP
                                        PK
                 2.  2 ADP + 2 PEP
   ->  2  ATP + 2  pyruvate
                                             LDH
                 3.  2 pyruvate + 2 NADH
     -> 2 lactate + 2 NAD
                                     20

-------
                       Neutralized Supernatant
                        (See Figures 5 and  8)
  ATP Assay
0.2 ml into cuvette
1.8 ml assay buffer
5 ul G-6P-DH, mix
      1
Read (340 nm)
  ADP, AMP Assay
0.2 ml into cuvette
1.8 ml assay buffer
   5 yl LDH, mix
   Read (340 nm)
5 pi glucose, mix
Immediately add
5 ul HK, mix
Read (340 nm)
after 20-min incubation
   5 ul PK, mix
Read (340 nm) after
 20-mln incubation
   5 yl MK, mix
                                                    Read (340 nm) after
                                                     20-min incubation
G-6P-DH - glucose-6-phosphate dehyrogenase

HK - hexokinase

LDH - lactate dehydrogenase

PK - pyruvate kinase

MK - myokinase
Figure 6.  Summary of the procedures for analysis of ATP, ADP, and AMP
           in adductor muscle tissue of M. edulis and whole N. incisa
                                     21

-------
       20.  To determine if any inhibitory effects of neutralized tissue


extracts on the nucleotide assay system occurred, known amounts of


ATP, ADP, and AMP were added to neutralized extracts as internal stand-


ards and assayed to check for inhibitory or enhancement effects by the


extract.  The following equations were used to calculate correction


factors (Cf):
                    [Sample + Internal Standard] - [Sample]
                                                                    (2)
               X% = 	

                          [Internal Standard]
               CfATP, ADP or AMP _   100%                           (3)

                                    100% + X%
       21.  A correction factor was not required for ATP since extracts


of M. edulis adductor muscle had a negligible effect on absorbance.


However, these same extracts increased absorbance which caused over


readings for ADP (112 percent) and AMP (111 percent).  Thus, a correction


factor was required for ADP (0.89) and AMP (0.90) to calculate accurately


their concentration.



                             Nephtys incisa



Collection, Culture, and Holding


       22.  Nephtys incisa is a marine polychaete worm which is indigenous


to the disposal area in Central Long Island Sound (CLIS).  Worms were


collected with a Smith-Mclntyre grab sampler (0.1 m^) from the South


reference site (Figure 1) at various times in 1983 prior to the test
                                    22

-------
periods and held In the laboratory for a short acclimation period.*




Tests were conducted with worms 3 to 4 cm in length.






Exposure Methods




       23.  Two 10-day suspended particulate tests (same treatments,




performed at different times) with separate collections of 1J.  incisa



were performed.




       24.  The suspended sediment experimental system consisted of




three modules:  the controlled dosing system, the dilution and distri-




bution system, and the test chambers (Figures 4 and 7).  Two identical




dosing systems, one for REF and one for BRH, provided a constantly




recirculating source of concentrated sediment slurry (in seawater)




passing by a three-way valve, that led to the dilution and distribution




system.  Argon gas was added to the reservoir of the dosing system to




minimize oxidation of the slurry (Figure 4).  The three-way valve was




controlled by a microprocessor programmed to deliver a pulse of slurry




at periodic intervals.  In the dilution and distribution system, the




concentrated slurry was mixed with seawater to the proper concentration




of suspended solids and distributed to the individual test chambers.




Actual concentration of suspended particulates in the test chambers




was determined (by dry weights) periodically.*




       25.  The test chambers were glass crystallizing dishes (150 by




75 mm), which contained 400 ml of sediment (2.5 to 3.5 cm deep).  Each




dish contained a smaller glass crystallizing dish (60 by 35 mm) in the











* See Lake et al. (1984) for complete details.







                                     23

-------
                 DOSING SYSTEM
                                •Suspended  Particles
      Spigot-*.
                         u
                   oooc
                               -^-Distribution Jar
                                'Stir Bar
               EXPOSURE SYSTEM
     Stir Bar
              \\\\
                                     'Exposure Container
Figure 7. Suspended sediment dilution system, distribution chamber, and
        exposure chamber used for acute toxicity tests with N, incisa

-------
center of the larger dish.   A Teflon®-coated stir bar was  placed  in  the

small dish, which received  the inflow water, to  keep the particulate

material in suspension.  The inflow water flowed out of the central

dish over the sediment surface, and overflowed the edge of the  large

crystallizing dish.

       26.  Exposure conditions for the solid phase portion of  the

suspended particulate tests were 100 percent REF or 100 percent BRH

sediment.  These two solid phase exposure conditions in combination

with the two suspended sediment exposures, REF or BRH at a nominal

concentration of 200 mg/1 (dry weight), gave a total of four treatments.

The measured concentrations are given in Table 2.  The exposure conditions

for these experiments were chosen on the basis of previous experiments

and were expected to be sublethal for the 10-day exposure period.

Survival of N.^ incisa was 100 percent in all treatments except the

BRH/REF treatment in the first test (Table  2).

       27.  The worms were fed prawn flakes  (ADT-Prime, Aquatic Diet

Technology, Brooklyn, N.Y.) in a suspension of seawater, which was

pumped by  peristaltic pump into the distribution chamber  of the dosing

system.  The amount fed was 127 mg  (dry weight)  per  test  chamber per

day.  This amount of food was  determined optimum in  prior  feeding studies

with N._ incisa.*

       28.  During  the tests,  all  dishes were examined daily for  the

appearance of any worms on the surface of the sediment, but none were seen.
 *  Personal  communication,  Paul  Schauer,  March 1983,  U.S.  Environmental
   Protection Agency.
                                      25

-------
                                Table 2


      Measured Concentrations (Dry Weight) of Suspended Particles

                        for Tests with N. incisa
    Treatment
 suspended/solid
Concentration (mg/A)
    x + SD
Dead**

REF/REF
BRH/REF
REF/BRH
BRH/BRH

REF/REF
BRH/REF
REF/BRH
BRH/BRH
Test
10
10
10
10
Test
10
9
11
12
I 02 Sept 83
211 + 87
171 + 53
211 + 87
171+53
II 20 Sept 83
199 + 73
226 + 47
199 + 73
226 + 47

0
3
0
0

0
0
0
0
*  Number of worms analyzed for AEG from a total of 30 worms;
   the remainder were used for other purposes.

** Based on a total of 30 worms per treatment.
                                    26

-------
On the last day of the test,  observations were made on the  burrows




visible through the sides of  the dishes,  and the depth of the  suspended




material deposited on top of  the solid phase was measured.   Worms




missing were presumed dead.  These results are reported elsewhere.




       29.  All tests were conducted with sand-filtered Narragansett




Bay seawater at 20°C and approximately 30 ppt salinity.  Flow rates




were about 35 ml/min.  The photoperiod was a 14:10 hr light-dark cycle.




Nephtys incisa were acclimated in REF sediment for a minimum of five




days at 20°C.






Adenylate Extraction




       30.  The worms from each treatment replicate were collected on




a fine mesh sieve  (0.9 mm mesh) and immediately anesthetized by immersion




of sieve and worms into  a 7 percent solution of MgCl2  in seawater for




2-1/2 min (Dean and Mazurkiewicz  1975).  The worms were washed by




immersion of the  sieve in clean seawater and the worms were removed




from the sieve and placed into a  Carolina dish (75 mm  diam.) containing




approximately  50 ml clean seawater.  One or  two anesthetized worms




(>0.1 g wet wt.) were placed on a millipore  filter pad (25 mm,  1.2  p)




and as much seawater  as  possible  was removed by vacuum.  The anesthetized




worms were gently removed from  the filter pad  onto a  labelled polythene




strip  and freeze  clamped (Figure  8).   The extraction  procedure  was




identical to that  used for nucleotide  extraction  from M^ edulis except




that  the  homogenized  tissues were doubly extracted with 6  percent  PCA




containing  0.33 percent  ethylenediaminetetraacetic acid (w/v)  (EDTA)




and the tissue extracts  were  assayed within 2 hr of  extraction.
                                    27

-------
   Sample:     One worm (3-4 cm; > 0.1 g wet weight)
               Freeze clamp in polythene film
               Store in liquid N£
               Weigh, homogenize in liquid N£;
               Add 1/2 total volume of 6% PGA (v/v) -0.33% EDTA (w/v)
               required to make a final dilution of 1:10 (tissue to acid)
               Thaw @ 0°C
               Centrifuge 20 min, 6000 g @ 5°C
   v
Pellet
Add 1/2 the total volume
of 6% PGA (v/v) -.33% EDTA (w/v)
as above.  Sonicate 30 sec.
Supernatant (1)
Centrifuge 20 min, 6000 g 
-------
The double extraction of whole N^. incisa with PCA-EDTA gave extraction




efficiencies consistently greater or equal to 96 percent + 0.3 percent.






Adenylate assay




       31.  The assay procedure for IJ. incisa extracts was identical




to that for M. edulis (Figure 6).




       32.  Inhibitory effects by the extracts of 1J. incisa on ATP




analysis were negligible thus requiring no correction factor.  However,




these same extracts increased absorbance which caused overreadings




for ADP (111 percent) and AMP (109 percent).  Thus a correction factor




(Cf) was required for ADP (0.90) and AMP (0.91) to calculate accurately



their concentration.






                          Statistical Analyses




       33.  Means and standard error were calculated for the concentra-




tions of the individual adenine nucleotides and the AEC.  Sample size




determinations were based upon a predetermined type I error of 0.05 and




a type II error of 0.20 with fixed differences of 0.1 or 0.05 unit for




AEC.  Non-pooled data were analyzed with analysis of variance (ANOVA)




methods to detect differences and determine the reproducibility of AEC




among treatments within a test.  If significant differences in AEC were




indicated by the ANOVA, then Tukey's  (HSD) test for pairwise comparison




of means between treatments within a test was used.  The reproducibility




between tests was determined with ANOVA by comparing the same treatment




between tests for all treatments.  This was followed by Tukey's test  if




the ANOVA indicated significant differences.
                                     29

-------
                           PART III:  RESULTS


                             Mytilus edulis


Extraction

       34.  Extraction procedures used in this and a previous study

(Zaroogian et al. 1982) for M. edulis adductor muscle tissue provided

high recovery efficiencies of 92 + 0.5 percent.  Low variability among

AEC values within treatments is evident in the small sample size required

to detect a 0.05 change in AEC values (Table 3).



                                 Table 3

          Sample Size Determinations for the Detection of Fixed

           Magnitudes of Differences in AEC for M. edulis after

         being Treated with BRH Sediment under Laboratory Condi-

             tions for 26 Days (Test I) and 28 days (Test II)
                    Magnitude of
   Variable	difference	Variance	N*

                            Test I  17 Nov 83

     AEC                0.1                0.00176          3
     AEC                0.05               0.00176          9

                           Test II  19 Mar 84

     AEC                0.1                0.00159          2
     AEC                0.05               0.00159          8
* N - Calculated sample size needed to detect a 0.1 or 0.05 unit change
      in AEC.  The power of the test = 0.80; a - 0.05 (Snedecor and Cochran
      1980).
                                     30

-------
Adenylate Energy Charge



       35.  Sample size determinations which were  calculated with esti-




mates of variation indicated that sample sizes  of  9 would allow detection




of a significant difference in AEG of 0.05.   Whereas,  sample sizes  of  3




are required to detect a 0.1 difference in AEG  (Table  3).  Also apparent



in Table 3 is the excellent reproducibility  in  sample  size  requirements




between experiments.  This is due to the similarity in variance in  the




two tests (Table 3).  Analyses of variance coupled with Tukey's test for




grouping of means indicated that the AEC for the treatment  50  percent




REF/50 percent BRH is significantly different from all other treatments




in test I (17 Nov 83) (Table 4).  In test II (19 Mar  84) these same




statistical analyses indicated that no significant differences occurred




among treatments (Table 4).  At the end of the  treatment period,  M. edulis




from all the treatments had lower AEC values than the AEC values  obtained




at the start of treatments in each test; however, the difference  was




statistically significant only for the 50 percent REF/50 percent  BRH




treatment in both tests (Table 4).  In order to determine the reproduci-




bility between tests, analyses of variance coupled with Tukey's test




were performed by comparison of the same treatment between tests  for all




three treatments.   It is evident in Table 4 that the AEC for the 50




percent REF/50 percent BRH treatment differed significantly between




tests.  No other significant difference occurred among the other treat-




ments between tests  (Table 4).  Although a significant difference  occurred




with the 50 percent REF/50 percent BRH treatment between tests, the data




support the fact that the reproducibility of AEC for  the same treatment
                                     31

-------
   between tests is excellent when variations in experimental conditions

   are considered  (Tables  1 and 4).



                                    Table 4

       The Response of Adenine Nucleotides in Adductor Muscle Tissue

        of M. edulis after Treatments with BRH Sediment for 26 days

         (Test I)  and 28 Days (Test II) under Laboratory Conditions
Treatment



Time 0*

100% REF

100% BRH
             umol/g Wet Weight Tissue

  n      ATP         ADP           AMP

             Test I  17 Nov 83
                                          AEC
 9   3.63(0.16)** 0.93(0.05)** 0.14(0.01)** 0.87(0.01)** A*

10   2.86(0.09)   1.07(0.08)   0.16(0.03)   0.83(0.01)   A

10   2.70(0.13)   0.97(0.06)   0.16(0.04)   0.83(0.01)   A
50%REF/50%BRH  10   2.38(0.11)   1.22(0.04)   0.36(0.04)   0.75(0.01)   B  aTt
Time 0*

100% REF

100% BRU
10

10

10
50%REF/50%BRH  10
       Test II  19 Mar 84

3.35(0.19)** 0.93(0.05)** 0.08(0.01)** 0.87(0.01)** A*

3.53(0.15)   1.21(0.07)   0.19(0.03)   0.83(0.01)  AB

3.36(0.19)   1.23(0.08)   0.17(0.03)   0.83(0.01)  AB

3.30(0.21)   1.35(0.03)   0.25(0.04)   0.80(0.01)   B  bt1"
 * Start of treatment.
** Mean value of each sample with standard error of mean in parentheses.
 t Means with different letters differ significantly within a test at
   a - 0.05.
** Means of similar treatments with different letters differ significantly
   between tests at o - 0.05 and means with no letter do not differ signifi-
   cantly.
                                        32

-------
                              Nephtys incisa
Anesthetization
       36.  Since preliminary AEG values were consistently low in N.




incisa extracts, possible ways were sought to increase them (Tables




5 and 6).  Initially, during the collection procedures, the worms were




extremely active which made handling them difficult once the sediment




was removed.  It was thought that this activity was depleting the energy




reserves and causing low AEC values.  In order to eliminate this activity




and facilitate handling, N^ incisa were anesthetized.  Anesthetization,




which was accomplished by immersion of worms in a 7 percent magnesium




chloride solution for 2 1/2 min, had a significant (a = 0.05) effect




on AEC (Table 5).  These conditions of anesthetization allowed handling




of worms without movement until they were freeze clamped.  Worms were




collected at the South reference site in Long Island Sound on 14 November




1982 and anesthetized and freeze clamped on board the vessel at the time




of collection.  In addition, anesthetization was not lethal to the worms




since recovery occurred within 20 min after immersion of the worms in




clean seawater.
                                     33

-------
                                   Table 5




                Adenylate Energy Charge and Adenlne Nucleotide




                 Concentrations in N. incisa with and without




               Anesthetization with Magnesium Chloride (n • 8)
                                      umol/g Wet Weight of Tissue




      Treatment                 AEC         ATP         ADP         AMP




With Magnesium chloride     0.64(0.01)A* 1.02(0.13)  0.65(0.01)  0.06(0.03)




Without Magnesium chloride  0.50(0.01)8  0.33(0.13)  1.36(0.03)  0.26(0.01)






* Means with different letters are significantly different at ex =0.05.






         37.  The data in Table 5 would also indicate that worms not treated




  with magnesium chloride were under stress since a charge of 0.50 was




  obtained along with a decrease in the concentration of ATP with corres-




  ponding concentration increases of ADP and AMP. The AEC was higher in




  anesthetized worms since all swimming activity was eliminated and energy




  was conserved.  Although the AEC values increased with anesthetization,




  they were not sufficiently high to indicate that the worms were not under




  stressful conditions (Table 5).  This suggested that the extraction of




  nucleotides from N. incisa was unsatisfactory or a PCA-resistant ATP hydro-




  lyzing enzyme (ATPase) was present in N. incisa.






  Extraction




         38. Neutralized tissue homogenates were analyzed for adenine nucleo-




  tides after storage at -20°C.  A loss of ATP was observed after 12 hr




  storage.  This indicated that a PCA-resistant ATPase was present in
                                       34

-------
N. incisa.  Thus, a series of experiments were initiated  to  test  trichloro-




acetic acid (TCA) at different concentrations  as  an extractant  for adenine




nucleotides from N. inciaa.




       39.  Six freeze-clamped wafers, each containing 25 individual




N. incisa, were broken into many small pieces  while frozen.   The  pieces




of each wafer were divided into two or three aliquots to  give a tissue




mass of approximately 0.2 g for each aliquot.   Each aliquot  of  the same




wafer was treated as a replicate for a particular treatment.  Thus,  there




were six replicates with approximately the same worm mass for each




treatment.  This enabled a comparison with the same worm mass between




treatments and eliminated a major source of variability inherent  when




using individuals.  The data in Table 6, series 1, indicate that  6  per-




cent PCA (v/v) is a better extractant of adenine nucleotides from N.




incisa than 7 percent TCA (v/v), which is reflected in the higher AEC




value.  Next, the concentration of TCA was increased to 10 and 20 percent




(v/v) and no difference was obtained between 6 percent PCA (v/v)  and 10




or 20 percent TCA (v/v) for extraction of adenine nucleotides from N.




incisa (Table 6, series 2).  Extraction of adenine nucleotides from N.




incisa tissue homogenates twice with 6 percent PCA (v/v)  containing 0.33




percent EDTA (w/v) gave much better results than extractions twice with




6 percent PCA (v/v) (Table 6, series 3).
                                    35

-------
u>
                                                     Table 6




                        Comparison of Perchloric and Trichloroacetic Acids for Extraction
of Adenine Nucleotides from N. incisa for AEG Determinations

Test
Series
1

2


3



Sample
Size
Treatment N
PGA 6% v/v
TCA 7% v/v
PGA 6% v/v
TCA 10% v/v
TCA 20% v/v
PGA 6% v/v
(PGA 6Z v/v) +
(EDTA 0.33% w/v)
6
6
6
6
6
6
6




Adenine Nucleotides
umol/g Wet Weight Tissue
ATP
0.64(0.18)*
0.10(0.03)
0.38(0.12)*
0.41(0.11)
0.51(0.08)
0.19(0.04)*
2.05(0.48)
ADP
0.36(0.05)*
0.19(0.03)
- 0.32(0.03)*
0.44(0.06)
0.49(0.06)
0.80(0.01)*
0.79(0.07)
AMP
0.08(0.009)*
0.03(0.005)
0.06(0.005)*
0.12(0.007)
0.13(0.02)
0.14(0.04)*
0.14(0.005)
AEC
0.73(0.01)*
0.49(0.01)
0.66(0.01)*
0.64(0.01)
0.59(0.01)
0.53(0.01)*
0.81(0.01)
              *  Mean  value  of  each sample with standard error of the mean in parentheses.

-------
       40.  Analysis of adenine nucleotldes within 2 hr after extraction




was another precaution taken for the preservation of adenine nucleotides




during their extraction from N^. incisa tissue.  This precaution appeared




to be necessary with II. incisa but not with M. edulis since neutralized




PCA extracts could be stored for 4 weeks at -20°C with no apparent loss




In adenine nucleotide concentration.




Adenylate Energy Charge




       41.  Since extraction of N. incisa with PCA-EDTA gave consistently




high recovery efficiencies of 96 + 0.3 percent, PCA-EDTA was selected as




the method of choice for extraction of adenine nucleotides from N. incisa.




In addition, low variability among AEG values within treatments is also




apparent with the small sample size required to detect a 0.05 difference




in AEC values (Table 7).
                                     37

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                                 Table 7

     Sample Size Determination for the Detection of Fixed Magnitudes

      of Differences in AEC for N. incisa After Being Treated for 10

            Days with BRH Sediment Under Laboratory Conditions
                Magnitude of
Variable        Difference             Variance            N*

AEC
AEC

AEC
AEC
Test I 02 Sept 83
0.1 0.00091
0.05 0.00091
Test II 20 Sept 83
O.I 0.00106
0.05 0.00106

2
7

2
6
*N - Calculated sample size needed to detect a 0.1 or 0.05 unit change in
     AEC.  The power of the test is 0.80; a = 0.05 (Snedecor and Cochran
     1980).
       42.  As indicated in Table 7, a sample size of 7 would allow

detection of a significant difference in AEC of 0.05.  Also apparent in

Table 7 is the excellent reproducibility in sample size requirements

between experiments.  Analyses of variance coupled with Tukey's test

for grouping of means indicated that the AEC for the treatment BRH/REF

was significantly different from all other treatments within a test for

each test (Table 8).  The fact that Nephtys from the treatment BRH/REF

in both tests I and II had an AEC value of 0.92, which was significantly

higher than AEC values for other treatments within a test, strongly

suggests that these differences are real.  This extremely high charge
                                     38

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would indicate that a highly oxidative and metabolically active state




existed in these individuals.   Nephtys were in a slightly less  active




metabolic state from other treatments, although AEG values obtained  for  all




treatments in both tests I (2 Sept 83) and II (20 Sept 83) are  indicative of




healthy individuals.  No other significant difference occurred among




the other treatments in either test I or II (Table 8).  These data lend




credence to the fact that reproducibility within a test is exceptionally




good.  In order to determine the reproducibility between tests, analysis




of variance coupled with Tukey's test was performed by comparison of the




same treatment between tests for all four treatments.  It is evident in




Table 8 that the AEC for treatment REF/BRH  differed  significantly between




tests.  No other significant differences occurred among the other treat-




ments between tests (Table 8).  Although a significant difference




occurred with the treatment REF/BRH between tests, the data support  the




fact that the reproducibility  of AEC  for the  same  treatment between




tests is excellent  (Table 8).




     43.  The data  in Appendix A indicate that  the AEC test is sensitive  to




changes in handling and acclimation procedures.

-------
                                   Table 8

       The Response of Adenine Nucleotides in N.  incisa After  Treatment
with BRH
Sediment
for
10 Days Under
Laboratory Conditions (Tests I and II)

Treatment
Suspended/
Solid n
REF/REF
BRH/REF
REF/BRH
BRH/BRH
REF/REF
BRH/REF
REF/BRH
BRH/BRH
10
10
10
10
10
9
11
12
1
1
1
1
1
1
1
1
umol/g Wet Weight Tissue
ATP
.36(0
.33(0
.37(0
.36(0
.42(0
.30(0
.17(0
.37(0
.04)*
.09)
.04)
.10)
.06)*
.16)
.07)
.05)
ADP
0.35(0
0.19(0
0.35(0
0.42(0

Test
.02)*
.02)
.01)
.02)
Test
0.31(0.01)*
0.20(0
0.24(0
0.34(0
.04)
.01)
.01)
AMP
AEC
I 02 Sept 83
0.06(0
0.02(0
0.07(0
0.02(0
.005)*
.001)
.006)
.009)
0.87(0
0.92(0
0.86(0
0.87(0
.01)*
.01)
.01)
.01)
A**
B
A at
A
II 20 Sept 83
0.04(0
0.03(0
0.03(0
0.05(0
.01)*
.005)
.003)
.005)
0.88(0
0.92(0
0.89(0
0.87(0
.01)*
.01)
.01)
.01)
A**
B
A fa1"
A
*  Mean value of each sample with standard error of the mean in parentheses.
** Means with different letters are significantly different within a test
   at a » 0.05.
t  Means of similar treatments with different letters differ significantly
   between tests at a - 0.05 and means with no letter are not significantly
   different.
                                     40

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                          PART IV:   DISCUSSION






     44.  Adductor muscle tissue of M. edulis  and whole 1J.  incisa were




freeze clamped immediately after collection and stored in liquid nitro-




gen  since it is important to inactivate the enzymes of the tissues




very quickly to prevent degradation of ATP (Holm-Hansen and Booth 1966;




Patterson et al. 1970; Ivanovici 1980).  In addition, the choice of




extraction method to maximize the extraction of adenine nucleotides is




also important for the determination of their in vivo concentrations



and AEC (Lundin and Thore 1975; Karl et al. 1978; Larsson and Olsson




1979; Karl 1980; Mendelssohn and McKee 1981).  When working with N.




incisa it is also important to anesthetize them to maximize the ATP




concentration and AEC by eliminating all swimming activity once re-




moved from the sediment prior to freeze clamping.  Pamatmat (1982)




reported that the polychaete Neanthes virens also showed unpredictable




alternating periods of hyperactivity and rest when deprived of sediment.




Skjoldal and Bamstedt (1977) reported that zooplankton underwent meta-




bolic stress during capturing evidenced by a marked  lowering of the ATP




concentration and AEC.



     45.  We have had success in using PCA 6 percent  (v/v) to extract




adenine nucleotides from M. edulis as exemplified by high concentrations




of ATP  (> 2.80 ymol/g wet wt) with high AEC values  (0.88) which were




obtained consistently with untreated laboratory-held M.  edulis  (Zaroo-




gian et al.  1982).  However, this  extraction procedure did not  appear




to be as suitable for extraction of adenine nucleotides  from JN.  incisa




since low concentations of ATP  (0.52-0.57  ymol/g wet wt) with  low AEC
                                   41

-------
values (0.73 - 0.74) were consistently obtained with both field-col-




lected and laboratory-held worms.  Initially, the extraction of adenine




nucleotides, particularly ATP, was thought to be incomplete.  However,




during extraction trials with various concentations of PCA and TCA,




loss of ATP with a corresponding decrease in AEG occurred in neutralized




N. incisa homogenates which were stored for 1 week at -20°C.  This indi-




cated that ATPases were not being inactivated during the extraction




process with either PCA or TCA at concentrations as high as 20 percent




(v/v).  Skjoldal and Bamstedt (1977) reported that 96 percent of the ATP




in frozen zooplankton stored at -26°C degraded to AMP in 8 days.  Wijsman




(1976) found that when M. edulis tissues were homogenized in PCA, only




part of the ATP was recovered and that recovery was dependent upon the




time between horaogenization and assay.  He also determined that the ATP




was not hydrolyzed by PCA itself.  We did not see any decrease in ATP or




AEG upon storage of M. edulis neutralized, PCA-extracted adductor muscle




tissue for as long as 4 weeks at -20°C in this or in a previous study




(Zaroogian et al. 1982).  Ivanovici (1980) reported that ATP was stable




for 4 weeks in neutralized PCA extracts of an estuarine mollusc (Pyrazus




ebininus) when stored at -30°C.  Wijsman (1976) used the total soft




parts of M. edulis in his study, whereas in this study we used adductor




muscle tissue and Ivanovici (1980) used columnar muscle tissue.  The ATP




degrading enzymes that Wijsman (1976) reported to be resistant to PCA




inactivation may be found in tissues other than adductor muscle.  Such




remaining ATP degrading enzymes (ATPases) in PCA extracts have also been




reported when PCA was used with microorganisms (Davison and Fynn 1974;




Lundin and There 1975; Swedes et al. 1975).
                                     42

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       46.  Wijsman (1976) found TCA to be a better  extractant  of  adenine




nucleotides than PGA from M. edulis with no accompanying  loss of ATP.




In contrast, Wadley et al. (1980) found PCA the superior  of  four tested




methods (PCA, TCA, H2SC>4, and boiling bicarbonate  buffer) when  used with




one gastropod and two bivalve species, whereas TCA yielded low  AEC values.




Trichloroacetic acid has been used to extract adenine nucleotides  from




microbial cell suspensions (Lundin and Thore 1975; Larsson and  Olsson




1979) and has also been found to yield high adenine nucleotide  concentra-




tions with marine zooplankton (Ikeda and Skjoldal 1980; Skjoldal  1981)




and M. edulis (Skjoldal and Barkati 1982).  Our study corroborates that




of Wadley et al. (1980) in that PCA extracted more adenine nucleotides




than TCA in equimolar concentrations.  However, higher concentrations  of




TCA (10 and 20 percent v/v) did yield concentrations of adenine nucleo-




tides equal to those obtained with 6 percent PCA (v/v).




       47.  Adenine nucleotide extraction procedures which used heat were




not considered for use intentionally since Karl and La Rock (1975) reported




thermal gradients were likely to be established in fluid-solid mixtures




due to variations in the kinetics of heat flow.  Therefore, rapid enzyme




inactivation and complete extraction of ATP would not occur.




       48.  Lundin and Thore (1975) reported that the enzymes responsible




for the loss of ATP could be irreversibly inactivated by EDTA in  combina-




tion with PCA, TCA, or heat and suggested that EDTA acts by destabilizing




the enzymes by complexing metal co-factors.  Methods  incorporating EDTA




for extraction of adenine nucleotides have been used with plants  (Guinn




and Eldenbock 1972; Mendelssohn and McKee  1981),  bacteria (Chappelle  and




Levin 1968; Klofat et al. 1969; Lundin  and Thore  1975; Thore et al. 1975),
                                    43

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zooplankton  (Skjoldal 1981), mussels (Skjoldal and Barkati 1982), and




polychaetes  (Karl et al. 1978).   In each case, highest concentrations




of ATP were  reported for the respective tissues analyzed.  We also found




this to be true in this study since the highest concentrations of adenine




nucleotides  were extracted when EDTA was included.  The data, however,




do not indicate that the ATP degrading enzymes are inactivated by the




EDTA as suggested by Lundin and Thore (1975) since no differences in ADP




and AMP concentrations occurred between the same tissue extracted with




PGA or PCA containing EDTA.  If, in fact, ATP concentrations were due to




degradation  of ATP, then an equivalent increase in ADP and/or AMP should




occur if no  AMP degrading enzymes are present.  Thus it would appear




that EDTA is facilitating the extraction of adenine nucleotides.  Since




additional work is required to elucidate the effect of EDTA in the extrac-




tion and stabilization of adenine nucleotides from N. incisa, the possi-




bility of EDTA inactivating ATPases cannot be disregarded.  The fact




that ATP was lost in our neutralized extracts of 1J. incisa when stored




at -20°C lends credence to the ATPase tenet and the effect of EDTA during




storage remains to be tested.




       49.   A low energy charge most likely indicates a poor extraction




of adenine nucleotides in tissue samples from individuals collected from




a non-limiting environment.  Thus, the intent here was not to quantify




the adenine  nucleotide concentrations but to develop an extraction




procedure for Ifl. incisa that consistently produces AEC ratios at levels




representative of the in vivo levels previously reported for actively




metabolizing cells in a non-limiting environment (Chapman et al. 1971).




Uniformity within and among the extraction procedure is extremely important,
                                    44

-------
as this will minimize the variability associated with  nucleotide extrac-




tion among treatments and tests.   This in turn would be  reflected in




greater accuracy and reproducibility.  Literature concerning  AEC and




polychaete worms is scant.  However,  Karl et  al. (1978)  determined  the




AEC ratios in the polychaete worm (Euzonus mucronata)  and reported  a




value of 0.80.  They used sulfuric acid as an extract  and extracted



tissue homogenates once in contrast to our twice.  Schottler  (1979)




reported AEC values for three species of polychaete worms (Nereis)  to be




0.88 to 0.90 for control worms under nonlimiting environmental conditions,




Although he used dry weights, his concentrations of adenine nucleotides




(ATP, 1.29 umol/g; ADP, 0.32 umol/g; AMP, 0.04 pmol/g) were comparable




to ours when using a wet-to-dry ratio of 7:1 to calculate concentrations




on a wet weight basis.  He also extracted the tissue homogenates twice




with PCA.




       50.  The AEC values for N^ incisa from both tests I and II were




indicative of actively metabolizing cells in a nonlimiting environment.




Small differences  in AEC  (0.03 in this study) between treatments and




tests that are significantly different can be detected with this method




because of the sensitivity and low variability  associated with the AEC




values.  However,  although these small differences are statistically




significant,  the biological  implications are not always meaningful.




Thus, the statistically significant  difference  between  tests  for the




REF/BRH treatments does not  necessarily  infer  a difference in health




condition.
                                    45

-------
       51.  During the tests, the worms appeared to burrow preferentially

in the REF sediment regardless of treatment or position of the REF sedi-

ment in relation to the BRH sediment.

       52.  When BRH sediment was suspended over the REF sediment the

worms were actively burrowing and feeding, thus they were metabolically

active and the highest AEC values were obtained with this treatment.   It

would suggest that BRH is somewhat higher in nutritive value than the

REF sediment.

       53.  The AEC values reported for M. edulis taken from a non-limiting

environment are: 0.91 (Wijsman 1976), 0.90 (Skjoldal and Barkati 1982),

and 0.85 to 0.88 (Zaroogian et al. 1982).

       54.  Mussels from all treatments in this study had lower AEC values

than those obtained at the start of treatment.  However, the AEC values

obtained with mussels from the 100 percent REF and 100 percent BRH

treatments in test I (17 Nov 83) and with mussels from all treatments in

test II (19 Mar 84) were representative of actively metabolizing cells in

a nonlimiting environment (Chapman et al. 1971).  Although the AEC values

in these treatments indicated a healthy condition, the values were at the

lower end of the spectrum for this health condition.  However, that the

mussels treated with REF sediment had lower AEC values than those at the

start of treatment could indicate the following:

       a_.  Holding conditions are not entirely adequate.

       b_.  Food quality is poor.

       c_.  Food supply is inadequate.

       d_.  Because of the particulate load (sediment plus food) the mussels
           are expending more energy (faster filtration rate) in relation
           to the energy return from the amount of food assimilated.
                                     46

-------
       55.  The fact that a greater decrease was observed in the AEC

value after 26 days of treatment with 50 percent BRH and 50 percent REF

sediment than with 100 percent BRH sediment in  test I  (17 Nov 83) and

test II (19 Mar 84) at first appeared to be a paradox, but upon further

examination seemed consistent with observations reported by others

(Davenport and Manley 1978; Giesy et al. 1983). Mytilus edulis is able

to undergo anaerobic respiration for long periods  of  time  (5 to 7 days)

(Wijsman 1976).  Shell closure and anaeroblosis may enable M. edulis to

avoid continuous ingestion of toxicants which  they are able  to  sense.

The lower BRH sediment concentration used in this  study may  have contained

contaminants whose concentrations were below  the  threshold concentration

at which the mussels closed to avoid exposure.  To suggest or  imply  that

the mussels treated with 100 percent BRH closed their shells and  survived

anaerobically for 26 days would be inane.  However,  it is  feasible and

possible that these mussels opened their shells intermittently  for

feeding and excretion.  This is supported by  the  observation during  test

I (17 Nov 83) and test II (19 Mar 84) that mussels were filtering  less

in the 100 percent BRH treatment than in the  other two treatments.*

       56.  The reproducibility between tests was excellent with  identical

AEC values for the same treatment between tests except for the 50 percent

REF/50 percent BRH treatment which differed significantly.  This  difference

is an accurate reflection of the metabolic state of  the mussels since it

is obvious in Table 1 that mussels in the 50 percent REF/50 percent BRH

treatment in test I (17 Nov 83) experienced higher concentrations of
  Personal communication, Dianne Black, March 1984, U.S. Environmental
  Protection Agency.
                                     47

-------
suspended particulate than their counterparts in test II (19  Mar  84).   In




addition, 13 percent mortality was recorded for the 50 percent REF/50




percent BRH' treatment during test I (17 Nov 83) as opposed to no  mortality




for the same treatment during test II (19 Mar 84) (Table 1).  These facts




also helped to explain the lower AEC value (0.75) obtained with mussels




from the 50 percent REF/50 percent BRH treatment in test I (17 Nov 83)




when compared to the AEC value (0.80) obtained with mussels from  the same




treatment in test II (19 Mar 84).  Although a casual interpretation of




the data would suggest that improvement in reproducibility of AEC




measurement for the 50 percent REF/50 percent BRH treatment is wanting,




an in depth study of the data indicated otherwise, in that the AEC




accurately reflected the variation in conditions that existed within and




between the two tests (Table 1).  Thus it would appear that the metabolic




state and health condition of the mussels in the 50 percent REF/50 percent




BRH treatment from test II (19 Mar 84) are better than their counterparts




in test I (17 Nov 83) as indicated by the AEC.
                                    48

-------
                           PART V:   CONCLUSIONS






       57.  The ABC appears to be a highly sensitive method  for  measuring




stress in £._ incisa and M._ edulis treated with dredged material  under




laboratory conditions.  The low variability associated with  AEC  measure-




ments and the excellent reproducibility between tests would  support this




conclusion.




       58.  Adenylate energy charge is an excellent representation of the




actual metabolic state and health condition of 1^ incisa and M^ edulis.




       59.  Both JJ^ incisa and M^ edulis are excellent species with which




AEC can be used to accurately assess their metabolic state and health




condition when exposed to sublethal environmental perturbations.




       60.  This investigation is the first phase in developing field-




verified bioassessment evaluations for the Corps of Engineers and the




US Environmental Protection Agency regulatory program for dredged




material disposal.  This report is not intended for regulatory purposes;




appropriate assessment methodologies that are field verified will be




available at the conclusion of this program.
                                    49

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                                    54

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    APPENDIX A:  EFFECTS OF HANDLING AND ACCLIMATION PROCEDURES ON AEC






     The data in Table Al indicate that the AEC test is  sensitive to




changes in handling and acclimation procedures.  Although the charges




in the 2 September 83, 20 September 83, and the 10 November  83 tests




indicate healthy individuals, the results of the 10 November 83 test




were different from the results of the 2 September 83 and 20 September




83 tests which were essentially identical.  Therefore, strict adherence




to uniformity in handling and acclimation of N. incisa is imperative




since the AEC measurement is sensitive enough to detect differences due




to these parameters.  This sensitivity of AEC to alterations in test




procedures would also be reflected in the reproduclbility of tests.




Analysis of variance and Tukey's test for pairwise comparisons indicated




that AEC values for each treatment in the 10 November 83 test were signif-




icantly different from the respective treatments in both the 2 September




and 20 September 83 tests.
                                     Al

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                                 Table Al

             The Response of Adenine Nucleotides in N. incisa

              After Treatment with BRH Sediment for 10 Days

                  under Laboratory Conditions (Test III)
Treatment

suspended/
  solid     n
REF/REF

BRH/REF

REF/BRH

BRH/BRH
10

10

10

10
             pMol/g Wet Weight Tissue
           ATP
                 ADP
AMP
AEC
                      Test III 10 Nov 83
1.63(0.33)*  0.59(0.05)*  0.06(0.01)*  0.82(0.01)* A**

2.11(0.38)   0.62(0.05)   0.11(0.01)   0.83(0.01)  A

1.68(0.27)   0.60(0.03)   0.08(0.01)   0.82(0.01)  A

1.66(0.33)   0.54(0.04)   0.11(0.01)   0.82(0.01)  A
*  Mean value of each sample with standard error of mean in parentheses.

** Means with the same letter are not significantly different at a = 0.05.
                                     A2

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