EPA-600/4-80-
April 1980
BIOLOGICAL MONITORING
OF TERRESTRIAL ECOSYSTEMS
USING HONEY BEES AND EARTHWORMS
A Workshop Summary
and Panel Reports
J.J. Bromenshenk
Workshop Coordinator/Chairman
I
Environmental Studies Laboratory
Department of Botany
University of Montana
Missoula, Montana 59812
J.B. States
Co-Chairman
Ecological Sciences
Batelle Pacific Northwest Laboratories
i P.O. Box 999
Richland, Washington 99352
Contract No. 68-03-1526
Project Officer
E.M. Preston
Terrestrial Systems Division
Corvallis Environmental Research Laboratory
Corvallis, Oregon 97330
CORVALLIS ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CORVALLIS, OREGON 97330
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EPA-600/A-80-
Aprll 1980
BIOLOGICAL MONITORING
OF TERRESTRIAL ECOSYSTEMS
USING HONEY BEES AND EARTHWORMS
A Workshop Summary
and Panel Reports
J.J. Bromenshenk
Workshop Coordinator/Chairman
Environmental Studies Laboratory
Department of Botany
University of Montana
Missoula, Montana 59812
J.B. States
Co-Chairman
Ecological Sciences
Batelle Pacific Northwest Laboratories
P.O. Box 999
Richland, Washington 99352
Contract No. 68-03-1526
Project Officer
E.M. Preston
Terrestrial Systems Division
Corvallis Environmental Research Laboratory
Corvallis, Oregon 97330
CORVALLIS ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CORVALUS, OREGON 97330
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DISCLAIMER
This report has been reviewed by the Corvallis Environmental Research
Laboratory, U.S. Environmental Protection Agency, and approved for publication.
Approval does not signify that the contents necessarily reflect the views and
policy of the agency, nor does mention of trade names or co=ercial products
constitute endorsement or recommendation for use.
i
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ACKNOWLEDGEMENTS
I wish to thank the workshop participants for their willingness to
expend their energy and devote long hours to the production of two separate
research projects to “prove” the utility of terrestrial biological monitoring.
1 am indebted to J.B. States and E.L Preston for their assistance in
conducting the workshop.
I wish to acknowledge the members of the work groups:
Soils — J.B. States (Chairman), B.S. Ausmus, K. Cromack, Jr., C.D. Drewes,
E.F. Neuhauser, and T.R. Seastedt.
Honey Bees — J.J. Bromenshenk (Chairman), D.M. Burgett, R,W. Ferenbaugh,
Y.I. Lebner, L. Rogers and H. Shimanuki.
M. Ginevan, B.A. Kahn and E.M. Preston worked with both groups.
‘ 14.
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FORE9ORD
PRESTON WILL WRITE, ATTACH IN CORVALLIS
1.14
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ABSTRACT
One goal of EPA’s Anticipatory Research Program is to identify and to
characterize emerging environmental problems before they become serious.
In view of this objective, EPA’s Corvallis Environmental Research Laboratory
will initiate a research program during fiscal year 1980 to develop biological
monitoring methods for terrestrial ecosystems. Approximately half of the -
initial effort will be directed tovards determining trends in the presence
and amounts of toxic chemical residues in biota from a limited geographical
area. If feasible, this endeavor could be expanded to a national monitoring
network. The other half of the effort will focus on correlating tissue
residue levels of these chemicals with physiological and ecological responses
and will assess the utility of such responses for a “biological effects
monitoring” program.
In March, 1980, a workshop was held in Corvallis, Oregon to discuss the
Implementation of a pilot program of biological monitoring using honey bees
and soil invertebrates. Specialists in the fields of ecology, soil biology,
apriculture, environmental problems, physiology, and biochemistry representing
government, university, and private research affiliations convened to consider
research and development needs, classes of chemicals and types of sites to be
monitored, sampling designs, and methods of sample collection, handling, and
analysis. A phased research program was designed for & short—term (one year)
field, laboratory, and paper studies and for a long—term (four to five years)
fully scaled field and laboratory endeavor. Specific projects were established
for the first year effort, while guidance was provided at the level of detail
necessary to establish goals, priorities, and direction of a long—term program.
An important aspect of the program is an emphasis on linking toxic chemical
levels to quantifiable physiological responses and to measurable effects on
the structural and functional status of biological systems. The program will
be reviewed frequently and the usefulness of biological monitoring organisms
and methods reassessed.
This report was submitted in fulfillment of Contract No. 68—03—1526 by
University of Montana under the sponsorship of the U.S. Environmental
Protection Agency. This report covers a period from March 6, 1980 to April 4,
1980, and work was completed as of April 4, 1980.
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Acknowledgement. . . . . . . , . . . , . . . . .
Forward. • • • • • , • . • • • • • • • • , • ,
Abstract . . . . . . . . . . . . . . . . . . . .
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35
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References . . • . . . . . . . . . • . . . . . . . . . . . . • . . . .
CONTENTS
• . I • I I • • I •
• . I • S S • S I •
• S I • • I S • I • •
1. Introduction .
2. Conclusions and Reco=endations. • • • . . . . . . . • • . . .
3. Program Overview • • . . . . . . . . . . . . . . • . .
Selection and use of biological monitors
Interfaces with other programs, agencies and industry.
Program management..
4. Monitoring the Air Medium. . . . • . . .
Rationale for use of bees
Purposes of using bees as monitors • • .
Monitoring concerns. • • . . . . . .
Phased research program. . . . • . •
Phase I — field studies. • • • . • .
Phase I — exposure monitoring. . , . . . .
Phase I — effects monitoring . . . . . . .
Phase I — cost estimates • . • . . . • •
Phase II — field and laboratory studies.
Phase II — exposure monitoring . • • . .
Phase II — effects monitoring. • . . . .
Pbase III. • • . . . . . • . . . . .
Pbase IV •
5. Monitoring the Soils Medium. • • . . . . .
• • • . . S • S I
Phase I — laboratory and paper studies demonstrating
“proof of concept” . • . •
Phase II — laboratory studies correlating biological!
functional soil monitors . . . . . . . . • . .
Phase III — field studies demonstrating combined biological/
functional monitors in active and passive modes.
Phase IV — application to a national network . • . . . . .
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SECTION 1
INTRODUCTION
One of the goals of EPA's Anticipatory Research Program Is "to Identify
emerging environmental problems before they become serious or Irreversible,
In keeping with this objective, EPA's Corvallls Environmental Research
Laboratory will Initiate a research program during fiscal year 1980 to develop
biological monitoring methods for terrestrial ecosystems. An important aspect
of this program will be frequent review to determine the feasibility of
specific biological monitors for assessing environmental quality.
Biological monitoring is a term which includes a wide range of activities.
For the purpose of this report, biological monitoring is defined as the use of
living material (populations, organisms, tissues) for the systematic determination
of the presence and amounts of pollutants and/or the physiological and ecological
effects of these substances as a function of space and time. From an ecological
point of view, there are two types of biological monitoring: "active monitoring"
which generally refers to the introduction of standard, managed organisms into
ecosystems or communities to measure trends in the accumulation of hazardous
chemicals and/or the responses of the organisms to these chemicals; and "passive
monitoring" which usually relates to the use of indigenous organisms for similar
purposes. The coal miner's canary in a cage is a familiar example of an active
monitor. Plants such as pine trees, whose visible pathological symptoms often
serve as the first indicators of air pollution, are examples of passive monitors.
EPA's decision to pursue a pilot program of biological monitoring appears
to be a response to a rather sudden convergence of thinking and events. Each
year, more and more hazardous substances are added to the environment. A
list of the chemicals presently considered environmentally hazardous might
contain thousands of compounds, and new compounds are constantly being
introduced. Until recently, monitoring programs emphasized physical and
chemical instrumentation to detect and precisely measure specific ambient
contaminants. Refinements in analytical techniques have demonstrated that
many substances are more widely, distributed than previously reported: As
the capability to measure smaller and smaller quantities of substances in
the environment increased, the biological significance of these low concen-
trations became more and more uncertain. It also became apparent, that humans
and the environment are exposed not to single, independent substances, but
to complex chemical mixtures. It follows that the combined effects of
these substances can be examined only by looking at the responses of living
components of biological systems. In addition, the emphasis of environmental
research is, of necessity, shifting from observations of short-term, acute
perturbations to long-term studies of the effects of low level, chronic
exposures.
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Thus, there is a growing awareness that standards and requirements for
control technology must be based on information concerning the consequences
of introducing various contaminants into ecosystems and the reasons why
cologlcal perturbations should be kept within certain limits. In order to
accomplish this, the capability must be developed to determine which ecosystem
changes are “natural” and which are anthropogenically induced.
One indication of this àwarenesiwas the International Workshop on
Monitoring Environmental Materials and Specimen Banking held in Berlin (West)
in October, 1978. The conference was sponsored jointly by the Commission
of the European Communities, Brussels, the Federal Ministry for Research and
Technology, Bonn, the Federal Environmental Agency, Berlin, and the U.S.
Environmental Protection Agency, Washington, D.C. A follow—up workshop was
held in Cermantown, U.S.A. in December of the same year. Their major
conclusions were:
o Network systems should be established to monitor ecosystem exposure to
substances which have, or may have, adverse effects.
o Specimen banks should be established for retrospective analysis of
trends in exposures to previously unrecognized pollutants or pollutants
for which analytical techniques may at present be inadequate.
o A number of plant and animal species appear to be suitable for
biological monitoring and specimen banking.
o In the absence of effective monitoring programs, “the detection of
serious environmental contamination from pollutants may occur only
after critical damage ha been done,”
In 1979, EPA received two reports on the establishment of such a program:
Coals of and Criteria for Design of a Biological Monitoring System by the
Ecology Co=ittee of the Science Advisory Board to EPA, and a Biological
Monitoring Concept Paper presented by T.A. Murphy to the Blue Ribbon Select
Committee on Monitoring. The two reports debated biological monitoring Issues
such as selection of species and sites, trophic levels, the general usefulness
of any biological monitoring program, and monitoring of trends in hazardous
pollutant residues versus biological effects.
Both reports concluded that biological monitoring procedures, on a
selective basis, deserve considerably more attention by EPA. The ecology
Committee of the Scientific Board commented that biological, physical and
chemical monitoring are needed to examine changes in the structure and
function of ecosystems because such biological monitoring is an “essential
component of any monitoring program.” The unique advantage of biological
monitoring is that environmental regulations are designed to protect living
organisms, not simply to limit chemical levels in the environment. Only
biological monitoring can assess attainment or non-attainment of biological
goals, including an effect such as toxicity or transport and fate processes
such as accumulation and concentration.
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As regards the monitoring objectives of EPA, these reports pointed out
that biological assessments serve the same functions as chemical and
physical determinations.
o Identification and definition of environmental problems — to reveal,
detect, or anticipate environmental problems and to determine their
nature, extent, magnitude and causes.
o Regulatory development — to provide the knowledge of causal relation-
ships necessary for effective and reasonable regulations.
o Enforcement of regulations — to determine whether legal requirements
are being met.
o Evaluation of regulations and programs — to evaluate the effectiveness
of programs, regulations, or other abatement or control activity.
Although the reports were very informative, tjiey did not provide specific
guidelines for a biological monitoring program. There was some consensus that
managed honey bees had iTwnediate potential for network biological monitoring
of toxic pollution exposures. Honey Bees were also identified as potentially
good biological monitors at the International Workshop on Monitoring Environ-
mental Materials and Specimen Banking. The two reports included little
discussion of soils, but there was considerable discussion of the need for
suitable soil monitors at the Berlin and Germantown Workshops. Earthworms
were recommended for initial monitoring and banking, while microcosnis and
soils were suggested for later, long—term programs.
To address these issues more specifically, a workshop sponsored by EPA’s
Corvallis Environmental Research Laboratory was held in Corvallis, Oregon,
March 18 -through 20, 1980. Participants In the workshop are listed in
Table 1. The purpose of the workshop was to design an Anticipatory Research
Program of Terrestrial Biological Monitoring. The resultant pilot program
is to assess the utility of specific biological tools (indicator species) for:
o Correlation of trends in toxic materials in the terrestrial environ-
ment and body burdens for use in a program of “bio-re Idue or exposure
monitoring” in a limited geographical area. This for 4 seeably could be
expanded to a national monitoring network.
o Correlation of tissue residue levels of toxic materials with physio-
logical and, if possible, ecological responses (changes in ecosystem
structure or function) for eventual use in an “effects program.”
o Acquisition of knowledge and understanding of the possible physiological
mechanisms by which responses to a pollutant or pollutants are brought
about and of the ecological significance of these responses.
o Determination of the feasibility, usefulness and accuracy of biological
monitors for exposure monitoring, effects monitoring, and assessment of
the condition or state of ecosystem health.
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The pilot program is based upon a phased approach over four to five years.
Phase I consists of short—term (approximately one year) field, laboratory and
paper studies on the use of biological monitors (honey bees and earthworms)
to determine trends in toxic pollutant residues in the environment and on
physiological responses to these materials and some population responses.
Expanding Phase I objectives, Phase It will begin to examine ecosystem effects
at the level of functional and structural components. Phase III begins field
tests and verifications of combined exposure and effects monitoring, using
active and passive modes. Phase IV initiates the use of biological monitors
in limited geographical areas for specific pollutant categories, thus providing
background for possible national networks. Phases I and II were outlined by the
workshop. Phases III and IV reflect long—term objectives, the specifics of
which will depend on the outcome of Phases I and II.
The program will emphasize trends or links between toxic chemical exposure,
‘7 physiological responses and ecosystem responses (changes in structure or
function). Dose—response tests and correlations of body burdens with levels
of pollutants in the environment and with observed effects are also important
aspects of the pilot study.
The long-term program depends upon a reiterative process to review the
potential usefulness of specific biological monitor species and methods.
Workshops, research and development efforts, feasibility studies, and
cooperative efforts through informal and formal contracts are important
elements of Phase II, III, and IV. To ensure the continuity and direction
of the long—term program, a core group of researchers, derived in part from
the participants at this workshop, will oversee the program. Provisions were
made for Program Advisory and Peer Review Groups.
This report serves as the proceedings of the workshop and presents
discussions and conclusions reached in defining the phases of a pilot program
of Terrestrial Biological Monitoring. Section 2 swnrn rizes the overall
workshop conclusions and recottunendations. Section 3 presents a suary of
general issues and long—term objectives, Sections 4 and 5 summarizediscussions
specific to each topic area: honey bees and soil organisms, respectively.
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TABLE 1. TERRESTRIAL BIOLOGICAL MONITORING WORKSHOP PARTICIPANTS
Participant Affiliation
Dr. .Beverly S. Ausmus Batelle Columbus Laboratories
505 King Avenue
Columbus, Ohio 43201
Dr. Jerry 3. Brotnenshenk Environmental Studies Laboratory
Workshop Chairman! University of Montana
Coordinator Missoula, Montana 59812
Dr. D. Michael Burgett Department of Entomology
Oregon State University
Corvallis, Oregon 97331
Dr. Kermit Cromack, Jr. Forestry Science Research
Oregon State University
Corvallis, Oregon 97331
Dr. Charles D. Drewes Zoology Department
Iowa State University
Ames, Iowa 50010
Dr. Roger V. Ferenbaugh Group H—8 Mail Stop 490
Los Alamos Scientific Laboratory
P.O Box 1663
Los Alamos, New Mexico 87545
Dr. Michael E. Ginevan Division of Biological and Medical
Research
Argonne National Laboratory
Building 202
9700 South Case Avenue
Argonne, Illinois 60439
Dr. B. A. Kahn Alberta Oil Sands Environmental
Research Program
15th Floor, Oxbrldge Place
9820—106 Street
Edmonton, Alberta T5K 236
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TABLE 1. Concluded
Participant Affiliation
Ms. Yolanda Lehner North Central Bee Research
436 Russell Laboratory
University of Wisconsin
Madison 1 Wisconsin 53706
Dr. Edward F. Neuhauser Department of Environmental and
Forest Biology
State University of New York
Syracuse, New York 13210
Dr. Eric H. Preston U.S. EPA
Project Manager Corvallis Environmental Research
200 SW. 35th Street
Corvallis, Oregon 97330
Dr. Lee Rogers Ecological Science
Batelle Pacific Northwest Laboratories
P.O. Box 999
Richiand, Washington 99352
Dr. Timothy R, Seastedt Department of Entomology and
Institute of Ecology
University o Georgia
Athens, Georgia 30602
Dr. Eachiro Shimanuki Bioenvironmental Bee Laboratory
Plant Protection Institute
Science and Education Administration
USDA Agricultural Research, NE Region
Beltsville Agricultural Research
Beltsville, Maryland 20705
Dr. James B. States Ecological Science
lJorkshop Co—Chairman Batelle Pacific Northwest Laboratories
P.O. Box 999
Richiand, Washington 99352
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SECTION 2
CONCLUSIONS AND RECOMMENDATIONS
Recommended research projects for Phases I and II of a pilot biological
monitoring program are listed in Table 2, which also presents specific
experiments for each Phase I project. Details of Phase II projects are
contingentupon the results of Phase I. Phases III and IV are covered in a
more general manner because these stages depend to a high degree on the
results of the first two phases and upon the level of funding.
It should be emphasized that the initial phases of the Terrestrial
Biological Monitoring Program (TBHP) are highly exploratory. The concensus
of the workshop participants was that honey bees and earthworms have a
high potential as specific tools for this purpose because they are
manageable and bioaccumulate many chemicals, Another selling point is
that the information available on all aspects of their biology, as veil
as their interactions with pollutants, probably equals or surpasses
that of any other invertebrates. Still, even for these organisms,
experience with pollutant interactions has been limited mainly to
acute exposures of a few toxic chemicals, and experience with long—term
low level exposures to individual and chemical complex mixtures is
almost non-existent.
Toxic heavy metals are emphasized in Phase I projects because small scale
pilot demonstration projects should, of necessity, be relatively simple, easy
to perform, informative, and cost effective. Other chemicals such as gaseous
effluents, radioactive wastes, organic pesticides, and other organics including
aliphatic, alicyclic, aromatic, and heterocyclic compounds received high
priorities for examination in Phase II.
Any organisms recommended for biological monitoring after the early stages
of the TRMP should be considered the best available at present, given the
constraints of our knowledge and the resources available for feasibility
studies. As our information base develops and our understanding of the use
of biological monitors becomes more discriminating, other biological monitors
may become more appropriate.
Specific recommendations and conclusions concerning the overall aspects
of the TBMP are:
o A pilot program of biological monitoring of exposures and effects is
needed and is technically feasible.
o Honey bees (Apie meilifera) and earthworms (Eisenia foetida) should be
utilized in the initial phases of the program as biological monitors for
two terrestrial media — air and soils, respectively.
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o The integrity of the research packages is essential. Each package
is aimed at providing information ranging from trends of toxic
(includes mutagenicity, teratogenicity, carcinogenicity) pollutant
residues in the environment, and resulting body burdens, all the way
to the biological effects and their significance at the ecosystem
level of organization.
o Research which provides integrated data from several levels of biological
organization, in both structural and functional terms, represents an
optimal approach.
o Initial validation of biological monitoring is contingent upon parallel
experimentation with a functional ecosystem monitor and a biological
monitor (e.g. earthworm/soil nutrient flux; honey bee! pollination).
o Passive monitors are needed in addition to active monitors. Developing
both types of monitors should provide effective tools for monitoring
short—term perturbations (in the active mode) and long—term perturbations
(in the passive mode) in the major ecosystems of concern.
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TABLE 2. TERRESTRIAL BIOLOGICAL MONITORING RESEARCR PROJECTS
AIR MEDIA
SOILS MEDIA
PHASE I Field Tests — Honey Bees
o Bloaccuinulation: Arsenic
—-Sampling Variance
—-Receptor Compartments
——Correlations of Levels in
Media and Bees
——Other Heavy Metals
Field & Lab Tests — Honey Bees
o Dose Response: Arsenic
——Reproduction/Mortality
——Hoarding/Honey Production
——Enzyme/Stress Indicators
PHASE II Field & Lab Tests—Honey Bees
o Bloaccuinulation: Organics/Inorganic
o Refine Phase I Methods
o Pollutant Transport/Cycling
o Colony & Ecological Responses
——Simulation Modelling
——Pollination Syndrome
——Effects of Pollutant Cycling
——Food Chain Transfers
——Stress Indicators
PHASE III Field & Lab Tests
o Passive Monitors — Native Bees
—Exposure Monitoring
—Population Effects
—Stress Indicators
——Simulation Nodelling/Megachilidac
o Active Monitors
—Field Validations of Techniques
PHASE IV Network Monitoring
o Active and Passive Monitors
PHASE I Lab Tests - Worms
o Dose Response: Heavy Metals
—-Body Burden
——Population Effects
— Growth & Reproduction
——Neural
— Action Potential & Synaptic
Transmission -
o Soil Nutrient Flux—Concept Paper
——SNY as Ecological Monitor
——Coupling Biological/Ecol. Monitors
PHASE II Lab Tests — Litter/Soil
o Dose-Response: Heavy Metals
--C0 2 , DOC, P0 1 —P or N11 3 -N, N0 3 —N
——Sensitivity Analysis of SNF vs.
Worm
PHASE III Field and Lab Tests
o Passive Monitors: Pollution Gradient
——Soil/Litter Sampling
— Elemental Analysis
——Sampling of Worms
— Elemental Analysis
— Neurophysiological Tests
o Active Monitors: Pollution Gradient
——Soil/Litter Sampling
— Elemental Analysis
— Exposure Analysis — Worms
— Effects Analysis — Worms
— Functional Analysis — Soils
(SNF With & Without Worms)
—-Other Species of Worms
——Field Validations of Predictions
PHASE IV Application to National
Network
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SECTION 3
PROGRAM OVERVIEW
The Terrestrial Biological Monitoring Workshop was to provide specific
recommendations for a pilot monitoring program. Throughout the workshop,
several issues or concerns of a general nature were raised, and they are
summarized in this section.
3.1 CURRENT STATUS OF TERRESTRIAL BIOLOGICAL MONITORS
The first issue was the relatively undeveloped state of the art for
using biological monitors in environmental assessments. Too often, biological
monitors seem to have been selected in a serendipitous, unfathomable manner.
Not only do we lack appropriate monitors for environmental problem solving
but our ability to interpret the significance of observed responses usually
is limited, even when considering monitors that have proven valuable. For
example, the presence or absence of lichens can provide early recognition of
pollutant exposure problems, but the significance of this response to ecosystem
or human ve1faz - is unclear.
Biological monitors must supply the scientific and technical information
needed by EPA and other users; especially information ranging from trends in
pollutant concentrations in the environmental media and resultant body burdens
all way to biological effects and their significance at the ecosystem
level. For the most part, these information needs are not met by current
biological monitors. Based upon discussions of these issues, the workshop
participants arrived at the following realizations:
o Better science needs to be applied to biological monitoring and
environmental problem solving. Most present techniques are -
inadequate and have very limited usage. A rigorous, organized
approach establishing clear statements of objectives and tests of
suitable hypotheses will significantly improve our capability
to develop useful, quantitative monitors.
o Biological monitoring should be based upon a new perspective, one
that clearly identifies the biological level of organization from
which inferences will be made and which focuses on links that can
be made between levels of exposures to pollutants and effects upon the
the structural and functional status of biological systems of inter’est.
o There is a need to produce quantitative information that can be used
to judge the significance of observed responses and which can be entered
effectively into the decision making process.
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3.2 SELECTION AND USE OF BIOLOGICAL MONITORS
Workshop participants were concerned that the pilot program not merely
establish more monitoring tools or ones of limited use for a few chemicals
or sites. The goal of the TEMP is to significantly improve monitoring
capability such as increased sensitivity, earlier warning of pollutant
build—up or potential environmental problems, greater reliability, more
cost—effectiveness, and better information, particularly on biological
effects.
When selecting and using biological monitors, it is important to establish
the objectives of monitoring: Is it to provide a biological indicator which
displays a rapid, un tnbiquous response to contaminant levels below those which
cause general environmental damage, or is it to Identify quantifiable
responses that can be related to the responses of the system and which
correlate in an understandable manner to pollutant levels? Desirable
properties of the first (often termed biological indicators) may not be the
same as those of the latter (termed biological monitors). The two functions
are not mutually exclusive, but they may not be perfectly correlated. For the
purposes of this workshop, discussions were directed towards developing
biological monitors, not just indicators.
Workshop participants were asked to examine organisms and tests in the
context of exposure and effects monitoring used in passive and active modes.
In addition, two types of monitors were considered for each anthropogenic
input (pollutant): taxanomic and ecosystem monitors. A taxanomic monitor
provides information about possible responses of a taxon (species, family,
order) regardless of ecosystem context; for example, exposure monitoring of
DDT in the eggs of birds. An ecosystem monitor provides information about
structure or function of an ecosystem (a particular assemblage of interacting
species) for each ecosystem type; for example, pine trees as monitors of
coniferious forests. These categories are not riutually exclusive. In general,
for taxanomic monitors, inferences usually can be made only as far as the
population level, while for ecosystem monitors, inferences can be made to the
ecosystem level.
The level of biological organization of interest affects the inferences
that can be made, Generally, if -we wish to have explanations for what is
observed, we should examine the next lover level of biological organization
and make observations at that level. On the other hand, if we are to make
statements concerning the significance of our observations, we should look
to the next higher level of biological organization. The problem is to identify
information requirements and then to design studies and interpret the data
appropriate for the level of organization about which the inferences will be
made.
The following definitions were agreed upon at the workshop;
Exposure Monitors — used to establish that exposures to anthropogenic
pollutants have occurred and to establish trends over time and across
space. Generally, this involves determination of tissue residues
(body burden determinations) and incorporates three concepts: the types
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and amounts of pollutants that impinge upon the organism, the dose that
the organism receives, and resultant body burden, assuming the material
bloaccumulates.
Effects Monitors —— used to evaluate the effects of exposure on the
structural and functional status of biological systems of interest.
Exposure monitors can be classed as accumulators concentrators, and
magnifiers. Bioaccumulation is the ability of an organism to concentrate a
substance throughout its life so that the concentration continuously increases.
Bioconcentration is the active concentration of a substance by an organism as
a result of activities such as ingestion or absorption. Bioinagnification is
the step—by—step concentration of substances through successive trophic levels
of food chains,
Exposure monitors can be further catergorized by:
o The time period during which the sample is exposed. Tree rings are
useful for long—term, honey bees are useful for short—term (seasonal)
studies. Only the queen lives for more than one year.
o Range of chemicals for which the monitor is useable; a specific
chemical or a category such as inorganics, or many chemicals or
categories such as inorganics, organics, radionuclides.
Selection criteria for exposure monitors include:
o Cheaper and/or more effective than physical/chemical methods.
o Accumulator and preferably a concentrator or magnifier to increase
detection capability.
o Relatively insensitive to as many toxic substances as possible. An
organism that dies when exposed to low levels of toxic materials has
a very limited period of use.
- o Widespread distribution, preferably nationwide.
o Important structural or functional component of ecosystems. If
exposures can be linked to effects, so much the better.
Effects monitors can be categorized as:
o Threshold monitors or organisms which are extremely sensitive. If they
do not respond, it, generally is assumed that the system is not
responding.
o Quantitative monitors which display measurable responses that can be
related to the response of the system.
o Known or suspected responses to pollutant.
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o Occurs in the ecosystem of interest.
o Relevant to important ecosystem Structures or functions.
o Appropriate level of biological organization.
o Process or resource of interest to human welfare — desirable.
An organism ma ft i not bioaccumulate a substance, yet may be very susceptible
to harm such as poisoning. Conversely, many good bioaccumulators are relatively
insensitive to injury from a variety of toxic substances, Thus, a good exposure
monitor may not be a good effects monitor and vice versa, but if the two can be
linked, that is a bonus.
The format presented in Figure 1 was used to guide workshop discussions
for selection of candidate monitoring organisms and methods and as an aid in
visualizing the relationships of the various concepts.
Figure 2 presents a flow chart depicting how information provided by a
biological monitoring program can be incorporated into the decision making
framework. Anthropogenic pollutants may cause perturbations to ecosystems
which change measurable ecosystem proper E es or attributes. The responses
of indicators are a function of the ecosystem properties, which can be
described by process equations, i.e. f(x,y)’s and g(x,y)’s. All information
flows ultimately lead to decision makers such as administrators, managers,
scientific and technical staffs, and public interest groups. To insure that
this occurs in an effective manner, the information obtained must answer the
proper questions, including what needs to be known to facilitate environmentally
Sound decisions, how to design and how to apply the best monitoring techniques
available so as to properly address specific statements of objectives, and how
to put the information obtained into a useable format and in a timely manner so
as to effectively influence decisions.
3.3 INTERFACES WITH OTHER PROGRAMS, AGENCIES, AND INDUSTRY
Each year, millions of dollars are spent on physical and chemical
monitoring and environmental impact assessments. Numerous applied and basic
research programs are attempting to develop the capability to predict the
responses of ecosystems to various perturbations. The basic question is to
provide questions about the consequences of introducing various quantities of
pollutants into the environment,
For economy of personnel and financial resources, it is important to
integrate EPA’s terrestrial biological monitoring effort with other ongoing
research. The information value of data gathered for the biological monitoring
program will be greatly enhanced if the project were carried outin an area
where extensive physical and chemical monitoring and/or ecological monitoring
is being conducted. This would facilitate the understanding of interactions and
relationships between the various aspects of the system and in doing so should
increase the potential predictive value.
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Figure 1. Selection of biological monitors.
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TERRESTRIAL BIOLOGICAL MONITORING PROGRAM
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ANTHROPOGENIC EXPOSURE MONITORS D
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ABIOTIC VARIBLES ‘ C
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Figure 2.
Biological monitoring in the context of the decision
making framework. Solid lines represent energy flows,
dotted lines information flows.
L____ 5 _
—
Ba
EFFECTS MONITORS
iNDICATORS OF
BIOTIC VARIABLES
Bb
INDICATORS OF
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The TBMP is based upon a conceptual framework for international and
national network monitoring of man’s influence upon his environment (Luepke,
1979; Murphy, 1979; and Orians, 1979). The purpose of the ThMP is to demon—
etrate the utility of this concept through development of specific monitoring
tools for use in such networks, One assumption made is that it is possible to,
select a minimum number of monitoring sites, ‘!not an exclusive set,” that
provides a representative cross—section of major ecosystems of the United —
States (Botkin, 1978). Thus, site selection and characterization will become-
a major consideration at the point of applying these tools to network systems.
Fortunately, that is precisely the issue being addressed by the Pilot Program
f or Long-Term Observation of Ecosystems (LTER) sponsored by the National
Science Foundation.
EPA has several ongoing programs aimed at problew pollutants, and their
biological effects including acid rains (CERL), toxic substances (TOSCA),
protocols for terrestrial ecological assessments (IERL—RTP), human effects
monitoring (HEMP), pesticides (OPP), and solid wastes (OSW).
Department of Energy programs, especially as regards synthetic fuels and
alternative energy technologies, have ongoing studies, several of which have
established sites for long—term assessment of pre—operational and post—operational
conditions. In cases such as these, DOE could profit by the addition of specific
biological monitoring capabilities, while EPA would benefit by not having to
locate sites or to characterize them.
There can ‘be no doubt about the need for a capability to monitor accumulation
and effects of anthropogenic pollutants. We now possess a conceptual framework
for establishing network monitors. The initial strategy is to “prove” the utility
of the concept by focusing on the most promising biological monitors for each of
the two terrestrial media — honey bees in air and earthworms in soils. The
objectives are to (1) correlate trends in.toxic materials in the environment
with body burden trends, (2) identify the biological responses to these trends,
(3) understand the underlying biochemical and physiological responses, and
(4) couple monitoring of important functional processes. If we can demonstrate
that the technique works for bees and/or earthworms, it seems that a virtual
flood of support for future development would appear from potential users.
3.4 PROGRAM MANAGEMENT
Proper program management is essential to maintain the focus on the purposes,
goals, and objectives of the ThMP. Figure 4 outlines a proposed management
Btructure. It is reconmiended that a core group of individuals from this work-
shop be retained in research and in a peer review or advisory capacity so as to
preserve the integrity and continuity of the program and to provide direction
and frequent review thzough each of the phases. The interagency advisory group
will include potential users. This should guarantee that the information needs
of the decision makers are addressed. It is the responsibility of the EPA
project officer in cooperation with the policy advisory group to set and review
program objectives. The project advisory group will meet semi—annually and will
be composed of four to six principal investigators. Membership is expected to
change as new projects are added and older ones are completed. The function of
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the peer review group is to critically assess products in view of stated
objectives and to provide guidance in these matters.
PROGRAM MANAGEMENT
INTERAGENCY
ADVISORY
GROUP
EPA PROJECT MANAGER
I _______
I
IPROJECT xJ IPROJECT Yj IPROJECT Z
Figure 3,
Management scheme for the Terrestrial Biological
Monitoring Program.
I
tAll
PEER REVIEW
GROUP
PROJECT ADViSORY GROUP
I
I
17
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SECTION 4
MONITORINC THE AIR MEDIUM
Members of previous monitoring workshops and advisory groups proposed
honey bees as good potential biological monitors, particularly for network
monitoring of trends in toxic pollutant residues. Honey bees have been used
for this purpose on a limited scale in the United States and in Europe.
Because of these recoendations, a TBMP work group was formed, consisting
of specialists in apiculture and environmental problems, the latter having
experience with honey bees or native bees as biological monitors. After
careful consideration, this group concurred that honey bees were ideal as
part of an exploratory program to demonstrate the utility of the biological
monitoring concept.
However, there are limitations to the use of honey bees. Honey bees are
man-managed and as such can be deployed as active monitors. But the skill
and knowledge of the person handling the experimental animal may greatly affect
the colony. For example, poor management can lead to disease problems, low
yields of honey and wax, or even death of the colony. Thus, during pilot
phases of the monitoring program, a standardized agricultural methodology
and experienced individuals are essential. In addition, many of the sampling
techniques in a monitoring program might, at least for short periods, disrupt
colony function.
4.1 RATIONALE FOR THE USE OF BEES
Despite these lfmitations,the consensus was that honey bees could greatly
enhance a pilot bio] gica]. monit,pring program because:
o Bees (or other organisms) as biological monitors are preferable to
chemical or physical instrumentation alone because they not only
serve as bioaccumulators but also give some indication of physiological
effects and provide information about the transfer of toxic materials
through food chains and ecological effects.
o Since honey bees are man—managed, they can survive or thrive in nearly
any biome. In addition, genetic variability can be reduced by using
inbred lines and artificially inseminated queens. Thus, the honey
bee possesses desirable characteristics for an active monitor.
Jtr’’/ - -
o - Bees magnify the level of many chemicals in their surroundings and
increase the ability to detect pollutants.
ó There is a potential for the transfer of harmful chemicals (toxins,
uiutagens, carcinogens) to humans via honey or pollen which should be
addressed.
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o The honey bee is economically important as a producer of honey and
beeswax and for pollination service. A monitoring system using
bees does not require transfer of the information (extrapolation
or translations of the data) to be readily understandable in terms
of consequences to mankind.
o The obvious link of honey bees to ecosystems is the pollination
syndrome.
o Subtle effects of stressors, such as pollutants on bee populations,
are often overlooked by scientists and beekeepers. A constant
monitoring program should enable us to determine if changes in the
productivity and efficacy of honey bee colonies occurs as a result
of low level exposures to environmental contaminants.
4.2 PURPOSES OP USING BEES AS MONITORS
It became apparent early in the discussions that honey bee^could
provide two different services as biological monitors. Honey bees, as monitors,
can provide an early warning of both pollutant accumulation and effects
signaling possible harm to other organisms or to ecosystems. Especially
Important are interfaces with ecological processes. Another reason for using
these organisms is to monitor hazards to the bees themselves and in turn to
the beekeeping industry. As such, bees are a rather unusual biological
monitor because they are not only sensitive bioindicators, but they themselves
are a valuable resource.
A.3 MONITORING CONCERNS
A major difficulty in discussing the use of honey bees was that so much
is known about them that it was not easy to prioritize the extensive list of
possible parameters which could be utilized or the numerous methods or
approaches.* What the group needed to identify were simple, cheap and
effective measurements useful in quantifying the biological effects of
pollutants. Particularly important was the identification of parameters
that control honey bee populations.
Dr. Shimanuki provided a particularly helpful tool —a simulation
model of honey bee populations developed at the Bioenvironmental Bee Laboratory,
Beltsville, Maryland. This model utilizes 22 parameters which can be readily
quantified. Using the model and the collective experience of the work group
participants, the discussion focused on interfacing the monitoring program
with.model development by providing data about pollutant effects on longevity,
mortalities, reproductive cycles and foraging profiles. Testing the sensitivity
of the various bee parameters that control the population model and providing
the opportunity for basic studies to gain a better understanding of inter-
actions are obvious benefits of working cooperatively on model development and
monitoring. Once confidence is developed in the model, it can be used as a
research tool to predict the impact of various chemical pollutants on populations
of bees, impacts which could have consequences at the ecosystem level. Thus,
the model became both a means of prioritizing the selection of parameters to be
examined and a key element for later research phases of the TBMP.
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A Biological Monitoring Research program would allov the addressing of
pollutant effects on bee populations (commercial or managed honey bee denies),
which may result in regional depressant effects. Long—term investigations
of “sublethal,” subtle, or chronic effects of exposures to anthropogenic
materials may establish potential blonomic and economic depressant effects
upon the bee industry and agriculture, such as increased costs of colony
replacement, commercial pollinator shortages, predisposition of diseases,
reduced honey and wax production, etc. (Figure 4)...
An underlying assuntption of the use of bees as monitors is that bees
affected by a chemical will in turn reflect the fate of the colony.
Examination-of this assumption lead to two realizations:
o Evaluation of effects should be done on two levels —— the effect of
the candidate chemical or chemicals on the individual bee and the
effect on the colony.
o Except for the queen, the individual members of the hive do not reflect
effects of the environment over long periods (greater than a year).
Thus, measures of the colony appear to be more appropriate than measures
of individuals for monitoring impacts of chemicals over extended periods, The
colony reflects more accurately direct cumulative effects.
In essence, when the honey bee is considered as a biological “tool,”
it should be remembered that the colony is the organism . The ultimate goal
is to understand interactions of the pollutant with the colony as a whole
and not just individual compartments (foragers, nurse bees, pupae, honey).
For some purposes, such as exposure monitoring, one compartment may be of
greater interest or utility than others, such as the use of honey versus bees,
but, the colony generally is the basic unit of study.
Another monitoring concern discussed was that tests are needed which can
easily and rapidly screen exposures and/or effects and be used in follow—ups
on more specific concerns. Specific biochemical or physiological parameters
could be selected to indicate the general health of bee parameters which
remain relatively constant under a variety of climatic conditions. By
establishing baseline values for these parameters and for the range of
alterations in clean and polluted environments, we might be able to identify
general physiological or biochemical stress indicators whose measurable
alterations would indicate environmental hazards. Then, other specific tests
could be used both on the bees and in the environment to delineate dangers.
These stress indicators should remain relatively constant in bees even at low
levels of honey production and/or pollination efficiency.
The use of honey bees as an environmental monitoring tool implies that
some index of environmental conditions based on honey bee responses or
activities would be developed and used in a predictive manner. Ecological
indices generally require calibration before being used in this manner and
this is not a trivial concern. Ebuchert et al, Batelle Pacific Northwest
Laboratories were suggested as one of the major authoritative groups working
on problems of this nature.
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The discussions concerning honey bees as monitors focused on observable
responses, their causes, their significance as indicative of ecosystem effects
and the usefulness of all of these to decision makers. However, there was a
concern expressed that we should not ignore possible threshold monitors
that do provide an early warning of environmental perturbations. Honey bees
may be excellent indicators in this sense, but the workshop discussions
did not address this topic in any detail. That should not be construed to
mean that this use of a biological monitor is not important.
Throughout these conversations, considerations of statistical design,
adequate precision, statistical respectability, chemicals of interest, siting
criteria, and interactions with other agencies and industry were addressed.
Specifics of these discussions appear under the description of the short—term
study plan in this section and in Sections 3 and 5.
ç__ ____ [ BEES
POLLiNATION OF
COMMERCIAL CROPS
AND INDIGENOUS
VEGETATION
PHYSIOLOGICAL
EFFECTS
Figure 4. Conceptual model of key interactions between bees, the
environment, and man.
ECOLOGICAL
STRESS
HONEY
POLLEN
WAX
lJ
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4.4 PHASED RESEARCH PR0GRA !
The bee work group produced a phased, four year research plan to perfect
the use of honey bees and native bees as monitors of pollutant acct=ulations
and effects. Major objectives of the program are to:
o Correlate trends in toxic substances in the environment with body
burden trends (exposure monitoring).
o Identify biological responses to these trends (effects monitoring).
o Ascertain the physiological and biochemical response mechanisms
involved,
o Provide integrated information concerning responses to pollutants at
several levels of biological organization from the biochemical to
the ecosystem.
o Develop models to examine and to predict the impact of various
contaminants on populations of bees, on structural and functional
status of ecosystems, and on valuable resources.
The work group believes that these objectives are attainable based
on their collective expertise in the use of insects in exposure and effects
monitoring, modelling of bee populations, bee life cycles,and population
dynamics, bee diseases, bee physiology, biochemistry, and toxicology, insect
ecology, and apiculture.
In order to achieve this goal in a timely manner, the initial effort should
focus on an approach that has a high probability of success, yet which will
address as many of the major objectives as possible.
Monitorin.g accumulation and effects of inorganic metallo—compounds in
honey bees located near a known emission source seems to be a good starting
point for demonstrating the utility of the biological monitoring concept.
In addition, it provides an ideal opportunity to establish,a small scale
monitoring program in the field.
Arsenic, cadmium, lead, and other metals build—up in bees, pollen, and
honey (Bromenshenk, 1980). There are reports dating to the early 1900’s of
severe bee kills near smelters and other arsenic sources. Although toxic
effects of heavy metals are well known, little information exists about
sublethal effects on colony health. Arsenic may be a continuing problem
to the beekeeping industry. Members of the bee work group recently examined
suspected chronic and/or acute arsenic poisoning of bees near industrial
sources or areas in which arsenical defolianta had been applied. -
The recognition of arsenic as a major environmental pollutant is growing.
Bencko, 1977, reviewed the history and present status of knowledge concerning
carcinogenic, teratogenic, and mutagenic effects of arsenic in humans.
Thus, a demonstration project focused on arsenic could provide valuable basic
information concerning a serious environmental threat.
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Hopefully, the first—year monitoring program will successfully define
exposure levels of individual honey bees (or groups of foragers) and of the
colony as a whole, give a more complete picture of arsenic’s mode of action,
and show precisely what effect various levels of arsenic have on bees and
colonies; i .e. critical dose—response relationships such as affects on
mortality, longevity, and reproduction. The long—term project will address
effects of arsenic and other pollutants at the population and ecosystem
levels and eventually will examine regional impacts via network monitoring,
at least on a limited scale, Beginning with Phase II , other che-micals such as
organic compounds, radionuclides, and/or gases will be included. By Phase III,
native bees will be incorporated as passive monitors to compliment honey bees
as active monitors.
4.5 PHASE I — FIELD STUDIES
In electing to pursue a field trial, the work group expressed the belief
that it already was feasible to utilize honey bees as exposure monitors, at
least for certain chemicals. Earlier workshops had identified honey bees as
promising tools for network toxic residue monitoring. However, honey bees
have only been used for this purpose in a limited number of cases and usually
for relatively short periods.
4.6 PHASE I — EXPOSURE MONITO ING
The purpose of Ph 6 I (Table 3—page 28) exposure monitoring tests is not
just to show that bees accumulate arsenic or other heavy metals. The major
objectives are to evaluate questions of deployment, sampling inter— and intra—
colony variability, smallest representative samples, minimum levels of
pollutant detectability, points most appropriate to monitor (foragers, nurse
bees, pupae, pollen, or other), and correlations of pollutant levels in bee
colonies with levels in the ambient environment.
Cbemicals of Interest
Arsenic was selected as the primary chemical of interest for the initial
effort because:
o It is magnified by bees.
o It is toxic but with a range of accumulation sufficient to allow
monitoring of fairly long—term, sublethal effects.
o It is distributed throughout the hive compartments — so that
accumulation and effects can be identified, studied, and quantified
at several points for a better understanding of the interactions
with bees and the colony as a whole.
o There is the possibility of food chain transfer of arsenic via
pollen and honey.
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Other heavy metals are of interest and should be included in analyses
for the same samples, if cost. restraints permit. Organic chexnic, .s, particularly
inicroencapsulated inse icides including Pennca -M Penncap.-EI!Y, and
encapsulated Diazinon(. .)and the—carbaryl Sevin 1 .. ) received a high priority
for inclusion to the program because these chemicals severely damage bees.
Although ongoing programs are examining these chemicals in bees, the
identification of encapsulated versus emulsified formulations of parathion and
diazinon is very difficult. Insecticide usage usually results in hot Spots
or localized problems which may occur as a single event (e.g. grasshopper
control which usually depends Qn one application during the growing season)
or as a series of events (e.g. aphid control which may rely on weekly spraying).
However, it should be feasible to produce a paper showing the role of bees as
effects monitors of lethal and sublethal levels of insecticides based on a
literature survey of current papers.
Other organic compounds such as PCBs, PARs, or airborne hydrocarbons
associated with the conversion of coal to synthetic fuels are of great interest.
Many toxic, mutagenic, or carcinogenic organic chemicals of great
concern to human health have never been examined in bees either from the -
viewpoint of possible bloaccumulation or adverse effects. We need to demon-
strate the efficiency of bees in monitoring these other types of chemicals
and sources. The work group felt that it was essential to design a bee
monitoring program for a wide spectrum of pollutants and sources, not just
a few chemicals or particular sites.
Criteria for Site Selection
The choice of the site for the short—term study should take into
account the following:
o Known source of arsenic and other inorganic toxins. Sixteen
copper smelters, a couple of chemical plants, and some geothermal
sources are possible candidate sites.
o Point source clearly definable and surrounded by a relatively
“pristine” environment so that sampling can be done along an
exposure gradient within an area, of similar climate and vegetation.
o Site well characterized in terms of available biotic and abiotic
data. Many of these types of point sources have been studied over
long periods of time and may have ongoing research and.physical
monitoring programs, especially of air quality,
Site selection becomes even more important in designing ‘long—term exposure
or effects monitoring programs. Sites must have integrity (protected from
the public) and must be accessible. For long—term studies, permanent sites
are essential. ‘Availability of support data affects not only expenditures,
but also the types of questions that can be addressed. Given the costs of
characterizing complex systems, ties with other programs could be
invaluable. As an example, there is an ongoing ecological monitoring program
throughout the Northwest at several of the research natural areas. Other
sites have been established at existing or planned fossil fuel and other
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technological development areas. The honey bee monitoring and possibly the
soils (earthworm) monitoring could greatly enhance these programs at minimal
expense to EPA.
Use of sites in agricultural areas versus less disturbed areas was debated
at length. One participant commented that a demonstration of high pesticide
levels within honey bee colonies located next to areas frequently sprayed:
was not going to impress anyone. But pesticides are hazardous to beekeeping,
to human health, to insect pollinated crops. For these reasons, pesticides
and agricultural areas deserve attention, especially as regards long—term effects
of low level exposures. Also, agricultural systems may be the most relevant
ecosystem for monitoring by honey bees because of the close interactions
of bees and commercial crops. Unfortunately, pesticide impacts are complex
issues that pose difficult problems in planning field exposure tests.
Field Design of Exposure Experiment
The recommended field design for the short—term exposure study (Table 3)
calls for a 15 week study using honey bee colonies at a minimum of three sites
arranged along an exposure gradient relative to an arsenic point source, such
as one of the nation’s sixteen copper smelters. At each site, colonies will be
established in new equipment using package bees from a “clean” area. Sister
queens, in bred—lines, and artificial insemination could minimize genetic
variability. Samples of bees, pollen, and honey will be taken at three—week
intervals — the development period from egg to adult. Five colonies per site
were recommended, but the final number should be based upon literature reports
and/or preliminary sampling of existing colonies in the study area to determine
the number of observations (colonies sampled) needed to obtain a mean arsenic
value with a desired confidence interval (prescribed base).
Phase I exposure studies rely upon the analysis of biological samples
for arsenic content. These may reflect the fact that foraging bees visit
different sites and as such are subjected to widely varying levels of arsenic
contamination. Further, this contamination may be non—randomnly distributed
through time, space, and within the colony.
The total number of bees or amount of pollen or honey which can be
removed without seriously disrupting the colony is limited. The statistical
problem is to apportion and to minimize both inter— and intra—colony
variability and to determine the number of individuals (bees, pollen pellets,
etc.) required to form a sample unit which adequately reflects the level of
arsenic in the compartments of the colony. It is expected that as the number
of bees in a sample increases, the average level per bee viii converge to the
average of the colony. - -
Similar statistical questions apply to the number of colonies sampled per
site and the number of replications per compartment (e.g. bees, pollen, honey).
Again, the problem is to determine what number (N) is needed to produce an
acceptable standard error (S.E.). Published data (Broinenshenk, 1980) suggests
that as regards the colonies at a site, the S.E. is a constant function of the
mean arsenic content and that hive to hive variability may be a major source of’
error.
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Specific statistical suggestions discussed by the work group included
logarithmic transformation of data, the approach described by Pielo’u, 1969
for selection of quadrate size in environmçntal sampling, and the application
of analysis of covariance and anova models to the data.
The work group recommended that at least four compartments of each colony.
be sampled for each site and date. These include foraging bees, nurse bees,
pupae with pigmented eye apsuii? and pollen. Other compartments of interest
include larvae, eggs, and both capped and unèapped honey. Honey is of
particular interest because of potential food chain transfer of arsenic to
humans. Workshop participants reported that there was no reliable method to
accurately determining arsenic concentrations in honey. If this analytical
problem can be resolved, honey should be sampled. It might be advisable to
collect and store honey for analysis at a later date.
Pupae and nurse bees will be collected from the brood frames within each
hive. Pollen and foragers returning from the field will be obtained from
traps attached to hive entrances. Top entrances are recommended for obtaining
samples free of debris. Combination traps for both bees and pollen could be
left in place on the hive with a baffle to direct bees into or past the trap.
At least one colony per site will be intensively sampled at a late enough
date in the season that bloaccumulation should have occurred. A recormnended
procedure is to take a large sample, 1200 bees for example, at the entrance to
a hive, In the laboratory, using tables of randonn iumbers, twelve samples
of 100 bees could be drawn for analysis, Following a similar procedure, the
bees could be recombined (jackknifed) into samples of. 200, 300, 400, or 600 for
analysis, The purpose of the exercise is to determine for each compartment of
the colony the smallest sample necessary for representativeness, accuracy, and
precision,
At each site, air, water, and vegetation will be sampled. Soil samples
could be included, but the work group did not think that soils would contribute
much to the transfer of arsenic from the environment into bee colonies, except
possibly by rersuspension under windy conditions.
Ambient arsenic is assumed to be an important entry route into bee systems.
Air samples will be obtained using samplers such as Hi—Vols. The work group
suggested utilizing at least four per site arrayed in a square, one sampler
at each corner of the square, the hives at the center. Each air sampler should
be located within the forage area of the bees, probably not more than one
kilometer from the hives. Specifics of the air sampling program such as
deployment, number of samplers, frequency of sampling, period of exposure, and
even the type of sampler utilized, are left to the investigator performing the
experiment since site factors will have to be incorporated in the design of an
appropriate sampling scheme.
Water and vegetation are assumed to be other major routes of entry.
Water and floral resources utilized by bees are easy to identify by bee
flight activity. Water and vegetation will be sampled at least every three
weeks. Vegetation sampling will focus on the flowers visited by the bees,
especially at points near the colonies and each of the air samplers.
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For any environmental sampling, the program must be fully designed before
the first sample is obtained from populations in the field in order to obtain
a truly representative sample. Individual, population, pollutant, and site
characteristics all interact in a dynamic framework, and each produces
intrinsic variability. Thus, the value of any sample is limited to the extent
to which it is truly representative of the Bet from which it is obtained,
In addition, procedural sources of error introduced during collection, handling,
storage, and treatment of samples before analysis must be adequately addressed
to assure valid results. This quality assurance cannot be over—emphasized but
the work group could only underscore its importance and identify major concerns
given the time constraints of a three day meeting to address a four to five
year program.
The research project proposed for Phase I is intended as a guideline for
the minimum acceptable sampling program. Lower limits were set based on
projected cost constraints. Fewer sites, co1oni e—coflections, or compartments
could negate the experiment’s value. This project must be reasonably funded to
assure quality. More sites are strongly recommended and options such as more
colonies at three or four sites or three colonies at more sites should be
carefully evaluated. Also, Hi—Vols may not be the best physical monitors for
arsenic. It was suggested that copper smelters may release appreciable amounts
of aEsine. If this is correct, some type of bubbler sampler may be needed
to assess air quality.
The major objectives of Phase I exposure monitoring are to determine:
o How p’bes deploy and calibrate honey bees as biological monitors.
o Which compartments within the colony should be monitored.
o How physical measurements of environmental quality compare to biological
measurements.
If this can be accomplished, procedures can be refined in Phase II, tested for
other heavy metals, and hopefully extended to other chemicals of interest such
as organics.
4.7 PHASE I — EFFECTS MONITORING
C,
Lethal dose values of ar/enic are well—established by field and laboratory
studies, but it should be reembered that little is known or understood about
the effects of these materi la upon bee populations. This is especially true
for dosages less than “acute.” Therefore, any monitoring program which
attempts to define effects must have as its antecedent a laboratory studies
program which attempts to define under carefully controlled conditions the
causal relationships between pollutant and colony health. If individual bees
or colony health are to be utilized as a measure of effects of a given pollutant
in a given environment, it will be very important to measure baseline health
parameters in the same or an identical environment free of the pollutant.
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Laboratory Studies of Population Effects
Based on ease of manipulation, ability to control conditions, reduction
of sources of variance, and cost, a laboratory approach is recommended for
Phase I effects studies (Table 3). Follow-up field studies and field validations
are strongly recommended for Phase II.
TABLE 3. RECOMMENDED BEE RESEARCH PROJECTS
PRASE I
EXPOSURE MONITORING
Field Studies of Arsenic Exposures
and Resultant Body Burdens
(1)
Sampling Variance
—Intra- and intercolony
—Sites
—Dates
Receptor Compartments of Colony (1)
—Foragers
—Nurse Bees
—Pupae
—Pollen
—Smallest Representative Sample
Levels in Ambient Environment (1)
Versus Bee Systems
—Air, Water, Vegetation of Colony
Bioaccumulation of Heavy Metals (2)
—Cadmium, Lead, Copper
EFFECTS MONITORING
Field Studies of Effects of Arsenic
Mortalities (Dead Bee Traps)/ (1)
Population Size (Estimates)
Numbers of Eggs/Larvae/Pupae (2)
Weight of Colony (Honey Yield) (2)
Laboratory Studies of Physiological
or Biochemical Responses to Arsenic
(Graded Dosages)
Pyruvate Metabolism (1)
Activity of Detoxifying Enzymes (2)
Laboratory Studies of Population
Responses to Arsenic (Graded Dosages)
Mortalities/Longevity (1)
Numbers of Eggs/Larvae/Pupae (1)
Hoarding Behavior (2)
Longevity of Field Exposed Bees
Ranking of Priorities: (1) Highest, (3) Lowest.
The laboratory study focuses on critical dose-response relationships:
mortality, longevity, reproductive cycles, and food storage. No work on
arsenic clearly shows what effects chronic levels have on the bee. The most
fundamental research needs to be done on mortality and longevity of adult bees
at graded doses. Colony effects also need to be established because longevity
of adults by itself may not be an adequate Indicator of damage. Either the
queen or the younger stages may be more susceptible to arsenic levels than the
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workers. Thus, reproductive processes Including numbers of eggs, larvae,
and pupae will be measured. Harm to the queen could be reflected by depressed
fecundity, fewer viable eggs, or a greater proportion of drones to workers
(-if sperm was affected). Lethal factors affecting any of the development
stages are expected to alter the number of individuals reaching the next
development stage (e.g. egg - larvae, larvae - pupae, pupae - adult).
Initial screening will be done In a greenhouse using a linear or logaritlunic
progression of ten doses of arsenic mixed with the food source (pollen or honey).
The doses will range from zero to nearly lethal. Colonies will probably be
4-frame "nucs" made up of one pound of bees. Mortality, longevity, numbers of ,
eggs, larvae, and pupae, and hoarding activity will be determined bi-weekly
for eight to twelve weeks. Hoarding is a laboratory measure of syrup storage
which indicates the colony's inclination towards gathering and storing honey.
It Is the colony as a whole that is responsible for honey production and
pollination. Reproductive failure or mortality results in smaller field
forces available for gathering nectar and pollen and for pollination service.
In terms of energy balance, these effects would require the colony to allocate
more energy to producing workers and less to provisioning the hive.
The proposed Phase I laboratory study did not directly address resultant
body burdens. This might be a worthwhile addition. Currently, a master's
thesis is scheduled for completion at the University of Montana, Spring, 1980
that specifically examines graded doses of arsenic, LDSO'8* and resultant
body burdens. In addition, an Intensive study of uptake, partitioning, and
cycling of arsenic and/or radioisotope tracers Is projected as part of Phase
II or III Investigations.
Field Studies of Population Effects
Although Phase I effects monitoring emphasises a laboratory study, the
work group thought it desirable to carry out a few, relatively inexpensive, easy
to perform tests In the field which would couple the field exposure monitoring
project to the laboratory effort. Colony responses to arsenic in a field
setting may differ radically from greenhouse responses. In the field, arsenic
has multiple routes of entry and variable levels of exposure across time and
space. Also, many more abiotic and blotic stressors Impinge upon the colonies
under field conditions.
Phase I field tests consist of population estimates, mortalities as
reflected by numbers of bees in dead bee traps, and numbers of eggs, larvae,
and pupae. In the field, the numbers of eggs, larvae, and pupae can be
determined quickly by photographing the brood frames so that counts can be
made at a later, more convenient time.
A lower priority project for Phase I which Is recommended If funding
constraints permit, is a longevity test In which frames of brood exposed at
the field sites will be brought into the laboratory, hatched under controlled
conditions (growth chambers), and longevity from time of/hatch-measured.
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The work group also strongly recommended weighing the colonies on each
sample date to obtain an estimate of honey storage. By itself, this data
may be of little value, but if a long—term exposure monitoring program were
initiated at these sites, than the first year’s data could be an important
point of reference. Portable scales are readily available for this purpose.
It is even possible to set each hive on a scale as a base, although this
increases costs substantially.
Laboratory Studies of Physiological/Biochemical Responses
As mentioned before, we need general biochemical or physiological stress
indicators whose measurable parameters could be altered to monitor situations
in which pollutants are either unknown or extremely mixed. Candidate “health”
parameters include enzyme levels (Ache, transaminases, aconitose), itnmunoglobin
patterns as an indicator of disease resistance, and measures of reproduction
such as egg hatch or sperm production. Unfortunately, not much work has been
done in this area with respect to bees and pollutants.
A first step is to identify the indicators which are most sensitive. For
this, we can draw upon the mountain of data available from work on vertebrates.
Few colonies of bees will be needed, and many of the assays involved are already
simplified and available in kit form as a result of vertebrate research. As
such, the screening for sensitive biochemical and physiological indicators
should be relatively straightforward and relatively inexpensive,
Phase I activities include a concept paper synthesizing current knowledge
or mode of action and on sensitive biochemical and physiological indicators for
heavy metals.
Two lines of investigation seem to be particularly relevant and varrent
immediate laboratory study —— effects on pyruvate metabolism and on microsoinal
enzyme detoxification systems.
We know arsenic is a stomach poison and that it uncouples oxidative—
phosphorylation — a vital process of intracellular respiration, which occurs
within the initochondria of the cell. Rat studies demonstrate that arsenic
affects pyruvate—dehydrogenase functioning, not by inhibiting the enzyme, but
by working on the substrate. This can be examined by measuring ratios of
effects on pyruvate metabolism. What is needed is transfer of rat liver and
intestinal methodology to bees, which should be fairly easily accomplished.
An interesting and possibly more useful test is to assay the activity of
detoxifying enzymes associated with inicrosomes. Again, rat studies indicate
that arsenic at low levels confers some tolerance to pesticides, presumably
by stimulating the activity of these enzymes. This suggests the possibility
that low levels of arsenic may affect the ability of bees to tolerate other
stresses. The techniques appear to be available to examine this phenonenon
with respect to arsenic and bees and may be transferable to other compounds.
If successful, this might provide a very useful, general stress measure.
We already know that many of the vertebrate assays can be modified for
use with bees. Conversely, alterations in physiological or biochemical
30
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parameters indicative of the general health of the bee may eventually be of
great utility not only for effects monitoring using bees but also for yielding
insights into general mechanisms of biochemical and cellular level action of
pollutants in vertebrates.
PHASE I — COST ESTIMATES
The bee work group did not attempt to produce detailed cost estimates.
Field exposure trials may involve 1,000 to 5,000 or more analyses of samples
for a single element — roughly estimated at $14.50 to $16.50 per sample for
arsenic alone; $10.00 to $16.00 for each additional element. Another major
outlay would be for 16 or more El—Vol samplers at $375 to $650 each. The
16 to 20 honey bee colonies, including bees and equipment cost $95.00 to
$190.00 each, depending on whether honey supers are included. Electric power,
stands for Hi—Vols, possible site rental, travel, and per diem are major field
expenses for approximately 20 weeks. During the field period, two technical
staff members would be needed, one to care for and sample bees, the other to
maintain the air samples on a daily basis,
Given the cost outlays to set up the experiment and to obtain samples, the
work group recommends analysis for more than one heavy metal. Often the same
preparation and digestion procedures can be utilized for several metals.
The laboratory studies performed at a properly equipped institution
are relatively inexpensive. Both of the Phase I laboratory projects could
be performed by graduate students under the close supervision of a principal
investigator. Rough cost estimates for measuring the population responses to
graded doses of arsenic were from $7,500 to $10,000. The enzyme study would cost
approximately the same in terms of staff and general equipment. The cost of
a centrifuge was projected as a major equipment outlay of from $5,000 to
$10,000.
PHASE II — FIELD AND LABORATORY STUDIES
Phase I activities are designed to mesh together, combiningexposures,
bioaccumulation, physiological/biochemical modes of action, and population
effects.
Phase II (Table 4) addresses the significance of these biological responses
in terms of honey bee systems and ecosystems. Phase I financial constraints
precluded experimentation with many chemicals or with a functional monitor
(pollination) in parallel with the biomonitor (honey bee). Nor was it
possible to pair an active biomonitor (honey bee) with a passive biomonitor
(native bee).
PHASE II — EXPOSURE MONITORING
During Phase II, chemicals other than inorganics will be included, If
bees are to be useful exposure monitoring tools, we must be able to use them
for a broad spectrum of chemicals. There are ongoing Btudfes of bloaccumulation
of radionuc].ides in bees and honey at Los Alamos Scientific Laboratories,
Batelle Pacific Northwest Laboratories, and Oak Ridge National Laboratories.
The bee work group recoends cooperative effects with these laboratories.
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TABLE 4. RECO1NENDED BEE RESEARCH PROJECTS
-J
EXPOSURE MONITORING
Field and Laboratory Studies
o Chemicals other than Metals (1)
——Pesticides
——Aliphatic, Alicyclic, Aromatic
Heterocyclic Compounds
—Radionuclides
—Cases
o Refinements of Phase I
Techniques
——Temporal and Spatial
—-Sampling Variability
—-Ratios of Change
o Mass Transport, Cycling of
Pollutants from Environment to
Bees
——Radiotracers
—Electric Charges on Bees and
Pollutants
EFFECTS MONITORING
Field and Laboratory Studies of Effects
on Honey Bee Systems
o Whole Colony
——Honey Bee Sii ulatipnfModel (1)
Adaptation and Testing
——Impacts of Cycling of (1)
Hazardous Chemicals
——Food Chain Transfers to Man of (1)
Toxic or Mutagenic Materials
o Compartments/Individuals (2)
Field and Laboratory Studies of Effects
on Ecological Systems
o Pollination Syndrome (1)
o Honey Bee Simulation Model/ (1)
Adaptation and Testing
o Impacts of Cycling of Hazardous (1)
Chemicals
o Biochemical/Physiological (1)
Responses to Various Classes
of Chemicals.
Ranking of Priorities: (1) Highest, (3) Lowest.
Organics are another high priority class of substances to add to Phase II.
Whether pesticides (insecticides or herbicides) or organics such as those
released by new energy conversion technologies should be utilized vas unresolved.
There are many good reasons for examining any of a great number of anthropogenic
chemicals. The workgroup assumed that the project advisory group or other
workshops would deal with these specific questions before initiating the long—
term program. Part of Phase II efforts should concentrate on refinements of
Phase I methods. Ongoing field verification and validation are important
aspects of a program to demonstrate.tbe utility of the biomonitoring concept.
PHASE II
(1)
(1)
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An area of particular relevance for understanding efficacy of bees as
environmental samplers is an understanding of mass transports and cycling of
pollutants from the environment to bees. Drs. R. Ferenbaugh and E. Cladney,
Los Alanios Scientific Laboratory, are interested in establishing bee colonies.
in a greenhouse large enough that they can be maintained without being allowed
to forage outside. The bees would be supplied with water and/or sugar solution
spiked with radioisotope tracers. Up—take of these tracers and subsequent
partitioning within the hive as veil as effects on the colonies would be
monitored. For elements such as arsenic that have no useable radioisotopes,
studies on non—radiactive istopes could be conducted.
A similar line of investigation is currently being carried out at North
Central Bee Research Laboratory, Wisconsin. where they are looking at
electrical charges on bees and materials such as pesticides and how the charges
affect the likelihood of contaminants being transported back to the colonies.
Both of these research areas attempt to explain more clearly how materials
are moved from the environment onto and through bee systems — invaluable
information if bees are to be used as samples of their surroundings.
PRASE II — EFFECTS MONITORING
The effects of the uptake and partitioning of contaminants by bee colonies
will be another means of linking effects and exposure monitoring.
Although ecosystems warrant a high priority for Phase II, the transfer of
ziutagenic, carcinogenic, or toxic substances not only from the environment into
bee colonies but also through hives and/or pollen to human food chains was a
major concern of several of the work group members. Exploratory research aimed
at hazardous chemicals in bee systems, which have never been looked at before,
more or less obligates us to look at the potential for their transfer to humans.
Many of the Phase I activities are conceptually coupled using the honey
bee population simulation model. Phase II emphasizes a cooperative effort with
the Bioenvironmental Bee Laboratory to adapt the model to pollutant impacts and
to conduct field tests of the sensitivity of the various bee parameters that
control it. The model appears to have a high potential for use as a research
tool for predicting the impact of pollutants.
During Phase II, the emphasis will continue to be at the level of the
colony, although at the level of the individual bee there are concerns that
should be addressed. Given the priority.of examining effects to bee systems
and ecosystems, the types of measurements which might be made at the colony
level include; foraging activity, honey crops, survival of colonies in a given
ecosystem, and possibly requirements for adaptive bee management to offset
pollutant effects such as the need for more sugar syrup, the use of pollen
substitutes, or movement of colonies. On the level of the individual it may
be useful to examine: predisposition to diseases, shifts in microflora, changes
in longevity, or alterations in physiology.
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The obvious link to the pollination syndrome is the numbers of foragers
available, Pollutant effects on colony foraging activity will depress floral
reproductive success, However, the native bee populations are also a
.component of the pollination subsysteto which needs to be assessed Th a
portion of the effort constitutes Phase III.
PHASE III
Phase III actIvities (Table 5) do not necessarily begin during the
third year. The emphasis of Phases l and II are on the honey bee and the
work plans are ambitious, even with strong financial support during the
second year. Studies of effects at the ecosystem levels, particularly as
regards the pollination syndrome, vi i i be labor intensive, Phase III sets out
to develop Passive Monitors using native bees and to better address the
ecosystem level (pollination competition between native and domestic bees).
If Phase III could be started along with Phase II, so much the better.
Essentially, Phase III consists of following the same steps to develop
native bees for use as monitçrs as were takenfor honey bees, Hopefully, not
every test will have to be repeated, but native bees may respond differently.
The native bees are much more susceptible to many pesticide residues than
honey bees.
TABLE 5 • RECOMMENDED BEE RESEARCH PROJECTS
PHASE III
E OSURE MONITORING
Field and Laboratory Studies
o Passive Monitors
--Osmia, LaBBtOgZOSBUS
——Bioaccuniu].ation
—-Sampling Variability
—CorrelationB of Pollutant
Levels in Bees and Environment
o Field Validations of Monitoring
Techniques Derived from Phases I
and II
EFFECTS MONITORING
Continuation and Expansion
Studies Concerning Impacts
Systems and to Ecosystems
Field and Laboratory Studies of Passive
Monitors
o Dose-response Tests
——Population Dynamics
——Reproductive Cycles
——Physiological Responses
o Adaptation and Testing of Megachilidae
Slmulation Model
o Validation of Effects to Pollination
Systems
of Phase II
to Honey Bee
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Native bees also remove some of the problems associated with the “manage—
merit” of the bees. Also, native bees are a structural and functional part of
the ecosystem in which they occur, while honey bees are often inserted and may
±n some ways be out of context,
Currently, there is a Megachilidae model being developed at Oregon State
University. Hopefully, it will be available by Phase III and can be used 1Ü’
much the same manner as the honey bee simulation model,
Phase III, In addition to developing the use of native bees as passive
monitors to compliment honey bees as active monitors, will continue to
develop, test and validate the honey bee monitoring methods which should be
applicable to small scale petwork test by this time.
PHASE IV
After passive and active monitoring tools have been proven in the laboratory
and adapted to the field, their use can be extended (Table 6) to actual monitoring
efforts. At this time, site selection becomes a major issue. Regional or
national networks require sites that provide a representative cross—section of
major ecosystems, including both those relatively undisturbed (wilderness areas)
and disturbed agricultural a.nd urban regions.
TABLE 6, RECOMMENDED BEE RESEARCH PROJECTS
PHASE IV
EXP
OSURE MONITORING
EFFECTS MONITORING
o
Expansion to localized or
Field Validations of Effects Monitoring
o
Regional Monitoring Networks
Bibliographies Relevant to
Techniques for Active and Passive
Monitors.
Methods, Chemicals, Bees as
Exposure Monitors.
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SECTION 5
MONITORING THE SOILS MEDIUM
This report outlines a research package demonstrating the utility of
the concept of network biological monitoring through development of specific
tools. The adopted strategy focuses on species identified in earlier work-
shops as the most promising biological monitors of the soils media — earthworms
and alms at*
o Correlating toxic material trends in the media with body burden trends
(exposure monitoring).
o Identifying possible biological responses to those trends
(effects monitoring).
o Gaining insight into possible physiological mechanisms by which these
responses may be brought about.
In addition, the soils work group felt strongly that this monitoring of
important ecological components should be coupled with monitoring of important
functional processes such as nutrient flux. Given limited resources in the
first year and uncertain resources in succeeding years, the soils work group
has developed a phased program potentially leading to implementation of a
national network at the end of four years.
5,1 PHASE I — LABORATORY AND PAPER STUDIES D }1ONSTRATING “PROOF OF CONCEPT”
There can be little doubt about the need for a capability to monitor
accumulations and effects of man—induced pollutants. To date, however,
efforts in this direction have mostly involved theorizing rather than research
to develop the tools. The soils work group believes that even one demonstration
of a workable technique might well release a virtual flood of support for
further development. Thus, “proof” of the utility of the conceptual frame-
work already developed is a necessary first step in developing an effective
monitoring network within that framework. The group gathered at this work-
shop represents a bringing together, perhaps for the first time, of the
expertise on earthworm life cycles, bloaccumulation, toxic effects and
ñeurophysiology, as well as expertise in ecological and soil sciences, needed
to provide reasonable assurance bf success.
Rationale
ven given this assurance, the extensive debate of preceeding workshops
suggests a need to express the assumptions under which Phase I research is
believed to fit the requirements and address the concerns of monitoring network
36
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theory. A nonprioritized listing of these assumptions is as follows
o The approach recommended does not have to be the only or even the
best approach for the soil medium, only a way that our collective
experience suggests will work,
o The initial effort should be as focused as is possible, while still—
covering enough bases to promise a useable product. Thus, it may- -
be adequate to identify only one or a few species with the potential
for accumulating one or more chemicals likely to cause measurable
effects that have significance to man and his environment.
o Soil components and processes are extremely important as monitoring
candidates because:
— Soil is the medium in terrestrial ecosystems which controls
productivity.
—— Soil is the ultimate depository/repository of most pollutants
reaching terrestrial ecosystems.
—— Decomposition of organic matter returning basic nutrients to the
soil is a critical point of convergence in terrestrial ecosystem
functioning. Decomposition processes may therefore, provide
an excellent index to ecosystem health.
— While many studies of pollutant effects upon ecosystem level
processes (e.g. nutrient cycling) exist, it is difficult to
monitor accumulation in soils without a biological model that
can act as an exposure model.
o Previous workshops and work groups’ experience suggests that body
burdens of -trace metals in earthworms, particularly accumulations
of cobalt 60 may be the best point of beginning. Earthworms are one of the
most important organisms responsible for mechanical mixing of the soil
and play a major role in maintaining physical soil characteristics and
processes such as aeration, water permeability, and mineral turnover
(Van Hook, 1974). Earthworms are key components in natural food chains
providing a food source for many small mamnals and birds. Earthworms
have been demonstrated to exert a significant effect on redistribution
of cadmium, carbon, and cesium in soils. Due to this redistribution
effect and the earthworm’s ubiquitous occurrence in nature, these
invertebrates may exert a significant influence on the distribution
of trace elements in soils and in food chains by altering concentrations
in tissues t1 rough bioaccwnulation (Van Hook, 1974). The earthworm
provides the only logical monitor for exposure (if not effects) in
soils at this stage because:
—— Distribution growth requirements and life history are laiown.
—- Earthworms can be worked with inboth the laboratory and field.
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—— Their physiology an taxonomy has been characterized.
—— They are large enough to permit segregation and analysis of tissues.
—— They have a defined structural Importance in terrestrial ecosystems
and at least a conceptually significant functional role (that Is
mixing, microbial turnover, trophic transfer).
—— A partial data base exists linking physiology, population dynamics,
and some soil processes.
o There Is a general principle that the explanation for an observed
phenomemon (such as a population crash) must be sought at the next
lower level of organization (lover egg production) and its significance
Is best appreciated at the next higher level of biological organization
(community structure). An effective monitoring network must provide
information ranging from trends in pollutant concentrations for environ-
mental media, and resulting body burdens, all the way to biological
effects and their significance at the ecosystem level of organization.
Consequently, a research approach which provides integrated data from
several levels of organization, in both structural. and functional
terms, would represent an optimum approach.
o Earthworms appear to meet the above criteria in several ways:
—— They are one of the only organisms that is sufficiently large and
widely distributed to offer quantities of experimental material
at a variety of locations. -
—— They are proven accumulators.
—— Lethal and sublethal responses to environmental exposures and tissue
accumulations have been demonstrated.
— The techniques for relating organism and population responses to
physiological mechanisms and to ecosystem function (such as
nutrient flux in the soil column) are available.
—— If causative mechanisms can be shown in earthworm neuromuscular
physiology at the cellular level,.the results may have broad
applications to other animals.
— We may be able to relate effects on structure (earthworm population)
to effects on function (nutrient cycling).
o At the same time there are some limitations to be kept in mind:
—— Earthworms are found only in certain types of soils and their
use in the passive mode is limited to regions where such soils
occur.
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—— The relationships between environmental exposure and body burdens
will not be simple ones. For example, distribution of pollutants -
in the soil medium does not necessarily equal worm exposure due to
variations in, for example, sorption on clays, chelation and micro-
bial exclusion.
— Changes from the laboratory (nutrient enriched) to the field
(nutrient normal) will radically affect the above exposure.
variables as veil as process rates (microbial activity, for
example) and will make direct extrapolations to the field difficult.
—— Laboratory experiments initially focusing on effects to a single
age class must eventually address populations with normal age
distributions before the move to passive monitoring in the field
can be made.
—— Even with the experience available in the work group, there are too
many unknowns to permit moving with confidence directly into the
field. The strategy must be to first “proof” the concept in the
laboratory before moving to the field.
—— Field populations subjected to pollutant stress over long periods
may accotnodate or adapt to ambient concentrations and therefore
fail to respond in the ways laboratory studies would lead us to
expect. Thus, once laboratory experiments have demonstrated the
utility of the approach, it must be validated in the field for both
active monitoring (standard organisms and soils placed in the field
and their responses to pollution gradients measured) and passive
monitoring (organisms and soils sampled in the field along existing
pollution gradients).
Testing the Earthworm as a Biomonitor
Given the above advantages and constraints, the expertise available in the
work group was applied to laboratory experiments investigating the feasibility
of using earthworms as a biomonitor. Design of these experiments is based on
a simplified conceptual model (Figure ) of key interactions between the
earthworm, Eisenia foetida, and its environment. Our observations indicate
that E. foetida Ia primarily a inicrovore that, under field conditions, feeds
In concentrated organic sources such as manure. Organic matter Is consumed
along with microbes; however, sterile organic matter cannot support the worms.
Thus, accumulation of heavy metals is assumed to be primarily the results of
consumption at a single trophic level. liowever, a simple interpretation is
complicated by (1) “recycling” of heavy metals by microbial uptake from feces
and (2) possible cuticular uptake of metals obtained from the substrate
(indicated by dashed flow from manure to the worms). The former problem
will be partially controlled and the substrate levels of heavy metals
maintained at near—steady state levels by placing the worms in a new food
source every two weeks.
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I
I
I
I
I
I
IHARVEST
I
$
S
I
I
$
S
S
L i
INPUT >1 MANURE FOOD SOURCE
Figure 5. Conceptual model of key interactions between the earth-
worm, Eisenia foetida, and its environment.
Exposure, Bloaccumulation and Population Effects——
The easiest and most relevant parameters to measure in earthworms regard-
ing biological effects monitoring would be growth, reproduction and neural
functioning. The species of worm we propose to use in the study would be
Eiaenia foetida, due to the large amount known about this worm. The following
research plan is designed to show how these parameters can be measured.
Recent work shows that growth and reproduction of earthworms can be used
as a model system. Preliminary evidence has demonstrated that they both
accumulate and are sensitive to heavy metals, with regards to growth and
reproduction.
Initial screening will be done with five levels of heavy metals within a
broad range such as 0—10,000 ppm. The material to be tested will be mixed
with the food source. The earthworms used in this procedure will all be
newly hatched worms ( <6mg) and mortality and weight changes will be determined
bi—veekly for 8 weeks. A narrower range of heavy metals will be selected which
represents levels from minimal effects to nearly lethal dosages.
40
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Five levels using sublethal concentrations can nov be tested for growth
rates and total body burdens. Total body burden would be done by digesting
the worm and assaying the heavy metals using AAS.
In addition, long—term growth and reproductive studies, (24 ‘weeks),
would be carried out using sublethal concentrations of each heavy metal,
Rate of cocoon production would be an indication of the stress placed upon
a population due to the heavy metals,
Neural Effects——
The monitoring of neurophysiological effects caused by heavy metals is
desirable because nervous system function is known to be greatly impaired by
such substances and because the earthworm’s nervous system control the
animal’s locomotory behavior in the soil.
Recent work has demonstrated the electrical activity arising from
identifiable neurons, as well as from associated muscle units can be easily
detected with an array of extracellular recording electrodes in contact with the
ventral surface of completely intact earthworms, Such an approach offers a
unique advantage for non—invasive and long—term monitoring of neurophysiological
parameters. Given this advantage it should be possible to obtain a detailed
assessment of impairment of neural function caused by the presence of known
amounts of heavy metals.
Animals will be reared at the same five sublethal levels of heavy metal
described in the previous section on exposure versus bloaccumulation and
effects. Neurophysiological parameters to be examined on a bi—weekly basis
include: (1) action potential conductibn velocity in single nerve fibers,
(2) effectiveness of central nervous system synaptic transmission, and
(3) effectiveness of neuromuscular synaptic transmission. From such
experiments ‘we can establish dose—response relationships for each of the
neurophysiological parameters. In addition, we can identify specific loci
and modes of action of the metals within the nervous system.
Our understanding of the impairment of neural function by toxicant
accumulation in the earthworm may have relevance to and lead to a better
understanding of the adverse neural effects of toxicants on other soil
invertebrates, which are not as amenable to neurophysiological study. In
addition these studies could yield insight into general mechanisms of cellular—
level action of heavy metals of vertebrate, as well as invertebrate nervous
systems, Finally, and perhaps most important, the impairment of neural and/or
muscle function by heavy metals could be a critical factor in modifying
ecologically significant locomotor activities of the animal, such as escape
withdrawal from predators or rates of burrowing in the soil.
Using Soil Nutrient Flux as a Functional Monitor
Rationale—
Biological monitoring at the species or population level alone allows for
generalizations to be directed at only one level of ecological organization
above that studied. The use of an earthworm population as a biomonitor allows
us to make inferences on the structure of the litter—soil community. Inferences
41
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to the functioning of this co=unity are tenuous at best due, in part, to
potential compensatory mechanisms of biota under perturbed conditions (e.g.
Edwards, 1965). At the same time, however, there exists an ample and expanding
data base to suggest that the direct links between a dominant structural
component of an ecosystem or subsystem and functional characteristics of the
system are strongly correlated and predictable, at least over an
time frame,
Ecosystem level behaviors are not just the stnn of subsystem behaviors
but also the result of interactions between subsystem components. Thus, the
measurement of an ecosystem property includes not only the effects of our
biological monitor — in this case the dominant soil invertebrate of many
economically important systems — but also the monitor’s interactions with
other components of the system. Validation of biological monitoring is
therefore contingent upon establishing the functional attributes of the
monitor within an ecosystem perspective and requires, at least initially,
concurrent analysis of the functional response of the litter—soil system to
those same conditions experienced by the biomonitor.
Concept Paper Synthesizing Current Knowledge——
Limited resources available during Phase I may preclude experimentation
with a functional monitor (nutrient flux) in parallel with that on the
biomonitor (earthworm) even though such pairing would be ideal (perhaps
saving a year in implementation of a monitoring network). However, much of
the research necessary to demonstrate the utility of such a pairing has
already been done. A highly useful Phase I exercise would be to produce
a concept paper synthesizing current knowledge on the use of soil nutrient
flux as an ecological monitor of pollutioâ. Examples of the material useful
to this state—of—the—art s i11m ry follow:
o Early papers by O’Neill, Reichle and Shugart showing that,
theoretically, nutrient cycling processes are extremely
sensitive to stress and are potentially the primary functional
compensatory mechanisms to stress.
o Water, Air, Soil Pollution. 1977 O’Neill et al.
Describes laboratory and field experiments showing ecological
responses to heavy metal pollution; results in all cases were
that nutrient loss increased at a lower dose than microbial
or invertebrate physiological or population parameters changed.
o Series of papers from Mehillo, Vitusek on Hubbard Brook,Croinack nd
Sollins on Coniferous forests, and Waide at Georgia supporting
theory. -
o Series of papers showing functional stability measures compared
to traditional diversity stability measures.
o Series of lab and field experiments using As, Cu, Cd, Pb,
hexachlorobenzene, fluidized—bed effluents, phenols, fly ash, uranium
mill tailings, strontium, plutonium — most measure populations,
physiological microbial indices as well as nutrient export.
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Thus, the purpose of the concept paper would be to furnish (1) a
state—of—art review of a terrestrial “ecomonitor,” and (2) a statement of
the advantages of coupling thiB with a biomonitor (such as coupling trends
in environmental pollution with trends in tissues, physiological and
population effects, and measuring partial contribution to effects noted at
the ecosystem level.) This paper would therefore provide a theoretical
test of the hypothesis that an active monitoring system using (1) a biomonitor
(e.g. earthworm) to address exposure and at least first generation effects ——
on physiological and population parameters and (2) and ecomonitor (e.g. litter—
soil cores) to address functional (e.g. nutrient—cycling) effects on the
ecosystem would provide an adaptable approach to short—term and long—term
characterization of a site under baseline or perturbed conditions.
Phase I Cost Estimates
The following are very rough minimal estimates for each of three activities
over the course of one calendar year:
Exposure, Bioaccumulation and Population Effects
1, analysis of heavy metal by AA (1000 samples) 10,000
2. salaries — one full—time technician 12,500
3. supplies 2,000
4. publication costs 250
5. travel — includes one trip to Iowa St. and 1,000
one scientific meeting
6. indirect costs —100% of salary above 12,500
38,250
Neural Effects
1. salaries — one half—time iesearch assistant 6,250
one half—time technician
2. hourly labor — approve 6 hours/week at mm. 6,250
3.10/hour
3. equipment — oscilloscope, with amplifiers 3,500
environmental chambers — 2@ 1000 ea 2,000
4. publication costs 250
5. travel —includes one trip to Syracuse and one 1,000
science meeting
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6. supplies — photographic, electrical, 2,500
chemical, glassware
7. indirect costs — 70% of salaries above 8,750
31,500
Concept Paper
1. salaries 7,500
2. clerical and publication 1,000
3. indirect costs 7,500
16,000
Net Cost 85,750
PHASE II — LABORATORY STUDIES CORRELATING BIOLOGICAL AND FUNCTIONAL SOIL
MONITORS
The concept paper suggested for Phase I would be the primary basis for
designing Phase II laboratory studies. These studies would employ the same
materials used for a food source or contaminant at the same concentrations
of pollutants used for earthworms, this time as input to an intact litter—
soil system. Experiments would monitor the output of C0 2 , DOC, P0 1 ,—P or
NH 3 —N, N0 3 —N as a function of dose. Efforts could be made at this time to
look at other pollutants such as airborne hydrocarbons and pollutant
interactions. Sensitivity of this system could be compared against effects
on earthworms as a basis for extending the laboratory work to field
situations.
PHASE III — FIELD STUDIES DEMONSTRATING COMBINED BIOLOGICAL/FUNCTIONAL
MONITORS IN ACTIVE AND PASSIVE MODES
The laboratory and paper studies of Phase I and II will provide the
data base for designing Phase III studies adapting the combined biological
and functional monitors to field conditions in both active and passive
anodes. Although the details of this research must await the data base,
there exists sufficient information to offer a skeleton design and research
plan, (Table 7) and to suggest a promising site. Because the adaptation
from laboratory to field is more direct for the active mode, it will
be given the most attention here.
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TARLE 7. PHASE III FIELD STUDY EXPERIMENTAL DESIGN
Passive Monitoring
o Sampling of litter and soil
along environmental gradient:
element analysis
o Sampling and collection of
• earthworm species along
gradient:
—— Element analysis
—— Neurophysiological testing
Active Monitoring
o Collection of litter and
soil samples along gradient:
removal to laboratory
—— Introduce laboratory species of
worm in half of samples
— exposure analyses
— effects analyses
—— Functional analysis of samples
with and without earthworms on
other half of samples. Measure-
ment of nutrient flux through
samples.
Exposure, Bloaccumulation and Population Effects
Development of the active dual monitor would involve going to a test site
and analyzing for heavy metals as veil as physiological effects. This would
give the ambient level of heavy metals found in the native population and the
corresponding physiological impact.
The soil from this field site would then be tested with the same species
of worms found in the field site but using worms that would not have been
exposed to the high levels of heavy metals found in the test site. These
“clean” worms could then be grown in the test site soils and later analyzed for
heavy metal content as well as physiological effects.
This experimental design would then give a correlation between the amount
contaminant found in a soil and (1) the amount found in a worm over a
and short exposure period and (2) physiological state of the animal.
would be a method of judging how well laboratory experiments correlate
field conditions.
Since the first phase of this research plan focuses on obtaining a data
base for organismal and population effects of heavy metals on a single
earthworm species, we propose to further expand our biological data base to
include a number of different earthworm species representing a variety of
natural geographical and soil horizon distributions. Some of these species
include: Alloiobophora ionga, A. turgida and Lwnbrzcus ribeiiua. Such
studies would provide baseline information regarding species variation in
heavy metal effects on growth, reproduction and physiological parameters.
The general methods for carrying these studies have been described in the
initial phase. -
of a
long
This
with
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Proposed Site
Selection of a site for evaluating the proposed active and passive
bio— and ecomonitoring system is extremely important to the overall sucéess
of this research. To illustrate this importance we considered the advantages
of a previously studied lead smelter on the Crooked Creek watershed near
Ralla, Missouri. The smelter has been in operation for alniost 15 years
emitting detectable amounts of Pb, Cu, Ck, Zn and Cd. This site was intensively
studied under a NSF research grant (Rann) during 1971 to 1974 on vegetation,
litter dynamics and hydrology. Models are available for plume dispersion,
loading, and hydrological transport. This was the site of initial studies
leading to many papers sited in the state-of—the—art review (Phase I). It
would allow field evaluations to be done along a gradient which has a known
history and could be quickly determined chemically at a new time. Earthworms
are established at the site and would allow bonus determination of (1) accumu-
lation (2) adaptive physiology over the hundreds of generations exposed, and
(3) maximal concentrations of metals which can be adapted to by the genetic
potential of the populations or their food sources.
Since much of the ecosystem theory suggesting nutrient export as a monitor
of ecosystem level response to stress came from this area, a bonus would be the
reevaluation of the theory’s prediction. The prediction is that when the site
is reevaluated, if the metal inputs have remained, that each effect would be
measurable at greater distances than in the early 1970’s. The importance of
this is that, if proven, it shows that an ecomonitor (e.g. nutrient cycling)
is a predictive indicator of structural changes in the system as a result of
stress continuing over a finite time period.
Thus, the series of experiments outlined offers a way of:
o Evaluating the combined biological and functional monitoring tools.
o Demonstrating population and physiological responses to short—term
perturbations (in the active mode) and long—term perturbations
(in the passive mode).
o Measuring the relative sensitivities and adaptive resilience of both
monitors over short and long time frames.
PHASE IV - APPLICATION TO A NATIONAL NETWORK
Once these monitoring tools have been proven in the laboratory and
adapted to the field at a specific site their use may be broadened as part
of a national or even an international monitoring network. Because earthworms
are not found everywhere, some substitute that serves a comparable role in
soil litter breakdown may have to be sought in arid and other extreme
environments. The functional monitor proposed will likely serve at all sites.
Given the necessary tools, selection of appropriate sites becomes the
next important hurdle. Current thinking (Luepke, 1979) suggests a need for
a minimum set of monitoring sites that would provide a representative cross
section of major ecosystems of concern. Such a network should include areas
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