%EPA
United States ~
Environmental Protection
Agency
Office of
Office of Acid Deposition.
Marine and Estuarire Protection Environmental Monitoring, and*
lA/"ipb*i.**«»*.A_ f\/«» t+f\ A f*n . J>N . i-. x *
Washington DC 20460
OHE? EPA-430/9-88/003
Quality Assurance
EPA-60Q/4-83-Q13
Water/Research and Development March 1988
Methods for Use of Caged
Molluscs for IN-SfTU
Biomonrtoring of Marine
Discharges
CVJ
en
HEADQUARTERS LIBRARY
ENVIRONMENTAL PROTECTION AGFMr"
WASHINGTON, D.C. 20460
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-v
t
EPA-600/4-88-013
March 1988
METHODS FOR USE OF CAGED MOLLUSCS FOR IN-SITU BIOMONITORING
OF MARINE DISCHARGES
Edited
by
Cornelius I. Weber, Ph.D.
Chief, Biological Methods Branch
Environmental Monitoring and Support Laboratory - Cincinnati
March 1988
DEVELOPED JOINTLY
BY
OFFICE OF RESEARCH AND DEVELOPMENT
OFFICE OF ACID DEPOSITION, ENVIRONMENTAL MONITORING, AND QUALITY ASSURANC;
ENVIRONMENTAL MONITORING AND SUPPORT LABORATORY
U. S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
AND
OFFICE OF WATER
OFFICE OF MARINE AND ESTUARINE PROTECTION
U. S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, DC 20460
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•f
)•
DISCLAIMER
This report has been reviewed by the Environmental Monitoring and
Support Laboratory, U.S. Environmental Protection Agency, and approved
for publication. The mention of trade names or commercial products does
not constitute endorsement or recommendation for use.
ii
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FOREWORD
Environmental measurements are required to determine the quality of
ambient water, the character of effluents, and the effects of pollutants
on aquatic life. The Environmental Monitoring and Support Laboratory-
Cincinnati conducts.research to develop, evaluate, standardize and
promulgate methods to:
0 Measure the presence and concentration of physical, chemical and
radiological pollutants in water, wastewater, bottom sediments,
and solid waste. ,
0 Concentrate, recover, and identify enteric viruses, bacteria, and
other microorganisms in water.
° Measure the effects of pollution on freshwater, estuarine, and
marine organisms, including the phytoplankton, zooplankton,
periphyton, macrophyton, macroinvertebrates, and fish.
° Automate the measurement of the physical, chemical, and
biological quality of water.
0 Conduct an Agencywide quality assurance program to assure '.
standardization and quality control of systems for monitoring
water and wastewater. :
This manual provides monitoring methods for use in determining the
biological effects of discharges to coastal and saline estuarine waters,
and was prepared primarily as a source of methods for use by permittees
under Section 301(h) of the Clean Water Act of 1977, as amended by the
Municipal Wastewater Treatment Construction Grant Amendments of 1981.
However, the methods are also applicable to other types of sources where
there is a need to determine the bioaccumulation of toxic substances, to
detect acutely toxic conditions in the plume, and to measure the degree
of stress (sublethal toxicity) to which the test organisms may have been
subjected. . !
Thomas A. Clark
Acting Director
Environmental Monitoring and Support
Laboratory - Cincinnati
111
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PREFACE
This manual was prepared with the assistance of the following Workgroup
members.
a. U.S. Environmental Protection Agency
Robert Bastian, Office of Marine Discharge Evaluation, Washington, DC
Bruce Boese, Environ. Res. Lab., Corvallis, OR
Donald Phelps, Ph.D., Environ. Res. Lab., Narragansett, RI
Richard Swartz, Ph.D., Environ. Res. Lab., Corvallis, OR
b- Southern California Coastal Water Research Project.
David Brown, Ph.D., Long Beach, CA
Henry Schafer, u
c. National Oceanographic and Atmospheric Administration
Paul Dammann, Ocean Acoustics Lab, Miami, FL
Alan Mearns, Ph.D., Office Mar. Poll. Assessment, Seattle, WA
d. California Department of Fish & Game
John Ladd, Sacramento, CA
Michael Martin, Ph.D., Monterey, CA
e. Tetra Tech
Thomas Ginn, Ph.D., Bellevue, WA
f. Dantes & Moore, Marine Services.
David Young, Ph.D., Los Angeles, CA
Although not a member of the original Workgroup, Dr. Donald
Baumgartner, U. S. Environmental Protection Agency, Corvallis, Oregon, also
provided valuable assistance in the preparation of the manual.
Cornelius I. Weber, Ph.D.
Chief, Biological Methods Branch
Environmental Monitoring and
Support Laboratory - Cincinnati
iv
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ABSTRACT
This manual describes methods for use of caged molluscs in
biomonitoring programs in coastal and saline estuarine waters. Molluscs
collected at relatively contaminant-free locations are placed in cages and
exposed for one month at a minimum of two stations: (1) in the plume,
within the zone of initial dilution, and (2) at a nearby reference
(control) station, outside of the area of immediate influence of the
discharge. At the end of the exposure period, the organisms are
retrieved, checked for mortality, analyzed for toxic substances, and
examined for indications of sublethal biological effects, including scope
for growth, and the distribution of toxic metals in the detoxification
system.
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p. vi - blank page
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CONTENTS
Foreword ill
Preface iv
Abstract v
Figures ix
Acknowledgments . . x
1. Introduction ' 1
2. Exposure Site Selection 3
3. Exposure Depth 4
. 4. Exposure Systems 5
Anchors 5
Line 5
Buoys 5
Exposure Cages/Bags . . 6
Gear Configuration 6
5. Test Organisms 8
Recommended Species 8
Size 8
Source and Condition 8
6. Sample Exposure and Retrieval 10
Exposure Procedures . . .' 10
Exposure Period 10
Sample Retrieval ..... 10
Field Observations 11
Sample Preservation and Transport 11
7. Chemical Analyses 13
Water and Wastewater Analyses ..... 13
Tissue Analyses 13
Sample Preparation 13
Priority Pollutant and Pesticide Analyses ... 13
Metabolite Analysis 14
Analysis for Toxic Substances in the Cytosol . . 15
8. Biological Analyses . 18
Fouling 18
Mortality 18
Incremental Growth . ., 18
Condition Factor ...... 18
Gonadal Index 19
Histopathological Effects . . . . . . 19
vii
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Scope for Growth 19
Experimental Design 19
Summary of Methods 20
Clearance Rate 21
Respiration Rate 21
Food Absorption Efficiency .... 22
Ammonia (Nitrogen) Excretion Rate 25
Calculation of Scope for Growth (SFG) Value ... 25
Statistical Analysis 26
Oxygen:Nitrogen Ratio 26
9. Quality Assurance 27
10. Data Analysis, Interpretation, and Reporting 28
Chemical Data 28
Water and Wastewater Quality 28
., Priority Pollutants in Tissues 28
Metabolites in Tissues 29
Distribution of Metals in the Cytosol 29
Biological Data 29
Fouling 29
Mortality 29
Incremental Growth 30
Condition Factor . 30
Gonadal Index 30
Histopathologic Effects 31
Scope for Growth "31
Oxygen: Nitrogen Ratio 32
References 33
vi ii
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FIGURES
Number
Page
7
1. Examples of exposure systems
2. Sephadex G-75 elution profile 17
3. Apparatus for measuring clearance rates and assimilation .,
efficiency 23
4. Exposure chamber with stirring bar for measuring respiration rates 24
ix
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ACKNOWLEDGMENTS
Sections of this manual were provided by the following contributors:
Scope for Growth - William Nelson, Science Applications International
Corporation, Environmental Research Laboratory, U.S. Environmental
Protection Agency, Narragansett, Rhode Island; Metallothionein Analysis -
Or. David A. Brown, Southern California Coastal Water Research Project, Los
Angeles, California, and Dr. Kenneth D. Jenkins, California State
University, Long Beach, California; Metabolite Analysis - Dr. David A. Brown
and Richard W. Gossett, Southern California Coastal Water Research Project,
Los Angeles, California.
In addition to the materials provided by the Workgroup members listed in
the Preface, and the contributions listed above, many helpful review
comments were received from the following: Philip A. Crocker, U. S.
Environmental Protection Agency, Dallas, Texas; Joseph Cummins, U. S.
Environmental Protection Agency, Seattle, Washington; Thomas J. Fikslin, U.
S. Environmental Protection Agency, Edison, New Jersey; Delbert B. Hicks, U.
S. Environmental Protection Agency, Athens, Georgia; William H. Pierce, U.
S. Environmental Protection Agency, San Francisco, California; H. Ronald
Preston, U. S. Environmental Protection Agency, Wheeling, West Virginia, and
Dr. Steven Ferraro, U.S. Environmental Protection Agency, Newport, Oregon.
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SECTION 1
INTRODUCTION
1.1 Information on the distribution and biological effects of toxic
substances discharged to marine waters is required for Section,301(h) of the
Clean Water Act of 1977 (Public Law 95-217), as amended by the Municipal
Wastewater Treatment Construction Grant Amendments of 1981, and other Agency
and state regulatory activities. Accordingly, monitoring systems are needed
which identify the chemicals of biological significance and provide
meaningful data on biological effects.
1.2 Many approaches have been used in monitoring for the presence and
adverse biological effects of toxic wastes discharged to marine waters,
including studies of the structure of natural planktonic and b^nthic
communities, the bioaccumulation of toxic substances, and physiologic and
histopathologic effects. Each approach has advantages and disadvantages.
Changes in the Structure of indigenous communities of organisms may be more
easily determined, but may not be as sensitive to pollution as changes in
the health of individual organisms, which can be adversely affected at low,
chronic levels.of exposure to toxic chemicals. Marine organisms do not
bioaccumulate all chemicals equally. Some chemicals may be in low
concentration or even below detection limits in wastewater, but accumulate
to high and/or.toxic levels in marine organisms. Conversely, some materials
in high concentration in effluents may not be bioconcentrated.
1.3 Extensive use of filter-feeding bivalve molluscs during the past decade
to determine the distribution and persistence of toxic substances in marine
waters, and to.detect and measure adverse effects of pollutants on aquatic
life, has resulted in the development of methodology which is suitable for
use in marine biomonitoring programs (Bayne et a!., 1978, 1981; Davies and
Pirie, 1980; Goldberg, 1975; Goldberg et al., 1978; Phelps and Galloway,
1980; Phelps et al., 1981; Phillips, 1976, 1977a, 1977b; Stephenson, et al.,
1979, 1980, 1981; Middows et al.. 1981; Bayne, 1985; Bayne et al., 1985;
Widdows, 1985a). In recent years, attention has focused on only a few
species, principally in the genus Mytilus.
1.4 This manual describes methods for the exposure of molluscs to
discharges to determine the bioaccumulation of toxic substances, to detect
acutely toxic conditions in the plume, and to measure the degree of stress
(sublethal toxicity) to which the test organisms may have been subjected.
In this protocol, caged molluscs are collected at a non-polluted site,
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exposed for one month at a minimum of two stations—in the zone of initial
dilution and at a reference station—and analyzed for toxic substances in
tissues and for sublethal biological effects.
1.5 Caution must be exercised in the use of caged molluscs in biomonitoring
programs. Differences in environmental conditions, such as temperature,
salinity, depth, and available food, will affect mussel growth and
condition, and may mask the effects of toxic discharges. These conditions,
therefore, should be similar at all stations selected for a given study.
1.6 Some organic contaminants which have a low octanol:water partition
coefficient are not bioconcentrated by organisms (Gossett et al., 1982).
Organisms in the plume may accumulate organic contaminants reflective of
historical rather than current discharges (Young et al., 1976). A
significant portion of contaminants (e.g., 39% for Copper, Phillips et al.,
1980) found in molluscs may be associated with sediments in the gut that may
not be absorbed. Thus, measurements of metals in undepurated organisms may
not give a true measure of'actual bioaccumulation of contaminants. Also,
often in these organisms there are large (greater than 3-fold) variations in
concentrations of organic contaminants related to both the stage in the
reproductive cycle, which varies seasonally, and the amount of upwelling of
contaminants from sediments (Brown et al., 1982d), which might make it
difficult to see differences between stations. Seasonal changes also occur
in histology (Reynolds et al., 1980), and in the rates of metabolism and
detoxification of contaminants (Brown et al., 1982d). These factors must be
taken into account when designing the field studies and interpreting the
data.
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SECTION 2
EXPOSURE SITE SELECTION
2.1 A minimum of two exposure sites must be used: (1) one in the plume,
within the zone of initial dilution (ZID), and (2) a reference site,
"upstream" from the zone of initial dilution and outside of the area
affectedly the discharge;
2.2 The plume exposure apparatus should be placed as close as possible to
the outfall diffuser (i.e. at the center of the ZID). Additional exposure
sites may be necessary or desirable to define contaminant gradients in the
vicinity of the outfall, and in the case of receiving waters that are
already stressed, to determine the contribution of other pollutant sources
to bioaccumulation levels.
2.3 The control site must have hydrographic and water quality
characteristics similar to those at the outfall. The test organisms are
sensitive to salinity, and the use of more than one control site (i.e., such
as upstream and downstream) may be required in estuarine environments where
salinity gradients are present.
2.4 Exposure locations to be avoided include shipping lanes and dredging
sites. Swift currents may preclude the use of some stations, but exposure
gear has been successfully maintained in currents as high as 5 knots.
2.5 Station positions may be established by use of surface buoys, visual
sighting (shore transects), use of fathometers to fix depth, acoustic
transducers (pingers), Loran C navigation aid, satellite navigation aids,
and portable navigation aids (e.g., Motorola MinirangerR).
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SECTION 3
EXPOSURE DEPTH
3.1 Test organisms are exposed at a minimum of one depth at each station.
At the discharge, exposure cages are placed in the ZID, but at least one
meter above the bottom to avoid the overriding influence of toxic substances
released during the exposure period by the sediments, which might have been
deposited by historical pollution not representative of the current
discharge. At the control station, cages are placed at the same depth(s) as
are used at the discharge. Under some circumstances, it may be desirable or
necessary to expose the organisms at additional depths to determine
concentration gradients or to detect the release of toxic substances from
the sediment. In cases where the plume depth is expected to vary during the
exposure period, it may also be appropriate to use multiple exposure depths
to ensure plume exposures.
3.2 To provide meaningful information, it is necessary for exposures to be
conducted at a depth which will ensure maximum potential plume contact. It
is the dischargers responsibility to demonstrate that exposures were
actually conducted in the effluent plume. The spatial distribution of the
plume may be determined by field water quality measurements (e.g., NH3,
turbidity), remote sensing (e.g., acoustic backscatter: Prom" et al., 1976;
Proni and Hansen, 1982}, or by mathematical models (See Tetra Tech, Inc.,
1982a and 1982b for examples and application). If models are used,
site-specific water density data (i.e., temperature and salinity) for the
exposure period should be used as input.
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SECTION 4
EXPOSURE SYSTEMS
4.1 Gear that has been used successfully in the past is described below.
For additional information see Stephenson et al. (1979) and Phelps and
Galloway (1980).
4.2 Because of the potential for loss of exposure gear due to natural
events (e.g., storms, ice flows) or vandalism, it is recommended that two
arrays be placed at each exposure site. The overall cost increase
associated with an additional array is considerably less than that required
for repeating the entire exposure if the test organisms cannot be recovered.
4.3 ANCHORS
4.3.1 Anchors that have been successfully used by various programs include:
(1} Train wheels (with axles removed), 340 kg.
(2) Degreased automobile engine blocks (use a commercial degreasing
firm). Two blocks are used on each line, chained together.
(3) Cast concrete blocks, 25 - 160 kg. •
(4) Fence anchors, auger-type, 1 - 2 m length (for use in soft
bottoms).
4.4 LINE
4.4.1 Sixteen millimeter (5/8 in.) polypropylene line or 8-mm (5/16 in.)
polypropylene encased steel cable (Rolyan PermaflexR) is recommended
for surface buoys. Smaller line (6 mm; 1/4 in.) may be used for
subsurface buoys. The line should be kept bagged and off the deck of the
surface vessel to prevent contamination.
4.5 BUOYS ;
4.5.1 Surface buoys are used primarily as station marker buoys, whereas
subsurface buoys are used to support the mussel cages and/or bags to
reduce losses due to ship damage and vandalism. Surface buoys placed in
navigable waters must be Coast Guard approved (e.g., RolyanR 1352).
The use of spar buoys is recommended.
4.5.2 Submerged buoys, such as a 30-cm diameter inflatable,
phosphorescent orange, plastic float, plus a 20-cm diameter
non-collapsable'float, can be used to support the mollusc cages.
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4.6 EXPOSURE CAGES/BAGS
4.6.1 Enclosures recommended for use with molluscs include polypropylene
or nylon test tube baskets and bait bags.
(1) Test tube baskets - use non-contaminating material, such as
polypropylene.
(2) Bags -
(a) Nylon mesh bags - 8 cm x 1 m (3 X 36 in.) nylon bait bags,
12 mm (1/2 in.) mesh, 20 kg test (Nylon Net Company, P.O. Box
592, Memphis, TN 38101).
(b) Polypropylene mesh bags - (VexarR), 15 cm X 225 cm, 12 mm
(1/2 in.) mesh.
4.7 GEAR CONFIGURATION
4.7.1 Examples of gear configuration are shown in Figure 1. A commonly
used configuration is where a USCG approved special purpose buoy (similar
to Rolyan 1352&) is attached by 8-mm (5/16 .in.) polypropylene encased
steel cable (Rolyan PermaflexR) to a 150-kg concrete anchor. Nylon
lines (6-mm) are run about 6 m to satellite moorings of 25-50 kg each to
which 6-mm polypropylene line is attached with 20-cm diameter hard
plastic floats used to suspend mussel baskets 1 m above the surface of
the sediment. A float placed about 6 m up the mooring cable prevents
entanglement with the subsurface floats, and baskets can be hung on the
cable itself for profile work. Bags containing mussels can also be hung
from a pipe framework as shown,in Figure le.
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1MT
Mil Mil CMTimit
msstts
. it m rgtmgrncic UN
. UKOt
UIFICE »UST
TM meet i it
HIISMFlCt WOT
nutic
Mll*MfTI.III I HI
WtStl USICTS
iwtn
1"
-0=
•UHll WMTl
IKMttM f«i
Figure 1. Examples of exposure systems (not to scale): (A) from Stephensen
et aU, 1980; (B) from Young et al.f 1976; (C) provided by
D. Phelps, USEPA, Narragansett, RI; (D) from Phelps and Galloway,
1980; and (E) provided by T. Fikslin, USEPA, Edison, NJ.
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SECTION 5
TEST ORGANISMS
5.1 RECOMMENDED SPECIES:
Mussels: Mytilus callfornianus - West coast
Mytilus edulis - West coast, East coast, and Gulf of
Mexico
Oysters: Crassostrea virginica - East coast, and Gulf of Mexico
" gigas - West coast
5.2 SIZE:
Mussels: 5-7 cm
Oysters: 7-10 cm
5.3 SOURCE AND CONDITION
5.3.1 Test organisms should be collected from an area that is relatively
free of contaminants. If no previous data are available on the level of
contaminants in tissues, and the physiological condition of the organisms in
the proposed collection area(s), representative samples should be collected
and tested. The proposed source of organisms selected for use should be
reviewed by the permitting authority before organisms are collected for
transplanting to the exposure sites. It should be noted that a state permit
may be required for collecting test organisms.
5.3.2 In the source area, test organisms should be collected from
approximately the same depth, preferably below mean-tidal level. Test
organisms can be collected by dredge or removed from rocky substrates with
stainless steel pry bars. Organisms should not be collected from steel or
man-made wooden structures. Collectors should wear clean polyethylene
gloves at all times. Care should be taken to avoid contamination of the
organisms during collection and transport. Organisms should be of
approximately the same size, to minimize the natural variation in chemical
and biological parameters.
5.3.3 A random subsample of 20-25 organisms should be removed from the
collection to determine mean length and weight, the condition factor, the
incidence of parasites and disease, the stage in the gametogenic cycle, and
body burden of toxic substances.
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5.3.4 Upon collection, the organisms should be triple bagged in 4-mil
cleaned polyethylene bags and placed in ice chests. The polyethylene bags
and ice chests should be cleaned with detergent (MicroR) and triple rinsed
with distilled water prior to use.
5.3.5 The molluscs should be transplanted to the exposure sites within 48 h
after collection.
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SECTION 6
SAMPLE EXPOSURE AND RETRIEVAL
6.1 EXPOSURE PROCEDURES
6.1.1 At the exposure site, the test organisms are placed in cages or mesh
bags. The bags are constricted every 6-8 in. with nylon cable ties to
ensure uniform exposure of the organisms to the surrounding water. Four
bags/cages, each containing 25 individuals (total of 100 individuals), are
exposed at each depth {Figure 1).
6.1.2 The test organisms should be protected from surface contamination by
enclosing them in cleaned 4-mil polyethylene bags until they are hauled
overboard and lowered below the surface. The protective bag is then removed
underwater.
6.2 EXPOSURE PERIOD
6.2.1 Organisms are exposed for one month. The choice of dates during
which exposure should take place may vary with location. If pronounced
seasonal changes occur, more than one exposure period is recommended. If
only one exposure period is used, it should be the period of maximum
exposure, i.e., when the sexual organs are well developed, the water
temperature is such that the animals are metabolically active, and there is
the least dilution of the discharge.
6.2.2 The period of maximum stratification and least dilution usually
occurs in late summer. However, contaminant concentrations in tissues
during this period may be the lowest of any time during the year because of
spawning. For this reason, it may also be advisable to expose organisms
during the winter. Exposures during periods of rapidly changing density
gradients should be avoided because of uncertainties in maintaining plume
exposures at a given depth.
6.2.3 Exposure periods greater than one month may be necessary at
discharges where certain toxic substances with relatively slow uptake rates
(e.g., Hg and Ag) are of concern.
6.3 SAMPLE RETRIEVAL
6.3.1 The potential for successful retrieval of exposure arrays is enhanced
by the use of electronic navigation aids during deployment and retrieval.
The use of such aids is especially important if subsurface buoys are used.
10
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It is also recommended that an acoustic transducer (pinger) be attached to
each array to aid in location during retrieval. Transducers are inexpensive
and operate for 6 months. The use of acoustic releases is not generally
recommended because they are expensive. However, they may be the best
alternative for some applications.
•i
6.3.2 For arrays with subsurface buoys, retrieval can be accomplished by a
combination of electronic positioning and acoustic location, followed by
diver retrieval of the exposure apparatus. The practical limit for diver
retrieval is about 36 m. In situations where the subsurface buoy must be
placed below diving depth, or in situations where diver retrieval is not
feasible for other reasons, .the exposure array may be retrieved by snagging
a bottom line attached to the anchor (Figure 1), or use of an-acoustical
release device.
6.4 FIELD OBSERVATIONS
6.4.1 Fouling
6.4.1.1 If fouling is severe, the flow of water to the molluscs may have
been sufficiently reduced to interfere with feeding. The degree of fouling
is observed and reported as the estimated percentage of mesh openings
occluded by fouling organisms (Stephenson et al., 1980).
6.4.2 Mortality
6.4.2.1 Conditions in the ZID may be acutely toxic. Therefore, the
percentage of test organisms surviving to the end of the exposure period
should be determined for each exposure site/depth.
6.5 SAMPLE PRESERVATION AND TRANSPORT
6.5.1 Contamination from substances in the surface film can be avoided by
placing the molluscs in polyetheylene bags before surfacing. When
retrieved, the organisms may be held briefly in cleaned ice chests until
further processing. Excess water should be drained from the organisms on
ship or after removing to shore.
6.5.2 Metal Analyses
6.5.2.1 Samples collected for trace metal analysis are placed in cleaned
ZiplocR bags, immediately frozen on dry ice and transported to the
laboratory in the frozen state. In the laboratory, samples are stored at
-20C until analyzed.
6.5.3 Priority Organic Pollutants and Metabolite Analyses
6.5.3.1 Samples collected for organic analysis are double-wrapped in
precleaned, hexane-rinsed aluminum foil. The aluminum wrapped samples are
then placed in ZiplocR polyethylene bags, immediately frozen on
- 11
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dry ice and transported to the laboratory in the frozen state. In the
laboratory the samples are maintained at -20C until analyzed.
6.5.4 Cytoso_l_Ana lysis
6.5.4.1 Samples collected for cytosol analysis are placed in cleaned
ZiplocR bags, and immediately frozen on dry ice. They are stored at -80C
upon return to the laboratory (experiments have shown that metallothionein
is stable at this temperature, but not at -20C; Oshida, 1982).
6.5.5 Biological Analyses
6.5.5.1 Samples collected for biological analyses returned to the
laboratory in cleaned polyethylene ica chests.
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SECTION 7
CHEMICAL ANALYSES
7.1 WATER AND WASTEWATER ANALYSES
7.1.1 Methods for water and wastewater analyses are described in USEPA,
1979, 1982.
i . •• .
7.2 TISSUE ANALYSES ;
7.2.1 Whole organism (soft part) composite samples are used for analyses of
toxic substances. Three replicate composite samples (15-20 organisms per
sample) should be analyzed from each exposure site/depth. Tissue samples
are analyzed for (1) the full list of 129 priority pollutants and six
pesticides, (2) for metabolites of toxic organic substances, and (3) for the
distribution of toxic metals and organics in the cytosol ;
(metallothionein/enzyme/glutathione pool). A subset of the priority
pollutants and pesticides may be analyzed if it can be demonstrated that
only those substances occur in the effluent. Data are reported in ug/g or
ng/g dry weight, with a wet weight conversion factor. ;
7.2.2 Sample Preparation
7.2.2.1 Immediately prior to analysis, frozen mussels are removed from the
bags, one at a time, scrubbed in deionized water to remove debris (use
polyethylene gloves), and thawed in polyethylene, borosilicate glass, or
stainless steel trays. The adductor muscle is severed with a clean,
stainless steel scalpel, the gonad is excised, and the renainer of the soft
parts are placed in a preweighed acid-cleaned containers. The quantity of
tissue required for analysis is approximately as follows: (1) Hg - 1 g; (2)
remainder of metals - 5 g; (3) organic priority pollutants - 50 g; (4)
metabolites - 5 g.
F
7.2.3 Priority Pollutant and Pesticide Analyses
7.2.3.1 Methods for the tissue analysis for priority pollutants and
pesticides are described in USEPA, 1981. The percent lipid (USFDA, 1970)
also should be determined for each sample because it may help explain the
variability in the concentration of organics. The moisture content of an
aliquot of tissue (dry weight conversion factor) is determined,by drying at
105C for 12 h (Stephenson et al., 1980).
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7.2.4 Metabolite Analysis
7.2.4.1 Recent studies indicate that metabolites represent the major form
of xenobiotic organics in marine organisms (Brown et a!., 1982b,c,d). In
addition, it appears that chronic effects of organic compounds are caused by
their metabolic products, while acute effects, which would occur under only
the most extreme circumstances, are caused by parent organic compounds (Sims
and Grover, 1975; Young et al., 1979; McKinney, 1981; Livingston, 1985).
However, most studies on the presence of organic contaminants in the
environment do not report levels of metabolites. These omissions may occur
because most metabolites cannot be extracted by normal procedures since they
are bound to proteins, DNA, glutathione, glucuronic acid, and other
substances in organisms (Reid and Krishna, 1973; Roubal et al., 1977;
Varanasi and Gmur, 1980; Miller and Miller, 1982). Therefore, to determine
their levels, they must first be released from substances to which they are
bound by a heat-catalyzed base hydrolysis (Miller and Miller, 1966; Miller
1970; Gingell and Wallcave, 1974; Gold et al., 1981; Brown et al., 1982b).
Results obtained by Brown et al. (1982b), indicate the recovery of
metabolites from tissues may be increased by one to two orders of magnitude
when this procedure is used. Since metabolites appear to be the predominant
form of xenobiotic organics in organisms, usually representing over 90% of
the total of parent compounds and their metabolites, it is important that
these analyses be included in programs designed to measure the
bioaccumulation of organic compounds. In fact, it may be that those
compounds which are rapidly metabolized after biological uptake may not be
detected by normal procedures.
7.2.4.2 The methods for extraction of metabolites are similar to EPA
standard procedures (Federal Register, 1979; USEPA, 1981), but with the
addition of a step in which the extract is heated to 90°C for 30 minutes
after extraction of the base/neutral extractable fraction and before
extraction of the acid extractable fraction. The procedure is as follows:
(1) Homogenize 5 g (wet weight) of tissue in 20 ml of deionized (DI)
water in a blender. Rinse the blade twice with DI water.
(2) Dissolve 1.2 g NaOH in the sample homogenate.
(3) Extract the homogenate three times with 50 ml of hexane/
acetone (1:1, V:V). Centrifuge if necessary to obtain complete
separation of the layers.
(4) Take the hexane (top layer) as the base/neutral extractable
fraction and analyze for parent organic compounds.
(5) Heat the remaining aqueous phase to 90C for 30 min to hydrolyze
possible conjugates (Gingall and Wallcave, 1974; Gold et al.,
1981).
14
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(6) Allow the solution to cool, adjust to pH 1 with 6N HC1, and extract
three times with 50 ml of methylene chloride with centrifugation if
necessary.
(7) Take the methylene chloride (bottom layer) to dryness with a
roto-vaporizer.
(8) Add 10 ml of methylating agent (5 mg 3-methyl-l-p-tholyl-triazene per
ml diethyl ether) to the dried sample.
(9) Blow-dry the sample under a stream of nitrogen.
(10) Redissolve the sample in methanol.
(11) Analyze the final methylated extract for the presence of metabolites
using 6C/EC, GC/FID or GC/MS (Brown et al., 1982b).
7.2.4.3 The distribution of metabolites between-a site of detoxification,
the glutathione-containing (GSH) pool, and sites of toxic action, including
the metallothionein-containing (MT) pool and the enzyme-containing (ENZ)
pool, can be determined by analyzing the composited cytosolic pools using
the above method, starting at (2) above (Brown et al., 1982b).
7.2.4.4 Both metals and organics share a common site of toxic action, the
ENZ pool, while organic metabolites also appear to act adversely on the MT
pool, reducing metal-binding and detoxification by this pool (Brown et al.,
1982b; Jenkins et al., 1982b). When all three cytosolic pools are analyzed
for both metals and organic.metabolites, it is possible to determine which
specific contaminants are present at sites of toxic action and therefore
responsible for direct toxic effects. When this procedure is used in
combination with general stress indices, such as scope for growth, it is
possible to ascertain both the sum total of direct toxic effects and
indirect effects related to the metabolic cost of detoxification.
7.2.5 Analysis for Toxic Substances in the Cytosol
7.2.5.1 The following simple procedures are used to determine the
partitioning of trace metals between a site of detoxification, the
metallothioneinrcontaining (MT) pool and a site of toxic action, the
enzyme-containing (ENZ) pool (Brown et al. 1982a).
(1) Tissues are thawed and individuals (when practical) or composites
of 15-25 organisms'are suspended in three volumes of chilled
buffer (0.05 M Tris-HCl, pH 7.4).
(2) Suspensions are homogenized with an antoxidant
(2-mercaptoethanol) for 15 sec at high speed in a Sorval Omnimix
homogenizer at 4C. The homogenate is centrifuged for 10 min at
10,000xg in a refrigerated centrifuge, and the resulting
supernatant is recentrifuged for 60 min at 100,000xg. The final
15
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supernatants (cytosols) from each sample are combined and
rehomogenized for 5 sec to ensure homogeneity. At this point,
cytosols can be stored at -80C until further processing.
(3) Frozen cytosols are thawed, vprtexed and 7 ml applied to a 1.6 x 70 cm
column packed with Sephadex G-75 gel. The sample is eluted with
0.05 Tris-HCl (pH 8.2) at a flow rate of 28 mL/h, and 3-mL fractions
are collected for metal analysis (Jenkins et al., 1982c). A standard
solution of proteins of known molecular weights, such as albumin,
should be used to characterize the Sephadex column.
(4) Fractions are analyzed for metals using flame atomic absorption
spectrophotometry when possible (e.g., usually Zn and Cu), or by
graphite furnace atomic absorption spectrophotometry when necessitated
by low metal levels (e.g., usually Cd and Ag}.
7.2.5.2 The first peak to elute, as located by the metal profiles, is the
high molecular weight enzyme-containing (ENZ) pool; the second peak is the
medium molecular weight metallothionein-containing (MT) pool; and the third
peak is the low molecular weight glutathione-containing (GSH) pool
(Figure 2). To save time for metal analyses, the location of these pools
can be determined by doing a Zn profile, and then combining fractions
constituting each of these pools for the remainder of the metal analysis.
7.2.5.3 A more rapid procedure has been developed, utilizing HPLC. Whereas
each Sephadex G-75 column run takes about 8 hours, HPLC runs take only
40 min. In the HPLC procedure, 0.1 - 0.5 mL samples are injected on a Toya
Soda TSK SW 3000 column (5 mm x 600 mm) and eluted at 1 nt/min with
0.2 M Tris HC1 (pH 7.4). One-mL fractions are collected and analyzed for
metals as described above (Jenkins et al., 1982b).
16
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Zn
FRACTION
foot
6 10 1C
ENZ
20 25 30
MT GSH
Figure 2. Typical Sephadex G-75 elution profile for a control mussel (Mytilus
californianus) liver showing the concentrations of Zn, Cu and Cd in
individual fractions constituting each of the ENZ: high molecular
weight enzyme-containing pool which contains Zn and Cu as essential
components of metalloenzymes, but is a site of toxic action for
excesses of metals; MT: medium molecular weight
metallothionein-containing pool which serves a
storage/detoxification function for essential (e.g.* Zn and Cu) and
non-essential (e.g., Cd) metals; and GSH: low molecular weight
glutathione-containing pool which serves as a site of detoxification
for organic metabolites (from Brown et al., 1982d).
17
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SECTION 8
BIOLOGICAL ANALYSES
8.1 The recommended biological observations and analyses, arranged in
approximate order of complexity and level of effort required, are as follows:
° Fouling
o Mortality
0 Incremental Growth
0 Condition Factor
0 Gonadal Index
0 Histopathological Effects
0 Scope for Growth
0 Oxygen:Nitrogen Ratio
8.2 FOULING
8.2.1 Fouling is determined in the field at the time of sample retrieval.
The degree of fouling is reported as the estimated percentage of mesh
openings occluded by fouling organisms (Stephenson et a!., 1980).
8.3 MORTALITY
8.3.1 The percentage of test organisms surviving to the end of the exposure
period is reported for each exposure site/depth.
8.4 INCREMENTAL GROWTH
8.4.1 The mean length of the shells (to the nearest 0.1 mm) is determined
(Riisgard and Poulsen, 1981) before and after exposure at the reference site
and in the plume'to determine the change in length of the shells during the
exposure period.
8.5 CONDITION FACTOR
8.5.1 The condition factor is the wet weight of the soft body expressed as
a percent of the total organism weight {Bayne and Thompson, 1970; Boalch et
al., 1981).
18
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8.6 GONADAL INDEX
8.6.1 The gonads are removed and weighed, and the (gonad weight)/(soft body
weight) ratio is calculated (Ouellette, 1978; Giese and Pearse, 1974). In
M. edulis, the gonad develops within the mantle so that physical separation
of the two tissues is difficult. Therefore, the entire gonadal/mantle
complex is taken as gonadal tissue (Lobel and Wright, 1982).
8.7 HISTOPATHOLOGICAL EFFECTS
8.7.1 Histopathological analyses will provide useful information regarding
the condition of the organisms and the site of toxic action, which could not
be determined ,by other means. Methods for tissue preparation are found in
Yevich and Barszcz (1981)
8.8 SCOPE FOR GROWTH
8.8.1 The physiological index, scope for growth (SFG), is a measure of the
energy available (Joules/h) to an organism for growth and reproduction, and
therefore is reflective of the health of that animal.. This index has been
found to be statistically correlated with the concentration of toxic
substances in tissues and is considered to be a sensitive method to detect
sublethal, adverse biological effects {Bayne et al., 1981; Phelps et a!.,
1981; Widdows et al., 1981; Martin et al., 1982a; Widdows, 1983; Nelson et
al., 1984; Lack and Johnson, 1985; Martin, 1985; Widdows, 1985}.
8.8.2 SFG is an integrated index which requires the measurement of four
parameters; clearance rate, respiration rate, assimilation efficiency, and
ammonia excretion rate. These variables are then transformed Into energy
equivalents, Joules/h (J/h), and substituted into the following equation:
SFG • (C x A) - (R + E) (1)
Where:
SFG = Energy available for growth and reproduction
C = energy consumed
A = Absorption efficiency
R = energy lost through respiration
E = energy lost through excretion
8.8.3 Experimental Design
8.8.3.1 If the data generated are to undergo rigorous statistical analysis,
the assumptions and limitations of the statistical test used must be
understood and followed. A review of this topic can be found in a recent
paper by Hurlbert (1984).
19
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8.8.3.2 There should be sufficient replication of cages at each station
to establish significant differences that are meaningful. A preliminary
experiment conducted in Narragansett Bay determined the variability
associated with the SFG index to establish the sample sizes required to
find a statistical difference (a = 0.05, p = 0.8) of 5 J/h between
stations {Nelson, unpublished). It was determined that a sample size of
five replicates, with two mussels from each cage, at each station was
sufficient.
8.8.3.3 Additional mussels are included in the cages for other
measurements and to compensate for any mortality. If possible, a
preliminary experiment to determine the variability at each monitoring
site is advantageous to determine sample sizes. If this is not
practical, the variability of the SFG index should be monitored over time
to determine whether sample sizes should be changed.
8.8.3.4 The size of the test organisms and stage of the gametogenic
cycle are important sources of natural variability observed in the SFG
measurements (Bayhe et al., 1981). One way to reduce some of the natural
variability present in the SFG index is to use mussels of similar size
(length). Collection of mussels of similar length from the same area may
be the best method available for obtaining mussels of similar tissue
weight and reproductive condition, both of which effect SFG.
8.8.3.5 In addition to the size of the animals, the environmental
conditions at each station should be as similar as possible with respect
to the physical parameters (i.e., temperature, salinity, depth, food
availability, etc.). If these conditions are too dissimilar, any
observed SFG differences may be attributable to an acclimation response
by the animal.
8.8.4 Summary of Methods
8.8.4.1 Calculation of the SFG value for M. edulis requires the
measurement of four parameters: clearance rate respiration rate, food
absortion efficiency, and ammonia excretion rate.
8.8.4.2 All SFG measurements for a given treatment should be completed
in the order shown below as soon as possible after collection of field
samples. A comparison between field and laboratory measurements carried
out with two mussel populations showed that differences between
populations were maintained in the laboratory over a period of 24 h
(Widdows, 1983). For the sake of consistency, the physiological
measurements are completed in the following sequence for each group of
organisms tested:
Day 1: AM - Collection
PM - Clearance rate
Day 2: AM - Absorption efficiency
- Respiration rate
PM - Ammonia excretion rate
20
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8.8.5 Clearance Rate
8.8.5.1 Clearance rate Is defined as the volume of water completely
cleared of particles larger than 3 urn in some unit time (Widdows et al.,
1979). This is measured by placing mussels into individual chambers
(Figure 3} through which 1-um filtered seawater flows at a rate of
75 ml/min.
A monocultured unicellular algae of good food quality, such as Ispchrysis
aff. galbana (T-Isg) or Tetraselmis suecica, is added to the filtered
seawater to deliver an incoming cell concentration of approximately
0.5 mg/L to each chamber. Each chamber should be gently aerated to
ensure that the algae are completely mixed and do not settle out.
Mussels are allowed to acclimate in the chambers for at least 1 h prior
to any measurements. The incoming and outgoing particle concentrations
for each chamber are then measured (this is facilitated by the use an
electronic counting device such as a Coulter Counter) and substituted
into the following formula to determine clearance rate:
Clearance rate * [(Ci - C2)/C2] X F (2)
Where:
C] and C£ = Incoming and outgoing particle
; concentrations, respectively
and: F = Flow rate in L/h through
the chamber.
8.8.6 • Respiration Rate
8.8.6.1 Respiration rates are measured by isolating each mussel in a
glass respirometer vessel (Figure 4) fitted with an electrode designed to
measure the partial pressure of oxygen (POg). . The electrode is
connected to an oxygen meter, such as a Radiometer Model PHM71 or
equivalent, which is in turn connected to a strip chart recorder. Each
mussel is allowed to acclimate for about 30 min in the vessel prior to
respiration measurements. Seawater containing algae is pumped;into the
vessel during this acclimation period at a rate of 80 ml/min to ensure
that food is present in the chamber and that the metabolic rate is
allowed to stabilize. At the end of the acclimation period, the flow of
seawater is stopped and the decline in P02 is recorded on the strip
chart recorder for approximately 30 min. Respiration rate is calculated
using the following formula:
MMHG RESVOL - MUSVOL 60:
ML02/H * = X SAT02 X X —
160 1000 02TIME
-------
Where:
ML02/H = Oxygen consumed per hour by the mussel, ml
MMHG - Change in partial pressure of Q£ over time, mm mercury
SAT(>2 = Oxygen saturation level of seawater at that
temperature, ml/I
RESVOL = Respiration vessel volume, mL
MUSVOL = Volume of the mussel, ml
02TIME = Time period of the measurement, min
8.8.7 Food Absorption Efficiency
8.8.7.1 After completion of the respiration rate measurements, all fecal
material should be removed from each feeding chamber. This ensures that
only the algae consumed during the SFG procedures are used in the
absorption efficiency measurements. At the food concentration used in
the SFG measurements (approximately 0.5 mg/), no pseudofeces are
produced. The mussels are allowed to feed overnight in the chambers.
Fecal pellets are collected from each chamber with a Pasteur pipette, and
filtered onto a 1-um NUCLEPORE polycarbonate filter and rinsed with
isotonic ammonium formate to remove any salts. The filter is removed to
a watch glass and a few more drops of isotonic ammonium formate are added
to facilitate removal of the fecal pellets. The fecal pellets are then
scraped off with a plastic spatula, deposited onto small pre-weighed
aluminum pans (1 cm') and placed in a drying oven at 100C for 24 h.
Pellets and pans should be weighed using a balance accurate to 0.01 mg,
such as a Perkin Elmer antobalance .(Model AD-27) or equivalent. Pellets
are then ashed at 500C for 4 h, and reweighed to determine the ash-free
dry weightrdry weight ratio for the feces. A similar procedure is
completed with the cultured algae to obtain the ash-free dry weight:dry
weight ratio of the food. Food absorption efficiencies are calculated
for each mussel according to the method of Conover (1966) using the
following formula:
F ' E (4) •
Food Absorption Efficiency = X 100 * '
(1 - E) X F
Where:
F = Ash free dry weight:dry weight ratio of the food
E = Ash free dry weight:dry weight ratio of the feces
22
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FILTERED SEAWATER «
ALGAE
MIXING CHAMBER
Figure 3. Apparatus for measuring clearance rates and assimilation
efficiency (figure provided by William Nelson).
23
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OXYGEN
METER
CHART
RECORDER
RESPIRATION RATE APPARATUS
WATER BATH
Figure 4. Exposure chamber with stirring bar for measuring respiration
rates (figure provided by William Nelson).
24
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8.8.7.2 This technique allows the calculation of a food absorption
efficiency for each mussel. In previous experiments cited iVthe
literature, fecal material was collected on pre-ashed glass fiber filters
which weighed a great deal more than the dried and ashed fecal material.
The great differential between the weight of the glass fiber filter and
the weight of the dried and ashed fecal materials appeared to introduce
an artifact into the data. The substitution of the light aluminum pans
resulted in a 50 percent reduction in fecal weight variability and the
subsequent absorption efficiency difference between individual mussels
(Nelson et al., 1984).
8.8.8 Ammonia (Nitrogen) Excretion Rate
8.8.8.1 Mussels are placed individually into HC1 stripped beakers
containing 300 ml of 1-um filtered seawater for a period of 3 h. The
mussels are then removed and a 0.45-um filtered, 50-mL sample is
collected from,each beaker, deposited into acid stripped polyethylene
bottles, and stored in a freezer at -20C until analyzed. Ammonia
analyses should be completed in duplicate for each sample according to
the method of Bower and Holm-Hansen (1980).' •
8.8.9 Calculation of Scope for Growth (SFG) Value
8.8.9.1 After completion of the physiological measurements, the length
and volume of each mussel is measured, and the soft parts are removed
from the shell, dried for 24 h at 105C, and weighed. The clearance
rates, respiration rates, and ammonia excretion rates are standardized to
mean dry weight of the mussels. The weight standardized rates for each
mussel are then used to calculate the SFG of each individual by
substitution into the following equation:
SFG = (C X A) - (R + E) (5)
Where:
' t
C - Energy (Joules/h) consumed (clearance rate X surrounding food
concentration X energy of food)
A = Absorption efficiency (%}
R - Energy lost through respiration
E » Energy lost through nitrogen excretion
8.8.9.2 The following energy conversions can be used to calculate SFG:
1 mg of T-Iso * 4.5 X 10 cells (Nelson et al., 1984)
= 19.24 J
1 ml Og respired = 20.08 J (Crisp, 1971)
1 mg NH4-N = 24.56 J (Elliot and Davidson, 1975)
25
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8.8.9.3 The energy content of any algae can be determined by filtering a
volume of the algae onto preweighed glass fiber filters, drying them at
100C for 24 h, and reweighing them to determine algal dry weight. The
filters are then analyzed vising the dichromate wet oxidation method of
Maciolek (1962) to determine oxygen consumed and the resultant energy
content.
8.8.9.4 SFG can be standardized for a 1-g animal. Calculation of
standardized rates is only recommended when using animals that vary
widely in length. If the lengths of the animals fall within a narrow
range, weight-specific rates should be calculated for clearance,
respiration, and ammonia excretion before calculating the SFG.
8.8.10 Statistical Analysis
8.8.10.1 Differences in physiological data and the resultant SFG values
between stations may be tested using one-way analysis of variance
(Snedecor and Cochran, 1978). Tukey's studentized range test or another
comparable range test can be applied to determine between-treatment
differences. Again it is important to satisfy all the assumptions
(random sampling, true replications, etc.) of the statistical test
employed so as to make proper statements about differences. To be
significant, differences between SFG values for organisms at different
stations must exceed experimental error.
8.9 OXYGEN:NITROGEN (0:N) RATIO
8.9.1 The 0:N ratio, which is the ratio of oxygen consumed to nitrogen
excreted, is another useful physiological index of stress. This value
can be calculated from the above data as follows (Bayne, 1975; Widdows,
1978b):
0:N
Where:
mg 0?/h mg NH4-N/h
£ -«_^w—^L^_^_ •
16 ' 14
ml 02/h * mg 02/h X 1.428
26
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SECTION 9
QUALITY ASSURANCE
9.1 A quality assurance plan must be prepared as an integral part of the
301(h) monitoring plan. Factors in the field that will affect the
quality and utility of the data include the condition, uniformity in
size, and stage in the gametogenic cycle of the organisms, the care taken
in avoiding contamination and injury of the organisms-during collection
and transport of the test organisms, the attention given to the depth and
positioning of the exposure gear, and the water quality conditions, such
as salinity, at the exposure sites.
9.2 Laboratory quality assurance practices include the regular
calibration of instrumentation, the use of duplicate analyses (i.e.,
every tenth analysis) and reference materials, and participation in
interlaboratory studies such as round robins and performance
evaluations. Detailed laboratory quality assurance guidelines are
described in USEPA (1979, 1982). Reference materials for water,
wastewater and tissue analyses are available from the Quality Assurance
Branch, Environmental Monitoring and Support Laboratory, U.S.
Environmental Protection Agency, Cincinnati, Ohio.
27
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SECTION 10
DATA ANALYSIS, INTERPRETATION, AND REPORTING
10.1 The overall objective of the caged mollusc biomonitoring program is
to provide the permittee and the regulatory agency with data useful in
identifying discharges that cause adverse effects on marine life or -
otherwise pose a threat to the marine environment. Differences observed
in the chemical and biological parameters at reference and plume stations
must be analyzed for statistical significance to account for natural
variations in chemical and biological data, which are often large. The
use of composite samples is generally not recommended because it obscures
the variation in the individual organisms, and prevents an adequate
determination of the precision of the analyses. However, compositing is
sometimes necessary to obtain sufficient material for analysis, or to
reduce an otherwise overwhelming analytical burden. The selection of
test organisms of similar size and stage of gametogenesis will tend to
reduce the variation in the biological data (Bayne et al., 1981), and
enable the investigators to detect smaller differences in population
responses between stations than otherwise possible.
10.2 Upon the completion of the statistical analyses (t-test, ANOVA,
etc.), parameters which fail to show a significant difference between the
reference and exposed stations are reported, together with an appropriate
discusssion if the results were unexpected or otherwise unusual.
Parameters which are significantly different are further evaluated to
determine the magnitude of the difference, whether any FDA, EPA, or state
criteria for standards have been exceeded, and what reduction in the
concentrations must be achieved to reach acceptable biological conditions.
10.3 CHEMICAL DATA
10.3.1 Water and Wastewater Quality
10.3.1.1 Water quality data should be reported to document conditions at
the reference and plume stations, and should be evaluated in terms of the
environmental requirements of the test organisms and confounding effects,
if any, on the interpretation of the biological data.
10.3.2 Priority Pollutants in Tissues
10.3.2.1 Data on priority pollutants in tissues of organisms exposed at
the reference and plume stations should be compared and evaluated in
28
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erms of differences in biological responses at the stations. FDA-action
levels should also be considered if test species are being harvested for
human consumption from the polluted zone.
10.3.3 Metabolites in Tissues
10.3.3.1 As mentioned above, it appears that chronic effects of organic
compounds are caused by their metabolic products, while acute effects, which
would occur under only the most extreme circumstances, are caused .by parent
organic compounds (Sims and Grover, 1974; Young et al., 1979; Livingstone,
1985). Body burdens of metabolites should be checked against data on
toxicity of metabolities and parent compounds.
10.3.4 Distribution of Metals in the Cytosol
10.3.4.1 The metal levels in each pool of cellular proteins are added and
expressed as an amount of metal per unit weight of tissue. The metal
concentration in the MT pool can be compared to the loading capacity of this
pool as determined by laboratory exposures. In this way, the degree of
utilization (saturation) of the detoxification capacity of the organism can
be determined (Brown et al., 1982a). Using this information, predictions
can be made as to how much additional metal could be loaded into the biota
before spillover of trace metals from the MT pool to the ENZ pool would
occur, with resultant toxic effects. These toxic effects occur because
excesses of essential metals or non-essential metals in the ENZ pool result
in disruption of normal enzyme function.
10.3.4.2 It should be noted that a certain amount of Cu and Zn will always
occur on the ENZ pool because these metals are essential components of
metalloenzymes (Brown and Chatel, 1978; Jenkins et al., 1982c; Viarengo,
1985). However, essential metals present in excess of that required in the
metalloenzymes must be partitioned onto MT or they will have a toxic
effect. Further discussions of the analytical methods and significance of
the metallothionein data can be found in the following references: Brown et
al., 1982c; Jenkins et al., 1982b,c,d,e; Kohler and Riisgard, 1982;
Noel-Lambot et al., 1980; Piscator, 1964; Shiakh and Lucis, 1971; Simkiss
and Taylor, 1981; Simkiss et al., 1982; Squibb et al., 1974; and Viarengo
et al., 1980, 1981;
10.4 BIOLOGICAL DATA
10.4.1 Fouling
10.4.1.1 If severe fouling is observed at the end of the exposure period,
the flow of water to the molluscs may have been sufficiently reduced to
interfere with feeding. The degree of fouling, therefore, should be taken
into consideration in evaluating data on growth and condition.
10.4.2 Mortality
10.4.2.1 The percentage of test organisms surviving to the end of the
exposure period is reported for each exposure site/depth. Conditions in the
29
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ZID may be acutely toxic. If less than 90 percent of the organisms exposed at
the reference site survive, the test would be considered invalid and
should be repeated. If survival at the reference site exceeds 90 percent, but
survival at the plume site(s) is less than 90 percent, acute toxicity may be
present. Under these circumstances, the bioaccumulation data would be
invalid. To obtain adequate bioaccumulation results, it would be necessary to
repeat the test using additional exposure sites.
10.4.3 Incremental Growth
10.4.3.1 This index is simply the increase in the mean shell length during
the exposure period. Shell growth is dependent upon water temperature,
available food, and other environmental factors, in addition to the presence
of pollutants. Under normal conditions, a growth of several mm would be
expected in 30 days. Riisgard and Poulson (1981), starting with organisms
2.26 mm in length, reported a increase in length of as much as 6.6 mm in M.
edulis in 18 days. Additional observations on the growth of M. edulis were
reported by Kautsky (1981). If the mean increase in shell length of organisms
exposed in the plume is significantly (P = 0.05) less than at the reference
station(s), the likelihood of adverse environmental conditions is indicated.
10.4.4 Condition Factor
10.4.4.1 The condition factor is the wet weight of the soft body expressed as
a percent of the total organism weight (Bayne and Thompson, 1970). Boalch et
al., (1981) observed a mean condition factor of 6% in a composite of 20 M.
edujis with a mean length of four cm. In their study, the condition factor was
significantly correlated (P = 0.05) with the logio of.the metal
concentration for all metals except copper. They observed that the use of a
composite sample reduced the variation from three orders of magnitude, for
individual organisms, to approximately 50%. Stephenson et al. (1980), using a
slightly different form of the condition factor, (soft body weight)/(length),
observed that mussels with the highest condition factor were collected away
from heavily industrialized areas.
10.4.4.2 A statistically significant (P = 0.05) decline in the condition
factor ratio during the exposure period, or a significantly lower CF at the
plume site compared to the reference site(s), would indicate the likelihood of
adverse environmental conditions at the exposure site.
10.4.5 Gonadal Index
10.4.5.1 The gonadal index varies with the stage in the gametogenic cycle of
the organism. Within the gametogenic cycle, the proliferation of gonadal
tissue and maturation of the gametes will be affected by the physiological
condition of the organism, which in turn will be determined by the
availability of food and other environmental conditions, including the
concentration of toxic substances. Stephenson et al. (1980) reported gonadal
indices ranging from 0.21 to 0.39. The low indices observed at some stations
in their study were assumed to be related to high metal concentrations in
tissues.
30
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10.4.6 Histopathological Effects
10.4.6.1 Histopathological analyses provide information on the general
condition of the organisms and the site of toxic action which could not
be determined by other means (Barry and Yevich, 1975; Lowe and Moore,
1978; Lowe et al., 1981; Mix and Schafer, 1979; Mix et al., 1977; Mix et
al., 1979a; Mix et al., 1979b; Reynolds et al., 1980: Thompson.et al.,
1978; Yevich and Barszcz, 1977). This information includes:
(1) Identity of specific tissues affected by the toxic substances.(site of
toxic action).
(2) Whether.the effects are reversible or irreversible.
(3) The sex, stage in the reproductive cycle, and condition of the gonads.
(4) Whether the poor condition of organisms that appear to be stressed was
caused by parasites, pollutants, or nutrition.
•i !i
10.4.7 Scope for Growth
10.4.7.1 "Scope for Growth" (SFG) is a measure of the amount of energy
available to an organism for growth and reproduction. Positive SFG values
indicate that under the conditions of the test, the organisms are using less
energy than they are taking in, and a surplus of energy is available for
growth and reproduction. Negative SFG values indicate that the organisms
are using more energy than they are taking in, and have an energy deficit.
If the latter condition persists long enough, death will result. However,
in the absence of lethal conditions, a decline in SFG may adversely affect
populations by reducing growth rates and reproductive potential (Bayne et
al., 1975, 1978; Bayne and Widdows, 1978; Bayne and Worrall, 1980).
SFG values have been found to be inversely related to the concentration of
toxic substances in mollusc tissues, and the test is considered a sensitive
method to detect sublethal, adverse biological effects of toxic substances
on molluscs (Phelps et al., 1981; Widdows et al., 1981; Martin et al.,
1982a; Martin, 1985; Widdows, 1985a).
10.4.7.2 The SFG is affected by environmental conditions, such as
temperature, salinity, depth, and available food, reproductive :;Stage, etc.,
and it is not possible to predict the actual SFG values for the organisms
under natural conditions. However, since the organisms from all stations
are tested under the same laboratory conditions, it can be assumed that the
differences in SFG values are due to the differences in the condition of the
organisms, which in turn are a reflection of the conditions to which the
organisms had been exposed in their natural environment. A depression in
the SFG values of organisms collected from areas subjected to pollution,
therefore, serves as a "flag" that they have been adversely affected by
environmental conditions.
10.4.7.3 The biological mechanisms which result in a lowered SFG may not
always be apparent. A decline in SFG was noted along a pollution gradient
in Narragansett Bay (Widdows et al., 1981; Widdows, 1985a). Gillfillan et
al. (1976) observed an inverse correlation between SFG and the concentration
of aromatic hydrocarbons in tissues. A similar relationship was reported
31
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between SF6 and the concentration of the water-accommodated fraction of
North Sea crude oil by Widdows et al. (1982), and between SF6 and tissue
burdens of metals and organics by Phelps and Galloway (1980). In a
recent study in the New York Bight {Phelps et al., 1983), histological
examinations revealed that test organisms with reduced SFG values
suffered from a pathologic condition known as "atypical cell
hyperplasia," .which could have caused decreased clearance rates and
lowered SFG values. Inverse relationships between GFG and the
concentrations of metals and organics have also been reported by Martin
et al. (1984), Martin (1985), and Widdows (1985a).
10.4.7.4 Typically, SFG values are subjected to statistical analyses to
determine if differences between stations are statistically significant.
However, statistical significance does not always imply biological
significance. A more extensive discussion of factors related to the
collection and analysis of SFG data can be found in Bayne et al. (1981)
and Widdows (1983).
10.4.8 Oxygen:Nitrogen Ratio
10.4.8.1 The Oxygen:Nitrogen (0:N) ratio provides information on the
relative utilization of protein in energy metabolism compared to other
carbon sources. A high rate of protein utilization, compared to
carbohydrates and lipids, results in a low 0:N ratios, which are
generally indicative of a stressed condition (Widdows, 1978). According
to Bayne (1973a,b), low 0:N ratios (i.e., 20 or less) may result from low
food concentrations (starvation), whereas at high food concentrations
(1.5 mg/L or greater), 0:N ratios will fall in the range of 40 to 50 in
the absence of other adverse environmental conditions. They indicated
that food levels available during most of the year support 0:N ratios in
the range of 25-30. Widdows et al. (1981), however, stated that 0:N
ratios of less than 30 were indicative of organisms that were very
stressed. They observed 0:N ratios of 50-75 in organisms that were well
nourished and living under generally favorable environmental conditions.
Although some variability in the 0:N data is indicated, they may be
useful in detecting stress.
32
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