EPA 903-R-98-022
CBP/TRS 215/98
October 1998
Standard Operating Procedures
for Conducting Sub-chronic Aquatic
Toxicity Tests with Sago Pondweed
Potamogeton pectinatus:
A Submersed Aquatic Angiosperm
Chesapeake Bay Program
EPA Report Collection
Regional Center for Environmental Information
U.S. EPA Region HI
Philadelphia, PA 19103
Primed on Recycled Paper for EPA by CBP
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Regional Center for Environmental Information
US EPA Region III
1650 Arch St.
Philadelphia, PA 19103
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Standard Operating Procedures
for Conducting Sub-chronic Aquatic
Toxicity Tests with Sago Pondweed
Potamogeton pectinatus:
A Submersed Aquatic Angiosperm
October 1998
University of Maryland
Agricultural Experiment Station
Wye Research and Education Center
Anne Arundel Community College
Environmental Center
Chesapeake Bay Program Infer'••"^'
410 Severn Avenue, Suite 109 1Q5Q A^ ''•• '-'• -
Annapolis, Maryland21403 Philac!.;-. -, - :
1-800-YOUR-BAY
http://www.chesapeakebay.net/bayprogram
Pnnted by the U.S. Environmental Protection Agency for the Chesapeake Bay Program
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FINAL REPORT
October, 1998
Standard Operating Procedures for Conducting Sub-chronic Aquatic
Toxicity Tests with Sago Pondweed Potamogeton pectinatus:
A Submersed Aquatic Angiosperm
Lenwood W. Hall, Jr.1,
M. Stephen Ailstock2 and Ronald D. Anderson1
University of Maryland
Maryland Agricultural Experiment Station
Wye Research and Education Center
Queenstown, Maryland 21658
2Anne Arundel Community College
Environmental Center
101 College Parkway
Arnold, Maryland 21021
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FOREWORD
Presently there are few estuarine toxicity test protocols
available for Chesapeake Bay resident aquatic species. The
submersed aquatic macrophyte, Potamogeton pectinatus, was
recommended as a test species for SOP development based on an
extensive literature review and synthesis of data from 25 candidate
species found in the Chesapeake Bay (Ziegenfuss and Hall, 1993).
This manual outlines standard operating procedures (SOP) for
conducting sub-chronic toxicity tests with sago pondweed. The U.S.
Environmental Protection Agency and Maryland Department of the
Environment provided the funding for development of this document.
We would like to acknowledge Dr. Elgin Perry for suggestions on
statistical analyses of the toxicity data and Sharon Horstman for
technical illustrations.
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TABLE OF CONTENTS
Page
FOREWORD i
1.0 INTRODUCTION 1
2.0 CLASSIFICATION AND DISTRIBUTION 2
2.1 CLASSIFICATION OF AQUATIC PLANTS 2
2.2 DISTRIBUTION OF SUBMERSED AQUATIC ANGIOSPERMS 6
3.0 LIFE CYCLE OF POTAMOGETON PECTINATUS 7
4.0 TAXONOMY 10
5.0 TERMINOLOGY 10
6.0 SUMMARY OF TEST PROCEDURES 11
6.1 CULTURE SYSTEM 12
6.2 NUTRITION 13
6.3 HABITAT REQUIREMENTS 13
6.4 ENDPOINTS 14
6.4.1 GROWTH MEASUREMENT 15
6.4.2 WEIGHT MEASURES 16
6.4.3 MORTALITY 20
6.4.4 RESPIRATION RATE 21
6.4.5 PHOTOSYNTHESIS 21
7.0 INTERFERENCES 24
8.0 HEALTH AND SAFETY 24
8.1 GENERAL PRECAUTIONS 24
8.2 SAFETY EQUIPMENT 25
8.3 GENERAL LABORATORY OPERATION 25
9.0 QUALITY ASSURANCE 26
9.1 INTRODUCTION 26
9.2 FACILITIES AND EQUIPMENT 26
9.3 TEST ORGANISMS 28
9.4 CULTURE AND DILUTION WATER 32
9.5 TEST SUBSTANCE HANDLING 32
9.6 TEST CONDITIONS 32
9.7 ANALYTICAL METHODS 33
ii
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TABLE OF CONTENTS (continued)
9.8 CALIBRATION AND STANDARDIZATION 33
9.9 ACCEPTABILITY OF TOXICITY TEST RESULTS 33
9.10 REFERENCE TOXICANTS 34
9.11 RECORD KEEPING 34
10.0 APPARATUS, EQUIPMENT, AND MATERIALS 35
10.1 FACILITIES 35
10.2 CONSTRUCTION MATERIALS 35
10.3 MATERIALS FOR CULTURING AND TESTING 35
10.4 TEST CONTAINERS 39
10.5 CLEANING 39
11.0 CONTROL AND DILUTION WATER 39
12.0 ORGANISM CULTURE PROCEDURES 40
12.1 INTRODUCTION 40
12.2 TEST INITIATION 40
12.3 SALINITY, TEMPERATURE, AND PHOTOPERIOD 41
12.4 RENEWAL OF CULTURE WATER 42
12.5 CULTURE RECORDS 42
13.0 TOXICITY TEST PROCEDURES 42
13.1 EXPERIMENTAL DESIGN 42
13.2 RANGE-FINDING TEST 43
13.3 DEFINITIVE TEST 43
14.0 DATA ANALYSIS 43
15.0 RESEARCH RECOMMENDATIONS 46
16.0 REFERENCES 48
APPENDIX A - Summary of Potamogeton p&ctinatus growth and
rhizome tip number from initial toxicity
tests.
APPENDIX B - Relative sensitivity of Potamogeton pectinatus
to selected toxic substances.
111
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1.0 INTRODUCTION
Submersed aquatic angiosperms occupy an important niche in
shallow water environments of lakes, rivers and marine coastal
communities. Economic as well as ecological value is derived from
their ability to provide food and/or habitat for numerous organisms
including many commercial and recreationally important species of
shellfish, finfish and waterfowl. These vascular plants reduce
wave action along shorelines to minimize erosion and flooding.
They also contribute substantially to water quality improvement by
providing oxygen and dissolved organic carbon, sequestering
nutrients and filtering sediments suspended in the water column.
Submersed aquatic angiosperms are acknowledged as key indicator
species (keystone species) of health and vitality of these
ecosystems. Existing populations are protected by legislation at
the federal and state level (Gorsuch et al., 1991).
Despite the importance of these plants, no standardized
toxicity test protocols have been developed for any of the
submersed aquatic angiosperms species. The availability of such a
test is critical since the goal of the ^Chesapeake Bay Basinwide
Toxics Reduction Strategy7' is to reduce input of toxic substances
to levels which do not result in toxic impact on living Chesapeake
Bay resources. Plant tests are particularly important for testing
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herbicide toxicity since this trophic group will be more sensitive
than animals (Solomon et al., 1995). This document describes a
standard operating procedure (SOP) for the sago pondweed,
Potamogeton pectinatus L. , one of the most widely distributed
submersed aquatic angiosperms in the Chesapeake Bay estuary. The
goal of this document is to provide a detailed procedure for
culturing and toxicity testing of sago pondweed in single or
multiple chemical water column laboratory sub-chronic tests. These
methods also have application for both effluent and ambient
toxicity tests if various physical, chemical and biological factors
are consistent among control and test conditions. Additional
research is needed however, in areas dealing with physical,
chemical and biological factors (e.g., nutrient standardization
within effluents or among ambient locations) before these protocols
can be developed specifically for these type of tests (see Section
15).
2.0 CLASSIFICATION AND DISTRIBUTION
2.1 Classification of Aquatic Plants
Considerable confusion exists in the scientific literature
over the classification of submersed aquatic plants because the
three most commonly used inclusive terms: submersed aquatic plant,
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submersed aquatic vegetation, and submersed aquatic macrophyte, are
botanically imprecise (Cook et al., 1974; Fassett, 1960; Godfrey
and Wooten, 1974; Godfrey and Wooten, 1981). The descriptor
submersed aquatic has been widely interpreted as applicable to
several types of plants ranging from species adapted to having at
least some parts consistently immersed in standing water, those
which are capable of survival when they are completely immersed for
extended periods and those which complete their life cycle
completely submersed. The nouns plant, macrophyte and vegetation
are specific references to members of the plant kingdom. These
terms are frequently used to include any large underwater
photosynthetic organism; hence, numerous species of algae are often
included in these groups.
The term which best describes the group of underwater plants
which are critical to the ecology of aquatic environments like the
Chesapeake Bay is submersed aquatic angiosperms. These plants,
listed in Table 1, are native herbaceous perennial or annual
flowering plants. Those listed as annuals are weak perennials which
survive as annuals over much of their range, including the
Chesapeake Bay. These populations grow yearly from seeds beginning
in the late winter early spring. Some, like Zannichellia
painstris, horned pondweed, complete vegetative growth, flower, set
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Table 1. Dominant Submersed Aquatic Angiosperms of the Chesapeake Bay
Common
Name
Common
Waterweed (M)
Coontail or
Hornwort (D)
Curly pondweed
(M)
Eelgrass (M)
Eurasian
watermilfoil (D)
Horned
pondweed (M)
Hydrilla (M)
Naiads
(M)
Redhead grass
(M)
Sago pondweed
(M)
Slender
pondweed (M)
Water stargrass
(M)
Widgeon grass
(M)
Wild celery (M)
Family
Hydorcharitacea
Ceratophyllacaea
Potamogetonaceae
Zosteraceae
Haloragaceae
Zannichelliaceae
Hydorcharitaceae
Najadaceae
Potamogetonaceae
Potamogetonaceae
Potamogetonaceae
Pontederiaceae
Ruppiaceae
Hydorcharitaceae
Scientific
Name
Elodea canadensis
Ceratophyllum
demersum
Potamogeton crispus
Zostera marina
Myriophyllum
spicatum
Zannichellia palustris
Hydrilla verticillata
A/a/as spp.
Potamogeton
perfoliatus
Potamogeton
pectinatus
Potamogeton
pusillus
Heteranthera dubia
Ruppia maritima
Vallisneria
americana
Flowering
Time
Summer
Mid to late
summer
Lspring/
Early summer
Spring
Late summer
Early spring
Mid-summer to
fall
Summer
Early to mid-
summer
Early summer
Late summer
Summer
Late summer
Late summer
Overwintering
Structure
Stem and fine
stolons
Stem tips
Vegetative
buds
Slender rhizome
Roots and lower
stems
Rhizome
Tubers and turions
Roots
Resting buds on
rhizomes
Turions
Winter buds on
stems
Seeds, stems or
stem tips
Root-rhizome
Tubers
Salinity Range
Fresh
Water
Fresh
Water
Fresh to
Slightly
brackish
High
Fresh to
moderately
brackish
Fresh to
moderately
brackish
Fresh or 6-9
ppt
Fresh to
slightly
brackish
Fresh to
moderately
brackish
Fresh to
moderately
brackish
Fresh to
slightly
brackish
Fresh water
Wide range of
tolerance
Fresh to
moderately
brackish
M = Moncotyledonne
D = Dicotyledonae
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seed and die by early summer. Others, like Heteranthera dubia,
Water stargrass, also begin growth in the spring but will flower
more or less continuously from mid-summer to early fall. Hardy
perennial species may arise from seeds deposited over previous
years or from overwintering vegetative propagules. These plants
grow vegetatively from rhizomes, horizontal underground stems,
which continue to produce new rhizomes and photosynthetic shoots as
long as their ambient environment is favorable for growth. If
detached from the parent plant, these rhizome fragments will root
if they come in contact with appropriate sediments and thus
establish additional colonies of plants. Alternatively, if
conditions become temporarily unfavorable to growth, these
perennials may exhibit cycles of dieback and regrowth, where the
existing photosynthetic stems die and abscise during the period of
stress and are replaced with new photosynthetic stems as conditions
again become favorable. When plants reach maturity, whether or not
any dieback has occurred, resource allocation shifts to sexual
reproduction.
For annual plants, flowering and seed production mark the end
the life cycle even though plants may continue to grow vegetatively
while environmental conditions are favorable. In contrast, the
hardy perennials continue vegetative growth after flowering and
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then begin forming their overwintering vegetative structures
(Table 1). These structures, which are buried in the sediments,
contain meristematic tissue and abundant carbohydrates. The
former is the source of the next year's plant while the latter is
used for maintenance and supports the initial growth the following
season. However, even plants such as the exotic Hydrilla
verticillata which produce conspicuous overwintering structures
functionally persist as annuals if conditions are not conducive to
the formation and storage of abundant carbohydrates in their
tubers.
2.2 Distribution of Submersed Aquatic Angiosperms
Submersed aquatic angiosperms are found worldwide in diverse
shallow water habitats. Their presence in these habitats is
influenced by a number of environmental factors which include water
clarity, chemistry, physical/chemical sediment composition and
habitat energetics. Where conditions are otherwise favorable for
submersed aquatic angiosperms, salinity has proven to be a useful
indicator of the general distribution of individual species. Some,
like Thallasia testutudinum, Haladula wrightii or Zostera marina,
are obligate halophytes requiring salinities comparable to ocean
water (35 ppt). These plants are often classified as seagrasses to
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distinguish them from those occupying more freshwater environments.
One difficulty with the use of this term is the interpretation of
brackish halophytes such as Ruppia maritima or Potamogeton
perfoliatus which are frequent in the higher salinity regions of
estuaries, but may also be found in high or low salinity
environments. Still, seagrass is a useful term to distinguish the
obligate halophytes from those species which primarily occur in
freshwater. The general distribution of Chesapeake Bay submersed
aquatic angiosperms according to their general salinity preferences
are listed in Table 1.
3.0 LIFE CYCLE OF Potamogeton pectinatus
Potamogeton pectinatus L., sago pondweed, was selected as the
experimental organism for developing a standardized toxicity
testing protocol for the following reasons: (1) ecological
significance to the Chesapeake Bay, (2) feasibility of year round
culturing and testing, and (3) sensitivity to toxic chemicals
(specifically herbicides) (Ziegenfuss and Hall, 1993) . The life
cycle and habitat requirements of sago pondweed were found to be
most representative of the 14 other species of submersed aquatic
angiosperms resident in the Chesapeake Bay and its tributaries.
Sago pondweed is a hardy perennial which produces a discreet
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overwintering structure called a turion. It is naturally
propagated vegetatively by turions, by division of the rhizome
system, and sexually by seeds (Figure 1). In Maryland, as well as
other areas of Chesapeake Bay, P. pectinatus has a long growing
season, often appearing in late April and persisting until mid to
late October. Height of the plants is determined by nutrient
availability and water depth. It is one of the most tolerant
species of submersed aquatic angiosperms to seasonal salinity
changes in the range of 0-22 ppt; however, it is most abundant in
water having salinities of 0 to 6 ppt. The wide salinity tolerance
range, which represents most estuarine areas in northern Chesapeake
Bay, is a positive feature for selecting this test species.
Like many species of submersed aquatic angiosperms, the only
exception to life underwater occurs during flowering. Flowers,
technically inflorescences or clusters of single flowers, are
formed and develop beneath the water surface. Upon maturity most of
these flowers are elevated 1-4 cm above the water surface,
presumably to facilitate pollination by wave action, wind, and
insects. Most flowering occurs in late summer and fall, a time
when flower predation by waterfowl is common.
Once seeds are set, they ripen quickly and detach from the
parent plant. Although seeds may float for a short time, most sink
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Figure 1. Life Cycle of Potamogeton pectinatus, sago pondweed
Flower
Inflorescence
Photosynthetic stems
Turion
Leaves
Winter
Spring
Summer
Fall
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quickly and are therefore deposited near the parent colony. The
efficiency of seed dispersal is speculative; however, some long
distance spread by waterfowl or current is thought to occur.
Length of seed viability is unknown. Since seed formation
constitutes the major mechanism for imparting genetic variability,
sexual reproductive success is of substantial interest.
Unfortunately, almost no information is available concerning this
important aspect of submersed aquatic angiosperms life cycles.
4.0 TAXONOMY
Although there has been considerable disagreement in the past
among systematists over the taxonomy of submersed species of
angiosperms including P. pectinatus, the family designations
appearing in Table 1 are now widely accepted. As with all higher
plants, the placement of a species within a family is based on
floral structures, while genus and species are distinguished
largely by differences in vegetative attributes (Brown and Brown,
1984; Pieterse, 1985a,b).
5.0 TERMINOLOGY
Standard measures of chronic toxicity usually include
mortality and sublethal effects such as growth and reproduction,
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with exposures spanning at least a tenth of the life span of a
species. The 28-d exposures that are used for sago pondweed are
classified as sub-chronic in duration. It is appropriate, however,
to derive a chronic toxicity value with a 28-d test by using the
standard method of calculating the geometric mean of the No
Observed Effect Concentration (NOEC) and the Lowest Observed Effect
Concentration (LOEC). The measurement endpoints used in this test
are wet weight, dry weight, rhizome buds and photosynthetic stems
quantity.
6.0 SUMMARY OF TEST PROCEDURES
Separate groups of individual sago pondweed transplants (each
with one rhizome tip and two shoots) are exposed to various
concentrations of test solutions for 28 d. Control treatments are
used as a measure of acceptability of the test by providing
information about the quality of test plants, dilution water and
the suitability of test conditions (nutrients, light regime, etc.).
Control water consists of nutrient enriched distilled water
adjusted to a desired salinity matching the test solution. Test
treatments consist of a series of treatment conditions (contaminant
concentrations) in geometric progression.
Various endpoints are evaluated statistically for significant
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differences among control and treatment groups. Wet and dry weight
endpoints are used to evaluate plant growth while photosynthetic
stems quantity and total rhizome buds per plant are measured as
more qualitative indicators of growth or stress. The important and
unique contributions of submersed aquatic angiosperms to shallow
water habitats require their inclusion in mandates designed to
evaluate the effects of toxics on resident biota. However, there
are various differences between plant and animal bioassays which
must be considered in the design of experimental protocols (see
sections below).
6.1 Culture System
In natural systems, submersed aquatic angiosperms are primary
producers and support rich and diverse populations of microscopic
heterotrophs. They also serve as a substrate for numerous species
of phototrophs. In closed systems, these communities of organisms
often undergo population explosion, thereby competing with
submersed aquatic angiosperms for light, inorganic carbon, and
nutrients. They may also influence the exposure of submersed
aquatic angiosperms to test contaminants by serving as physical
barriers or as metabolic sinks for the compounds being evaluated in
the bioassay. Since these organisms may significantly affect
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submersed aquatic angiosperms endpoints both directly or
indirectly, their presence must be avoided for accurate assessment
of toxic effects. This requires the establishment and maintenance
of axenic culture systems (free of other species) for the bioassay.
6.2 Nutrition
Autotrophs, unlike animals, do not participate in feeding
relationships but rather meet their nutritional needs by absorbing
simple organic compounds from their immediate environments. These
nutrients are then used in assimilatory pathways using an external
energy source. In laboratory tests designed to evaluate the
toxicity of specific chemicals or chemical combinations on plants,
both nutrient concentrations and light are easily standardized when
using replicate treatments by using prepared media formulations,
calibrated sources of photosynthetically active radiation (PAR)
using common photoperiods, and uniform aeration with ambient air or
ambient air enriched with the same concentrations of carbon
dioxide.
6.3 Habitat Requirements
A number of animal bioassays have been developed to assess
particular components of aquatic habitats, sediment, water, and the
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surface microlayer. Organisms, or that part of an organism's life
cycle selected for these studies, usually have an obligate
requirement for the specific habitat feature. For example, the
coot clam embryo larval test is conducted in the water column
because early development of this species occurs in the water
column in approximately 6-8 days (Hall et al., 1994). Testing of
older life stages of coot clam juveniles occurs in sediment since
this is the media where this life stage is resident (Burgess and
Morrison, 1994).
In contrast, submersed aquatic angiosperms typically live as
a continuum which interconnects each of these habitat components.
These features, like nutrient levels, are easily standardized in
laboratory tests of specific chemicals by using artificial
sediments and prepared water column solutions with a uniform light
source and calibrated carbon dioxide delivery system.
6.4 Endpoints
The two types of endpoints used in aquatic toxicology and
ecological risk assessment are assessment endpoints and measurement
endpoints. Assessment endpoints are explicit expressions of actual
environmental values for protection (e.g., fish populations).
Measurement endpoints are measurable responses to a stressor (EC50
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and LC50s). Growth, mortality, reproduction, and respiratory rates
are common measurement endpoints used to assess the effects of
toxics in animal bioassays. Some of these endpoints, particularly
vegetative endpoints, are also used as measures of effect in plant
bioassays. However, some unique sources of error exist which must
be considered. In addition, rate of photosynthesis as measured by
either O2 evolution or CO2 uptake are common endpoints used for
measuring the effects of toxic compounds on phototrophs like
submersed aquatic angiosperms and aquatic algae.
6.4.1 Growth Measurement
Unlike animals, where growth is distributed over the entire
organism, cell division and the growth regions of herbaceous
perennial plants are isolated in tissues called meristems, which
occur at the tips of roots and stems. Throughout the growing
season, these meristems produce new cells which enlarge and then
differentiate into additional roots, rhizomes, photosynthetic
shoots and eventually flowers and overwintering structures. As new
plant structures are produced, older parts senesce and die. The
sequence and rate of death of these parts are determined by the
particular structure, age of the structure, and the degree of plant
stress. Leaves and roots usually experience higher rates of
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turnover than rhizomes and turions, which are most persistent. In
addition, at the end of each growing season, the photosynthetic
shoots normally die, leaving rhizomes or turions to overwinter.
Thus, the loss of parts, and their replacement and dormancy are
survival features of submersed aquatic angiosperms which are not
found in bioassays employing higher animals. These normal features
of submersed aquatic angiosperms life cycles and their response to
environmental stress must be considered when establishing and
interpreting endpoints in plant bioassays.
Growth measurements are made at the end of the 28 d test by
removing plants from their culture vessels and placing them in
shallow light colored, preferably white, dissecting pans filled
with 1-2 inches of tap water (Figure 2) . The number of
photosynthetic stems and rhizome buds are then counted and
recorded. The former measure is an excellent indication of vigor
while the latter is a useful measure of vegetative reproductive
capacity. The process of counting also helps remove components of
the artificial sediment in preparation for dry weight
determination.
6.4.2 Weight Measures
Reductions in wet and dry weight are frequently used as
measures of stress in plant bioassays. Application of these
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Figure 2. Individual Test Chambers
Manifold assembly
Tubing
Metal lid
Glass tube
P. pectinatus-
2 oz autoclavabl
glass jar
Autoclavabie foam plug
1 quart Mason jar
750 ml Bioassay Culture medium
(Table 2)
12 inch sand
Artificial substrate
(Table 2)
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parameters for toxicity tests employing submersed aquatic
angiosperms are potentially complicated in two ways. First, since
these plants have parts which naturally senesce and die but persist
in the living plant, endpoint measures of weight will include mass
of both living and dead tissue. Further, submersed aquatic
angiosperms which are exposed to lethal concentrations of a
contaminant may not die as quickly as animals, and they may even
experience some significant in vitro growth prior to death. Thus,
these biomass measurements may show increases even though the test
plant is dead at the end of the test. In both cases, the
complication can be avoided by normalizing all endpoints relative
to the control groups.
A second unique feature of weight measurements as endpoints
for assessing the effects of toxics on submersed aquatic
angiosperms is their ability to become dormant under stress
conditions. Dormancy as a stress response is distinct from the
complication of varying amounts of dead tissue attached to living
plants, although the persistence of dead tissue is a factor.
Submersed aquatic angiosperms exposed to a sublethal stress may
experience a dieback whereby the photosynthetic stems extending
into the water column turn brown, die, and then often detach from
the subterranean rhizomes. The rhizomes remain alive but may not
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resume growth or continue to form photosynthetic shoots. Often,
growth will only resume after the removal of the stress factor.
However, this new growth may be vigorous depending upon the length
of dormancy and the availability of food reserves. Thus, these
plants have the capacity to recover from stresses which result in
significant dieback of photosynthetic structures. The difficulty
lies in how the toxic effect is to be interpreted in light of
conflicting endpoints, i.e., low biomass but high survivorship.
Since multiple endpoints, which include growth measurements,
are recommended for evaluating the response of P. pectinatus to
contaminants, dry weights are determined following counts of
photosynthetic stems and rhizome buds (Section 6.4.1). If weight
measurements are the only parameter taken, plants are removed from
their culture vessels at the end of the test period and placed in
shallow pans of water. The artificial sediment is then removed by
gentle agitation and rinsing. The plants and detached parts are
then blotted with absorbent towels and placed in pre-weighed foil
pans for fresh weight determination. After weighing, the plants
are dried in their foil pans to a constant weight at 55C in a
drying oven. After 48 hours, plants are again weighed to determine
the dry weights of the samples.
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6.4.3 Mortality
Mortality is a viable endpoint for assessing toxic effects of
contaminants on submersed aquatic angiosperms. However, because
submersed aquatic angiosperms lack movement and have structures
embedded in sediment, mortality determinations can be difficult.
When plants are removed from sediment, loss of structural
integrity, turgor, and chlorophyll are key indicators of death.
Occasionally, microscopic examination of cells may be required,
especially for subsoil structures such as rhizomes, which are more
resistant to degradation. Cytoplasmic streaming and cell membrane
integrity are the parameters best suited for determining mortality
under these conditions.
Mortality determination is further complicated by the
submersed aquatic angiosperms's natural tendency to retain dead
tissue when they are placed under stress conditions. For
chlorophyllous tissues, the quantity of dead tissue relative to
unstressed controls can be measured directly as chlorophyll/unit
weight, or estimated as percent green. Both measures have been used
in toxicity test protocols; however, the former is best interpreted
within the context of other endpoints such as growth or
photosynthesis, while the latter is best suited to more informal
range finding tests. Due to the various complicating factors
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described above, the use of mortality as an endpoint is not
recommended in this SOP.
6.4.4 Respiration
Respiration rate can be used as an endpoint for measuring
contaminant effects on submersed aquatic angiosperms by the same
techniques employed in animal bioassays (Mehrle and Mayer, 1985),
except oxygen consumption must be determined with plants held in
the dark to avoid the complication of oxygen production by
photosynthesis. The most common method used is to incubate plants
in opaque Winkler bottles for 2-3 hours prior to measuring oxygen
consumption with ion selective electrodes. Since respiration is a
short term endpoint, effects on respiration in sub-chronic tests
are adequately reflected by changes in biomass accumulation;
therefore, we do not recommend the use of this endpoint.
6.4.5 Photosynthesis
Change in the rate of photosynthesis is a commonly used
endpoint for measuring the effects of toxic chemicals on aquatic
plants. Two methods can be used to measure this rate: oxygen
production and CO2 consumption. Measures using oxygen consumption
must discriminate between oxygen evolution by photosynthesis and
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concurrent oxygen consumption by plant respiration. The methods
most frequently used for this analysis require incubation of the
plants in transparent (light) and opaque (dark) Winkler bottles.
Gross photosynthetic rate is determined by measuring O2 evolution
by submersed aquatic angiosperms incubated in the light and
subtracting the oxygen consumed by similar plants incubated in the
dark. Rates are normalized by time and either fresh or dry weight
of test plants.
For example:
Gross Photosynthesis Determination
Test conditions Incubation Plant O2 measured
Period weight gms ppm
Light bottle + SAA 1 hr 1.0 15
Dark bottle + SAA 1 hr 1.0 _2
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Rate = 13 ppm O2/hr/gm fresh weight
Net photosynthesis is calculated by comparing O2 production of
submersed aquatic angiosperms incubated in the light against a
blank (uninoculated control). Rates are normalized for incubation
period and differences in plant weight.
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Net Photosynthesis
Test conditions Incubation Plant O2 measured
Period weight gms ppm
Light bottle +SAA 1 hr 1.0 15 ppm
Blank bottle 1 hr 1.0 7 ppm
8 ppm
Net photosynthesis = 8 ppm O2/hr/gm fresh weight
The use of oxygen production as a measure of photosynthesis
has been criticized because of the chemical reactivity of oxygen
with abiotic culture components, the competition for O2 by biotic
contaminants such as bacteria, competition for O2 by other plant
metabolic pathways, and differences in the rate O2 is transported
from the plant to the incubation medium. Where there are concerns
over these sources of interference, CO2 consumption is used to
determine photosynthetic rate.
As with oxygen determination, plants are incubated in a closed
system free of ambient air contamination. Measurements of CO2 are
made in the light and normalized by incubation time and weight of
the test plants. Rate is expressed as CO2 fixed/unit time/unit
weight. This is a more sensitive measurement of photosynthetic
rate because of the lower reactivity of CO2 compared to O2 and the
23
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absence of competition for CO2 by heterotrophic biota. A source of
error does exist if submersed aquatic angiosperms are contaminated
with other phototrophs such as epiphytic algae. Due to the various
complicating factors mentioned above, the use of photosynthetic
endpoints are not recommended.
7.0 INTERFERENCES
Toxic substances may be introduced by contaminants in
substrates, dilution water, and testing apparatus. Adverse effects
of extreme temperature or pH ranges may mask the presence of toxic
substances. Pathogenic and/or epiphytic organisms in the dilution
water or test water also may affect test organism survival.
Inadvertent introduction of contaminants during water quality
measurement may confound test results. Artificial substrates may
sequester toxic substances and also affect test results.
8.0 HEALTH AND SAFETY
8.1 General Precautions
Conducting toxicity tests may involve differing levels of
risk. Personnel conducting tests protect themselves by taking all
safety precautions necessary to avoid inhalation or absorption of
toxic substances through the skin and to prevent asphyxiation due
24
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to lack of oxygen or presence of volatile noxious substances.
8.2 Safety Equipment
Personnel use safety equipment, as required, such as
disposable rubber gloves, lab coats and/or aprons, respirators, and
safety glasses. Laboratory safety equipment includes a proper
ventilation system, first aid kits, fire extinguishers, fire
blanket, and an emergency eye wash and shower unit.
8.3 General Laboratory Operation
Work with samples containing suspected toxic substances is
performed in compliance with accepted rules pertaining to the
handling of hazardous materials. Toxicity tests with volatile
compounds are conducted under a ventilation hood. Because the
chemical composition and toxicity of samples are usually poorly
understood, samples are considered potential health hazards and
exposure to them is minimized.
The laboratory is generally kept clean and orderly to
promote safety and reliable test results. Containers used in the
laboratory are always labeled to indicate their contents and
prevent contamination. Guidance on safe practices when conducting
toxicity tests is available from general industrial safety manuals
25
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including U.S. EPA (1977).
9.0 QUALITY ASSURANCE
9.1 Introduction
The following quality assurance (QA) section is adapted from
U.S. EPA (199la). Quality assurance practices for conducting
toxicity tests with sago pondweed should address all aspects that
affect the integrity of the final data, such as: (1) contaminant
handling and storage; (2) quality of dilution water; (3)
condition of test plants; (4) condition and operation of
laboratory equipment; (5) test conditions; (6) instrument
calibration; (7) replication; (8) use of reference toxicants; (9)
recording data and observations; and (10) data evaluation. For
more information on quality assurance and good laboratory practices
related to toxicity testing see: FDA (1978), U.S. EPA (1975,
1979a, 1980a, 1980b, 1991b), Dewoskin (1984), and Taylor (1987).
9.2 Facilities and Equipment
Separate culture and toxicity test areas are necessary to
avoid possible cross contamination which could result in the loss
of cultures. The laboratory should be equipped with a ventilation
system to prevent recirculation of contaminated air from testing
26
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areas, sample preparation and storage areas, and chemical analysis
areas. In addition to space and ventilation requirements,
temperature control equipment must be capable of maintaining test
temperatures with minimal variation, programmable lighting is
required to simulate day-night conditions, and an oil-free
mechanical air supply is needed for both toxicity testing and
culture areas.
For these plant toxicity tests, supplemental air enriched
with C02 is regulated in two ways. CO2 is proportioned with ambient
air using a Visablend gas proportioner to provide a final 1-3% CO2
concentration at flow rates of approximately 2500 ml/hr to the
manifold system (Figure 3) . Gas delivery to individual culture
chambers (Figure 2) is adjusted manually by changing the depths of
the glass tubes to equalize pressures.
9.3 Test Organisms
Test organisms must be identified to species. The organisms
used in toxicity testing experiments must appear healthy, vigorous,
and have low mortality in cultures, during holding, and in test
controls. Plants should be cultured at approximately the same
salinity (within 3 ppt) as the test salinity. All transplants in
a test should be the same life stage (one rhizome tip and two
27
-------
0)
"55
O
C/J
(0
O
,
.a
a>
CO
2
&
eo
I
C3)
LC
28
-------
shoots). In most instances, tests are initiated with tip plus two
shoots due to the ease of handling this life stage.
Axenic test organisms are initiated as clonal lines from
sterilized turions (Ailstock, 1986). Turions are rinsed in tap
water, exposed to 10% (v/v) solution of commercial bleach with 0.1%
(v/v) Triton X-100 (wetting agent) for 5 min and soaked in 10.0 g
I'1 of the fungicide Captan (active ingredient: (n-
trichloromethylthio)-4-cyclohexene-l,2 dicarboximide) for 24 h.
Subsequent work is performed in a laminar flow hood under sterile
conditions. Turions with an intact epidermis undamaged in the
previous treatments are again treated with 10% bleach solution for
5 min. After dissecting apical meristems (apices) from the turions
and removing their largest sheathing leaves, the apices are
sterilized in the bleach solution for 5 min and rinsed 3 times in
sterile distilled water. Depending on the plant sample, this
surface sterilization procedure is sometimes unable to eradicate
all bacteria, some of which appears to actually reside within the
plant tissue. These endophytic bacteria are isolated from
contaminated explants, (plants grown in culture tubes and
pretreated with antibiotics) and the Kirby-Bauer method is employed
to determine the bacterial sensitivity to various antibiotics
(Ailstock, 1986). A 12-h treatment of the dissected apices with
29
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10% (v/v) solution of the antibiotics nitrofurantoin, polymyxin B,
kanamycin, or novobiocin before their transfer to propagation
medium reduces the evident contamination to an acceptable level of
20%. When the explants are chopped in smaller pieces and cultured
on the basal medium enriched with sucrose, eradication of the
endophytic bacteria from the explants is confirmed by the absence
of explosive bacterial growth. These antibiotics have had no
observable effect on subsequent growth of the treated explants.
The axenic explants, plants consisting of a rhizome bud and
two photosynthetic stems, are then placed in 150-ml culture tubes
with 20 ml of the basal medium of Murashige Shoot Multiplication
Medium B, (Huang and Murashige, 1976) which is supplemented with 10
g I"1 sucrose to serve as the standard propagation medium (Table 2).
The medium pH is adjusted to 5.0 with 1 N HCL and 0.1 N NaOH prior
to autoclaving at 18 psi for 20 min. The cultures are maintained
in growth chambers at 20°C under constant illumination with cool
white fluorescent light at 70 ^mol m^s"1.
For the purpose of mass propagation to increase plant stock,
sterile rhizome fragments including at least one rhizome are
routinely transferred to sterile 1-1 autoclavable jars (Kerr Glass
Mfg. Corp., Sand Springs, OK) containing 500 ml of propagation
medium. Explants consisting of a single rhizome bud with two
30
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Table 2. Composition of media.
Murashiges Shoot
Multiplication Medium B
Bioassay Culture Media
Components
NH4N03
KN03
CaCl2 (anhydrous)
MgS04 (anhydrous)
KH2P04
FeNaEDTA
H3BO3
MnS04.H20
ZnS04. 7H20
KI
Na2MoO4.2H20
CuS04. 5H20
CoCl2.6H20
NaH2P04. H20
Adenine sulfate
IAA
ilnositol
Kinetin
Thiamine HCL
Sucrose
PH
mg/litre
1650.00
1900.00
333.00
181.00
170.00
36.700
6.20
16.90
8.60
0.830
0.250
0.025
0.025
170.00
80.00
2.00
100.00
2.00
0.400
10,000
5.0
H2O column
synthetic freshwater solution
Components mg/litre
NaHCO3 96.00
CaS04.2H20 60.00
MgSO4 60.00
KC1 4.00
Substrate Murashiges Minimal
Organic Medium
NH4N03
KN03
CaCl2 (anhydrous)
MgSO4 (anhydrous)
KH2P04
FeNaEDTA
H3BO3
MnS04.H20
ZnSO4.7H20
KI
Na2Mo04.2H20
CuS04. 5H20
CoCl2.6H20
ilnositol
Thiamine HCL
Agar
PH
1650.00
1900.00
333.00
181.00
170.00
36.700
6.20
16.90
8. 60/4
0.830
0.250
0.025
0.025
100.00
0.400
6000.00
5.6
Aeration
Ambient air enriched to a final concentration of 3% CO2 is
delivered thru a sponge stoppered lid via a 50 ,wl glass
microcapillary pipet attached to microtubing. Enriched air is
mixed using a Visablend Gas proportioner then delivered thru a
flow meter at a rate of 2500 ml/hour.
31
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subtending photosynthetic stems are placed in 25 mm X 150 mm
culture tubes containing 25 ml of propagation medium. After 4-5
wk, cultures are visually inspected for uniformity, with the few
(5-10%) showing exceptional growth then discarded. The remaining
cultures weighing between 1.0 and 2.0 g are used as experimental
material for evaluating toxic effects of test contaminants in the
bioassay protocol described in Section 13.0.
9.4 Culture and Dilution Water
Water used for culturing and testing purposes should be from
the same source. The water should be tested for chemical
contaminants (metals and organics) at least once per year.
Distilled water is recommended for these tests.
9.5 Test Substance Handling
Procedures for handling and storage should conform to the
conditions described in Section 8.
9.6 Test Conditions
The temperature of the test solution should be measured by
placing a thermometer or probe directly into the test solution or
a surrogate beaker containing the same volume of solution as the
32
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test beaker. Dissolved oxygen concentration, pH, and salinity must
be measured in the actual test solutions. Test condition
parameters should be measured at least initially, at day 14 and at
the end of the 28 d exposure duration (see Table 3, Section 13).
Periodic measure of water quality conditions during the test are
encouraged but the use of clean methods is essential to avoid
contamination.
9.7 Analytical Methods
All routine chemical and physical analysis (T, DO, Sal, pH,
etc.) of culture and dilution water and test solutions are
performed as outlined in U.S.EPA (1979a, b). Reagent containers,
chemical stock solutions, and working solutions are dated to assure
that the shelf life is not exceeded.
9.8 Calibration and Standardization
Instruments used for routine chemical and physical parameter
measurements are calibrated prior to use according to the
instrument manufacturer's procedures.
9.9 Acceptability of Toxicity Test Results
Mean control growth after 28 d at the optimum salinity for
33
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sago pondweed should be at least four to five times the initial
weight on day zero (Appendix A). For this reason, 24 h continuous
light is recommended to obtain such growth within 28 d. Typical
numbers are 20-25 mean numbers of rhizome buds and 40-50 mean
numbers of photosynthetic stems. Within test comparisons of
control and treatment conditions are used to determine statistical
differences.
9.10 Reference Toxicants
Reference toxicants are used to establish the validity of
toxicity data generated from toxicity tests. The reference
toxicants provide information regarding the relative health and
sensitivity of the plants used in toxicity testing. Presently no
generally accepted reference toxicant chemical exists for plant
toxicity tests. A readily available herbicide such as atrazine may
prove to be a logical choice as a reference toxicant chemical for
plant studies.
9.11 Record Keeping
Proper record keeping is very important. Bound notebooks are
used to maintain detailed records of culture maintenance, equipment
maintenance and calibration, receipt and storage of contaminants,
34
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test conditions employed, and results. Annotations are made in ink
to prevent the loss of information. All data from the toxicity
tests are kept in either bound notebooks or bioassay data sheets
(Tables 3 and 4).
10.0 APPARATUS, EQUIPMENT. AND MATERIALS
10.1 Facilities
The bioassay laboratory should consist of separate and defined
toxicity testing and organism culture areas. The laboratory should
be equipped with an oil-free air supply with CO2 enrichment (see
Section 9.2), programmable lighting for day-night simulation, and
controlled temperature. A water treatment system, such as
Millipore Milli-Q, Super-Q, or equivalent, is required to deliver
contaminant-free freshwater. Water supply lines should be
constructed from PVC or other non-toxic plastic.
10.2 Construction Materials
Glass is used for construction of equipment that comes in
contact with the plant species.
10.3 Materials For Culturing and Testing
• Refractometer, pH meter, dissolved oxygen meter, and
35
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Table 3. Sample data sheet for recording sago pondweed water
quality parameters.
SAGO PONDWEED SUB-CHRONIC TEST WATER QUALITY PARAMETERS
Test I.D. : Date:
Contaminant: Date test began:
Concentr at ion
(ag/L)
Salinity
(ppt)
Temperature
(C)
PH
Dissolved O2
(mg/L)
36
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Table 4. Sample data sheet for recording sago pondweed endpoint
measurements.
Treatment
A
B
Sample
1
2
3
4
5
6
7
8
9
10
1
2
3
4
5
6
7
8
9
10
Photosynthe t ic
Stems
Rhizome
Buds
Wet Wt.
Final
Dry Wt.
Final
37
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thermometer are required for measuring routine physical
and chemical parameters in culture water and test
solutions.
• Light meter capable of reading photon flux density for
Photosynthetically Active Radiation (PAR) (umol/m2/sec).
• Adjustable pipetts, 0.2, 1.0, and S.OmL with disposable
tips are used for mixing test solutions.
• Waterproof photostable markers are used for labeling
containers in the laboratory.
• Synthetic sea salts (HW MarineMix, Hawaiian Marine
Imports, Inc., Houston, TX) are required for salinity
adjustments of culture water and test solutions.
• Reagents needed for routine physical and chemical water
quality parameters include pH calibration buffers (4, 7,
and 10) and electrode filling and storage solutions,
dissolved oxygen probe membranes and filling solution,
and salinity standards for refractometer calibration.
• Laboratory glassware required for preparation of
standard, chemical stocks, test solutions, and dilutions
include beakers (150 mL - 2.0 L) , volumetric flasks, and
graduated cylinders.
• Manifold assembly as in Figure 3.
38
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Laminar flow hoods, autoclave, filtration capable of 0.02
10.4 Test Chambers
Chambers used for testing sago pondweed are one L autoclavable
glass jars (quart Mason jars) which contain the plants rooted on
artificial sediment (Figure 2). Air is delivered by inserting a
glass tube through a hole in the metal lids which are sealed from
ambient air by autoclavable foam plugs. Individual test chambers
are then connected to the manifold assembly as shown in Figure 3.
10.5 Cleaning
All glassware used to prepare stock solutions, test
solutions, and contain organisms during toxicity tests, are cleaned
before use according to procedures outlined by the U.S. EPA (1985)
and ASTM (1980). Glassware is washed with detergent and rinsed
with tap water, 10% nitric or hydrochloric acid, deionized water,
and pesticide-free acetone, followed by a minimum of three rinses
with deionized water.
11.0 CONTROL AND DILUTION WATER
Deionized or distilled water is used as the source of control
and dilution water. The salinity can be adjusted with a good-
quality commercial seasalt such as HW MarineMix (Hawaiian Marine
39
-------
Imports, Inc., Houston, TX). Salinity adjustments are necessary
for determining the effect of salinity on single or multiple
chemicals.
12.0 ORGANISM CULTURE PROCEDURES
12.1 Introduction
Techniques for establishing and propagating axenic clonal
lines of Potamogeton pectinatus have been described by Ailstock
(1986), Ailstock et al. (1991), Fleming et al. (1988, 1994), and in
Section 9.3 of this document. Turions used for establishing the
working lines of test plants can be harvested from local
populations in the fall after flowering, when plants begin their
winter die back. Generally, 6-10 turions from each population are
sufficient to establish axenic cultures using the method detailed.
The advantage of this approach is that local ecotypes are used for
contaminant testing. Alternatively, if the use of local ecotypes
is unimportant, established axenic clonal lines may be available
from other groups.
12.2 Test Initiation
Axenic plants weighing between 1.0 and 1.5 grams grown in 25
X 150 mm tubes containing 25 ml of the propagation medium are used
40
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for conducting all toxicity tests (Table 2). Generally, there are
10-15 photosynthetic shoots and an average of 3-5 rhizome buds for
1.0 to 1.5 grams of plant. These plants are transferred to the
bioassay medium listed in Table 2. All media and glassware are
sterilized by autoclaving at 260°F at 15 psi. All subsequent plant
manipulations are carried out using sterile technique in a laminar
flow hood.
Roots and rhizomes are pressed into the agar substrate
contained in the 2 oz specimen jars. The roots and rhizomes are
then covered with % inch of sterile sand which holds the plants in
place as they are immersed in 750 ml of the water column solution
(Figure 2). The cultures are aerated with ambient air or ambient
air supplemented with CO2 using a manifold system shown in Figure
3. Test concentrations of contaminants can be added immediately or
anytime up to 72 h after culture initiation.
12.3 Temperature, Photoperiod and Salinity
Plant cultures used for toxicity tests are grown for a minimum
of 4 weeks at 20-23°C under full spectrum florescent lighting
providing about 70 /umol/m2/s PAR. Photoperiod is largely a matter
of choice; however, a cycle of 24 h light has consistently yielded
excellent results, producing 4-5 fold increase in biomass of
41
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controls within 4 weeks. Salinity can be increased by the addition
of a salt mixture as previously discussed. P. pectinatus, while
tolerant of salinities of 12 ppt, grows best in the freshwater or
low salinity bioassay medium. Results from preliminary salinity
experiments show enhanced growth for sago pondweed at 1 and 6 ppt
when compared to 12 ppt.
12.4 Renewal of Culture Water
Plants begin utilizing the nutrients listed as bioassay media
components when cultures are first established (Table 2). Over the
28 d test period, no observable loss of vigor has been observed in
control plants. Hence, media renewal to replace nutrients in tests
of this duration is probably not necessary.
12.5 Culture Records
Details on the culture history and daily culture maintenance
operations are recorded in a laboratory notebook.
13.0 TOXICITY TEST PROCEDURES
13.1 Experimental Design
Sago pondweed bioassays are initiated with plants weighing
between 1.0 and 1.5 grams (=4-5 weeks old). A total of 10 plants
42
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(one per 1 L chamber) are tested at each test condition and control
in static tests for a 28 d period. A summary of test conditions is
listed in Table 5.
13.2 Range-Finding Test
Contaminant concentrations for range-finding tests should span
four orders of magnitude (1, 10, 100, 1000 x/L) for single or
multiple chemicals. A 28-d test using 24 h continous light is
recommended for the range-finding test.
13.3 Definitive Test
Toxicity tests usually consist of one or several control
treatments and a series of toxicant dilutions (i.e., 100, 56, 32,
18, 10, and 5.6% of concentration causing a significant effect for
an endpoint). The dilution series is determined from the range-
finding test. Each dilution, except for the highest concentration
and the control, is at least 50% of the next higher one. The
control treatments use the same conditions, procedures, and plants
as are used with the contaminant treatments.
14.0 DATA ANALYSIS
The data are tabulated and summarized. The toxicity endpoints
43
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Table 5. Recommended Test Conditions For Sago Pondweed.
1. Temperaturea:
20-25C
2. Lighting:
3. Photoperiod:
70 yumol/m2/s PAR
24 h continuous light
4. Size of Test Vessel:
I L
5. Volume of Test Solution:
750 mL
6. Age of Test Plants:
4-5 weeks (=1-1.5 g)
7. No. of Plants per Test
Vessel:
8. No. of Replicates per
Concentration:
10
9. Feeding Regime:
Culture Media in Table 2
10. Aeration:
Yes, with CO2 enrichment
11. Dilution Water:
Distilled water
12. Test Duration:
13. Effect Measured:
28 d
Growth (wet and dry weight) ,
number of rhizome buds,
photosynthetic stems.
44
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are based on reductions in number of rhizome buds, number of
photosynthetic stems, dry weight, and wet weight at the end of the
test. Normality and homogeneity of variance is determined with the
Kolmogorov-Smirnov Test and Levene Median Test, respectively. If
data transformation is necessary to satisfy assumptions of ANOVA,
various data transformation techniques are available. If the data
still do not satisfy assumptions of ANOVA after transformation, a
one way ANOVA on ranks is used (methods such as Dunnetts test).
Determination of the no-observed-effect-concentration (NOEC)
and the lowest-observed-effect-concentration (LOEC) for multi-
concentration tests is accomplished with hypothesis testing.
Endpoint data from each concentration are compared to the control
values using a one-tailed multiple comparisons method such as
Dunnett's Test. The NOEC is the lowest concentration that is not
significantly different than the control value. The LOEC is the
lowest concentration that is statistically different (p <0.05) than
the control value. A chronic value is determined by calculating
the geometric mean of the NOEC and LOEC values.
In a case where any treatment mean may have a value that is
greater than the control, a two-tailed multiple comparisons design
such as the Student-Newman-Keuls Method is used in addition to the
one-tailed test to determine if the contaminant may have a
45
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stimulatory effect. Standard 28 d EC50 values could also be
calculated using procedures described in U.S. EPA (1991a).
Computer programs for analyzing toxicity test data are
available by contacting: Western Ecosystems Technology, Inc., 1402
South Greeley Highway, Cheyenne, WY 82007-3031, telephone number
307-634-1756; and Jandel Scientific Software, P.O. Box 7005, San
Rafael, CA 94912-8920, telephone number 415-453-6700.
15.0 RESEARCH RECOMMENDATIONS
The following research recommendations are suggested to
improve the sago pondweed SOP:
• Standardization of physical, chemical and biological
conditions for ambient or effluent toxicity tests. This SOP
developed for single and multiple chemical water column
toxicity tests with sago pondweed provides a high degree of
experimental control over the physical, chemical, and
biological variables common to submersed aquatic angiosperms
habitats. Therefore, the relative toxicity of different
chemicals can be accurately determined. In order to use this
test for either ambient or effluent toxicity testing, research
is needed to determine how to standardized the various
46
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biological, physical and chemical (e.g., nutrients) conditions
found in ambient or effluent samples in order to determine the
effects of contaminants. These type studies should be
conducted with environmentally realistic exposures of multiple
chemicals (e. g. pesticides) found in the geographic area of
interest.
Development of a reference toxicant for plants such as
submersed aquatic angiosperms. Reference toxicants are used
to establish the validity of toxicity data generated from
toxicity tests. Reference toxicants provide information
regarding the relative health and sensitivity of species used
in toxicity tests. Reference toxicity tests are used
routinely with animals (e.g., cadmium chloride, copper
sulfate, sodium dodecyl sulfate). However, reference
toxicants have not been identified for plant toxicity tests
such as submersed aquatic angiosperms. These reference
toxicity tests should be developed for sago pondweed . One
possible reference toxicant candidate for sago pondweed is the
herbicide atrazine. Atrazine toxicity data are available for
this species (Hall et al., 1995, Appendix B).
47
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16.0 REFERENCES
Ailstock, M.S. 1996. Clonal propagation of Potamogeton pectinatus
in axenic culture. In; Proceedings of the Thirteenth Annual
Conference on Wetlands Restoration and Creation. F.J. Webb,
Jr. (ed.), May 15-16, 1986, Hillsborough Community College,
Plant City, FL.
Allan, S.J. and R.E. Daniels. 1982. Life table evaluation of
chronic exposure of Eurytemora affinis (Copepoda) to Kepone.
Mar. Biol, 66:179-184.
American Society for Testing and Materials (ASTM). 1980. Standard
Practice for Conducting Toxicity Tests with Fishes,
Macroinvertebrates and Amphibians. ASTM E 729-80,
Philadelphia, PA. 25 pp.
Bradley, B.P. 1975. The anomalous influence of salinity on
temperature tolerances of summer and water populations of the
copepod, Eurytemora affinis. Biol. Bull. 148:26-34.
Brown, M.L. and R.G. Brown. 1984. Herbaceous Plants of Maryland.
Port City Press, Baltimore, MD. 1127 pp.
Burgess, R.M. and G.E. Morrison. 1994. A shoot-exposure
sublethal, sediment toxicity test using the marine bivalve,
Mulinia lateralis: Statistical design and comparative
sensitivity. Environ. Tox. Chem. 13: 571-580.
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Bushong, S.J., L.W. Hall Jr., W.E. Johnson, W.S. Hall and M.C.
Ziegenfuss. 1987. Acute and chronic toxicity of tributyltin
to selected Chesapeake Bay fish and invertebrates. Final
Report, Johns Hopkins University Applied Physics Laboratory,
Shady Side, MD.
Chesapeake Executive Council. 1989. Chesapeake Bay Basin-wide
Toxics Reduction Strategy. Annapolis, MD.
Cook, C.D.K., B.J. Gut, E.M. Rix, J. Schneller and M. Seitz. 1974.
Water Plants of the World, Dr. W. Junk b.v., Publishers, the
Hague.
Correll, D.L. and T.L. Wu. 1982. Atrazine toxicity to submersed
vascular plants in simulated estuarine microcosms. Aquat.
Bot. 14:151-158.
Daniels, R.E. and J.D. Allan. 1981. Life table evaluation of
chronic exposure to a pesticide. Can. J. Fish. Aquat. Sci.
38:485-494.
DeWoskin, R.S. 1984. Good laboratory practice regulations:
comparison. Research Triangle Institute, Research Triangle
Park, NC. 63 pp.
Fassett, N.C. 1960. A Manual of Aquatic Plants. The University
of Wisconsin Press, Madison, WI.
Federal Drug Administration. 1978. Good laboratory practices for
49
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non-chemical laboratory studies. Part 58. Federal Register
43(247):60013-60020, December 22, 1978.
Finney, D.J. 1978. Statistical method in biological assay. 3rd
edition. Charles Griffin and Co. Ltd., London. 508 pp.
Fleming, W.J., M.S. Ailstock and J.J. Momot. 1993. Net
Photosynthesis and Respiration of Sago Pondweed (Potamogeton
pectinatus) Exposed to Herbicides. In: Third Symposium on
Environmental Toxicology and Risk Assessment: Aquatic, Plant,
and Terrestrial, ASTM STP. J. Hughes, G. Biddinger and E.
Mones (eds.), American Society of Testing amd Materials,
Philadelphia, PA. pp 1-14.
Fleming, W.J., M.S. Ailstock, J.J. Momot and C.M. Norman. 1991.
Response of Sago Pondweed, a Submerged Aquatic Macrophyte, to
Herbicides in Three Laboratory Culture Systems. In: Plants
for Toxicity Assessment: Second Volume, ASTM STP 1115. J.W.
Gorsuch, W.R. Lower, W. Wang and M.A. Lewis (eds.). American
Society for Testing and Materials, Philadelphia, PA. pp. 267-
275.
Fleming, W.J., J.J. Momot and M.S. Ailstock. 1988. Bioassay for
phytotoxicity of toxicants to sago pondweed. In:
Understanding the Estuary-Advances in Chesapeake Bay Research.
Proceedings of a Conference, March 29-31, 1988, Baltimore, MD.
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Chesapeake Research Consortium Publication 129. CBT/TRS
28/88.
Gaedke, V. 1990. Population dynamics of the calanoid copepods,
Euryteiaora affinis and Acartia tonsa in the Ems-Dollart
Estuary: A numerical simulation. Arch. Hydrobiol. 118:185-
226.
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56
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APPENDIX A
Summary of Potamogeton pectinatus control growth, photosynthetic
shoot number, and rhizome tip number from two 28 d studies in 1994.
Date
July 28
July 28
July 28
December 6
December 6
December 6
July 28
July 28
July 28
July 28
July 28
July 28
December 6
December 6
Light Salinity Endpoint
Cycle L:D (ppt)
24:0 1
24:0 6
24:0 12
16:8 1
16:8 6
16:8 12
24:0 1
24:0 6
24:0 12
24:0 1
24:0 6
24:0 12
16:8 1
16:8 6
Mean Rhizome Tips
Per Plant
Mean Rhizome Tips
Per Plant
Mean Rhizome Tips
Per Plant
Mean Rhizome Tips
Per Plant
Mean Rhizome Tips
Per Plant
Mean Rhizome Tips
Per Plant
Mean Photosynthetic
Shoots Per Plant
Mean Photosynthetic
Shoots Per Plant
Mean Photosynthetic
Shoots Per Plant
Mean Wet Weight
Growth
Mean Wet Weight
Growth
Mean Wet Weight
Growth
Mean Wet Weight
Growth
Mean Wet Weight
Results
27.4
25.3
24.0
16.0
19. O1
19. 51
56.7
41. 31
34. 61
10.3 g
8.7 g
5.01 g
3.5 g
2.8 g
Growth
A-l
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APPENDIX A (continued).
Date
December 6
July 28
July 28
July 28
December 6
December 6
December 6
December 6
December 6
December 6
Light
Cycle L:D
16:8
24:0
24:0
24:0
16:8
16:8
16:8
16:8
16:8
16:8
Salinity
(Ppt)
12
1
6
12
1
6
12
1
6
12
Endpoint
Mean Wet Weight
Growth
Mean Final Dry
Weight
Mean Final Dry
Weight
Mean Final Dry
Weight
Mean Final Dry
Weight
Mean Final Dry
Weight
Mean Final Dry
Weight
Mean Dry
Weight Growth
Mean Dry
Weight Growth
Mean Dry
Weight Growth
Results
2.2 g
0.931 g
0.899
0.7111
0.376
0.362
0.353
0.235
0.225
0.213
g
g
g
g
g
g
g
g
Significant difference (p<.05) from 1 ppt control with same
date and endpoint.
A-2
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APPENDIX B
Relative sensitivity of Potamogeton pectinatus to various
contaminants in previous studies.
Chemical
Duration Endpoint Results
Reference
Atrazine
Atrazine
Alachlor
5 h
IC501
29 ppb
21-42 d O2 produc- inhibited at
tion 650
4 wk
%Biomass
increase2
Atrazine
Glyphosate
Paraquat
Acifluorfen 3 h
Alachlor
Atrazine
Cyanazine
IC501
112 § .001 ppm
104 @ .01 ppm
76 @ .1 ppm
79 @ 1 ppm
54 @ 10 ppm
104 @ .001 ppm
103 @ .01 ppm
50 @ . 1 ppm
23 @ 1 ppm
95 @ .001 ppm
105 <§ .01 ppm
96 @ .1 ppm
97 @ 1 ppm
99 @ 10 ppm
154 @ .001 ppm
121 @ .01 ppm
31 @ .1 ppm
23 @ 1 ppm
22 @ 10 ppm
>10,000 ppb
>1,000;<10,000
PPb
29 ppb
32 ppb
Fleming et al.,
1988
Correll and Wu,
1982
Fleming et al.,
1991
Fleming et al.,
1993
B-l
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APPENDIX B (continued).
Chemical
Duration Endpoint Results
Reference
Glyphosate
Linuron
Paraquat
Metolachlor
Metribuzin
Simazine
2,4-D
3 h
IC501
>10,000 ppb
70 ppb
240 ppb
>10,000 ppb
8 ppb
164 ppb
>10,000 ppb
Fleming et al.,
1993
xNet photosynthesis inhibited by 50%.
2Biomass increase is expressed as percent increase in weight over
4 weeks with control increase equal to 100%.
B-2
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