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
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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
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ISa. DECLASSIFIC ATI ON/ DOWNGRADING
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Approved for public release; distribution unlimited.
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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.
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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
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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
-------
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
-------
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
-------
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
-------
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
-------
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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