EVALUATING EXPOSURE AND ECOLOGICAL EFFECTS WITH
TERRESTRIAL PLANTS
PROCEEDINGS OF A WORKSHOP FOR THE us EPA EXPOSURE ASSESSMENT GROUP
28 AUGUST 1991
US EPA REGION 10
1200 SIXTH AVENUE
SEATTLE, WASHINGTON
PREPARED BY
LAWRENCE A. KAPUSTKA & MlNOCHER REPORTER
ecological planning and toxicology, inc.
5010 SWHout Street
Corvallis, Oregon 97333-9540
WITH CONTRIBUTIONS FROM
MILTON GORDON
University Of Washington
JOHN FLETCHER
University Of Oklahoma
STEVE KLAINE
Clem son University
DON MILES
University Of Missouri
Project Officer: Maggie Wilson
Tetra Tech, Inc.
10306 Eaton Place, Suite 340
Fairfax, Virgjma 22030
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DISCLAIMER ;;;
PREFACE iv
WORKSHOP AGENDA "v
ACKNOWLEDGEMENTS vj
WHY CONSIDER PLANTS? 1
SIGNIFICANT RESOURCE '.'" 1
LEGAL PROVISIONS 1
BIOLOGICAL IMPORTANCE 4
ROUTE OF EXPOSURE FOR ANIMALS (HUMANS) 6
AMENABLE TO MEASUREMENT 6
ECOLOGICAL RISK ASSESSMENTS 8
GENERAL APPROACH 8
ACCESSIBILITY CONCERNS 9
ENDPOINTS OF INTEREST 11
DEFINITIONS and ECOLOGICAL HIERARCHICAL LEVELS 11
TOXICITY ' 13
Endpoints , 13
Surrogate Species 15
Exposure Conditions 16
METHODS AVAILABLE 17
ECOLOGICAL MEASUREMENTS 17
General , 17
Positioning the Plots 19
Habitat & Community Structure '. 21
Populations & Individuals 21
Remote Sensing Methods 22
Direct Observational. Methods 23
Defined Area Sampling Techniques 24
Plotless Sampling Techniques 27
Summary Comments On Vegetation Sampling 32
TOXICITY TESTS 32
Class-l Tests 33
Seed Germination/Seedling Emergence 33
Root Elongation 33
On-Site Germination Test 34
Life-Cycle ...:' 34
Floating and Rooted Aquatic Plant Growth Tests 34
Class-ll Tests 36
Photosynthesis: Gas Exchange s 37
Photosynthesis: Fluorescence 38
Peroxidase ) 40
Polyamines 43
Dinitrogen Fixation 43
Genetic Toxicology Assays.. 43
Cell Culture Assays , 45
Community Terrecosm 48
INTERPRETATION '. 50
BIOLOGICAL FACTORS 50
Interactive Plant-Microbial Associations 50
Bacteria 51
Mycorrhizas 52
Bioconcentration Factor 53
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Action of Stenle Soil 53
Action of Non-sterile Soil 54
The Role of Plant Purifying Aqueous Environments 54
Overall Uptake and Metabolism of Xenobiotics by Plants 55
Absorption 55
Metabolic alterations 56
Deposition of xenobiotics in cell wall 57
Fate of xenobiotic during senescence of plant tissue 57
Metabolism of Xenobiotics in Genetically Engineered Plants 57
Resistance to Heavy Metals .^ 58
Use of Plant As Indicator of Ionizing Radiation 58
STATISTICAL FACTORS 59
Precision/Accuracy/Uncertainty .' 59
Plant Interspecies Variability 59
Lab To Field Variability 60
Statistical Approaches To Ecological Assessment 61
Multivanate analysis 62
Time series analysis 62
Geostatistical analysis 63
Environmental sampling and study design 63
Summary Comments on Statistical Approaches 63
CONCLUDING REMARKS 64
General ' 64
WORKSHOP SUMMARY 65
APPENDIX I 69
VEGETATION SAMPLING METHODS. CALCULATIONS 69
Equations For Defined Area Sampling 69
Equations For Plotless Sampling Methods 71
Line Intercept 71
Point-Quarters 72
APPENDIX II 74
APPENDIX III 86
SPATIAL ANALYSIS 86
Retrospective Study 86
Scoping Study 86
WORKSHOP ATTENDEES 92
LITERATURE CITED 94
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DISCLAIMER
The information in this document was developed for the United States
Environmental Protection Agency by Contract Number 68-DO-0100 to Tetra
Tech, Fairfax, Virginia. It has been subject to the Agency's peer and
administrative review, and it has been approved for publication as an EPA
document. Mention of trade names or commercial products does not constitute
endorsement or recommendation for use.
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PREFACE
This document explores the use of plants as indicators of ecological condition.
Of specific interest is the incorporation of plant processes as indicators of
exposure or effects that can be linked to toxic conditions found at hazardous
waste sites. Although the emphasis is on terrestrial plants in the field, there is
much to be learned from studies of plant processes in other settings.
Accordingly, extension of knowledge of selected aquatic and wetland plant
systems, experimental work in laboratories ranging from whole plant through
molecular events, and measurements that demonstrate either exposure or
effects are considered.
The theme of this document was the focus of a workshop held 28 .August
1991 at the US EPA Region 10 office, Seattle, Washington. Three objectives
were pursued. First, to identify the usefulness and value of incorporating
plants in the assessment process used in Superfund. Second, to provide
information to guide users toward methods that might be appropriate for
specific sites. Third, to identify potential near-term research activities that
could expand the application of plant analysis for Superfund assessments. A
working draft of this document was provided to the workshop participants. All
were invited to submit review comments. This final product incorporates
comments developed during the workshop as well as written review comments.
IV
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WORKSHOP AGENDA
EVALUATING
TERRESTRIAL
08:15-08:30
08:30 - 08:45
28 AUGUST 1991
EXPOSURE AND ECOLOGICAL EFFECTS WITH
PLANTS
08:45 - 09:00
09:00-09:15
09:15-09:25
09:25- 10:00
10:00- 10:20
10:20- 10.55
10:55- 11:30
11.30- 11:50
11:50- 13:00
OPENING REMARKS.
Workshop Objectives.
OVERVIEW
Ecological Risk Assessments
Forensic Ecology
Superfund
STANDARD METHODS
Ecological
lexicological
SPECIAL TOPICS
Introduction
Aquatic Macrophytes.
BREAK
Tissue Culture.
Fluorescence
QUESTIONS/ANSWERS
LUNCH
Anne Sergeant
Maggie Wilson
.Larry Kapustka
Larry Kapustka
Mino Reporter
Mino Reporter
Steve Klame
John Fletcher
Don Miles
13:00- 13:35
13:35- 13:50
13:50- 14:45
14:45 • 15:00
15:00 • 16:30
16:30- 16:45
Metabolism & Other Features.
Rhizobiologv
OPEN DISCUSSION
Round Table.
BREAK
FUTURE DIRECTIONS
Round Table
WRAP-UP
Milt Gordon
Larry Kapustka
Panel & Audience
Panel & Audience
Maggie Wilson
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ACKNOWLEDGEMENTS
Information presented in this document comes from many sources. Much of
the background material was initially considered in the 1988 workshop and
proceedings Ecological Assessment of Hazardous Waste Sites: A Field and
Laboratory Reference {EPA/600/3-89/013). Additional background material has
been adapted from notes of oral presentations delivered by L. A. Kapustka at
three Annual Superfund Workshops sponsored by the US EPA Environmental
Response Team-Edison, New Jersey. Portions of this document were adapted
from several published reports, papers, and manuscripts including:
Kapustka, L.A. & M. Reporter, lin review). Terrestrial Primary Producers. Chapter 16.
in P. Calow led) Handbook of Ecotoxicotoey. Blackwell Press,
Kapusixa, L.A., G. Under, & M Shirazi. 1990. Quantifying effects in ecological site
assessments* biological and statistical considerations, in H. Lacayo, R.J. Nadeau,
G.P Patil, & L. Zaragoza (eds) Proceedings: Workshop on Superfund Hazardous
Waste: Statistics! issues in Characterizing a Site.
Kapustka, L.A. 1987. Interactions of Plants and nonpathogenic soil microorganisms, in
D.W Newman & K.G. Wilson (eds.) Models in Plant Physiology and Biochemistry,
Vol III. CRC Press.
Kapustka L A &BA Williams. 1991. The conceptual basis for assessing ecological
risk (ram incineration facilities. Presented at the 84the Air & Waste Management
Assoc meeting; Vancouver, B.C 16-21 June 1991. 91-132 2: 12pp.
Under, G & L A Kapustka On prep) The use of spatial statistics to organize and
evaluate ecological risk at Superfund sites.
Specific contributions were made by Dr. Steve Klaine (aquatic test methods and
peroxidase). Dr. John Fletcher (tissue culture), Dr. Don Miles (chlorophyll
fluorescence), and Dr. Milton Gordon (metabolic responses, metabolism,
complications, and potentials}.
VI
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I. WHY CONSIDER PLANTS?
A. SIGNIFICANT RESOURCE
There are multiple reasons to use plants in the evaluation of toxicity in
ecological settings. The goods and services provided by plants, though largely
taken for granted, touch virtually all realms of human interest. Plants are
conspicuous as the centerpiece of croplands, rangelands, and timberlands,
where the plant products are traded as commodities in the traditional
marketplace. In wetlands, parklands and other natural areas the monetary
worth, though not as well defined economically, is significant.
As the most prominent of primary producers, green plants form the foundation
of virtually all ecosystems. The photosynthetic process of plants (and a
restricted group of microbes) represents the only significant means of infusing
bioavailable energy into ecosystems. Ultimately, all animals, bacteria, and fungi
(and the plants themselves) rely on this energy source obtained from light.
In addition to this crucial role, plants contribute many other important
ecological functions. The physical structure of individual plants and groups of
plants define habitat for wildlife. The plant canopy and root system afford
protection against soil erosion. Finally, plants are intimately involved in soil
nutrient dynamics. Plants contribute the bulk of the organic matter that
significantly defines soil fertility. The many interactive processes among
plants, bacteria, and fungi in the rhizosphere govern the flow of nutrients.
Despite such obvious prominence, plants have been under-utilized in the
establishment of regulatory policy and in the evaluation of actual and potential
adverse consequences of human activities. This likely stems from our cultural
heritage; during our formative years most of us are sensitized to animals
(especially birds and mammals), but are instilled with little appreciation for
plants. In not seeing the value of plants, toxicology has missed opportunities
to protect and improve environmental conditions. Perhaps this situation is
changing.
B. LEGAL PROVISIONS
Ecotoxicity assessments are performed in four related but operationally distinct
situations. Ecological and toxicological information is critical in defining and
selecting goals and options for site remediation and restoration. Toxicity tests
are used to evaluate potential adverse effects of pesticides and other toxic
chemicals prior to registration. Tests are incorporated in waste discharge
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permits and related monitoring activities. Finally, toxicity tests are conducted
as part of the baseline risk assessment at hazardous waste sites.
The language in the Comprehensive Environmental Response Compensation and
Liability Act (CERCLA, 1980) as amended by the Superfund Amendment and
Reauthorization Act (SARA, 1986) provides a basis for inclusion of plants in the
evaluation of hazardous waste sites.11- 2t This statute draws numerous
additional laws and regulations into the process by reference to "Applicable,
Relevant, and Appropriate Regulations" (ARARs). Federal and state listings of
rare and endangered species are among the ARARs referenced in the process.
Where wetlands are part of a site, the jurisdictional delineation of wetland
habitat involves plants. The determination of adverse impact to plants may
also be part of the resource damage assessment effort.
In addition to the clean-up focus of CERCLA/SARA, the US Department of
Energy has embraced the concept of ecological restoration. Major research
programs are in the early stages to develop and implement restoration efforts.131
Any restoration effort of a hazardous waste site must focus strongly on
vegetation parameters including phytotoxicity.
Within the conterminous states, the US has approximately 33,000 known
hazardous waste sites (see Figure 1). Many of these are sufficiently large and
located in environmentally sensitive settings to warrant detailed ecological
analysis. Others, due to their location in heavily industrialized zones, may
require a lesser effort to complete the ecological risk assessment. Over 31,000
sites have been reviewed by EPA. Some 19,000 are not considered
appropriate for federal action. Approximately 1,200 sites have been placed on
the National Priority List (NPL). Only 33 sites have been removed from the NPL
since the program began. Most sites have not had adequate ecological
assessments completed. Of these, only a small number have included
phytotoxicity assessment endpoints.
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Site Distribution
v
o
673 '
449
< 225
315
102
625
470
5.0
Figure 1. Distribution frequency of hazardous waste sites in the conterminous United States.
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C. BIOLOGICAL IMPORTANCE
Vegetation is the dominant biological component of terrestrial ecosystems, with
nominally ten biomass units of plants, to four biomass units of microbial
organisms, to one biomass unit of animals. Depending upon the species, soil
characteristics, and environmental stresses, 40% to 85% of the plant mass
resides below ground in contact with chemicals in the soil. On the macroscale,
plants are the biological source of energy as well as nutritional components for
animals. Furthermore, the structure of vegetation, in concert with the varied
abiotic landscape features, establishes habitat that animals rely on for
protection from adverse weather and predators.
Ecological risk assessment is a necessary component of contaminated
environment evaluation and remediation. This assessment is based on a good
understanding of both contaminant exposure and ecosystem response to this
burden. Plants play an important role in both of these processes. Macrophytes
may influence contaminant fate within the ecosystem in many ways. They
may act as a sink for non-phytotoxic chemicals effectively reducing the
exposure to other trophic levels. They may accumulate potentially toxic
compounds from sediments and soils and serve as a source to reintroduce them
into the food chain. In addition, the influence of contaminant stress in plants
on ecosystem stability is poorly understood. Thus the major features of plants
for ecological assessments include the following:
o they respond to stressors found in soils through altered
photosynthetic and respiratory rates;
o they harbor microbial populations in their root systems that
facilitate uptake and metabolism of various organic and inorganic
constituents including pollutants;
o they sequester and/or metabolize toxic substances in organs and
tissues both above and below ground;
o they serve as a conduit of toxic substances into the food web; and
o they stabilize soils agarnst wind and water-mediated sheet erosion,
thereby reducing mass transport of hazardous materials from the
site.
Plants should be considered an important component of any ecological
assessment of hazardous waste sites. To assess the full consequences of a
contaminated site, it is crucial that analyses of the vegetation be integrated into
the context of the landscape features surrounding the site. Furtt>ermofe, the
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plants growing in the contamination zone should receive careful consideration
as candidates for toxicity testing and monitoring studies since they have
already demonstrated a tolerance of the contaminants.
The vegetation growing on a site may be composed of cover crops planted
specifically to stabilize soil surfaces, naturally occurring vegetation (including
native and naturalized species), or some mixture of natural and planted species.
As the degree'of "naturalness" increases, so does the ecological complexity,
and thus greater levels of analytical sophistication are required to ascertain the
site's ecological condition. The impact of hazardous waste on vegetation may
be realized in a variety of ways and with different consequences (see Table 1).
Table 1. Generic Negative Impacts of Hazardous Materials on Plants that Influence
Vegetational Characteristics
Primary/Direct Impacts
o quantitative suppression of plant growth
o qualitative shift in community composition and/or shift in
community structure
Secondary/Indirect Impacts
o quantitative impairment of plant-microbial interactions affecting
energy flow and nutrient cycling processes (decomposition,
symbiotic relationships)
o altered animal use either for food or habitat
Ecological assessments of plants are often made under conditions that ignore
the critical, interactive influence of soil microorganisms. Not all measurements
need to consider the root environment, yet we should be cognizant of the
potency of nonpathogenic microorganisms to modify plant processes.
Plants distribute net photosynthate according to various species-specific,
developmentally regulated, and environmentally modulated allocation patterns.
Typically 40 to 85% of the net photosynthate is incorporated into root
tissues.141 The pattern of allocation is highly dependent upon the communities
of microorganisms inhabiting the rhizosphere and penetrating root tissues.
Under gnotobiotic conditions (i.e. free of all bacteria, fungi, or other potential
biota), the addition of nonpathogenic bacteria to grass seedlings can result in
overall changes in net primary production ranging from 40 to 370% of controls.
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with no apparent alteration of the shoot, but virtually all of the growth
response in the roots.151
For a perspective of the root environment, consider some general features of a
young, growing, herbaceous plant of average credentials with aerial portions of
the plant having a wet-weight mass of 10 g. We can expect, for simplicity, a
root mass of 20 g distributed in a soil volume of 1000 cc. This soil volume
harbors some 10 to 2000 billion microorganisms, 1 million nematodes,
thousands of insects in various stages of development, a few hundred of seeds
of potentially interfering plants, and roots of a few neighboring plants. If this
plant is to grow at a moderately high relative growth rate of 8%/day for 30
days, the aerial portion of the plant will increase tenfold. Most likely, so will
the roots extending into a proportionately new soil volume with its attendant
populations. During (and in response to) this growth, the microbial population
will multiply 3- to 25-fold per unit volume of soil.161 What makes the root
environment so crucial is that growth of the entire plant is dependent upon the
nutrients acquired and translocated to the foliage and active meristematic
zones. In order to maintain a consistent relative growth rate, the plant must
acquire proportionally larger amounts of nutrients per unit time to supply the
"demands" of the growing plant.171
D. ROUTE OF EXPOSURE FOR ANIMALS (HUMANS)
Plants often exhibit pivotal influence on the magnitude of toxic chemical
exposure to animals (including humans). Their ameliorating influence on wind
and water erosion can dramatically affect exposure estimates. Plants also
function as a conduit providing contaminants to animals via food chain
transfers.
E. AMENABLE TO MEASUREMENT
Plants, in general, can be measured, tested, and monitored more readily than
other biota. Ecological measures of distribution and abundance are relatively
simple. The sessile nature of plants eliminates many technical issues implicit in
most wildlife methods. The diversity of plant forms allows selection of plant
species representing short- (seasonal) to long-term (years, decades) intervals of
potential exposure. €xcept for endangered species or certain drug producing
species, plants carry no social or moral constraints impeding research or
monitoring activities. Overall ease of performing plant ecological measurement
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and plant toxicity tests contribute to the relatively low costs of plant
ecotoxicology methods.
Vegetation assessment relevant to contaminated sites can be achieved through
remote sensing, direct vegetation measurements, and selected functional (or
process-oriented) measurements. The objectives and values for each approach
vary:
Remote Sensing
o To gain current and historical information on land use and to
establish generalized perspectives of landscape interactions.
o To define generalized vegetation patterns (especially gross structural
attributes) suitable for habitat classification.
o To aid in defining the boundaries of impact (in some situations,
especially where plants exhibit stress responses to contaminants).
Direct Vegetation Sampling
o To verify patterns discerned from remote sensing.
o To provide community composition data (i.e., species identity and
dominance/density values).
Functional Processes
o To evaluate direct impacts on vegetation.
o To identify probable secondary impacts that may affect animal
populations (including human) or other ecosystem processes.
In addition to collecting the typical data for community descriptions, there may
be reasons to collect stem and root sections or cores. Annual rings can
provide direct evidence of changes in growth rates. Growth rates may be
compared to known trends for a species or against rates measured for plants
outside of the impacted area. Tissues may also be used to determine chemical
concentrations or isotope values for tissues spanning the temporal ranges from
pre-impact to present (or time of death of the individual).
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II. ECOLOGICAL RISK ASSESSMENTS
A. GENERAL APPROACH
Ecological Risk Assessments are designed to define actual and potential harm
to biological resources. The information must be structured so that regulatory
decisions and risk management options can be scientifically based. Ideally, the
risk analysis forms the critical foundation for selection of alternative
technological options. Ecology is an integrative discipline which draws upon
diverse sources of information [e.g. chemical, physical, geological, biological,
etc.} to describe the interactions of organisms, populations, communities and
ecosystems with each other and their surroundings. The challenge is to focus
on the critical and relevant ecological issues from the vast array of potential
ecological relationships and to do so in a manner that contributes to the risk
analysis.
Barnthouse181 has discussed the basic risk paradigm in terms of its use for
ecological risk assessments. In doing so, he distinguished two broad-use
categories. Traditional risk assessments are intended to predict the likelihood
of some event (i.e., adverse lexicological effect) occurring. This is
accomplished from analysis of the hazard or toxicity and exposure conditions.
In the strictest sense the risk assessment forecasts the probability of a given
effect. Barnthouse also recognized the common usage of ecological analysis
after an effect has occurred. He referred to this as "retrospective risk
assessment." Perhaps a better term would be forensic ecology: the evaluation
of measurable ecological endpoints in order to establish linkage between source
and levels of contamination and ecological effects.
In Superfund the bulk of ecological work is forensic in nature. Analyses of field
conditions and ecological endpoints are used to help define the extent of
contamination effects. Laboratory work compiled with field observation serve
to define the spatial boundaries of concern. Much of the information that is
collected in this forensic phase is useful in the predictive sense as well.
Estimations of concern can be evaluated in a site-specific context for prediction
of future impacts under no-action and remediation options.
The purpose of an ecological assessment of a-hazardous waste site is to
determine if an adverse ecological effect has occurred as a consequence of the
materials present at the site. The information gathered in the ecological
assessment should provide valuable msigtrts into spatial distribution, risk
modeling, and evaluation of remediation options. In this regard it should be
noted that an ecological risk assessment is not an ecosystem risk assessment.
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Rather the ecological features relevant to exposure are imbedded in the
determination of risk to selected resources.
Hazardous waste sites have restricted access due to legal, proprietary and
human health risk considerations. Restricted access imposes significant
constraints on ecological assessment and is the foremost reason for the paucity
of ecological information on existing sites. Precautions necessary to ensure
worker safety add significantly to the cost of collection site data. Sample
handling, chain of custody, and Quality Assurance/Quality Control requirements
add further to the special costs of assessing hazardous waste sites.
Collectively, these conditions lead to restricted, sometimes incomplete, data
sets upon which decisions must be made. Throughout a project, the site
assessment process must provide information that can feed into critical
decisions. These include determining the
o Magnitude and extent of current impact,
o causality/weight of evidence,
o estimation of future impacts,
o merits of remediation options.
Consequently, it is exceedingly important that careful planning be done to
ensure that the proper information is obtained in the correct fashion. Sampling
design and statistical assumptions must be considered early on to achieve
effective and efficient use of resources.
B. ACCESSIBILITY CONCERNS
Access to hazardous waste sites generally is restricted due to legal, proprietary
and human health risk considerations. Restricted access imposes significant
constraints on ecological assessment. However, vegetation can be analyzed in
ways that overcome such access limitations.
General landscape pattern and gross structural features of vegetation can be
inferred from conventional aerial photography. More sophisticated measures
can be derived through remote radiometric sensing. Photosynthesis responds
to environmental stress in ways that affect the spectral reflectance and
fluorescence radiance emanating from a plant, and this phenomenon provides
unique assessment opportunities for remote sensing. Remote sensing of
vegetation affords, access to restricted sites and can be used in limited cases
on archived radiometric data. No other ecological community is so amenable to
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passive, non-intrusive assessment. Indeed, because of the dependence of
other life forms on plants, quantitation of plant communities by remote sensing
may be the best means of acquiring preliminary estimates of impact for
dependent groups I i.e., habitat structure and other landscape ecology features
such as patchiness or connectivity may be useful in predicting animal use rates
and exposure levels).
The quality of vegetation assessment and the efficiency of data acquisition can
be greatly enhanced by gathering specific information early in the scoping
process. Key pieces of information such as base maps and photographs should
be gathered. Sources for contour maps include the U.S. Geological Survey;
vegetation maps accumulated from published reports and organizations, U.S.
Forest Service, Park Service, U.S. Fish and Wildlife Service etc.; aerial
photographs from the Agricultural Stabilization and Conservation Service.191
Considerable historical information may also be obtained through the original
land survey records, although caution must be exercised in using this
information.1101
Finally, advanced planning is needed to obtain all necessary collecting permits
from federal, state, local, and/or private entities. Site access permits should
also be obtained before sending any staff to the field. Access permits should
be obtained for potential reference sites as well.
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III. ENDPOINTS OF INTEREST
A. DEFINITIONS and ECOLOGICAL HIERARCHICAL LEVELS
Adopting the terminology of Surer,i11' there are several potential assessment
and measurement endpoints relevant to plant ecotoxicology. The endpoints of
interest vary depending upon the ecological level of organization to be
addressed. Potential endpoints listed in Table 2 are adapted from Suter's
chapter. Assessment endpoints are formal expressions of the actual
environmental values that are to be protected; the environmental characteristics
that can indicate a need for remediation or restoration; the highest value that
can be assessed operationally. Measurement endpoints are quantitative
expressions of an observed or measured effect; a measurable environmental
characteristic that is related to the assessment endpoint.
As a part of the identification process, the ecologist should develop a
generalized or conceptual model that relates the various biological resources to
one another. In this regard, the major functional groups are identffied and this
becomes the first cut effort to begin consideration of exposure pathways;
direct exposures, indirect exposures, as well as identifying possible habitat
influences that are independent of toxicity. There is general consensus that
measurement endpoints must be selected at the same level or one level of
organization below that of the assessment endpoint. The uncertainty
introduced as one extrapolates more than one level of organization beyond the
measurement endpoint is too large to warrant the exercise.
There is a growing persuasion within the ecological risk community to select
the most relevant (most significant to the specific setting) ecological resources
for characterization. This is in opposition to the suggestions of Suter to focus
on social relevance. The basis for rejecting the social relevance "filter" is that
the scientists should provide the strongest scientific case given the project
objectives; then, it becomes a risk communications issue to develop linkage
with socially relevant concerns that support -management decisions.
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Table 2. Potential Ecological And lexicological Endpoints.
ASSESSMENT ENDPOINTS
MEASUREMENT ENDPOINTS
Individual
Death
Growth
Fecundity
Overt symptomology
Biomarkers
Tissue concentrations
Population
Extinction
Abundance
Yield/production
Age/size class structure
Massive mortality
Population
Occurrence
Abundance
Age/size class structure
Reproductive performance
Yield/production
Frequency of gross morbidity
Frequency of mass mortality
Community
Market value
Recreational quality
Usefulness/desired type
Community
Number of species
Species evenness/dominance
Species diversity
Pollution indices
Community quality indices
Community type
Ecosystem
Productive capability
Ecosystem
Biomass
Productivity
Nutrient dynamics
Selection of methods appropriate for ecological risk assessment can be a
difficult task given the vast array of potential assessment and measurement
endpoints. Methods suited for research may require more technical knowledge
than is available for routine toxicity assessment or site evaluation. To help
guide the selection process, two categories of tests methods were estabNshed
in Warren-Hicks et al.1121 based on the relative degree of standardization and
the quantity of toxicity data supporting the method. The categories were
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identified as Class I and Class II. Expanding this concept, the following
operational criteria were are used throughout the rest of this document:
Class I. - A test or measurement having an accepted protocol; also
having a well defined and well characterized ecological,
physiological, or toxicological foundation.
a. Extensive data set from applied uses in toxicology or
environmental assessments.
b. Limited data set from applied uses in toxicology or
environmental assessments.
Class II. - A test method having well defined or characterized ecological
physiological, or toxicological foundation but lacking a
standardized protocol.
a. Method having widespread use in basic sciences; applied
science protocol in draft stage ready for inter-laboratory
validation.
b. Very promising method that may require additional basic
research to verify specificity, interference, or similar
technical issues before a draft protocol can be prepared.
Two recent books113- 141 presents excellent overviews of the relationship
between toxicology and ecology. Traditional vegetation measures, described in
detail in quantitative plant ecology books, provide essential baseline information
for ecotoxicological studies. Growth in plants is readily measured as a change
in height, length, or biomass. Individual plants or groups of plants in specific
plots are measured. In woody plants, relative growth can be inferred from
width of annual growth rings. Physiological endpoints, or biomarkers, range
from measures of photosynthetic rates, photosynthetic condition, total
respiration, dark respiration, and various specific enzymes. Reproductive
endpoints may include fruit set, seed set, or tiller production.
B. TOXICITY
1. ENDPOINTS
Phytotoxicity usually refers to an appraisal of an unfavorable plant response to
some substance or group of substances (even that resulting from growth
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substances, hormones, and secondary metabolites.1151 The response is
measured by some prescribed endpoint such as mortality, germination, growth,
or other relevant physiological entity. Typically, the substance or mixture of
substances is administered to control exposure at nominal or verified
concentrations. The response of different individuals at different
concentrations is summarized in a variety of standard formats such as No
Observable Effects Level (NOEL), Lowest Observable Effects Level (LOEL),
median effects concentrations (LC5o, £€50) or other levels of effects. The
more detailed phytotoxicity tests are coupled with measurements of media and
tissue residue levels (pre- and post-test) to verify exposure concentrations.
Unfortunately, relatively few reports present this more detailed analysts.
Phytotoxicity endpoints on tests with acute exposure conditions range from
quanta! measures of survival (mortality) through continuously distributed
measures such as growth. Growth may be reported as change in height or
length, biomass, percentage cover, or other suitable metrics. Although
generally not incorporated into standardized protocols of regulatory agencies,
measures of photosynthetic rate (gas exchange) or photosynthetic condition
(fluorescence) are also used. Specific metabolic enzymes, total respiration, and
dark respiration have been similarly used as measurement endpoints.
Generally, the chronic exposure tests rely more extensively on growth and
specific metabolic measures as the endpoints; although cursory examination of
metal toxicity reports for terrestrial plant species indicate that accounts
concerning chronic exposure tests are more typical than acute exposure tests.
Survival (mortality) and various biomarker metrics are often incorporated into
the investigations.
In recent years, much of the literature on phytotoxicity has been generated in
response to regulatory requirements in Canada, Europe, and the United
States.116- 17- 1S- 19- 20- 21- 221 by far, the majority of phytotoxicity research has
been focused on water quality issues.123- 24- 2S1 Consequently, much of the
information is from studies with various algae. To a lesser extent, duckweed
(Lemna minor) and a scattering of rooted macrophytes. have been examined.(26-
271 Terrestrial interests have been driven primarily by pesticide registration and
toxic chemical screening processes with much less emphasis on metal toxicity.
The preponderance of metal toxicity reports comes from investigations into the
safe disposal of sewage sludge.1261
A large portion of the phytotoxicity literature diverges from the established
regulatory presentation format. Consequently, comparison among reports is
complicated by differences in exposure, duration of tests, measurement
endpoints, and assessment endpoints. There is rarely sufficient information
presented in papers and-reports to permit recalculation in order to achieve
14
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comparable units. For example, one report may tabulate "significant toxicity as
being 10% or greater inhibition of growth relative to controls," while another
uses 30% reduction in growth as the endpoint; one will report nominal total
metal concentrations and another provides exchangeable metal concentrations,
and yet another provides tissue concentrations, without regard for exposure
conditions.
2. SURROGATE SPECIES
Phytotoxicity research has been restricted to a large extent to plant species
that are easily manipulated under laboratory conditions. Seed availability is
also an influential factor affecting choice of test species. Accordingly, a limited
suite of agronomically important, herbaceous plants have been used. The US
EPA Tier 1 test requirements for registration of pesticides1291 lists the following
plant species (Table 3):
Table 3. List Of Plant Taxa Identified In Sanctioned Toxicity Tests.
FAMILY
Solonaceae
Cucurbitaceae
Compositae
Leguminosae
Cruciferae
Umbelliferae
Poaceae
Poaceae
Poaceae
Liliaceae
SPECIES
Lycopersicon esculentum
Cucumis sativus
Lactuca sativa
Glycine max
Brassica oleracea
Daucus carota
A vena sativa
Lolium perenne
Zea mays
Allium cepa
. COMMON NAME
Tomato
Cucumber
Lettuce
Soybean
Cabbage
Carrot
Oat
Perennial Ryegrass
Corn
Onion
The Organization for Economic Cooperation and Development (OECD)
recommends a similar list of 16 herbaceous crop species representing four
taxooomic families.|3°- 311 Np_ standardized toxicitv tests use or recommend Ihg
use of a woody species.1321 This oversight is somewhat surprising, given the
level of attention afforded to forests worldwide, Except for the occasional
academic paper, site specific investigation of toxicity dealing with trees and
shrubs, or pesticide study involving control of woody weeds, the toxicological
literature is limited to herbaceous plants.
15
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3. EXPOSURE CONDITIONS
Direct exposure is achieved if the test soil or sediment is incorporated into the
test as soil or sediment. This provides a more defensible evaluation of toxicity
as it relates to potential exposure conditions. The major disadvantage is that
analysis of contaminant concentration is more difficult. Indirect exposure tests
are derived from some extraction of the test soil or sediment such as occurs
with elution; the eluate is then used as the test material. In most cases there is
a high level of uncertainty in the extrapolation of toxicity conditions inferred
between direct and indirect test methods.
No tests have been developed to evaluate volatile organics. During collection,
shipping, and handling of soil samples volatiles are likely to escape. For this
reason field observations may be more critical for sites having volatile organic
contaminants. It may be important to consider chronic exposures including life-
cycle tests. Special test methods may be modeled after the approach by
Mueller33 in his investigations of allelopathic properties of desert shrubs.
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IV. METHODS AVAILABLE
A. ECOLOGICAL MEASUREMENTS
1. GENERAL
Greig-Smithi3*) 'provided a detailed theoretical treatment of vegetation sampling.
Other excellent treatments of vegetation sampling, typically with fewer
theoretical considerations, are available.135- 3S- 37- M- 39- 4°i The distribution of
organisms in nature is governed by a variety of environmental, biological, and
behavioral factors. These distributions may result from reproductive
tendencies, success of germination and establishment, biological interactions,
and microhabitat variation. Three fundamental patterns of distribution are
recognized; namely, regular, random, and aggregate (See Figure 2.)
Combinations, such as random aggregates may exist also. In practice,
populations of various species in a community grade across all classical
distribution patterns. Highly disturbed sites present additional spatial
complications.
REGULAR
RANDOM
AGGREGATE
Figure 2. Plant Distribution Pattern.
The type of distribution one anticipates may dictate the specific sampling
regime adopted and introduce constraints on statistical analysis. Various
approaches to quantitative vegetation sampling can be used for hazardous
waste site assessments. Often, the details of the sampling procedure are
17
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varied to accommodate the structural and distributional features of vegetation
type.
Defined area sampling offers the greatest flexibility in subsequent treatment of
the date. That is to say more information may be gleaned from the numbers
such as quantitative indices of interspecies associations, comparative frequency
and density values and other characterizations by distribution. Density (the
number of individuals per unit area) is obtained directly. Frequency, an
indication of the uniformity of distribution, and the dominance or phytomass
per unit area are calculated easily. Before a defined area sampling technique is
undertaken, several questions should be resolved.
o What size plot will yield the most reliable data?
o What shape should be used; square, rectangle, or circle?
o How many plots are required for an adequate sample?
o How should the plots be positioned within the site?
Methods to measure species distribution and abundance have been developed
in many schools of quantitative plant ecology. Techniques widely used in the
basic sciences range from subjective approaches that yield general descriptions
of species presence supported by semi-quantitative values (e.g., the Relevee
method) through rigorous quantitative determinations using fixed plots as well
as variable plots. Applied fields of plant ecology including forestry, rangeland
ecology, and crop sciences have developed special variations of several
methods intended to focus on narrow, targeted endpoints of interest to these
disciplines. Much of the theoretical sampling information used in plant ecology
can be adapted to historical data with the appropriate cautionary caveats {land
survey work in the U. S.|41I> Photographic interpretation and remote sensing
also provide useful insight into spatial and temporal ecological patterns,
including plant stress.142-43-44-45-46- "•48)
For clarity the following definitions are used:
Trees are defined as erect, woody plants having a stem diameter .>. 10
cm at 1.4 m above ground level (Diameter at Breast Height, DBH).
Juveniles of tree species with lesser DBH' are typically scored in the
shrub category.
Shrubs are defined as erect or prostrate woody plants (including
individuals of tree species) <. 10 cm DBH.
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Herbaceous plants are all non-woody plants including bryophytes and
lichens.
a) POSITIONING THE PLOTS
If one assumes random distributions, the ideal method of data collection would
dictate random positioning of the plots. Though feasible under some
conditions, in most field situations it is difficult to impossible to determine the
location of a predetermined random locus. Generally, one of two approaches is
adopted.
(1) TRANSECT
The origin of a line is located in the site. The line is established following a
compass bearing. At predetermined regular or random intervals along the line,
a plot is delineated and sampling information recorded. The orientation or
bearing of the line may be selected randomly. Often, however, topographic
features are taken into account. The investigator may wish to establish the
transect perpendicular to ridges or parallel to the ridges, or parallel to some
other recognizable boundary. The major objective here ts to minimize sampling
bias.
(2) STRATIFIED-RANDOM SAMPLING
The area to be sampled is dissected into a grid system. Each cell within the
grid is identified by a unique number. Cells to be sampled are selected
randomly. Upon locating the approximate boundaries of the grid cell, the plot
is positioned through some unbiased "random" process (e.g. a random number
of paces north and west of the southeast corner of the grid cell).
After each of the above questions has been resolved, sampling may begin. The
information collected in each plot should include:
o the number of individuals of each taxa;
o some measure of the size of each individual (e.g. DBH, Height,
Canopy Cover, or Phytomass).
Generally, the summary data is presented in tabular form in one of two ways
The species list may be arranged according to life form (i.e., trees separated
19
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from herbs; grasses separated from forbs) and alphabetized. Alternatively, the
species are arranged in decreasing order of the Importance Percentage (IP).
This presentation permits rapid review of data and may be used for statistical
quantitation comparisons among areas or between sites (e.g. reference site and
target site).
Often, the conditions of a hazardous waste site preclude extensive reliance on
the direct techniques of vegetation sampling. The guiding principles for
suggesting the measurements described in this section were couched in the
following questions:
o Does the measurement provide information that allows one to
document or infer ecological impact?
o Can the measurement data be obtained rapidly (i.e., minimizing on-
site effort and exposure time of workers) while adhering to high
standards for accuracy and precision?
o Has the utility of the measurement for ecological assessment been
demonstrated?
Data summaries should be prepared for each discernable vegetation unit, both
off-site and on-site. For trees, this includes the calculated estimates of density
(number of individuals per hectare), basal area (the stem cross-sectional area
calculated from the measures of DBH, a surrogate value for dominance),
frequency (the percentage of plots having a particular species), and the
importance percentage (IP, the mean of the normalized density, basal-area, and
frequency values). These calculations, which are to be prepared for each
species, yield average values that should be accompanied by standard error
estimates.1*91 Comparable calculations are performed for the shrub and
herbaceous plants, Cover estimates or phytomass values are used in place of
basal area for shrubs and herbaceous plants. Typically in the herbaceous plant
sample methods, measures of density are not obtained.
The summary values acquired from sampling may be -used to calculate various
synthetic indices such as species diversity or coefficient of community.
Extreme caution must accompany any interpretation of such values, since
natural succession and stress affect the diversity of a community in non-linear
patterns. Also, the indices do not provide for inclusion of variance or precision
estimates. Furthermore, the effect of a hazardous waste site may be to elevate
or decrease diversity. Qualitative values of harm or benefit cannot be assigned
to fluxes in diversity in the absence of careful ecological analysis of the
features affecting a given change.
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bl HABITAT & COMMUNITY STRUCTURE
Generalized approaches to identify habitat type may be sufficient to
characterize plant resources and potential animal resources at risk. Such
general descriptions may also be used to define important biotic unit
boundaries. Cursory efforts, including simple reconnaissance surveys, may
provide valuable first-cut impressions of nominal conditions. Formalized
procedures are codified by the U.S. Army Corp for wetlands delineation.1501
Community structure analysis requires intermediate levels of characterization of
natural areas. The analysis may combine various types of data collected for
site characterization. More generalized, non-quantitative approaches include
descriptive treatment of life forms present. Various quantitative and semi-
quantitative measures of plant canopy cover for each major species or life form
are readily obtained. More sophisticated treatments of quantitative data permit
characterization of successional status, interspecies associations, and
calculations of indices of diversity or dominance.
Since some methods are rather time-consuming (and therefore expensive to
conduct), it is imperative that attention be given to data requirements and the
methods selection be performed early in the planning process. The vast array
of sampling methods and approaches, on the one hand present a seemingly
infinite array of options; or on the other, they also represent a rich opportunity
to achieve efficiency through selection of appropriate methods to satisfy
specific data requirements.
c) POPULATIONS & INDIVIDUALS
Generally, ecological risk assessments do not focus on individuals. However,
in plant ecology there are unique opportunities to evaluate environmental
condition at the individual level. Mortality of individuals can indicate localized
zones of contamination in air or soil. Laboratory and field toxicity
measurements accumulate information at the individual level, providing some
indication of statistical variation in response to given levels of exposure.
Besides death, a considerable number of quantitative plant ecology methods
can be used to assess rates of growth. Whether in field settings or in
controlled environments, endpoints of growth provide sensitive, and
ecologically relevant endpoints in ecotoxicology. We often think of the growth
measurements in rigidly controlled experimental conditions that permit
21
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determination of treatment effects. Growth can also be examined over longer
times through measurements of growth rings, twig length, radius of clones,
etc. Statistical trend analysis can be used as a tool to establish linkage to
environmental variables, including toxic substances.1511
On shorter time intervals, several measures that might be considered indicators
of plant health can provide sensitive indications of exposure to or effects from
toxic substances. Indications of morbidity may include incidence of diseases or
symptoms such as foliar abnormalities resulting from heavy metal toxicity,
dysfunctional root morphology resulting from metals, chlorosis from air
toxicants, or deformed reproductive organs. Sophisticated analysis of
photosynthetic activity or photosynthetic potential are also useful indicators of
stress effects in plants. Finally, there are substantial bodies of literature that
detail the accumulation of specific chemicals, especially metals, in various
tissues.
In the relatively brief history of ecological study, numerous techniques have
been developed to collect data to describe natural communities. The sampling
techniques vary in their thoroughness (accuracy) and in the time and therefore
cost required to execute properly. Generally the techniques that can be
performed rapidly in the field have inherent limitations on subsequent data
manipulation and interpretation. However, they may provide the desired
information and therefore are sufficient to do the job. Once the purpose of the
study'has been established the proper methods can be selected.
2. REMOTE SENSING METHODS
Remote sensing may be used advantageously iri a number of ways to assess
vegetation of hazardous waste sites. It was beyond the scope of this
workshop to address this topic adequately. Extensive efforts are underway in
the U.S. National Aeronautics and Space Administration and to a limited extent
in EPA to characterize regional patterns in vegetation. As this data
accumulates, it will become useful for some of the larger hazardous waste
sites. Primary sources of radiometnc data are the Landsat Multi Spectral
Scanner (MSS), the Thematic Mapper (TM), and the French Systeme Probatoire
d'Observation de la Terre (SPOT) data banks. Resolution is the major limitation
of these satellite imaging systems. Pixel resolution limits for the three types
are: MSS, 80m; TM, 30m; and SPOT, 20m. For improved resolution, the
satellite images may be supplemented with fixed-wing aircraft (including
ultralfghts) utilizing comparable sensing equipment. The flights may also
employ infrared and conventional photography. Coordinated work at individual
srtes for verification ("ground truTtimg") or for additional resolution can be
22
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performed from "cherry picker" booms with field model sensors. These
different levels of resolution provide the following opportunities:
o relatively unlimited accessibility;
o safe; non-intrusive assessment and monitoring; and
o through archived data (MSS since 1972; TM since 1982; SPOT since
1984; global coverage each 18 days), the opportunity to assess
large-scale seasonal and annual vegetational patterns.
Radiometric data have been used effectively to accomplish the following
objectives:'52' "•5*-55-56- 57>
o to map vegetational boundaries (detecting shifts in dominant canopy
species within a given forest type),
o to estimate net photosynthesis and net primary production,
o to estimate foliar nitrogen content,
o to detect drought stress,
o to detect effects from pest epidemics such as gypsy moth, and
o to assess forest decline due to air pollutants.
Conventional aerial photography should also be incorporated into the vegetation
assessment. Most of the continental United States has been photographed
repeatedly since 1938. Although the photographic record is incomplete and
sporadic, and technical limitations (such as varied camera angle and altitude)
are typically great, the photographic records contain valuable qualitative
information on vegetation and land use patterns over a 50 year time span.
Even subjective knowledge of generalized trends over five decades can offer
important interpretive perspectives to ecological assessment.
3. DIRECT OBSERVATIONAL METHODS
The contamination characteristics of a site may require special precautionary
steps to protect the personnel conducting on-site vegetational measurements.
Contamination characteristics should be the primary consideration in selecting
the detail of the measurement. The specific objectives of vegetation sampling
should be defined early in the assessment process since the objectives dictate
thoroughness and methodology options.
23
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The first phase of direct observations should be directed toward ground
truthing of the remote sensing information. This should be initiated with
analysis of the off-site, uncontaminated border regions associated with the
contaminated area. Clearly it is most desirable to validate the remotely sensed
data with field data from the contaminated site under study. However, it may
not be feasible to gain the required access to the site and the site may pose
unreasonable risk to the research personnel. Even if the only validation is from
adjacent border regions, the remotely sensed data will be valuable in assessing
the vegetation on the affected site.
a) DEFINED AREA SAMPLING TECHNIQUES
(1) PLOT SIZE
Ideally the plot size should be selected such that the data obtained fits (or at
least approaches) a normal distribution. At the same time, the plot should not
be too large, since a greater effort is required to tally the individuals and no
additional information is gained (See Figure 3). In fact for certain purposes
(e.g. statistically determining associations) the larger plot may obscure the
relationships. Plots for trees are commonly 100 m2; shrub plots generally
occupy 1-4m2; and herb plots range from 0.1-1.0m2. Generally as vegetation
becomes more dense, smaller plot sizes are favored.
Y= NUMBER OF PLOTS
WITH X-INDIVIDUALS PER PLOT
PROPER SIZE
PLOT
TOO
SMALL
01234567 01234567 01234567
NUMBER OF INDIVIDUALS PER SAMPLE PLOT
Figure 3. Frequency Distribution Comparisons To Select Proper Plot Size.
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(2) PLOT SHAPE
Since any plot established within a community will result in the possibility of
some individuals positioned on the boundary of the plot, some unbiased system
must be established to decide whether an individual is to be tallied or not. One
system may be to tally an individual if half or more of the plant stem is
anchored within the plot. Another is to count every other individual that falls
on the boundary, thus eliminating the need to decide how much of an individual
crosses the line. In the field it will become obvious that determining the
boundary is a difficult task.
For a given area, the boundary or perimeter of the plot is greatest for a narrow
rectangle, less for a wide rectangle, less for a square, and least for a circle.
Consequently circular plots should result in fewer "in-out" decisions compared
to squares. Squares should be better than rectangles. Wide rectangles should
be better than narrow rectangles. However site conditions, including
vegetation type, must be considered before making the choice. Establishing a
circular plot in thick vegetation is virtually impossible and will result in
excessive sampling error. Labor costs are greatly affected by the choice of plot
shape.
(3) SAMPLE SIZE
Several systems for determining sample size have been used in ecology. One
of the earliest is the "species-area curve" (See Figure 4). As sampling
proceeds, a graph is made by plotting the number of species encountered on
the ordinate and the area sampled on the abscissa. Eventually, within a given
vegetation type, a point is reached where all but the extremely rare taxa are
recorded. Thus additional sampling will not generate much information in terms
of species present.
25
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NUMBER OF
SPECIES
AREA SAMPLED
Figure 4. Species Area Curve.
Note that the species-area curve will not necessarily indicate adequacy of
sample regarding the density of individuals.
The best objective indicator of adequacy of sample for density is the formula,
n = (s2 t2| / d2
Where a is the sample size;
s is the variance;
t is the value from the students t statistical
table for the desired level of confidence
and the appropriate degrees of freedom;
d is the allowable error expressed in the
same units as s.
This may be used for the density of all species combined or for a given species
of interest in the study. In order to use this formula it is necessary to know, or
to be aote to -estimate, tne standard deviation and to know the level of
26
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accuracy needed. It is acceptable to make d a variable. For example, it may
be an acceptable to be within 10% of the actual mean density. Thus d. can be
expressed as 0.1X.
In vegetation sampling, one is often dealing with relatively small areas of
relatively high variance. Under these conditions the formula will be of little
value as it will indicate that virtually the entire area should be included in this
sample. When this occurs, if the area is sufficiently small, one may wish to
sample an approximate percentage (e.g., 10% or 20%) of the total area. In
plotless techniques, where densities cannot be extricated from the data,
adequacy of sample is often judged by plotting species-sample curves (same as
species-area curve, but here species-intervals sampled or species-pins
sampled). Alternatively, one could plot Dominance (Cover) vs. sample effort.
The following sections discuss vegetation assessment methods. Each of the
methods discussed should be considered a Class I test. Additional detail on the
methods, especially the equations used to summarize the data is found in
Appendix I.
bl PLOTLESS SAMPLING TECHNIQUES
Defined area sampling techniques utilize known areas within each sample plot,
but plotless routines as the name implies do not encompass an area. Generally,
the plotless methods require less time to perform and can be an effective
means of quantifying vegetation.
The following plotless sampling methods are widely used in basic and applied
ecology: line-intercept, point-frame (also known as pin-frame), point-quarters
and variable-radius. Although in theory any sampling method could be applied
to any vegetation type, the line-intercept and point sampling methods are
typically used in low growth, herbaceous habitat. The point-quarters and
variable-radius methods are used mostly in forested areas.
(1} GROUND TRUTH MAPS/QUALITATIVE ASSESSMENTS •• FLOHISTICS
Visiting the site is required to verify the community transitions/breaks indicated
in derial photos and to identify all prominent species. Depending on the site,
multiple visits at different seasons may be needed to capture the breadth of
species richness within the communities. Botanists familiar with the regional
and local flora should be employed to compile the floristics checklist and to
spot unusual gaps in the assemblages of species. The utility of synthetic
27
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community measures (such as the Species Diversity Indices, Indices of
Similarity, etc.) are affected greatly by the degree of taxonomic discrimination
associated with primary data collection.
(2) GROUND TRUTH MAPS/QUALITATIVE ASSESSMENTS -• RELEVEE
A semi-quantitative analysis of the vegetation may be sufficient to satisfy the
objectives for many sites (e.g., highly disturbed and biologically isolated
locales, sites that pose unacceptable risk to personnel, or sites that satisfy
criteria for remote sensing analysis and only require generalized "ground-
truthing"). The Relevee method1581 is in effect a structured, subjective
reconnaissance that uses flexible, loosely defined sampling areas (see Table 4}
and generalized ranges of cover estimates (see Table 5). Additional information
on growth habit (technically referred to as sociability), may be taken (see Table
6). Because of its subjectivity, the method may be the most cost-effective
means of detecting gross differences in community organization or species
assemblages associated with contamination. However, because Relevee is
highly subjective and only semi-quantitative, traditional parametric statistics are
inappropriate to analyze the data. It is important to remember that this
technique was developed to obtain information that could be used to classify
similar vegetation types in discernable groups. The method introduces a level
of discipline in the collection of data through an otherwise subjective
technique.
In the initial design, the investigator selects a "representative" site within a
particular vegetation stand. A single Relevee sample is recorded. Various
stands are sampled for the purposes of classifying vegetation types. The single
most important "assurance" of the quality of the data is the ability of the
investigator to select the representative site within the stand based on "prior
knowledge of what was typical" for the given vegetation.
For assessment of vegetation at hazardous sites, a series of Relevee samples
can be collected within the affected area and from adjacent unaffected zones.
These data sets can be then examined according to the traditional Braun-
Blanquet classification strategy.
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Table 4. Estimated Minimal Area For Each Relevee Survey For Selected Vegetation Types
VEGETATION TYPE
Temperate Forest
Trees
Shrubs/herbs
Grassland
Wetlands/Meadows
SURFACE AREA (M2)
200 - 500
200 - 500
50 - 200
50- 100
5-25
Table 5. Modified Braun-Blanquet Cover Class Ranges
COVER CLASS
1
2
3
4
5
+
r
RANGE, IN %
75 to 100
50 to <75
25 to <50
5 to <25
1 to <5
<1 to 0.5
Observed but so rare
as to not contribute
measurably
MEAN, IN %«
87.5
62.5
37.5
15.0
3.0
a Note: The algebraic mid-point of the cover class range is routinely used
in calculations, even though the values do not carry as many significant
figures as implied.
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Table 6. Braun-Blanquet Plant Sociability Classes
CLASS
CRITERIA
1
2
3
4
5
occurring in large, nearly pure stands
occurring in large aggregates, coppice or in carpets)
occurring in small aggregates, clusters, or cushions
occurring in clumps or bunches
occurring singly
(3) LINE-INTERCEPT
This technique offers a rapid means of assessing the relative importance of
predominant species. It may also be used to sample from images such as aerial
photographs, or microscope views. Typically, a line transect is established
along some bearing through the area to be sampled. At predetermined intervals
along the line a segment of the line is examined for contact with vegetation or
other objects to be sampled. The length o1 interval to be observed can be
determined just as plot size described earlier. In a low growing grassland, for
example, one might record the contacts along 1-meter segments every fifth
meter (See Figure 7 in Appendix I.).
(4) POINT FRAME
The point-frame or pin-frame consists of 10 pins mounted at uniform intervals
in channels in a frame. The pins should have a needle-like point. Theoretically,
the point has no dimension. Thus as a pin becomes blunt, and "acquires
dimension," the contact of the "point" is enhanced.- This leads to an over-
estimate of cover. Usually, the frame is supported by braces such that the pins
are angled at 45 degrees to the surface. The frame is positioned at a given
location and the pins are lowered through the channels. Because of these
nuances it is crucial to have the same technical staff using the same or
essentially the same sampling device to minimize bias.
Use of the point-frame technique is restricted for practical purposes to low-
growing herbaceous vegetation or cryptogams. Two major variations regarding
the type of date recorded are used commonly. These are aerial contacts and
30
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basal contacts. In the case of aerial contacts, each pin is lowered through the
canopy and each contact of the point of the pin with a plant part is scored.
Thus a single point may contact zero to several leaves or stems of one or more
species. To accomplish this procedure, there must be virtually no wind moving
the plants, since any movement will alter the potential contact loci. When
sampling basal contacts only, one scores only the objects touched by the point
of the pin as it rests on the surface. The information is recorded separately for
each frame (set of 10 pins).
As with any of the techniques, there must be some plan to locate the frame
within the area to be sampled and to determine the number of pins to be
scored. A common practice is to position the frame at predetermined intervals
along a transect. Some analyses suggest thar 1,500 pins might be needed to
acquire an adequate sample.1591 This, of course, is a function of variability of
the site and the accuracy required.
Calculations for the point-frame technique are identical to those for the line-
intercept technique. Simply substitute "pins" for "intercept length" and
"frames" for "intervals" in the several equations. Generally, however, one only
reports the Dominance (Cover) value, this may also be referred to as the
"Percentage Composition."
(5) VARIABLE RADIUS
Several methods have developed that utilize geometric relationships to estimate
plant densities. Instruments range from sticks with variable sized apertures
mounted at specific distances along the stick; to optical units with prisms and
range-finder adjustments. The fundamental relationship used in these tools is
that an object of a given size viewed from a distance occupies a percentage of
an arc. The methods use an aperture of given dimension placed at a fixed
distance from the eye. The tool is rotated through a full circle (360°) with the
eye "fixed" at the center of the circle. Objects that appear to fill the aperture
are tallied and used to calculate the density of trees or shrubs. The method is
used extensively in forestry, being particularly good in relatively even aged-
even sized stands.
(6) POINT-QUARTERS
The point-quarters is one of the most rapid, accurate and versatile sampling
techniques available. The initial use of the basic method was in the land
surveys conducted in the mid-1800's. Subsequently, the equations were
31
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developed to convert the data into the standard ecological terms, density,
frequency, and dominance. (See Figure 8 in Appendix I for additional
description of method.) Since no defined plot is established in this sampling
procedure, density is arrived at indirectly. The density is computed on the
assumption that the square of the mean point-to-plant distance represents a
measure of the area occupied by the plants sampled. The total density for the
sample is obtained by dividing the mean area per plant into the unit area of
which the density is to be expressed.
4. SUMMARY COMMENTS ON VEGETATION SAMPLING
Within each generalized method, the investigator has several options available
(e.g., position, plotless versus defined area plots, size, shape, number and
several other factors). The point-quarters method is by far the most efficient
way to quantify trees. For each point, the field data collected includes the
species, distance, and OBH of the four designated trees. If defined area
sampling is used,for each tree or shrub within the plot, there is a record the
species and some measure of size. The number of individuals or stems of each
species within each plot is recorded. An estimate of canopy cover may be
used as an estimator of dominance. For herbaceous plants, estimates of cover
or biomass are preferred. As an aid to estimating cover classes listed in Table
5 are often used. The cover value is recorded for each species present in each
plot. Alternatively, a harvest or clip-plot method is used to obtain aerial
phytomass values for each species within each plot. The vegetation is severed
at ground level and sorted according to species. The plant material is then
dried in an oven at 70 to 80 C for 24 hours (or until constant weight is
established. The material should be placed in a desiccator while it cools to
room temperature (especially in humid environments) and then the weight is
recorded. The raw data should be tabulated by plot and by species within each
plot.
B. TOXICITY TESTS
The most widely used acute phytotoxicity tests involving vascular plants are
the seed germination test (a direct exposure method) and the root elongation
test (typically performed with eluates). Interestingly, the seed germination
assay, often promoted as representing a sensitive, critical stage in the life
cycle, is rather insensitive to many toxic substances. The insensitivity results
from two factors: first, many chemicals may not be taken into the seed; and
second, the embryonic plant derives its nutritional requirements internally from
the seed storage materials making it in a sense isolated from the environment.
32
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Finally, from an ecological perspective, seed germination is relatively
unimportant for perennial plant species. Even for non-domesticated annuals,
extremely low percentages of seed germination are typical.1601
Short term tests with plants for toxicity testing were originally developed from
simple measurements used in plant physiology and weed science.1611 The tests
have been adopted to test single chemical and mixed chemical effects. More
recently they have been used to evaluate soil contamination. They are used to
test soils brought to the laboratory for ecological assessment of terrestrial
waste.162-63-64-65-6e-6?|
1. CLASS-I TESTS
al SEED GERMINATION/SEEDLING EMERGENCE
The seed germination test has been used extensively since standardized
protocols were introduced.|6e- 69- 70) Pre-sorted seed lots are exposed to test
chemicals in a soil matrix. Site soil or test chemicals are mixed with control
soils in a logarithmic series. Germination is made five days after initiating the
test. The effective concentration of the test soil to give a 50% decrease of
seed germination is used for determination of ECsQ. This test is considered as
a direct soil toxicity test. Species commonly used are chosen to cover four to
five types of plants. Alfalfa, beet, clover, corn, cucumber, lettuce, foxtail
millet, mustard, oats, perennial ryegrass, pinto bean, soybean, sorghum, radish,
and wheat have been reported most often.
b) ROOT ELONGATION
The root elongation test was developed as an indirect toxicity test. Roots are
exposed to water extracts and the soluble test soil constituents potentially
toxic to the growing roots. After incubation in a chamber with controls for
temperature and moisture, root length is measured. The ECso °f Tne test
group is calculated as the concentration of the extract that inhibits root length
of test samples by half that of the control samples. Preference seems to have
been given to lettuce as a test species.171- 7Z73-741
33
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ci ON-SITE GERMINATION TEST
A modification of the seed germination tests has was developed for field
use.1751 The on-site containers were kept under a canopy and shaded from the
sun and rain. Test performance was evaluated/against companion laboratory
tests. Biologically reasonable differences were obtained between field and
laboratory protocols with cucumber, lettuce and red clover but not with wheat.
The on-site version of the seed germination test requires special attention to
insure that quality control criteria are met. The principle advantage of the test
is the reduction of shipment and handling effort and their accompanying costs.
d) LIFE-CYCLE
Life-cycle bioassays are used to assess sublethal responses of plants to toxic
chemicals. Exposure may be either acute or chronic. The endpoints used to
quantify the effects of toxic chemicals include morphological and phenological
measurements that can be easily accomplished in greenhouse, growth
chamber, or field conditions. This system also allows examination of the roots
for morphological impact.
Two plant groups have been used in developing rapid life-cycle tests.
Arabidopsis™ and flrass/ca.1771 Arabidopsis is well characterized
physiologically and genetically and is ideally suited for laboratory assays.
Technical impediments arise from the prostrate growth habit and tiny seed size.
The small seeds virtually preclude measures of any parameter involving seed
counts (e.g., percentage germination, reproductive success). The rapid cycling
Brassicas have been developed by the Crucifer Genetics Cooperative of the
University of Wisconsin. This group of plants is gaining popularity as a model
system especially by molecular biologists and geneticists. The advantage of
Brassica compared to Arabidopsis include their upright growing habit and large
seed size. Relatively large variation in many growth parameters may limit the
utility of some potential endpoints. However, the short life-cycle permits up to
10 generations in a year. This offers good opportunity to investigate non-lethal
effects of considerable ecological import (e.g., reproductive potentially
reproductive success). These technical issues may preclude commercialization
of these life-cycle tests.
e) . FLOATING AND ROOTED AQUATIC PLANT GROWTH TESTS
Work performed with aquatic plants can aid the development of methods for
terrestrial and wetland ns'k assessments. The need for aquatic plant bioassays
34
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has been recognized for more than a decade1781. Duckweeds, floating vascular
plants of the Lemnaceae family, were used in the first true aquatic plant
toxicity bioassays. Single toxicant dose-response relationships have been
reported during the past decade,179- 801 Taraldsen and Norberg-King1811 recently
reported that for some effluents duckweeds were more sensitive than daphnids
or fish for determining effluent toxicity. Bioassay endpoints used in these
studies included reduction of frond production, reduction of root length,
biomass, 14C uptake, total Kjeldahl nitrogen and chlorophyll. Wang1821 has
shown that reduction of chlorophyll pigments can be a more sensitive indicator
of toxicity than frond production. These studies have shown that duckweeds
have utility as a bioassay organism.
The ease of culture and bioassay methods have been a good argument for the
use of duckweeds in aquatic bioassays. One problem with these organisms is
their inability to effectively sample contaminant bioavailability in interstitial
waters.However, duckweed has been used as a bioassay tool to detect
herbicide residues in saturated soils {personal communication L. W. Anderson,
US Department of Agriculture, Davis, CA). During this test, suspect soil or
sediment is placed in a petri dish and overlaid with a.film of water. Duckweeds
are placed on this film such that their roots are in intimate contact with the soil.
This test may have value as a rapid screening tool for terrestrial and wetland
soils. Nevertheless, rooted aquatic plant bioassays offer better promise for
evaluating sediment toxicity. Hydrilla verticillata Role (hydrilla), a common
aquatic angiosperm in the Southeastern'United States, is easy to culture and
handle, tolerant of a broad range of environmental conditions and has a fast
growth rate. Culture and bioassay methods have been reported and a variety
of endpoints evaluated.183-841 The most reproducible and toxicant related
endpoints were new root growth and peroxides activity. In addition, this plant
has been shown to play an important role in the uptake of sediment-
incorporated pesticides.1851 This may be an important route of chemical
mobility in the environment.
Hydrilla may prove to be a good sediment and water column toxicity bioassay
organism. Since it is an exotic species, however, it is impossible to use this
plant in the field of in-situ bioassays. This limits the ability to extrapolate
laboratory results to actual field sites. Other plants that have similar growth
and culture characteristics as hydrilla include E/odea canadensis, Myriophy/fum
spicatum and Potomogeton pectinatus. Following procedures similar to those
used with hydrilla Klaine has initiated laboratory bioassays with P. pectinatus,
and also begun development of in-situ sediment toxicity bioassays with P.
pectinatus. This work will determine how well laboratory bioassays predict the
response of the same organism in the field and examine how soil and sediment
sampling methods influence bioassay results.
35
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The Waterways Experiment Station (WES) of the U.S. Army Corps of Engineers
has developed methodology to quantify the uptake of heavy metals by marsh
plants.f86- "• 88- ••• 90- 91- "• 93- 94- 9S- 961 The methodology was developed for the
purpose of evaluating the suitability of dredged material for disposal on uplands
and for wetland construction. It is well documented that marsh plants
accumulate certain trace metals and that these metals may either cause toxicity
to the plant or may be passed along to higher tropic levels. This methodology
could be adapted for use in the evaluation of the level of toxicity in soils at
hazardous waste sites, and for predicting the effect of sediment associated
metal on plants established in a restoration effort. The methodology, in brief, is
as follows. Sediments to be tested are homogenized, air-dried, and placed in
containers. Specimens of selected species of plants are planted into the
sediment and allowed to reach maximum standing stock (normal duration - 90
days) under favorable growth conditions in greenhouse. The above ground
material is then harvested, extracted with DTPA, and analyzed for selected
metals. The biomass of the harvested material is also measured. Tests may be
conducted to evaluate phytotoxicity under reducing conditions (i.e., flooded).
The method has the advantage of being simple to run and indicative of the
effects of site specific soils on the plants that may colonize the system. Highly
repeatable results have been obtained by WES.
2. CLASS-II TESTS
Plant physiology endpoints provide a rich array of ecotoxicity options. For
persons able to perform Good Laboratory Practices, the test protocols are
relatively easy to learn. Unfortunately, the bulk of physiological methods have
been benignly neglected in protocols of regulatory groups (e.g., US EPA,
OECD). Potential tests for standardization could be chosen from those in the
United Nations Environmental Programme (UNEP) manual Techniques in Bio-
productivity and Photosynthesis.®^ A variety of biochemical and enzymatic
techniques are available. These biomarker techniques can be applicable to
acute or chronic toxicity endpoints and may be applied across several life-
stages. For a detailed discussion of biomarkers, see McCarthy and Shugart1981
Use of photosynthetic parameters to evaluate environmental condition has been
accepted conceptually as being important [the obvious linkage to higher level
ecological concerns]. However, it has been exceedingly difficult to develop
practical interpretations linking photosynthesis and plant yield.1991 Linkage to
environmental stress is equally difficult due to the many annual, seasonal,
diurnal variations compounded by differences among species.
36
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Faced with such problems, many are quick to dismiss photosynthetic analysis
as impractical. Much of the concern, and perhaps confusion over this issue,
comes from our general persuasion in ecology to emphasize differences among
species. Ecologically, plants exhibit wide differences in photosynthetic
efficiency, rates of carbon assimilation, adaptation to light conditions,
temperature, salinity, diurnal period, etc.; and there are various alternative
photosynthetic systems (i.e., C-3, C-4, CAM] adapted to different
environmental conditions.
a) PHOTOSYNTHESIS: GAS EXCHANGE
Uptake of C02 or 02 evolution are familiar biochemical techniques for studying
effects of chemicals on photosynthesis.t100-101-102-1031 Sophisticated methods
of analyzing photosynthetic condition are available.1104-105J Portable units can
be used to measure the "instantaneous" rates of net C02 uptake.
Modifications of the basic methodology also permit full canopy
measurements.11061 There are many technical considerations that require skilled
personnel to ensure reliability of the resulting data. If the proper precautions
are taken, however, excellent comparative data can be obtained to assess the
impact of stress imposed by hazardous materials on the photosynthetic
process. Relatively modest changes in protocols allow measurement of
respiratory rates of non-photosynthetic tissues or darkened photosynthetic
tissues.
Isotope discrimination can also be used to assess long-term ecological
conditions. The biophysical and biochemical features of leaves impose
resistance to the incorporation of C02.1107-108> 1091 As a consequence of this
resistance, plants discriminate among isotopes. This discrimination is
confirmed by a comparison of the natural abundance of 13C and 12C to the
abundance found in plants. Furthermore, the alternative photosynthetic
pathways among plants exhibit differing levels of discrimination. Basically, any
factor that affects the resistance of CO2 influx enhances the discrimination.
Thus stressors that affect stomatal opening can be expected to alter the
isotope discrimination. Peterson and Frye11101 provide an excellent discussion
of the processes of isotope discrimination and illustrate their uses for
ecosystem analyses through several case studies.
37
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bl PHOTOSYNTHESIS: FLUORESCENCE
The typical green terrestrial plant is well adapted to sensing and revealing
significant changes in its environment. This allows native plants growing in
natural settings to be used to assess changes which might be toxic to plant or
animal tissue. The basis of this bioassay is the chlorophyll molecule which
serves as an intrinsic fluorescent probe of the performance and capacity of
photosynthesis. Under normal conditions, 97% of the light energy absorbed by
chlorophyll is converted to biochemical forms of energy in photosynthesis.
Stress conditions can reduce the rate of photosynthesis, disturb the pigment-
protein apparatus, or block the light-driven photosynthetic electron transport in
the chloroplast. This results in an increased loss of absorbed light energy of 6
to 10% via chlorophyll fluorescence with a peak in emission at 683nm at
physiological temperatures. The inverse relationship between in vivo
chlorophyll fluorescence and photosynthesis has long been known as the
Kautsky Effect.
Light-induced chlorophyll fluorescence from dark adapted leaves can be
recorded with portable, sensitive instruments using intact leaves. This
nondestructive method essentially monitors the physiological well being of the
plant. Any stress including disease, nutritional stress, water, temperature,
radiation, and chemical stress can be quickly and accurately recorded. The
overall photosynthetic process can be thought of as a series of sensitive sites
connected to the fluorescent photosynthetic reaction center which respond to a
large number of different insults and report these effects as a change in
fluorescence. Chlorophyll fluorescence in intact native plants can be used to
assess toxicity in the environment or in a laboratory bioassay.
Fluorometric analysis of photosynthesis has gained wide acceptance as a
method to detect the genetic, biochemical, and physiological condition of
plants.i111- 112- 113- 114- 115- 116- 117' 118' Toxicological data specific to
photosynthetic systems has been collected on hundreds of chemicals and
several plant species over the past five decades. This rich assembly of
information makes chlorophyll fluorescence one of the most promising
biomarkers for detection of exposure and effects.
The chlorophyll fluorescence method is a good biomarker to evaluate
ecologically significant environmental stress. The test is sensitive, reliable, and
feasible. The method has great potential for use in pesticide and toxic chemical
risk assessment, hazardous waste site assessments and ecological monitoring
programs. The underlvtng science of plant fluorescence is better known than
that for most other biological method used to evaluate environmental effects.
The fundamental information regarding plant fluorescence dating to the 1930
was summarized by Franck & Loomis in 1943.f
38
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The early work, and that which followed from the 1950s through the 1980s,
was focused on dissecting the mechanisms of plant physiology in general and
photosynthesis in particular. The wealth of information acquired provided an
excellent opportunity for use in environmental stress biology. In the mid- to
late 1980s, applied uses began to receive attention.[12°- 121i In the two-year
period 1989-1990 some 73 articles on chlorophyll fluorescence have appeared
in scientific journals (D. Miles pers. comm.). Coincident with this broadening of
the subject into environmental topics, instrumentation has been developed to
facilitate field measurements. One such report described in considerable detail
the inner workings of a portable instrument and provided data on willow,
fireweed, scotch pine, corn, and birch leaves plus spinach chloroplasts.
Any of the Atrazine type herbicides bind at or near this site causing a block in
electron transport and an immediate response in chlorophyll fluorescence.
There have been several similar examples in the literature of the use of
chlorophyll fluorescence to monitor the presence of herbicides in the
environment.!122-123-1241
Other inhibitors of electron transport which affect fluorescence are heavy
metals.'1251 The effects of lead, cadmium, and mercury on photosynthetic
electron transport have been studied by Miles and co-workers.(126- 127- 128- 1291
These metals either increase or decrease the level of F^ or Fv. With limited
experimentations we can predict with some precision the site of interaction of
these compounds with electron transport.
Specific genetic mutants of photosynthesis in the higher plants have also been
very useful.[130' These genetic mutants have lesions in a variety of sites
throughout the photosynthetic process and each has a characteristic effect on
the fluorescence emission. By knowing the locus of the mutation, we can now
correlate change in specific photosystems with the emission characteristics of
fluorescence. Working in reverse, it is possible to measure an effect of any
type of stress on photosynthesis and with our available knowledge predict the
reaction or sets of reactions in photosynthesis that may be responding to this
stress.
This aspect of chlorophyll fluorescence has been used in a variety of
environmental studies. In the study of stress, the effects have been
quantitated by the use of chlorophyll fluorescence.1131- 132- 133- 1M1 Water
stress,'135- 1361 nutrient stress, high'137- 138' or low'139- 14°- 141> temperature
stress,(142- 1431 the effect of high light intensity11441 and or ultraviolet light, all
have been monitored through the changes in chlorophyll fluorescence emission
In addition, even gaseous pollutants affecting entire plants can have an effect
on the emission of light-energy in fluorescence. Studies of ozone damage m
leaves have utilized fluorescence monitoring1145'
39
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As with any good scientific study, the use of whole plant fluorescence requires
well selected control or reference plants. The controls must be measured under
the same conditions as the unknown. If a number of factors are stressing a
plant, these data can still be used provided the test plants only differ from
controls by a single factor. The selection of the reference plants is extremely
important. A detailed discussion of the method is presented in Appendix II.
c) PEROXIDASE
Peroxidase activity has been studied extensively in plant physiology
laboratories. The peroxidase assay shows some promise for use as a biomarker
for phytotoxicity assessment. Bioassays with Hydrilla on sediments from
Superfund sites on the Great Lakes (organics and metals) incorporated five
bioassay endpoints: shoot growth, root growth, chlorophyll a, dehydrogenase
activity and peroxides activity. Good correlation between sediment chemical
content and plant response was observed. Stepwise regression indicated
extremely good prediction (r2= 0.982) of peroxides activity based on the
sediment concentrations of Hg, Zn, Pb, Ag, and Cd. In addition, new root
growth was correlated with the combination of anthracene, fluoranthrene and
chrysene (r2 = 0.872). This large number and concentration of contaminants
in these sediments made it difficult to determine which organic(s) or metal(s)
were causing the biological stress. The bioassay, however, responded very
well to total chemical burden. Each site was ranked based on the response of
each bioassay endpoint. The sum of these endpotnt rankings was used to
reorder the sites from least toxic to most toxic. This corresponded rather well
with total metal concentration and total organic concentration (Table 7).
Considering that such metal or organic was considered equitoxic (no attempt
was made to determine toxic equivalents for each chemical species) the
similarity between the sediment rankings was significant. Work in proceeding
presently to determine toxic equivalent from single species dose-response
relationship.
Klaine and coworkers used this bioassay, and the same five endpoints, to
determine the effectiveness of remediation efforts on some riverine sites in
Ohio. Sediment samples from the Cuyahoga River, Black River and Toussant
Creek were compared with control sediments from the Old Woman creek in
Ohio and the Florissant River, Missouri, both before and after remediation
efforts. The Missouri control sediments from the Florissant River were provided
by US Fish & Wildlife, Columbia, Missouri. These sediments have been used as
controls in the laboratory for three years and provided a measure of the
40
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reproducibility of the plant bioassay A statistical analysis of the Missouri
controls for each endpoint indicates no significant difference between sampling
times (Table 8). Hence, there were no apparent procedural differences
between sampling times 1 and 2 that caused a change in the endpoints.
TABLE 7. Ranking Of Sediments From Least To Most Toxic Based On Cumulative Rank
For All Five Plant Bioassay Endpoints And On Total Metal And Organic
Concentrations.
ORDER
(least toxic)
(most toxic)
CUMULATIVE
RANKING
BR107
BR108
BR101
BR109
SR103
BR103
SR110
SR106
IH104
IH106
IH103
IH107
METAL
CONC.
SR110
BR109
BR108
BR107
BR103
SR106
SR103
BR101
IH104
IH103
IH106
IH107
ORGANICS
CONC.
SR110
SR106
BR108
SR103
BR109
BR103
BR107
IH104
IH103
IH106
BR101
IH107
The only indicates of toxicity in these assays was the peroxidase activity (Table
8). The severe reduction in peroxidase activity exerted by the Black River
sediments suggests that senescence was occurring. Sublethal chemical stress
on H. verticillata typically induces increased peroxidase levels such as those
seen in the second sampling. Single chemical burden data generated in this
laboratory indicate that this response is dose-dependent until plant senescence
begins to occur. Then peroxidase activity is generally less than that of the
control. Data from the second sampling are consistent with this observation.
The post-remediation sample exerted less stress than the pre-remediation
sample; but, the post-remediation sample still caused a significant sublethal
stress on the organism (peroxidase activity was significantly higher in the Black
River sediments than in the Missouri control or Old Women Creek sediments)
which suggests that remediation, while measurable, was not 100% effective.
-------
TABLE 8. Response of root growth, shoot growth, dehydrogenase activity, chlorophyll 0
concentration and peroxidase activity in Hydrilla vsrticillata to whole
sediments.*
ENDPOINT
ROOT GROWTH
Control
owe
BR
TC
CR
SHOOT GROWTH
Control
owe
BR
TC
CR
DEHYDROGENASE
Control
owe
BR
TC
CR
CHLOROPHYLL a
Control
owe
BR
TC
CR
PEROXIDASE
Control
owe
BR
TC
CR
FIRST SEDIMENTS
mean
10. 2a
9.2a
4.6a
13.0a
3. la
8.la
5.3a
8.6a
30.6a
46 3a
70.7a
50 2a
1 3a
0.9a
1.4a
1 3a
1.90a
203a
0.83b
1.90a
s.d.
6.8
1.7
2.3
3.1
1.7
4.9
3.4
2.2
4.3
12.1
22.4
82
053
003
041
022
0.09
0 35
0.03
042
SECOND SEDIMENTS
mean
16.5a
I2.3a
9.3a
7.3a
3.7a
6.2b
8.2b
3.5a
12.7a
12.4a
15. 5a
2.6a
1.29a
1.09a
1 52a
3.6a
2.40a
2.60a
4.lOc
3.80C
s.d.
11.7
2.6
2.3
5.9
3.3
1.8
2.0
2.5
8.7
3.9
6.3
1.8
0.41
0.33
0.07
1.80
0.40
0.33
0.62
0.24
Control = Missouri control sediments {Florissant River) from U. S. AF&W, Columbia, MO
OWC = Old women Creek. BR = Black River
TC = Toussant Creek CR = Cuyahoga River
"similar letters after means indicate those means not statistically different from each other
(p = 0 051
42
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di PQLYAMINES
The increase of polyamines in plants exposed to chemical can also be used for
phytotoxicity measurements.|146- 1471 Similarly, a test for detection of
glutathione-S-transferase shows promise.11481 Wettlaufer et al.11491 reported
changes in polyamine titer specific for each metal (Cr, Co, Cu, Hg, Ni, and Ag).
However the change in titer was relatively small compared to that for other
stresses. Therefore it is not a good candidate as a field biomarker. The
response could be useful in controlled laboratory investigations into
bioavailability provided other stresses are minimized or excluded.
e) DIMTROGEN FIXATION
Dinitrogen fixation assays provides multiple assessment endpoints. This
complex system is well characterized genetically, morphologically, and
biochemically for free-living and symbiotic systems.1150- 1511 Especially in the
symbiotic groups measurement endpoints include nodule number, nodule size
and various measures of dinitrogen fixation capacity. The Acetylene Reduction
Assay is rapid and easy to perform. Garten'152' compared dinitrogen fixation
assays with multiple toxicity test methods. Although his analysis reports only
moderate correlations with the other tests, it should be noted that some of the
dinitrogen fixation data used in his analysis came from measurements that were
not made according to routine precautions for this test method. The test
probably holds more promise than concluded by Garten.
f) GENETIC TOXICOLOGY ASSAYS
Numerous opportunities exist for genetic analysis. The karyotoxicity of
pesticides and fungicides m mitosis of root meristems has been well
documented, with recent reviews giving more than 270 references.1153- 154-155-
156> The choice of plants used vanes, but Tradescanfia plants have been used
for a wide variety of bioassays using the various endpoints for genotoxicity
listed betow. The length of exposure of the meristem depends on the celt cycle
duration but is usually limited to 6 hrs and not recommended above 48 hrs.
These studies require some experience in karyotyping. The effects observed
fall into four groups:
o Clastogenic changes or changes in the longitudinal integrity of the
chromosomes.
43
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o Aneugenic changes or disruption tn chromosomes during cell
division.
o Mutagenic effects, which are more convenient to observe in the
upper plant parts include changes in the color of petals or staminal
hair, in petunias, or the appearance of pollen in rapidly growing
plants such as Brassica or deficiencies in the pattern of chlorophyll
within the leaf blade.
o Unusual effects such as variation of nucleolar appearance, atypical
extension of the centromeres, reduction in the number of chiasmas.
These also account for clumping of mitotic figures, formation of
permanent mitotic figures which prevent cell separation, pycnosis of
the nucleus.
Often, the plants, following their short exposure to potential toxicants are
allowed to proceed to seed set and the quantity of seed sets noted. Pollen
cells are also studied for nuclear abnormalities.
The doses at which these aberrations are first noted usually provide upper
limits of cytotoxic thresholds. Statistical treatment of this data provides
validity for the cytotoxic thresholds. Rapid data processing using image
analysis for cytogenetic bioassays has been reported.11571 This may now be
developed further with "expert system" software to enhance use of this
method.
The limitations of these cytogenetic examinations often come from different
interpretations given by examiners on different tests. However, in a number of
test comparisons, different laboratories arrived at the same score for the same
test (e.g., with root systems). Often, the doses selected for these tests seem
to be selected without validity and the experiments terminated pre-maturely,
that is that the dividing cells are not allowed to go through recovery of their
cycle. This assay system also needs standardized conditions of plant growth,
estimate of the normal frequency of aberrations in control plants, as well as the
use of proper positive, negative and solvent controls.
At the population level, analysis of genetic diversity holds great promise.
Guttman and his students'16-8- 1591 have shown a trend for "genetic
bottlenecking" in populations subjected to stresses. The same principles of
selection are likely to hold for plants.
44
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Q) CELL CULTURE ASSAYS
The effect of pesticides, toxic chemicals and metals on plant growth and
metabolism has been investigated using plant suspension cultures (i.e. plant
cells growing in liquid nutrient medium as indicated in the many chapters in
Vasil.11601 Callus cultures (i.e. associated plant cells growing on solid medium
made with agar or other types of natural polymers and containing the test
chemicals) can also be used.
Preliminary testing of responses of plant cells in culture to xenobiotic
compounds permit analysis of plant toxicity.1161- 162] Because the cultures are
devoid of microbes, the response and the metabolism of the chemicals of
interest by plant cells alone can be studied. Should the occasion arise, the
combined effects of plant cells and the microbes commonly found in
association with selected plants can be studied in experiments where a single
variable is changed at a time.11631
In estimating the extent to which toxic wastes disrupt a plant community, or
in determining what remedial action is necessary to restore a natural plant
community, it is important to acknowledge that most natural plant
communities are comprised of a cross-section of physiologically diversified
taxa with variable responses to chemical insult. The second point is well
illustrated by summary data showing that similar response of two taxa to a
chemical only occurred when the taxa were in the same genus.116*1 Thus in
order to accurately evaluate the toxicity of contaminated soil to a natural
plant community, phytotoxicity testing must include a broad representation
of physiologically different taxa. Testing a large assortment of different
kinds of plants under greenhouse or growth chamber conditions can be very
costly. A simpler and more cost efficient approach is to use tissue cultures.
However, numerous questions are often raised as to whether or not tissue
culture cells are a true reflection of intact plants grown in soil. The
advantages and disadvantages of using culture cells for phytotoxicity testing
are discussed in this paper.
Numerous investigators have conducted studies to evaluate the use of
tissue cultures in phytotoxicity testing.'165- 166- 167- 168- 1691 In general the
various assay systems that have been described share many common
features. Established cell lines that have been in culture for several years
are used as test tissues. Defined medium tailored for the test tissue is
provided as either solid agar or liquid medium. Usually 15 to 40 ml of
medium is placed in flasks ranging in size from 50 to 125 ml. The test
chemical or mixture is usually provided in the starting medium but could be
aseptically added at some point during culture growth if so desired.
Phytotoxicity of a test chemical is determined by comparing the response of
test cultures ( + chemical) with that of control cultures (- chemical). At the
45
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simplest level, cultures exposed to toxic chemicals can be monitored for
growth alone. This is done by sampling known aliquots of cell suspensions,
centrifuging the cells in tared tubes and taking wet and dry weights.
Generally wet weights are found to be sufficient.1170- 171> In many cases,
where there is possibility that the cells under study are not multiplying in
culture but only enlarging by solute and water uptake, a ratio of wet and dry
weights is most useful. Several additional endpoints have been measured to
assess phytotoxicity such as: growth parameters (packed cell volume, cell
number); or cell or molecular events (precursor incorporation into
macromolecules, membrane permeability to fluorescein, and reduction of
triphenyltetrazolium).'172|-
(1) ADVANTAGES
The two main advantages of tissue culture tests versus whole plant assays
is that culture tests are relatively inexpensive and more reproducible in
comparison to the latter. Once the cultures have been started they can be
grown for one to four weeks without any maintenance expense such as the
addition of nutrients or water. A space measuring 4'x4'x4' will
accommodate approximately 540, 125 ml flasks or 1180, 50 ml flasks. In
contrast to these conditions, if plants are grown in a conventional
greenhouse it takes approximately 450 sq ft of space to maintain 1200
plants in 3" x 3" pots, and some degree of maintenance (watering, etc.) is
required almost daily.
The second major advantage of culture over intact plant tests is the high
reproducibility of the culture tests. The biological variation among the
replicate samples in a culture assay is minimal since the inoculi are taken
from a single genetic source, whereas plants grown from seeds are subject
to a greater degree of genetic variation. The nutrient, water, and
temperature conditions for cultural cells are uniform throughout the day and
year, whereas plants grown under greenhouse conditions in different parts
of the world will have much less uniform growth conditions, often times the
basis for substantial variability in test results. Seasonal variation in
photoperiod and light quality can lead to large variation in plant response if
grown under greenhouse conditions.
Although little oes been done to date, tissue culture techniques offer
excellent opportunity to evaluate toxicological impacts on endangered plant
species. Non-destructive methods are available that permit culturing of
tissues (including meristems for regeneration). Sensitivity of endangered
species to specific chemicals or site samples can be addressed. Similarly,
46
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slow growing perennial species can be tested once tissue cultures are
derived.
(21 DISADVANTAGES
The concern expressed most often about the use of plant cultures in
phytotoxicity testing is whether or not the chemical response of cultured
cells is an accurate reflection of the intact plant. Two arguments may be
used to defend culture, cells in this regard. The first is a theoretical
argument that most cell lines in use are rapidly growing non-photosynthetic
cells whose physiology and metabolism are typical of non-photosynthetic
root tissue which is the exposed portion of plants growing in contaminated
soil. The second argument is that research addressing the question has
shown that in general plant cultures when used properly do reflect the
phytotoxicity of chemicals to intact plants. In studies cqnducted by Zilkah
et al.,1173-174-1751 it was shown that in general there was a good correlation
between the response of seedlings, callus, and suspension cultures. The
exceptions were that cultured cells showed a response to some chemicals
which seedlings did not, and the chemical toxicity of photosynthetic
inhibitors was only detected by green cultures and seedlings.
Another important consideration comes from the common practice of
measuring phytotoxicity at a fixed time1 following chemical treatment. This
is an acceptable and reliable practice if the phytotoxic compound acts
rapidly and completely kills the tissue. However, if the phytotoxic effect is
more subtle and only slows or delays growth than treated cells may catch
up with control cells over time. Thus in using a tissue culture system it is
important to know the growth kinetics of the control cultures and compare
the growth increment of the treated and control cells over a period of time
when there is a continuous net growth of the control cells.
A disadvantage which is seldom mentioned in using cultured cells is that
studies conducted with several physiologically diverse taxa, as required in a
comprehensive test system, require a substantial amount of bookkeeping
and organization. For example several different media must be prepared to
satisfy the needs of different celt lines, different transfer and harvest dates
must be selected to match the growth kinetics of individual cultures, and a
concerted effort must be made to follow a regular schedule to insure the
uniform growth of cultures. In contrast when seedlings are grown from
seeds there are breaks in scheduling, the same soil mist and maintenance
procedures are used for all plants, and individual assays may be started and
stopped on convenient dates.
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Tests using plant cell cultures to assess the toxic effects of chemicals are
limited in that they do not predict what whole plant tissues would be affected.
In a number of cases, cells in culture can be manipulated to differentiate into
plants.1176-1771 This factor can be used in a chronic assay, with the number of
plants obtained from treated cells being compared to control cells. Plant cell
cultures can be maintained by regular transfers over many years to show highly
predictable growth parameters. Long term cultures have been preferred for use
in phytotoxic tests.1"8- 179- 1801 It can be argued that such cultures lack cell
variability which is a desirable condition for phytotoxic testing. Tests using
plant cell cultures can be improved by testing established cell tines from
specially selected plants together with cell lines which are stable but have been
in culture for only a few months.'1811
To take advantage of the wealth of physiological and genetic information
developed in the basic plant sciences it is appropriate to consider Arabidopsis
thaliana, Brassica napus (both from Brassicaceae) and Medicago sativa, (a
legume) for toxicity testing. These plants have been used in very limited
fashion for toxicity tests despite their versatility as experimental models.1182-
IBS. 184. IBS. IBS] yse Of these or other model systems commonly used in
physiology and genetics would provide a beneficial connection between the
basic and applied studies, especially where discovery of mechanisms of toxicity
are important. Arabidopsis has been recently selected internationally as a
representative plant for the determination ot its entire genome. Information on
all aspects of this plant is accumulating at a rapid pace
-------
relationships among plants affected by xenobiotics.'1871 The approach
developed by Pfleeger can be described as an experimental terrecosm.
Generally the impact on natural plant communities from the release of organic
chemicals into the atmosphere, both as applied pesticides and industrial waste
products, is not well understood. To study the potential impacts of such
stressors in a reasonable time, artificial plant communities were established
using soil containing the seed bank from an annually plowed field that had no
pesticide application for over ten years. The communities were grown in raised
beds producing a community area of O.B m2. Atrazine, 2,4-D and malathion
were applied at two concentrations, at or below the manufacturers'
recommended level except the high malathion treatment, with all treatments
done in triplicate. Measurements were made on eight major species, as well as
effects of interspecific competition on two target species. Cover by species
was monitored over time in nested neighborhoods of 10 cm and 20 cm around
individuals of Poa annua and Calandrinia cHiata. Neighborhood biomass and
total community biomass were harvested after all species began flowering.
Community production decreased with atrazine and 2,4-D treatments, but not
with malathion. All tested compounds modified species abundance. The most
notable effect was the alteration of dominance and the simplification in
communities treated with atrazine and 2,4-D and, to a lesser extent, malathion.
There were four general response patters exhibited by a species' biomass in
treated communities: it 1) decreased, 2) increased, 3) was unaffected or 4)
decreased only at the high concentration. In one significant exception, Erodium
was equally reduced by malathion at both concentrations. Organic chemicals
altered interspecific competitive relations for all treated communities. Chemical
treatment changed the identify of consistently competitive species (i.e., species
significant in at least three or four sampling times) and the timing of
interactions. Each target species had its own suite of competitors that
individually changed with chemical treatment. Ten cm neighborhoods had
more competitive interactions than the 20 cm neighborhood, when cover was
used as a predictor of competitive influence. However, when biomass was
used, the 20 cm neighborhood accounted for more interactions. Neighborhood
cover was a more useful predictor of target biomass than final neighborhood
biomass, because it was simple to use, indicated more species interactions, and
was nondestructive. This use of artificial plant communities to study the
effects of organic chemicals is simple and economical, and the experiments
generate small amounts of contaminated waste. Simple modifications of the
test method to incorporate site soil as the test variable can be made. The
method also uses non-domesticated plants, which is uncommon under current
federal regulations, but reduces the environmental heterogeneity common in
most field studies. The-method is amenable to transport and is appropriate for
studying other processes in plant communities.
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V. INTERPRETATION
The toxicity test exposure can be either direct or indirect. Test conditions vary
from field tests, glass house, growth chamber to culture flasks. Direct
exposure is achieved if the test soil is incorporated into the test as soil.
Indirect tests are those that are derived from some extraction of the test soil
such as occurs with elution; the eluate then being used as the test material.
There are advantages and limitations of either test approach. Direct tests
provide a more defensible evaluation of toxicity since they relate to potential
exposure conditions. However, the direct tests are more difficult to analyze
with respect to relevant contaminant concentrations, (i.e. , the soil solution
concentration rather than total matrix concentration]. In most cases there is a
high level of uncertainty in the extrapolation of toxicity conditions inferred
between direct and indirect test methods.
Mixed contaminants continue to confound efforts to evaluate toxicity.1188' 189'
With respect to metal toxicity, there has been some progress in understanding
the additivity effects especially in aqueous exposures. In the soil matrix there
are few guidelines to evaluate interactive effects due to metal contaminants.
Questions of availability (i.e. , what is actually in the exchangeable fraction) are
made more problematical by uncertainties of uptake and physiological
consequences of exposure to multiple contaminants. Clark et al.|190] exposed
plants to nutrient solutions with deficiencies of required nutrients and excess of
several metals to examine interactive effects of metals. Their information
illustrates significant difficulties of interpreting tissue concentration data as an
endpoint for toxicity assessment. Similar difficulties apply to organic toxicants
as well.
A. BIOLOGICAL FACTORS
i. INTERACTIVE PLANT-MICROBIAL ASSOCIATIONS
Both the seed germination and root elongation tests, as well as the majority of
other laboratory tests, as described herein, fail to consider the integration of
ecosystem processes; that is to say, the effects of the xenobiotic on the
rhizosphere. This zone, in the immediate vicinity of a root, contains microbes
and other biota which influence the root and are in turn affected by the plant
root.
The roots provide prime environments for bacterial and fungal populations, the
so-caJJed rhizosphere effect. Estimates11911 of the bacterial bJomass to a soil
depth of 30 cm range from 32 to 76 g nrr2 and of fungal biomass from 84 to
50
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117 g rrv2. Indirect evidence of root-mediated microbial activity can be
observed in bacterial methane production. In a water-logged soil, methane
evolution increases some 6-fold if rice plants are grown in the soil and 12-fold
during periods of illumination compared to control soils having no growing
plants.11921 Since plants are leaky, various chemicals escape the confines of the
root, and shoot tissues becoming ready sources of metabolites and essential
growth factors for microorganisms proximal to the root.11931 Bacteria live on
the organics liberated from roots. This "rhizo-deposition" occurs as soluble
organics (10 to 100 mg g-1 root) and as muciget plus root cap 20 to 50 mg g-1
root}. Fungi may derive sustenance from neighboring dead roots.11941
Environmental conditions, developmental stage, associated microorganisms,
and neighboring plants dramatically alter the quantity and quality of chemicals
exiting a plant.11951 This nutritive pool fosters or inhibits the growth of specific
heterotrophic organisms which span a continuum from lethal pathogenic forms
to obligate, mutualistic symbionts:
INTERACTIVE PLANT-MICROBIAL ASSOCIATIONS
Lethal Obligate
Pathogenic Neutral Mutualistic
-1.0 0.0 +1.0
I. I
The mutualistic associations involving higher plants and microorganisms
undoubtedly have their origins as victim-pathogen associations in evolutionary
time scales. What has evolved are highly regulated environmentally sensitive
partnerships with varying degrees of dependency and biochemically regulated
interactions.
al BACTERIA
Associative bacteria exist asymbiotically in the root zone of plants. Some
genera are capable of reducing atmospheric nitrogen to ammonia and
assimilating this nitrogen into organic forms. Several genera of bacteria, most
notably Pseudomonas associate with roots and influence the uptake of iron and
other nutrients.
Symbiotic relationships between plants and bacteria are typically recognized as
Legume-/?fi/2o6fL/m and actmorhizal associations. These dinitrogen fixing
51
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symbionts have been researched extensively. Detailed genetic maps and
physiological processes are well characterized. Whereas the legumes are most
prominent in agricultural settings, the actinorhizal associations are most
significant ecologically.
b) MYCORRHIZAS
Mycorrhizal infections were established on the earliest land plants. Under most
field conditions, the normal form of nearly all higher plants is in an association
with a suitable fungal partner. Several types of mycorrhizas are recognized
based on infection morphology and fungal taxonomy. The most widespread are
Vesicular-Arbuscular Mycorrhizal (VAMI, ectomycorrihizae, and
ectendomycorrhizae. The most widely accepted roles of mycorrhizae and (1)
facilitation of phosphate uptake and (2) increased tolerance to drought.|198-197-
1381
The VAM plants have increased access to phosphate resulting from (1)
alterations in the root morphology leading to increases in root mass, (2) hyphal
extension into soil zones otherwise unaccessible to the plant root, (3) increased
phosphatase activity, and (4) a lower shoot/root ratio.11991 Phosphate
availability determines whether the VAM association will be beneficial to the
plant.. At low phosphate concentrations, plant growth is reduced over that of
controls as the amount of phosphate available to the symbiosis is too low to
result in an increase in net photosynthesis. Intermediate levels of P favor the
association. High phosphate concentrations result in reduced plant growth as
the fungus tends to grow "out of control," becoming pseudopathogenic.1200'
The dynamics of carbon allocation in mycorrhizal plants has been studied in a
clover root-mycocosm system.1201- 2021 An excellent review of the relationships
between stresses and carbon allocation has been submitted for publication.>203>
The interplay between mycorrhizae and soil properties is summarized by Miller
& Jastrow.'204'
The rhizosphere dynamics influence fate and transport of toxic substances.
Limited work on uptake and metabolism of xenobiotics and metals has revealed
the importance of the plant-microbe relationships.1205- 2081 Comparisons among
mycorrhizal and non-mycorrhizal plants exposed to metals or pesticides exhibit
wide ranges of responses. Toxidty end points (growth or internal tissue
concentrations) may be inhibited or stimulated depending on which plants,
which fungi, and which toxic substance is involved.12071 No clear patterns of
the iGsponsus can t)e discerned at piesent. Consequently modeling uffui i& That
project uptake, transport, or fate of xenobiotics and metals are not likely to
predict real world responses. Advances in molecular genetics using
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taxonomically distinct probes are showing promise for use in identification and
monitoring of mycorrhizal populations in soils.l208-209'210'
2. BIOCONCENTRATION FACTOR.
When considering the uptake of contaminants from soil by plants, it is best to
express the concentration of the material in terms of the bioconcentration
factor (BCFJ which is the ratio of the amount of material present in the plant
tissue to the concentration of the material originally present in the soil. BCFs
calculated on the fresh weight basis of plant tissue are approximately ten-fold
smaller than those calculated on the dry weight basis. The ratio of course
depends upon the amount of water in the plant tissue. O'Connor, et al.'2ni
found that bioconcentration factor for dinitrophenol on the basis of 14C ranged
from about 0.001 to 0.64. They noted that the determination of BCF on the
basis of the partition of radioactivity does not take into account the multiplicity
of compounds which may form in both the soil and the plant tissue.
3. ACTION OF STERILE SOIL.
When first considering the action of plants on xenobiotics in the environment, it
is necessary to take note of the fact that even sterile soil is a very complex
organic material which frequently exhibits unknown and often unexpected
catalytic activities. It is quite common for various types of organic reactions to
be catalyzed on the surface of clay particles. There are many theories that
postulate that the initial formation of chirality in organic compounds and,
indeed, the initial formation of many complex organic compounds occurred on
clay under abiotic conditions and ultimately resulted in molecules complex
enough to be recognized as "living." Gordon and co-workers have noted that
many simple clays are able to catalyze the polymerization of phenols,
particularly catechols, to complex colored compounds amongst which are
presumably various types of diphenylene dioxyquinones. The abiotic role of
soil, thus, must be carefully considered. Anderson, et al.12121 have studied the
fate of a number of volatile compounds in sterile soil: methyl ethyl ketone,
tetrahydrofuran, chlorobenzene, benzene, chloroform, carbon tetrachloride,
xylene, 1,2-dichtorobenzene, c/s-1,4-dichloro,-2-buiene, 1,2,3-trichloropropane,
2-chloronaphthalene, ethylene dibromide, hexachlorobenzene, nitrobenzene,
and- toluene. They found that there was a rapid disappearance of these
compounds due to abiotic factors during the first seven days of application to
soil. They had great difficulty achieving mass balance and considered this
partially was due both to non-reversible absorption phenomena and some
storage conditions. Dec, et al.12131 examined the metabolism of 2,4-
53
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dichlorophenol which was incorporated into a synthetic humus prepared by
polymerizing dichlorophenol with a number of phenolic compounds in the
presence of horseradish peroxidase. They also prepared a humic acid complex
in which the dichlorophenot had been absorbed on the surface of naturally
occurring humic acid by means of horseradish peroxidase. Mixtures of free
dichlorophenol and humus were also prepared. They found, contrary to
expectations, that mineralization of dichlorophenol from the synthetic humic
acids was greater than free dichlorophenol. Thus, it appears that the catalytic
reactions which resulted in the binding of dichlorophenol to the humic acid
rendered the material more chemically reactive and subject to mineralization.
O'Connor, et al.12141 studied the behavior of dinitrophenol in soil in which
various types of municipal sludges had been added and found that the
degradation was rapid.
4. ACTION OP NON-STERILE SOIL.
Normal soil, as it is usually encountered, is teeming with complex forms of life.
A large percentage of normal soil consists of bacteria, fungi, and numerous
microscopic and macroscopic organisms. Many of these organisms react with
xenobiotics which are added to the soil and tend to modify the chemical nature
of the xenobiotics. In the case of many toxic compounds, it is well known that
there is selection of organisms which tend to break down the xenobiotic
materials. Soils, which have been used as dump site for many types of
chemicals, are often used as sources for bacteria which are able to break down
the material in question. In addition, plant life tends to modify the composition
of soils. Plant roots exude a number of components into the soil and some of
which tend to feed various fungi which greatly aid the growth of the plant. In
addition, plants elaborate a number of enzymes into the surrounding soil and
make biologically available various materials, such as phosphates which are
necessary for the growth of the plant. Thus, a plant growing in an enriched
humus represents a complex interactive and changing ecological system.
Furthermore, it is difficult to define the role, or the composition of this system,
since it varies depending upon temperature, humidity, oxygen content, distance
below the surface of the soil, drainage, etc.
5. THE RotE OF PLWJT PURIFYING AQUEOUS GWIRONMENrs.
The role of plants purifying aqueous environments has received some attention.
There are a number of sites around the world, such as in Vernon, British
Columbia, where popJar trees ate used to remediate a munJcipaJ sJudge and to
dispose of municipal waste water by transpiration. In a similar fashion, sweet
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gum trees are used on the East Coast to handle municipal waste water and
sludge. There are a number of experimental setups, a number of which have
been written up in the popular press, where a number of plants such as water
hyacinths, duckweed, and watercress are used in "living machines" to purify
municipal waste water.'215-216'
6. OVERALL UPTAKE AND METABOLISM OF XEIMOBIOTICS BY PLANTS.
A number of studies with xenobiotics, particularly pesticides, have indicated
that there are a number of phases in the fate of xenobiotics in plants. These
are:
o Absorption by the roots.
o Possible metabolic alteration in the root tissue. These processes can
include reduction, oxidation, or hydrolysis. Various conjugation
reactions are possible or the complete oxidation of the xenobiotics
can occur.
o Deposition and detoxification of xenobiotics by conjugation or
polymerization to cell wall components such as cellulose or lignin.
o Breakdown of the plant cell during autumn senescence followed by
the possible re-utilization by other plants or animals.
The details of these processes are summarized in the following four
subsections.
a) ABSORPTION
Xenobiotics enter plants through the roots along with nutrients and water.
They enter into the free space of the root tissue and then eventually make their
way either into the phloem or the xylem. The various membranes involved in
this process to some extent act as barriers to the entrance of the xenobiotic. A
number of studies have been made in an effort to obtain some predictive values
for the adsorption and translocation of compounds.'2171 It was proposed that
the adsorption and. translocation could be predicted on the basis of the
partitioning of the material between octanol and watei [log Kow]. Me Farlane,
and co-workers12181 have concluded that one can generalize that compounds
which possess log Kow values in the 1, 3 range, molecular weights less than
300, end pKe vetoes that tlo not favor ioniration at neutral pH values tend to
enter plants by passive diffusion, and move up in the transpiration stream.
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b)
METABOLIC ALTERATIONS.
A number of metabolic reactions and conjugation reactions have been
determined for xenobiotics in plants. As seen in Figure 5 taken from a review
of the molecular fate of 2,4-D'2191 there are a number of hydroxylation and
oxidation reactions which can occur, followed by conjugation of the oxidized
materials to glucose or by conjugation of the side chains to various amino acids
such as glutamic and aspartic acid.
a—-*—*— «-«,—«.
«
O —-01.—«IMI o—C4I.
Figure 5. Metabolic Pathway.
Further elaborations on this scheme are certainly known. A number of studies
on the glucosylation of xenobiotics in various plant cells"220- 2211 have been
undertaken.
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Cl DEPOSITION OF XENOBIOTICS IN CELL WALL.
A number of studies have shown that compounds such as dichlorophenol and
1,2-dichloroethane and trichloroethylene (Gordon, Perkins, and Ahmed,
unpublished) can be deposited in high molecular weight polymers. Pogany, et
al.12221 have shown that dichlorophenol and 4-chloroaniline are deposited in the
starch and lignin fractions of tomato and maize cells. The exact nature of this
deposition is not known. It is conceivable that phenolic compounds are
polymerized into lignin like compounds by peroxidative reactions involved in the
synthesis of lignin. In the case of ethylenic compounds, it is quite possible that
the compounds are metabolized by conjugation with glutathione followed by
further metabolism which results in the formation of alkylating agents, or the
compounds are activated by P450 systems to epoxide intermediates and
ultimately aikylate either cellulose residues or lignin. The complete
mineralization to C02 of a number of xenobiotics is known.
d) FATE OF XENOBIOTIC DURING SENESCENCE OF PLANT TISSUE.
The fate of the conjugated xenobiotics during the senescence and breakdown
of plant tissue has not been thoroughly investigated. The possible fate of
conjugates of xenobiotics as well as metabolic products fixed to cellulose,
lignin, or cell walls, has not been extensively explored in the literature and
certainly is deserving of much more attention. Pogany, et al.12231 have studied
residues of 4-chloroaniline and 2.4-dichlorophenol which were bound to
insoluble plant polymers. The bound residues in maize cultures were released
and could be further mineralized or bound onto soil organic matter. When the
grass, Lolium multiflorum, was grown on soil containing bound residues of 4-
chtoroaniline and 2,4-dichlorophenol, about 2% of the applied radioactivity was
taken up by the grass. No phytotoxic effects were observed. The authors
indicated that in field experiments, the uptake rate.s could be expected to
decrease by approximately 50-fold. There are no guides to the expected
recovery or persistence of compounds upon repeated cycles of utilization,
deposition into cell walls, breakdown, and re-utilization.
e) METABOLISM of XENQBIOTICS w G€N€Tte to transform fHants with bect«fia< gen«« which
are capable of completely mineralizing various xenobiotics. In the past few
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years there have been a number of reports of herbicide resistant plants which
have been produced by incorporating into the plants various bacterial genes.
The bacterium, Alcaligenes eutrophus, contains a plasmid which codes for a
sequence of reactions which completely demineralize 2,4-D. The first of these
enzymes, which converts 2,4-D to 2,4-dichlorophenol, has been incorporated
by two groups into tobacco plants1224-22S1 with the resulting resistance to 2,4-
D. Genes are available which will confer resistance to glyphosate,
sulfometuron methyl (Oust) and phospho and bromoxynil. Cotton plants which
contain herbicide resistance genes probably will be released shortly. Gordon
and co-workers have incorporated into tobacco two genes from Alcaligenes
eutrophus which convert 2,4-dichlorophenol to the corresponding catechol and
hence to the ring open compound as a means to enable plants to remediate
some toxic waste dumps.
f) RESISTANCE TO HEAVY METALS.
In a number of projects referred to above wherein poplars or sweet gum are
used to remediate municipal sludges and wastes, it is apparent that the trees
were tolerant to heavy metal toxicity. Plants contain a family of genes coding
for peptides known as phytochelatins. These are polypeptides of variable
lengths which have the general structure (gamma glutamic acid cysteine].I22e-
2271 Steffens has published a number of papers dealing with the binding of
cadmium, copper, silver, and zinc to phytochelatin. There is also a number of
reports that plants adapted to heavy metal contaminated soil show increasing
levels of phytochelatins, although the basis for heavy metal tolerance in plants
may not be as simple as increased levels of this metal chelating material. Misra
and Gedamu12281 found that it was possible to make heavy metal resistant
plants by incorporation of metallothioneins (a molecule not found in plants) into
Brassica plants. In these transgenic plants the metallothionein was under
control of the CaMV35S promoter. The metalothioneins are small peptides
with molecular weights of approximately 6,000 daltons and have a high
cysteine content. Up .to 30% of the amino acids in these peptides can be
cysteine. In many eukaryotic cells the metalothioneins are under control of a
promoter which is activated by heavy metals.
a) • USE OF PLANT AS INDICATOR OF IONIZING RADIATION.
An extensive review of work undertaken by Arnold Sparrow at Brookhaven
during the decades of the 1950s summarizes plant responses to ionizing
radiation.1229' In addition to a host of enzyme responses, there are multiple
visual observations that could be used to detect radiation responses. These
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include abscission, color change, dwarfing, sterility, early onset of flowering,
tumorous growths, abnormal vegetative proliferation in floral positions, fruit
color changes, leaf curling and others. The wealth of data presented in the
review should be explored for potential endpoints for other phytotoxic
stressors.
B. STATISTICAL FACTORS
1. PRECISION/ACCURACY/UNCERTAINTY
a)
PLANT INTERSPECIES VARIABILITY
In an analysis of toxicity among diverse plant taxa, Fletcher et ai.[2301 reported a
wide range of sensitivity to herbicides. For the herbicide prometryn there was
an approximate difference in sensitivity of 21-fold. Of 16 different classes of
chemicals, the smallest range of sensitivity was 3.5-fold -(for linuron) and the
largest range of sensitivity was 316-fold (for picloram). As expected, the
variation in sensitivity increased as the taxonomic distance increased.
Unfortunately, similar detailed analyses have not been generated for sensitivity
to metals and metalloids. Nevertheless, there are indications that the variation
among species with regard to metal toxicity and tolerance exhibits similar
ranges of response. Baker12311 cites work of W. Ernst on twelve species of
herbs showing the variation in metal uptake capability as expressed by plant
concentration to soil concentration.
Table 9. Interspecies Variation In Plants Toxicity.
HERBICIDE
LINURON
PICLORAM
PROMETRYN
VARIATION
4x
316 x
21 x
Adapted from Fletcher et al.12321
METAL
Cadmium
Copper
Lead
Zinc
VARIATION
273 x
9x
240 x
18 x
Adapted from Baker. i"3'
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b)
LAB To FIELD VARIABILITY
There is continued concern regarding the validity of tab-to-field extrapolations.
The best analysis of this problem was provided by Fletcher et al.12341 in which
they showed remarkable agreement (i.e, " two-fold variation). More than 40%
of the comparisons between greenhouse and field studies were essentially
identical in response (i.e., ranging 1.0 to 1.5x; see Figure 6).
FIELD - GREENHOUSE COMPARISON
ADAPTED FROM FLETCHER ET AL. 1990. ET&C 9:709-770
1.50
1.99
2.00
2.50
2.49 2.99
RESPONSE DIFFERENCE
3.00
3.49
3.50
3.99
Figure 6. Field to greenhouse comparison of phytotoxic responses.
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2. STATISTICAL APPROACHES TO ECOLOGICAL ASSESSMENT
Because waste sites and reference sites are nonrandom samples, most classical
approaches to statistical analysis (e.g., hypothesis testing and analysis of
variance) may not provide the methods of choice in ecological assessments for
hazardous waste sites.(23S1 Unless these potential flaws in quantitative analysis
are addressed, hazardous waste site assessment should rely on techniques
which are more appropriately identified as being exploratory data analysis in
character. Various statistical methods may be applied and yield a framework
wherein chemical, toxicologicai, and ecological information become integrated.
These component parts then become building blocks within the site-assessment
process. Depending upon the effort invested in gathering site information, the
resulting data should yield a framework for an ecological assessment for a
hazardous waste site.
The chemical, toxicologicai, and ecological information collected for a site may
be balanced or weighted among these component parts, depending upon site-
specific characteristics. Historically, for example, chemtcally-based methods
were the primary assessment tools applied to hazardous waste site evaluations,
regardless of whether the concerns regarded human health or ecological
effects. Causal linkages between adverse biological responses and
contaminant presence were assumed, and were based largely on extrapolation
from laboratory-derived single-compound toxicity evaluations to field settings
most frequently characterized by complex chemical mixture exposures.
However, if toxicity-based criteria and ecological survey data were considered
complimentary components to chemical analyses during site assessment, then
statistical methods could integrate these component data sets.
Management decisions regarding the environmental hazard associated with
chemical contaminants at the site could be developed using an integrated
assessment strategy and would not rely exclusively on chemical analyses; for
most environmental hazard assessments, toxicity-based criteria have become
increasingly important owing to the complex chemical 'mixtures characteristic of
environmental exposure. Toxicity assessments which evaluate adverse effects
through measurement of biological endpoints12361 and field surveys which
measure ecological endpoints indicative of higher level structure and
function12371 contribute to the environmental hazard assessment process and
enhance resource management during all pnases of The evaluation process. For
establishing these critical linkages among chgmicaJ. toxicologicai, and
ecological information, the quantitative methods most appropriate for these
integrations may be suggested by the date collections themselves, and may
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include various methods which have found past applications in applied ecology
and environmental impact assessment.1238-2391
a) MULTIVARIATE ANALYSIS
Independent of the applications apparent within the context of contaminant
ecology, applied multivariate techniques (e.g., direct gradient analysis,
ordination, and classification) have had a recurring role in ecological research,
and have been used within a variety of settings, including terrestrial and
aquatic habitats (freshwater, estuarine and marine as well as freshwater and
estuarine wetlands); historically, a wide variety of ecological endpoints (e.g.
populations and communities) have been the primary focus in these
applications which have classically evaluated vegetation, or microbial and
animal populations or communities which were subjected to naturally occurring
stressors (e.g., temporal and spatial habitat variation; environmental
perturbations such as fire) or anthropogenic sources of habitat alteration.
Many compilations and reference texts are available and provide starting points
for evaluating the past record of these techniques.1240- 241- 242- 243> Their
application to hazardous waste site assessment may be estimated from a
review of the applied literature, and these approaches should be adequate, if
judged pertinent to site assessment during the.early stages of work plan
development.
b) TIME SERIES ANALYSIS
While the time constraints of hazardous waste site work may preclude long-
term studies on any one site, various methods drawn from statistical time
series analysis may be applicable to site evaluation, particularly since the site
has been, and will continually be, "changing" with time. Indeed, the potentially
dynamic character of waste sites, particularly those cdnsidered from their initial
"discovery and listing" through various stages of "clean up and restoration,"
suggest various time series techniques (e.g., trend analysis) which may
repeatedly contribute to a specific site assessment during its "life history."
Additionally, the historical information which is available for a particular site
may afford the opportunity to conduct a variety of techniques drawn from time
series analysis; the application of time series analysis has found wide
application within basic ecological research, and numerous references are
available which should be considered within the setting of hazardous waste site
assessment.'244-24S-24S-247-248<
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Cl GEOSTATtSTICAL ANALYSIS
Recently, the description and interpretation of spatial distributions for waste
site contaminants have increasingly been applied to exposure assessments1249-
250. 251] an(j the coincidence in patterns which may be apparent between
contaminant and toxicity distributions has been tentatively applied toward
linking these measures within a site assessment.12521 Within waste site
settings, applied geostatistical analysis has found applications in soil and
sediment evaluations; while primarily applied to mapping exercises for plotting
contaminant distributions within landscape settings, the roles of variogram
analysis and kriging may be of greater value beyond that contribution which is
required in developing contaminant distribution maps.1253- 2M1 See Appendix III
for an illustration of this approach.
d) ENVIRONMENTAL SAMPLING AND STUDY DESIGN
Regardless of the statistical methods used in evaluating chemical, toxicity, and
ecological data collected for a site, the most critical problems which should be
considered in the site work plans revolve about field sampling and its design
and implementation. Without adequate, well-designed field sampling plans the
subsequent data analysis could become a secondary issue, particularly within
the context of litigation. Various references have been compiled which address
the problems of field sampling within an ecological context1255- 256- 257- 2581 and
recent efforts to delineate these issues within an applied context have
considered hazardous waste sites specif ically.1259-260-2611
e) SUMMARY COMMENTS ON STATISTICAL APPROACHES
Ecological assessments for hazardous waste sites should include acute toxicity
tests which most frequently measure mortality, and short-term tests which
measure biological endpoints other than death. Toxicity assessment tools,
then, may yield information regarding acute biological responses elicited by
site-samples as well as suggest longer-term biological effects (e.g., genotoxicity
or teratogenicity) potentially associated with subacute and chronic exposures
to complex chemical mixtures characteristic of hazardous waste sites.1262-263]
Toxicity evaluation methods which contribute to site assessment should reflect
site-specific demands implicit to the ecological assessment process, but toxicity
tests are but one component of an ecological assessment for a hazardous
waste site. Strongest inferences regarding the coincidence of contaminants
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and biological response may be derived from sampling plans which consider
both toxicity and chemical characterization, yet an ecological assessment must
also consider field components early in site evaluation. This becomes
particularly important when field sampling is considered, since integration of
toxicity assessments (be those in situ or laboratory-generated), chemical
analysis and field assessments requires a well-designed sample plan to establish
linkages among toxicity, site-sample chemistry and adverse ecological effects.
Spatial statistic techniques like kriging are finding increased applications in
linking toxicity with other elements of site-evaluation (e.g., field-sample
chemistry). Through kriging, for example, areal distributions for site-specific
toxicity and chemistry data sets may be derived; then, "distribution maps" for
toxicity and chemistry data may be overlaid. Patterns of coincidence apparent
in these distributions may then suggest linkages among toxicity, site-
contaminants, and adverse ecological effects. Similarly, multivariate
techniques, particularly direct gradient and cluster analysis, appear quite
relevant to hazardous waste site assessment. The applied ecological research
literature presents numerous case histories frequently developed from studies
concerned directly with habitat alteration consequent to anthropogenic
activities (e.g., mining and agricultural practices, as well as aquatic impact
assessments for effluent discharges into lotic systems), and these methods
may be pertinent to site assessment for aquatic or terrestrial sites. Time series
analysis, while not having a history in waste site assessment, offers numerous
techniques which would appear appropriate to site assessments; these methods
may be particularly significant, if the entire "life history" of the hazardous
waste site is considered during the early phases in work plan development.
VI. CONCLUDING REMARKS
General
For the most part ecological risk assessments are focused on the upper levels
of ecological organization (i.e. population and higher). Toxicology
measurement endpoints are generally restricted to the level of individuals.
Consequently, there is considerable uncertainty in risk assessments. The
general field of plant toxicology suffers from an inadequate understanding of
how laboratory bioassay results predict actual field response. Good dose-
response relationships exist for both aquatic and terrestrial plant bioassays;
little laboratory-to-field correlations have been attempted. Therefore, it is
difficult to use these or other bioassays in an ecological risk assessment
scheme. • The development of in situ bioassays is critical for continued
advancement in this field. There is also little knowledge regarding the role of
64
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plants in contaminant fate, mobility and bioavailability. A better understanding
of the processes controlling contaminant uptake, translocation and metabolism
in plants is necessary.
Phytotoxicity has been constrained by the extensive reliance on two rather
insensitive tests (seed germination and root elongation) that also have little
ecological relevance. Much opportunity exists to improve the integration of
ecological and physiologically knowledge. As ecotoxicity matures as an applied
science many limitations mentioned in this report can be erased.
The plant methods available to evaluate ecological and toxicology concerns are
widely known and readily available. There are arguably fewer problems
associated with plant test methods than with other more widely accepted tests.
Plant scientists need to do a better job of communicating the wealth of
knowledge available. They also need to focus on adapting well understood
procedures into streamlined protocols for non-experts. In an effort to initiate
dialogue toward this end, the following summary of test methods lists the tests
discussed in this report according to Class designation. Guidance as to the skill
level and experience recommended for the test is also provided. In any such
identification of skill requirements, there will be exceptions of advanced
personnel performing poorly or entry level personnel exceeding expectations.
Nothing substitutes for competent, educated, and trained specialists.
A.WORKSHOP SUMMARY
Attendance at this workshop (nearly fifty persons) indicates a high level of
interest in the subject. Throughout the discussions, there was one common
theme expressed by the plant scientist, namely there are many opportunities to
improve environmental analysis through the use of plants. The major
reservations expressed by Superfund practitioners centered on linkage of test
results to ultimate remediation decisions.
Measures are available to evaluate ecological status, physiological condition,
and phytotoxic response to anthropogenic stressors. Greater use of plant test
methods appears to be constrained at present by the limited awareness of the
utility of plant measurements. This limited awareness results from:
1) Scarcity of detailed protocols that have had supervised multi-
laboratory performance tests to document precision, accuracy and
other quality control parameters.
65
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2) A relatively small number of technical persons educated and trained in
the applied plant science disciplines (i.e., phytotoxicity,
ecotoxicology).
3) The small number of commercial facilities prepared to conduct plant
tests.
To assist practitioners in the selection of appropriate tests, a summary table of
available methods was generated (see table 10). In addition to designating the
level of development of the test, an effort was made to evaluate the general
skill and experience level needed to perform the tests successfully.
Table. 10. Recommended minimum skill levels as determined by education and work
experience. Most tests require support from chemist or biochemist in each
phase. The skill level of chemical support staff is approximately equal to the
plant science skill levels.
NAME
ECOLOGICAL
FLORISTICS SURVEY
WETLAND DELINEATION
PLOT SAMPLING
PLOTLESS SAMPLING
GENETIC DIVERSITY: ISOENZYMCS
GENETIC DIVERSITY: DNA PROBES/SEQUENCING
COMMUNITY TERRECOSM
[CONTINUED ON NEXT PAGE]
CLASSa
l.a.
1 a.
l.a.
l.a.
II. a.
II. a.
II. b.
SKILL LEVEL"
B
A
B
B
D
D
B
66
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Table. 10. (CONTINUED).
TOXICOLOGICAL
SEED GERMINATION
ROOT ELONGATION
LIFE-CYCLE TEST
TISSUE CULTURE
PHOTOSYNTHESIS: C02 FIXATION
PHOTOSYNTHESIS: FLUORESCENCE
PEROXIDASE
POLYAMINES
GENOTOXCITY: (e.g. Tradescamia system)
GENOTOXCITY: (DNA unwinding, adducts, etc.)
GENOTOXCITY: (Gene Induction, activation, etc.)
DINITROGEN FIXATION
TISSUE CONCENTRATION
l.a.
l.a.
l.b.
l.b.
II. a.
II. a.
II. a.
ll.b.
l.b.
II. a.
ll.b.
II. a.
l.a.
A
A
B
B
C
C
C
D
C
C
C
C
A
a Class designation as described on page 14.
b Recommended minimum skill level as reflected by education and work experience. All degree
listings are implied to be in the biological sciences and preferably with emphasis in the plant
sciences. For brevity equivalent degrees (e.g. 8.S., B.A., and A.B.) are not listed. In addition,
technical staff should be versed m GLP practices.
Skill Level A: Test Selection - Master of Science
Test Performance -- Bachelor of Science
Data Reduction and Interpretation - Master of Science
Skill Level B: Test Selection -• Master of Science plus three years experience
Test Performance •- Bachelor of. Sciences plus two years experience
Data Reduction and Interpretation -- Ph. D. or equivalent
Skill Level C: Test Selection •• Ph. D. or equivalent
Test Performance -- Bachelor of Science degree plus two years experience
Data Reduction and Interpretation - Ph. D. or equivalent
Skill Level D: last SftocTM?n - Pti.D. 0tus three years experience
Test Performance •- Master of Science or equivalent
Data Reduction and iTTrerprronon - Ph.D. plus ttifBe years experience
67
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There needs to be better dialogue between the technical experts and the end
users. Plant scientists must convey in more precise language what each test
method can contribute in the risk assessment process. With better definition it
will be easier for project managers to understand what to expect from any
given test. This should result in increased use of plant tests for superfund site
assessments.
Beyond the general educational realm, there were several pleas for increased
emphasis to fund plant projects. It is beyond the scope of this workshop
summary to develop the research priorities. However, in general terms the
candidate areas for consideration can be grouped into two categories
1) Preparation of draft protocols followed by inter-laboratory testing
2) Increased opportunity for demonstration grants to field test Class I
and Class II tests.
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VII. APPENDIX!
VEGETATION SAMPLING METHODS: CALCULATIONS
The sampling provides "raw" data on the species identity of individual plants as
well as some measure or estimate of size (mass, cover, diameter, etc.) for each
individual. The information is treated separately for each sampling element
(i.e., plot, interval, point, etc.) sampled. This data is then reduced to through
various equations that permit quantitative descriptions of the vegetation unit
being sampled. The calculations may then be performed for all taxa collectively
and/or individually. As with any collection of data, appropriate steps in data
management should be followed to permit expression of the values in statistical
terms (i.e., means, modes, variance, etc.).
The concept of dominance is based on the assumption that a species with the
greatest biomass exerts the most influence on the community. For trees,
dominance has been equated to basal area. By definition, the basal area is the
planar area of the tree trunk at 1.4 m (4.5 ft.) above the ground. This value is
calculated from the Diameter at Breast Height (DBH) which is standardized at
1.4 m. Frequency is an indicator of the dispersal of a taxon throughout the
sampling area. Often for comparative purposes, the values of dominance,
density, and frequency are normalized and expressed as a percentage of the
total. These normalized values may then be summed in an expression of
Importance Value or Importance Percentage (I.P.)
IMPORTANCE = Relative Density/3 +
PERCENTAGE Relative Frequency/3 +
Relative Dominance/3
N.B.: In earlier literature, the relative values were summed but not divided by 3.
The expression was referred to as the Importance Value or IV. In some sampling
routines, dominance or density information is not obtained. If one desires to
calculate the IP based on only 2 relative terms, the denominator is 2 instead of
3.
EQUATIONS FOR DEFINED AREA SAMPLING
DENSITY = (Number of Individuals!
(Area Sampled) / (Unit Area)
W.B..: The "Una Area" must, be aigebfjicatiy coropatU>te «with the daw. For
example, tree density is usually sampled in a plot of 100 m^ but expressed on a
69
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per hectare basis. Thus if 85 trees were tallied in a sample of 1.000 m2 area,
density would be 85 trees/11,000 m2/10,000 m2/ha) = 850 trees per ha.
FREQUENCY
(Number of Plots with Soecies X)
(Number of Plots Sampled)
DOMINANCE = (Total Species X Phvtomasst
(Area Sampled)/(Unit Area)
N.B.: Canopy Cover, Basal Area, or some other parameter may be used
instead of Biomass.
RELATIVE DENSITY =
RELATIVE FREQUENCY =
RELATIVE DOMINANCE =
(Density of Species X) x 100
(Total Density)
(Frequency of Species X) x 100
(Total Frequency)
(Dominance of Species X) x 100
(Total Dominance)
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EQUATIONS FOR PLOTLESS SAMPLING METHODS
LINE INTERCEPT
Data is collected along predetermined intervals. It consists of the portion of the
interval of the 1-dimensional space (line) occupied or intercepted by vegetation,
litter, soil, etc. as depicted in the following illustration (Figure 7).
100.0
8
Sp.A.: 8*15=23. Sp. B.: 7*9=16
Sp.C.: 5. Sp. D.: 4. Sp. E.: not present
Figure 7. Line-Intercept Sampling Method.
The summary calculations are performed on line-intercept data as follows:
RELATIVE DENSITY = (Total No. Individuals of So. X) x 100
(Total No. Individuals of All Species)
DOMINANCE
(Total Intercept Length for So. X) x 100
(Total Interval Length Sampled)
(Dominance may be called Basal Cover)
RELATIVE DOMINANCE =
FREQUENCY
RELATIVE FREQUENCY =
(Total Intercept Length of Sp. X) x 100
(Total Intercept Length of All Species)
(No. Intervals with So. X Present) x 100
(Total No. of Intervals Sampled)
(Frequency of So. X) x 100
(Frequency of All Species)
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POINT-QUARTERS
Data collected at each sample point consists of the taxonomic identity of the
plant, the distance from the point to the center of the plant stem, and the
diameter of the plant (as per convention at 1.4 m height for trees). Points are
often located at intervals along a transect. At the point, a perpendicular line is
projected through the transect, thus dividing the area into four quadrants (See
Figure 8.). In each quadrant, he plant (tree) nearest to the point is identified
and the point-to-plant distance and plant diameter are measured.
IV
Is
Figure 8. Point-Quarters Sampling Method.
SPA SP. B SP. C SP D
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The calculations are as follows:
TOTAL DENSITY = Unit Area
(Mean Point-to-Plant Distance)2
RELATIVE DENSITY =
DENSITY =
DOMINANCE =
RELATIVE DOMINANCE =
FREQUENCY =
RELATIVE FREQUENCY =
(No. Individuals of So. X) x 100
(No. of Plants sampled)
(Relative Density of Sp.X) x Total Density
100
(Density of Sp. X) x (Mean Basal Area of
Sp.X)
(Dominance of So.X) x 100
(Dominance of All Species)
(No. of Points with So. X Present)
(No. of Points Sampled)
(Frequency of Sp. X) x 100
(Frequency of All Species)
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VIII. APPENDIX II
The use of in vivo chlorophyll a fluorescence as a measurement of
photosynthesis is now being applied very frequently to a wide variety of
research areas in plant ecology and plant physiology.1264- 2651 The chlorophyll
molecule can be considered an intrinsic fluorescent probe of the photosynthetic
system in chloroplasts. Fluorescent probes can report to the researcher,
externally the physiological conditions occurring in the most basic biosynthetic
process of plants. In the leaf of higher plants or in algal cells, the yield of
fluorescent emissions is influenced in a number of ways by processes that are
either directly related to photosynthesis or indirectly influence photosynthesis.
This report will review the use of the chlorophyll fluorescence signal to monitor
the physiological well being of the individual plant or plant community. The
fluorescence emission by isolated leaf sections or by intact chloroplasts which
have been intensely studied will be discussed. These findings apply directly to
the fluorescence emission observed by entire photosynthetic organs, such as
stems or leaves. The fluorescent characteristics of the isolated chloroplasts are
much better controlled and more carefully studied than the entire leaf. The
basic interpretations of the changes in the fluorescent signal of the chloroplast
can be applied to the intact plant leaf or to a larger plant canopy, provided care
is taken to include adequate controls. In order to compare fluorescent emission
from one experimental situation to another, the conditions must be very clearly
defined. The description of the light emission system of photosynthesis and
what this fluorescence is revealing to the investigator about the state of the
photosynthetic process is described below. Examples of effects of chemical
stress and other well known environmental stresses on changes in fluorescence
will also be provided.
There is an extensive literature available resulting from the basic study of the
photosynthetic mechanism which can be applied to assessment of chemical
toxicity. Researchers have utilized a large number of different types of
inhibitors to dissect the photosynthetic system. In addition, there is a large
literature developing on environmental stress effects on chlorophyll
fluorescence and the use of fluorescence in characterizing these stresses. The
majority of the effects described here will be characteristic fluorescent emission
from plants in natural conditions of temperature on.a slow time scale (15-30 s).
The much faster microsecond or picosecond changes in chlorophyll
fluorescence are more closely related to the primary photophysical and
photo.chemical events of photosynthesis and will not be discussed here. The
fast time scale of fluorescence is much more difficult to measure and would
have less application to stress physiology or chemical toxicity studies.
74
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(1) GENERAL DESCRIPTION OF THE PHOTOSYNTHETIC APPARATUS.
Light energy utilized in photosynthesis by higher plants and algal cells is
absorbed by a number of photosynthetic pigments with absorption spectra
covering a large range of the available light energy. The most prominent
pigments which absorb this energy are chlorophyll a and chlorophyll b (Figure
9). The light energy which is absorbed by the chloroplast first excites pigment
molecules of the light harvesting chlorophyll proteins (LHC). These LHC
proteins transfer their energy to either Photosystem I (PSI) or Photosystem II
(PSII). These photosystems contain the reaction center pigments for the
conversion of absorbed light energy to oxidation and reduction potential to
drive dark electron transport. Light energy which was absorbed initially by the
LHC and transferred to the reaction centers is tost by a number of different
mechanisms. Approximately 3% of the light energy absorbed by chlorophyll
pigments is re-emitted from the first excited state as fluorescence. Figure 10
shows the typical fluorescence emission spectrum of leaves or whole
photosynthetic cells. At low temperature this fluorescent emission has a major
peak at 683 nmt a shoulder a 695 nm, and a broad second peak 735 nm. At
room temperature, light energy absorbed in photosynthesis is re-emitted and
observed at the 683 and 740 nm emission peaks. The light energy absorbed
by the reaction center drives photosynthetic electron transport through PSII and
PSI leading to -the oxidation of water, oxygen evolution, the reduction of
NADP+ to NADPH, membrane proton transport, and eventually to ATP
synthesis (Figure 11),
The loss of light energy from the reaction center as fluorescence comes
primarily from the PS II reaction. When the chloroplast or leaves have been
dark-adapted, the pools of oxidation or reduction intermediates for the electron
transport pathway return to a common level. Upon illumination of a dark
adapted leaf, there is a rapid rise in light emission from PS II fluorescence
followed by a series of slow oscillations. This is referred to as the Kautsky
Effect. Figure 12 shows the usual onset kinetics of fluorescent emission from a
typical dark adapted higher plant leaf. Changes in the fluorescent yield and the
kinetics of fluorescent emission from dark adapted.leaves are sensitive to
changes in the photosynthetic apparatus. Following many years of study of
chlorophyll fluorescence to analyze its relationship to photosynthesis and to
characterize photosynthesis, we know that any unusual change in overall
bioenergetic status of the plant can be detected .by a change in chlorophyll
fluorescence.12661 This includes all the reactions from the oxidation of water
through electron transport, development of the electrochemical gradient, ATP
synthesis and eventually the series of enzymatic reactions for CO 2 reduction to
casbohydsate in the leaf. Evert changes in the plant which affect stomata
opening and gas exchange with the atmosphere are reflected by changes in the
fluorescence characteristics as a leaf.
75
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o
o
• c
CO
.0
0.90
0.80
0.70
0.60
0.50
0.40
0.30
0.20
0.10
0.00
« Chi a
• Chlb
360 400 440 480 520 560 600 640
680
Wavelength, nm
Figure 9. The Absorption Spectrum Of Solvent Extracted And Separated Chlorophyll a And
Chlorophyll b.
1.197e+07 r-
0>
u
0
en
0>
c_
o
77°K
0.00000
650
700
750
800
Wavelength (nm)
Figure 10. Fluorescence Emission Spectrum Of Whole Zea mavs L. Leaves Excitation At 430
nm. (A) Is The Typical Spectrum At 25°C. (B) Is The Emission Spectrum At 77°K.
76
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liv
2H .Mi
Figure 11. Model For The Organization Of The Chloroplast Inner Membrane Showing The
Relationship Of PS I, PS II. The Cytochrome Complex. And The ATP Synthetase.
This Model Illustrates The Path Of Electron Flow From Water To NADP. The
Apparent Molecular Mass For Each Polypeptide Is Indicated In Kd By The Numbers
On The Protein.
D
>
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(2) MEASUREMENT METHODS
There are a variety of instruments which have been used to record the
fluorescent emission from chlorophyll in chloroplasts or plant leaves. The
requirements fbr these instruments are an actinic light source which will excite
any photosynthetic pigment and a method for measurement of the 683 or 740
nm emission peak of chlorophyll, while excluding the actinic illumination from
the detector. A typical laboratory instrument to measure fluorescence kinetics
of leaves or chloroplasts is shown in Figure 13A. In this instrument blue light
is provided by an tungsten light source through a blue glass filter with a peak
transmission of 430 nm. Fluorescence emission is measured with a
photomultiplier tube or amplified photodiode blocked by a red glass cut-off filter
(transmits 90% of the light over 670 nm). With this apparatus the dark
adapted leaf is oriented so that when the photographic shutter is open to allow
the actinic beam to excite chlorophyll, the yield of emission of fluorescence
from the leaf is recorded by the sensitive photomultiplier tube. The signal from
the photomultiplier tube or photodiode is amplified and recorded on a chart
recorder or for faster recordings, a storage oscilloscope. In a modern
instrument, the recordings can easily be made A/D input boards, analyzed, and
stored in a personal computer.
In addition to this laboratory instrument, which can be constructed simply, a
small number of portable field instruments are now available commercially using
photodiode light sources and solid-state photodectors (Figure 13B). These
instruments are very useful for environmental field work provided good controls
are used to obtain accurate measurements.l267-268-269'
The other general form of instrument used for recording fluorescence
characteristics of photosynthetic organisms is the spectrofluorometer. The
spectrofluorometer utilizes two monochromators in order to scan the exciting
wavelengths of energy or to measure the emission wavelengths. A standard
spectrofluorometer utilizes a high-intensity Xenon light source through grating
monochromators to provide precise wavelengths of actinic illumination to the
sample. The emission is measured from the sample through a precision
monochromator (usually double-grating) and detected on a wide-range,
sensitive photomultiplier over the 400-750 nm range. With this instrument it is
possible to measure excitation spectra for fluorescence at one wavelength or
emission spectra of the photosynthetic tissue over a wide range. The most
useful form of this spectrofluorometer contains a low temperature (liquid
nitrogen, 77K) sample holder in order to measure high resolution fluorescence
emission forms from the chloroplast12701 (Figure 10).
78
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Figure 13A. The Diagram Of A Laboratory Kinetic Fluorometer. LVDC. Low Voltage Power
Supply For The Actinic Lamp; AL, Actinic Lamp; SH Photographic Shutter; AF
Actinic Filter (Broad Blue Band); L, Leaf; BF. Blocking Filter (Red Light Transmitting);
PM, S-20 Response Photomultiplier (Extended Red Sensitive); HVDC. High Voltage
Power Supply; AMP, Photocurrent Amplifier; X-Y, Plotter; SS. Storage Oscilloscope.
Figure 13b. Illustration Of A Portable Chlorophyll Fluorometer.
79
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The simple fluorescence measurements of chlorophyll emission over 30
seconds from dark adapted leaves are considered here. In the measurement
shown in Figure 12, the typical response has been identified by a series of
phases.12711 Immediately following excitation, the chlorophyll fluorescence rises
to a point 0. From the initial point 0 there is a slower rise to a small peak12721
followed by a decline (D) and then the maximum level of fluorescence emission,
referred to as P for the peak. This peak is reached in the average instrument at
approximately 0.1 to 1.0 second after illumination. The timing for this series of
oscillations to the peak depends upon a number of factors including the amount
of chlorophyll, and the intensity of the actinic light. After the fluorescence has
risen to the peak in intact leaves, it now declines to a semi-steady state, S and
will rise in a second peak, commonly called M. Following the second smaller
peak, there is a further decline to a level similar to S now referred to as T, the
terminal level of fluorescence. In almost every photosynthetic system studied,
this same series of oscillations occur within the first 30 seconds of illumination
of a dark adapted leaf. With isolated chloroplasts the change in fluorescence
ends with P.
After years of intensive study, we have information about each of these
fluorescence changes. In order to compare the emission of one sample to
another, a series of standard measurement are usually made.12731 These
measurements are referred to as F0, for the initial level of fluorescence
followed by FM for the maximum level of fluorescence at P (Figure 14). The
difference between FM and FQ is the variable fluorescence (Fy). This FV is a
useful characteristic to follow the physiological state and photosynthetic
capacity of the photosynthetic apparatus. The variation of F0, FM, and Fv with
light intensity is illustrated in Figure 15. From this it is clear that FV/FM varies
tittle with light intensity and this parameter can be used as a universal
measurement of the physiological state of the chloroplast under different
conditions of light, pigment, age, etc. Measurement of Fv or FM alone are
highly light intensity-dependent.
The electron transport reactions in the chloroplast which are most important in
determining the level of in vivo chloroplast fluorescence have an effect on the
oxidation-reduction state of the initial stable electron acceptor of PS II (QA). In
the reaction center of PS It the primary chlorophyll, P-680, is excited by
absorbed light energy to P6SO*. P-680* quickly reduced a short lived
pheophytin a and eventually reduces the QA electron acceptor in the PS II
reaction center (Figure 11). QA is a special plastoquinone bound to- one of the
reaction center polypeptides of PS II. If this acceptor is oxidized, then it will
receive the electron from the reaction center and the level of fluorescence will
remain low {is quenched", therefore Q). If this electron acceptor is reduced
(QA-) then there is no immediate place for the electron from the reaction center
80
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to go and the excited states of the reaction center will collapse back releasing
their energy as fluorescent emission of the chlorophyll. The key to regulation
of the level of fluorescence of PS II (and therefore the entire chloroplast or the
photosynthetic apparatus) is the oxidation-reduction state of QA- Since QA~
can be oxidized by all of the electron carrier pool between PS II and
Photosystem I, then any change in the ability of the carriers between PS II and
PS I to oxidize QA- will affect the level of fluorescence of the leaf. This is why
we can use in vivo fluorescence to monitor all of the electron transport
reactions from PS II through the cytochrome complex to PS I. Through these
reactions that generate membrane potential, ATP synthesis, NADP+ activation
and reduction to NADPH (Figure 11), and eventually the utilization of this
reducing potential for C02 reduction, any change in the reactions will affect the
redox level of QA- This can be monitored as changes in the characteristics of
fluorescence from dark adapted leaves. Limitations of electron transport on the
oxidizing (water splitting) side of PS II between the PS II reaction center and
water will have the opposite effect on fluorescence. The level of fluorescence
will remain low rather than high. A limitation of electrons being donated to the
reaction center of PS II causes the fluorescence to remain at level near F0.
81
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100
Fm
**
.2
rx
0>
o
c
0)
o
V)
0)
o
3
tl
80
60
40
20
Fv
Fo
2 0
2 4 6 8 1012
Time (sec)
Figure 14. A Typical Fluorescence Of A 3 Minute Dark-Adapted Zea mays L. Leaf From Fo (0)
to FM IP).
100 200 300 400 500
LIGHT INTENSITY nmo1/m2/sec
600
Figure 15. Comparison Of The Effect Of Light Intensity On Chlorophyll Fluorescence
Parameters.
82
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(3) APPLICATION OF FLUORESCENCE
The characteristics of inhibition of photosynthesis allow us to use fluorescence
as a monitor of the overall rate of photosynthetic electron transport. >274- 27S-278'
Any alterations of electron transport on either the oxidizing or the reducing side
of PS II will cause a detectable change in the level and the emission spectrum
of fluorescence. This system is an extremely useful intrinsic fluorescent probe
of the bioenergetic status of the whole plant.
A typical effect of an inhibitor of photosynthesis is shown in Figure 16. Whole
plants or isolated chloroplasts exposed to a herbicide know to inhibit
photosynthesis 3-1277-278l-dichlorophenyl)-1,1-dimethyl-urea (DCMU) has a very
dramatic effect on the fast level of fluorescent emission.<279> DCMU blocks
electron transport just subsequent to the QA step. The only electron
acceptors available are the limited pool of QA. therefore, when treated with
DCMU, we find a very small change in variable fluorescence and a very high
yield of fluorescence. This small change reflects the available QA'S and the
high yield reflects the blocked overall process. This increases the emission of
fluorescence from the usual 3 % level to 6 to 10 % level. The specific site for
DCMU inhibition of electron transport is well known. '
The previous work in which chlorophyll fluorescence has been used as a tool in
general plant physiology'280- 2B1- 2821 has measured emission kinetics and
spectral changes of fluorescence at both room temperature and at low
temperature (77K). These changes can also be monitored to assess the effects
of environmental pollutants or chemicals on the state of photosynthesis. In
addition to fluorescence, there is a slow light admission (luminescence) from
the reaction center with a half time in the millisecond range.1283- 2841 This
luminescence or delayed fluorescence indicates the recombination of electron
acceptors and electron donors at the reaction center. Delayed fluorescence
has been very useful in monitoring the function of reaction centers in
photosynthesis and can also be useful to study the effects of environmental
changes on photosynthesis. However it is more difficult to measure being only
1 % of the fluorescence signal of PS II. Another method for monitoring changes
in the bioenergetic of photosynthesis is measuring the carotenoid band-shift in
the whole leaf. This band-shift occurs at 518 nm and is an important
characteristic of PS I and PS II.'2851 In addition to the measurement of
fluorescence, both the delay fluorescence and the 518 nm abscrbance change
are further markers which can be used to monitor photosynthesis in intact
plants and provide further information.
83
-------
V)
"c
TO
0)
u
c
o
u
(0
o
3
100
80
10 12
Figure 16. Chloroplasts Fluorescence Changes In Isolated Zea mays L. Broken Chloroplast The
Presences Or Absence Of The Inhibitor 3-<3.4-Dichorophenyl)-1.1 -Dimethyl-Urea. (DCMUK
-------
(4) UTILIZING THE WHOLE PLANT FLUORESCENCE
The development of our knowledge of chlorophyll fluorescence has been
important to the biochemist and the physicist in understanding the basic
reaction of photosynthesis. This information can be useful to environmentalist
working in the opposite direction to determine how the photosynthetic systems
have been altered. There are many sites in the electron transport chain related
to photosynthesis that will sense a variety of different chemical compound or
stresses. Any changes in lipid soluble compounds, or in highly reducing or
oxidizing compounds will affect different sites in the electron transport system.
The site can be almost immediately identified by monitoring the characteristics
of chlorophyll fluorescence. In addition, any change in the series of carbon
metabolism reactions of the chloroplast will eventually alter the level of the
reduced NADP-H pool and this will provide a characteristic change in the
chlorophyll emission.I286- 287< Changes as remote as those affecting the gas
exchange of the leaf'2881 will also be reflected in a change in fluorescence yield.
We can think of a higher land plant then as a monitoring system of the
environment. The plant is ideally suited since it has a massive root system
extending into the soil and ground water and taking up targe amounts of water
soluble compounds. This extensive root system will allow the plant to collect
and report on any chemical in its environment which is taken up. Any
compound taken up by the root, transported through the stem xylem to the
leaf, and finally to the leaf mesophyll cells, can have an effect on
photosynthesis. This would provide an immediate assessment, not only that a
substance is limiting photosynthesis, but how it may be limiting photosynthesis
and something about the chemical nature of the compound.
Presently new instrumentation is being developed to image whole plants or
groups of plants using solid state video cameras.12891 These instruments will
record fluorescence emission characteristics in real time using computer
technology. This approach holds real promise for the use of chlorophyll
fluorescence more widely to monitor any change in the characteristics of a
plant. At present this monitoring is being extended to the 30 meter range from
the plant but with laser excitation it appears feasible to monitor florescence
from a much greater distance. 1*90.291.292.2931
We have the possibility of not only being able to use chlorophyll fluorescence in
a well controlled system in the laboratory to assess toxicity of chemical to
biological systems, but we eJso can move that system into the field. We
should be able to use widely distributed sentinel plants to assess changes in
the environment either using the presently available portable instrumentation or
The remote sensing instrumentation in the near future.
85
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IX. APPENDIX III
SPATIAL ANALYSIS
RETROSPECTIVE STUDY
A hazardous waste disposal site located in central Oklahoma was studied in
1974.i294' Several toxic metals were present in the waste materials at the site.
In an attempt to reduce water volumes trapped in the disposal lagoons, a
sprayer was operated on dry, windy days when the prevailing winds were from
the south. The resulting spray mist with dissolved metals and other
constituents moved northward. During the study, plant samples were collected
from the land surrounding the lagoons and northward into a pasture. These
samples were analyzed for the metals Cd, Cr, Cu, Fe, Pb, and Zn. In this
application of GEO-EAS, similar information was obtained for each of the metal
concentrations. The semi-variogram plot of the ergotic (default parameter)
model (Figure 17) suggested a high degree of covariance. Subtracting the
covariance in the nonergotic model resulted in a semi-variogram plot
approaching the "ideal" form (Figure 18). In both the ergotic and nonergotic
models there was an apparent deviation manifested at 15 m. This would
appear to be a consequence of several "missing sample loci" from the lagoon
areas. The kriged map (Figure 19) illustrated a directional plume consistent
with what was known for the site, namely a unidirectional wind dispersal. The
maps for the other metals are not shown since they were fundamentally the
same as that for Cr.
SCOPING STUDY
AVENUE A PHOTO INTERPRETATION: PERCENTAGE VEGETATION COVER
The purpose of this study was to determine if the percentage vegetation cover
estimates from photos of the Avenue A site, Rosamond, CA exhibit patterns
that might be correlated with dispersion of contaminants from ash piles.
MATERIALS & METHODS: Nine aerial photos [20" x 24") of the Avenue A site
produced by EMSL-Las Vegas were provided by ERT-Edison. The scale was
nominally one inch = 60 feet.
Three registration marks were placed on each photo and three corresponding
registration marks were positioned on a gridded acetate sheet. The grids on
86
-------
the acetate sheet bounded one inch x one inch squares. A set of 150 random
X,Y coordinates was generated in LOTUS. These were sorted into groups
corresponding to the photos with the 0,0 position designated as the northwest
corner of the photo set.
Exploratory work with a Decagon Image Analyzer demonstrated that the soil
could be "zeroed out" of the image by adjusting the threshold setting. Optimal
focus during image acquisition required subjective judgement. Generally,
individual objects (here solitary shrubs) were focussed so that the right half of
the image on the display screen had a "halo fringe. "The "Dual Threshold"
option was used to acquire images. For these photos the settings 25-80 was
selected to capture canopy cover of shrubs; 25 -110 was selected to capture
canopy cover of total vegetation. The image edit option was used to trim the
image to precisely the area bounded by the one inch square grid selected for
analysis. Images were stored on disk for future reference. Calibration of the
image analysis mode was accomplished by using the "fill window mode.
"Images corresponding to one square inch through four square inches were
used as checks. The precision was determined to be 100 + 0.3%. The
minimum object sensitivity setting was 0.01 calibration inches. Each edited
image was measured to yield area of the image; the settings were such that the
area measured corresponded to percentage cover.
RESULTS: Of the set of 150 randomly selected sample grids, 38 were
eliminated because they corresponded to a road, obvious surface scar, photo
edge, or other feature that would bias the data. Thus 112 grids were
measured. Frequency distribution plots of percentage cover class show the
mode for total vegetation cover to be near 70%,; shrubs, 40%, and the
difference between total and shrub (nominally grasses) at 30%. Values ranged
from 4 to 86% for total cover; < 1 to 65% for shrub cover; and 4 to 45% for
"grass" cover.
These data were entered into the GEO-EAS program acquired from EMSL-LV.
The kriging estimates were generated with the circular distribution model
assumptions with default splining. Values shown on the contours are
percentage cover. The contour map for total vegetation cover (Figure 20}
shows a "valley" running from the mid portion of the map (roughly
corresponding to "ground Zero" of the site. Percentage cover values in this
valley are mid to low 30% range whereas surrounding areas are typically in the
50 to 60% range. SimiiarJy, the contour map of the stvub cover data (Figure
21) has a distinct valley from near the center of the map to the south east
corner. Shrub cover values in the "valley" are in the teens to lower 20% range.
The surrounding areas are m the mtd 20 through upper 30% range. "Grass"
87
-------
cover data (Figure 22) resulted in a different contour pattern. Basically, the
center of the map has a depression with cover values in the teens.
Surrounding areas are in the 20 through 30% cover range. A transect was
positioned from the northwest corner to the southeast corner. The resulting
contour profile (Figure 23) further illustrates the three patterns described
above.
DISCUSSION: This exploratory analysis provided some interesting information.
First, it appears that the image analyzing system can be used effectively if
proper caution is taken. Although this work was done from print photos,
negatives would be preferred for future work. The data collected has
considerable limitations. Foremost of these limitations is that no ground
truthing accompanied this data set. Thus the percentage cover reported may
not be accurate. Generation of contour maps always has subjectivity infused in
the process. Kriged maps reflect assumptions on relationships between and
among data, map resolution, etc. Accordingly, the maps should not be used as
showing absolute information; rather, they should be used to illustrate possible
(perhaps probable) patterns. In this case the contour maps suggest that
something is different in the vegetation cover in the southeast tract of the
mapped area. Given that this is associated with a known contamination zone
in the center of the area, it is tempting to forecast a cause-effect relationship.
Such forecasts must be posed only as hypotheses to be tested. The contour
map can serve as an important guide in laying out a field sampling plan.
Finally, if the information collected from the maps is real (i.e., there actually is
a tract with suppressed vegetation cover), the reason may or may not rest with
contaminants. Toxic wind dispersed material could be dampening the growth
of the plants. Alternatively, disturbance in the primary activity zone could be
enhancing wind erosion. The deposition of sands and finer soil particles could
be reducing plant growth by abrasive action and/or burial. It was concluded
that field sampling for vegetation impact (cover analysis, tissue contamination,
samples for toxicity tests) should concentrate on the southeast tract and that
areas to the northwest.should be an appropriate reference.
88
-------
I I
I
i. ». ». u. a. a. »
MttMC*
I I
i. i. i*. is. ». a.
IllUKI
Figure 17. Ergotic Semi-variogram plot. Figure 18. Non-ergotic Semi-variogram plot.
13. -
tl.
32.
*J.
norUiirty
Figure. 19. Kriging Estimates Produced From Crhomium Concentrations.
89
-------
1
i
Figure 20. Kriging Estimates Produced From Total Vegetative Cover.
Figure 21. Kriging Estimates Produced From Shrub Cover Data.
90
-------
Figure 22. Kriging Estimates Produced from "Grass" Cover Data.
AVENUE A VEGETATION COVER
KRIGED CONTOUR PROFILES
60
40
a
8
a • •
a
°o rr - --
------ V" a-i.""
& a
a a
aa
-fTTTT „„,,,., | I I 1
0 12 "M 36 43 60 72 &4
6 -IB 30 42 54 66 78 90
NORTHWEST - SOUTHEAST [MAP UNTSl
a
2
Figure 23. Contour Profile Derived From Figures 20-22. Series 1 Refers To Total Vegetative
Cover; 2. Shrub Cover; and 3 "Grassy" Cover.
91
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u>
ro
NAME
Nigel Blakely
Richard Blanche!
Mike Bollman
Jefl. Brand)
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Pal Cirone
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