EPA 560/5-75-008
TEST METHODS FOR ASSESSING THE EFFECTS OF CHEMICALS ON PLANTS
JUNE 1975
FINAL REPORT
OFFICE OF TOXIC SUBSTANCES
ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
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Document is available to the public through the National Technical Information
Service, Springfield, Virginia 22151
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EPA 560/5-75-008
Test Methods for Assessing the
Effects of Chemicals on Plants
By
R. Rubinstein
E. Cuirle
H. Cole
C. Ercegovich
L. Weinstein
J. Smith
Science Information Services
The Franklin Institute Research Laboratories
The Benjamin Franklin Parkway
Philadelphia, Pennsylvania 19103
June, 1975
Contract No. 68-01-2249
Project Officer
Elton R. Homan
Prepared for
Office of Toxic Substances
U.S. Environmental Protection Agency
Washington, B.C. 20460
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NOTICE
This report has been reviewed by The Office of
Toxic Substances, EPA, and approved for publi-
cation. Approval does not signify that the
contents necessarily reflect the views and
policies of the Environmental Protection Agency,
nor does mention of trade names or commercial
products constitute endorsement or recommenda-
tion for use.
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FOREWORD
The work described in this report was performed by the Environmental
Protection Group, Science Information Services Department, The Franklin
Institute Research Laboratories, under U.S. Environmental Protection
Agency Contract # No. 68-01-2249. The study was conducted under the
direction of Messrs. Alec Peters, Department Director, and Bernard E.
Epstein, Manager of the Environmental Protection Group; Mr. Richard
Rubinstein was the project manager. Dr. Elton R. Roman of the Office
of Toxic Substances, U.S. Environmental Protection Agency, was the
Project Officer.
This report was written by:
The Franklin Institute Research Laboratories
Richard Rubinstein
Eunice Cuirle
Dr. Herbert Cole - The Pennsylvania State University
Dr. Charles Ercegovich - The Pennsylvania State University
Dr. Leonard Weinstein - Boyce Thompson Institute for Plant Research
Dr. Jerry Smith - Academy of Natural Sciences (Philadelphia)
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TABLE OF CONTENTS
Section Title Page
FOREWORD ill
1 INTRODUCTION 1-1
1.1 Test Methods for Terrestrial and Aquatic
Pollutants 1-4
1.2 Test Methods for Airborne Pollutants 1-6
1.3 Test Organism Selection 1-9
2 SELECTION OF TEST METHODS 2-1
2.1 Introduction 2-1
2.2 Mechanisms of Toxicant Action 2-2
2.3 Fate of Toxicants in or on Plants 2-37
2.3.1 Aerial Portions of the Plant 2-37
2.3.2 Underground Portion of the Plant . . . . 2-39
2.3.3 Behavior of Substances Inside the Plant . . 2-39
2.3.4 Conclusions 2-41
2.4 Criteria for Selection of Test Methods .... 2-43
2.4.1 Scope or Extent of Procedure 2-45
2.4.2 Facilities for Test Procedure 2-45
2.4.3 Treatment Methods for Test Substance . . . 2-46
2.4.4 Evaluation of Factors Influencing Plant
Response and Chemical Activity. .... 2-46
2.4.5 Plant Age and Treatment and Exposure
Duration 2-48
2.4.6 Evaluation Criteria for Chemical Treatment . 2-49
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TABLE OF CONTENTS (cont'd)
Section Title Page
3 RECOMMENDED TEST PROTOCOLS 3-1
3.1 Procedure for the Evaluation of a Chemical's
Effect on the Soil Microbe Populations .... 3-13
3.2 Evaluation of a Chemical's Potential Toxicity to
Major Soil Nitrogen Cycle Bacteria 3-20
3.3 Evaluation of Chemical Phytotoxicity to Soil Fungi
of Importance in Cellulose Decomposition . . . 3-24
3.4 Additional Listings of Test Procedures for Soil
Microflora 3-30
3.4.1 Nitrification 3-30
3.4.2 Soil Algal Growth 3-31
3.4.3 Soil Fungi Growth 3-32
3.5 Preliminary Evaluation of Toxicity to Terrestrial
Plants through Laboratory Seed Germination and
Seedling Growth Testing 3-35
3.6 Greenhouse Evaluation of Toxicity to Terrestrial
Plants through Foliar Spray and Soil Amendment
Testing 3-47
3.7 Evaluation of Plant Toxicity through Field Testing . 3-69
3.8 Additional Listings of Test Procedures for Terres-
trial Tracheophyta for Laboratory and Greenhouse . 3-83
3.9 Additional Listings of Test Procedures to Determine
the Influence of Environment on Phytotoxicity in
Laboratory, Greenhouse and Field 3-89
3.9.1 Influence of Spray Droplet Size .... 3-90
3.9.2 Air and Soil Environment 3-90
3.9.3 Exposure Site 3-91
3.9.4 Pretreatment Environment 3-91
3.9.5 Vapor Transfer from Soil 3-91
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TABLE OF CONTENTS (con'.td) ,
Section Title Page
3.9.6 Persistence and Movement in Soil. . . . 3-91
3.9.7 Phytotoxic Interactions between Chemicals
in Soil 3-92
3.10 Additional Listings of Spray Application Equipment
for Phytotoxicants in Growth Chambers, Greenhouse
and Field 3-93
3.11 Measurement of Phytotoxicity Using Aquatic Plants . 3-95
3.11.1 Introduction 3-95
3.11.2 Algae 3-96
3.11.3 Vascular Plants 3-102
3.12 Procedure for Preliminary Evaluation of Potential
Toxicity of Algae through Laboratory Bioassay
Testing. (Adapted from the Provisional Algal
Assay Procedure—EPA), (PAAP) 3-104
3.13 Approach for the Evaluation of Toxicity of Aquatic
Vascular Plants . 3-117
3.14 Evaluation of Phytotoxicity of Airborne Substances . 3-121
3.14.1 Initial Screening . . . . . . . 3-121
3.14.2 Secondary Screeing 3-125
3.15 Problems in the Development of Screening Protocols
for Airborne Pollutants Test Chambers .... 3-129
3.15.1 Introduction of Pollutant to Chamber. . . 3-130
3.15.2 Monitoring of Pollutant 3-132
3.15.3 Dose-Response 3-134
3.15.4 Factors which Affect Response of the Plant
to a Pollutant ....;... 3-136
3.15.5 Indirect Effects on the Pollutant-Plant
Interaction . . 3-138
3.15.6 References 3-140
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TABLE OF CONTENTS (con'td)'
Section Title Page
4 SUMMARY 4-1
5 CONCLUSIONS 5-1
6 RECOMMENDATIONS 6-1
APPENDIX A INTERVIEWS A-l
APPENDIX B TAXONOMIC LISTING OF SUGGESTED TEST SPECIES . . B-l
7 BIBLIOGRAPHY 7-1
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I. INTRODUCTION
The purpose of this report is to indicate suitable test species
and methods for the determination of toxicity on plants. Plant ex-
posure to air, soil, and water contaminants have been considered. The
loss of a volatile compound into the atmosphere in the form of a gas or
aerosol during manufacture may indicate a potential hazard to plants
via the vapor phase, thereby necessitating air pollution testing.
Material was obtained both from published literature and
unpublished sources. Many knowledgeable members of the scientific
community were interviewed.
The implementation of the proposed Toxic Substances Control Act
will require safety evaluation of most substances commercially manu-
factured by the United States chemical industry. In the utilization of
some substances, every pound of material can be accounted, retained or
converted with no environmental release. Other substances will be used
under circumstances where significant quantities will be released into
the environment. Inevitably, some losses into the environment will be
sustained during manufacture. The modes of loss are described in Table
1.1.
Table 1.1 Soil, Water, and Atmospheric Pollutant Effects on Terres-
trial Plant Growth
I. Sources of toxicants
A. Point source release.
1. Air transport
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a. Direct effects on plants
b. Indirect effects by
(1) Uptake from soil
(2) Uptake from water
2. Water transport
3. Direct application
a. liquid, i.e., spray irrigation.
b. solids or semi-solids.
B. Non-point sources.
1. Loss from manufacture sites.
2. Release following consumption and utilization.
The ease of detection of phytotoxic effects with vascular plants
depends on severity and morphological expression. Gross morphologic
effects are usually readily apparent. Subtle changes in physiology
affecting crop yield can be detected only through ecological study of
the various populations within the community. This is also applicable
to changes in competition and reproductive succession in native plant
communities.
The extent of required testing should depend on the amount of
material manufactured and released into the environment. A material
that is manufactured in small quantities with complete conversions or
recovery and no environmental release may require no plant testing.
With an increase in the quantity manufactured and projected environ-
mental release, the degree of plant toxicity testing should also .
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increase.
Ideally, a series of test protocols should be developed and listed
as acceptable evaluation procedures. Because of the great variability
within the plant world, these should represent guidelines and acceptable
methods, not as rigid either/or procedures. The procedure and tech-
niques should begin in a rather uncomplicated fashion and increase in
complexity and sophistication with testing requirements.
Some herbicide chemists and plant physiologists believe that no
procedure is a precise guide to the potential for phytotoxicity.
Technically, this is correct. Plant species, age, dosage, chemical
formulation, exposure site and other factors will influence plant
response. These considerations might be taken to indicate that any
efforts to develop test protocols would be futile. However, even an
inadequate effort would be better than none, and a preliminary evalua-
tion could provide a basis for further work. It is essential to recog-
nize that simple laboratory or greenhouse procedures in phytotoxicity
evaluation represent a commencement in investigation. The end point
in testing is dependent on the extent and manner of chemical release.
The majority of plant toxicity studies has been performed by
herbicide chemists, physiologists, and air pollution specialists. The
techniques described and species are therefore those used for studying
known pollutants and chemicals synthesized for their herbicidal
(albeit differential) effects.
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1.1 TEST METHODS FOR TERRESTRIAL AND AQUATIC POLLUTANTS
The degree of complexity, sophistication, and expense of a parti-
cular test should be governed by the chemical's potential hazard to the
environment. This includes the amount produced, projected use (indus-
trial or consumer), waste disposal procedures, and the environmental
fate of the compound. Therefore, test methods have been arranged in
their order of complexity: laboratory (incubator), greenhouse, field
plot, and specialized. In the scheme (Figure 1-1, amplified below),
the toxicity of the previous test is considered. Therefore, detection
of toxicity in an early stage will necessitate intensive testing at
complex levels.
The following are sequential procedures for development of terres-
trial plant soil and water pollutant hazard evaluation (elementary to
complex).
1. Growth chamber or laboratory testing
a. seedlings in pots.
b. soil, water, or foliar spray exposure route for test
chemical.
c. standard plant species.
2. Greenhouse testing
a. seedlings grown to maturity.
b. soil, water or foliar spray exposure route for test
chemical.
c. standard plant species.
3. Preliminary field plot testing
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a. seedlings grown to maturity—yield data.
b. soil, water or foliar spray exposure route for test
chemical.
c. standard plant species and selected plant species as
likely to occur in release site.
4. Field plot testing
a. seedlings grown to maturity—yield data.
b. soil, water or foliar spray exposure route for test
chemical.
c. standard plant species and selected plant species as
likely to occur.
d. field evaluation on sites of likely release or introduc-
tion of test chemical in actual situation.
e. multi-year continued testing.
5. Progeny testing-Mutagenicity and F^ population effects from
"selfed" parents where possible.
6. Population studies in natural ecosystems where applicable
(especially with non-point source release materials). These
would be continuing studies on release sites.
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CONTROLLED-
CONTAINED
USE ONLY
NO / CONSIDERED
FOR LIMITED
USE
EPA COMPREHENSIVE
EVALUATION
Figure 1. Proposed Testing Scheme for Plant Toxicity Testing
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1.2 TEST METHODS FOR AIRBORNE POLLUTANTS
Research on the effects of toxic airborne contaminants on plants
began about 100 years ago in Tharandt, Germany where damage to forests
by smelter fumes came under investigation. A considerable number of
studies have been made in subsequent years to identify a number of
important atmospheric toxicants of anthropogenic origin. Unfortunately,
the toxicants studied are few in number; the most important ones are
sulfur dioxide, ozone, fluorides, nitrogen oxides, peroxyacyl nitrates,
chlorine, chlorides, ethylene, some particulates, and a few other chemi-
cals , including herbicides. Although these compounds represent the
great bulk of those emitted into our atmosphere by industry or by in-
ternal combustion engines, they represent only a small percentage of
the potentially toxic airborne substances.
Because the number of potentially toxic substances is almost un-
limited and there is essentially no body of information available con-
cerning relative toxicities, concentrations, and durations of exposure
to produce toxic effects, or a list of the plant receptors that are
useful as bio-indicators, only general approaches can be recommended at
this time. Each of these is based upon methods that have been employed
for the diagnosis or evaluation of injury from the more important air
pollutants.
1.2.1 Plants as Biological Monitors of Air Pollution
Both the higher and lower plants have been employed
extensively to monitor air pollution near industrial and
urban areas. Some plants are extremely susceptible to
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specific pollutants (e.g., Bel-W3 tobacco for ozone,
lichens for sulfur dioxide, gladiolus for fluoride, etc.).
To determine the validity of these and other indicators has
taken many years of controlled fumigation studies and field
observations. No species of plant can be recommended that
would be satisfactory for all potential toxicants and, at
our present level of knowledge, a different species of
plant cannot be recommended for each potential toxicant.
The use of plants as biological monitors would expose the
native vegetation in the field to dosages of the potential
toxicant that might occur in the atmosphere. This can be
done with open-top field chambers, supplied with either the
test chemical or unpolluted air. Where the toxic substance
is already emitted into the atmosphere, the same chambers
may be used to expose native plants to the ambient pollu-
tant and other plants to clean air following selective fil-
tration. Both approaches are presently being used in air
pollution research, but have been limited to studies with
the more common phytotoxicants.
1.2.2 Effects of Air Pollutants on the Histology of Plants
That air pollutants alter the histology of plant or-
gans has been known from studies at Tharandt, Germany during
the latter half of the nineteenth century. Attempts to use
comparative histology or histological profiles to diagnose
plant injury have been relatively common during the past 20
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years. In some cases, ultrastructural changes as seen with
the electron microscope have been documented. For the most
\
part, the use of histological profiling to discriminate
between the common air pollutants has been only moderately
successful, and injuries induced by other factors produce
similar histological syndromes.
1.2.3 Effects of Air Pollutants on Metabolism or Metabolite Pools
Although there have been many investigations on altera-
tions in physiological and biochemical processes of plants
by air pollutants, no methods of real diagnostic value have
been developed even for the common phytotoxic air pollu-
tants. Of the possible methods available, the development
of an isozymic profile for a number of phytoxicants might
be the most successful. But, even this method would have
value only for the few toxicants studies and not for the
bulk of potential toxicants.
1.2.4 Effects of Air Pollutants on Cytogenetics
This approach might have value for possible chemical
mutagens, but relative utility of various test species has
not yet been found. Sparrow, at Brookhaven, however, has
developed what may become a very sensitive assay for muta-
genesis using the stamen hairs of Tradescantia, but this
work has not advanced sufficiently to be useful.
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1.3 TEST ORGANISM SELECTION
Considerable attention was given to the reasons for selecting any
given organism. Respondents in personal interviews differed according
to their orientation and organizational mission.
Studies with pesticides and herbicides have generally utilized
crop and weed plants. There appears to be a "core" of test crops with
some variation according to locale. Generally, industry was more prag-
matic in fixing the number of species based on "cash" crops. Industrial
protocols were well-defined as to the series of tests to be performed
for any given chemical. From academic scientists, there were more
variations in both tests and species.
In air pollution testing, the species was determined by the pollu-
tant. These investigators have determined which plants are sensitive
to the most common air pollutants and have designed their protocols and
tests accordingly.
Aquatic testing was limited mainly to algae and aquatic weeds.
Inasmuch as the latter are target organisms, most testing has been done
for "efficacy" rather than for plant protection.
Generally, cultivated species selected for study were from uniform
strains. Weeds presented a particular problem with respect to uniformi-
ty and simulation of natural conditions. Many interviewees responded
that weed seeds should be exposed to extreme cold before planting to
simulate natural conditions. Cultivated plants however were easily
maintained under laboratory conditions. More were selected for economic
importance rather than ecological significance. Several were selected
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for ease of maintenance or testing.
In the description of tests in this report were included those
species which have been used and, on occasion, should be used. In
general, the most pragmatic considerations have been followed. Plants
of economic concern include or represent important crops. However, one
must consider the particular community which is threatened. Two im-
portant considerations for selection within any mixed plant-animal
community are species dominance and position in the animal-plant food
chain. Damage to a dominant plant species can result in succesion
changes within a community. Destruction of a particular food plant
could affect animal as well as plant succession. The uptake, meta-
bolism, and/or biological magnification of a chemical by a plant species
also presents a hazard to species in the surrounding communities.
Selection of species, strains and stages of development for testing has
been based on these considerations. When a large volume pollutant
release affects predominant or important species, comprehensive testing
of that particular species should be undertaken.
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2. SELECTION OF TEST METHODS
2.1 INTRODUCTION
The bulk of the knowledge regarding test procedures for chemical
effects on plant growth has arisen as a result of the search for chem-
ical configurations with herbicidal or growth regulatory properties, or
a result of investigations on the response of the plants to air pollutants
emitted by industries or motor vehicles. Because of the prominence of the
screening procedures developed for herbicides, the greater part of this dis-
cussion will be directed to the absorption and movement of the substances in
plants. Much of this work has been done in the laboratories of the major
chemical manufacturing firms throughout the world. Some has been done by
public research agencies including the United States Landgrant University
Experiment Stations and the U.S.D^A. Agricultural Research Service.
Various methods have been published in the scientific periodical lit-
erature. Others reside only in the laboratories and libraries of the
chemical companies.
An axiom often repeated and demonstrated in this research is:
"The correct question must be asked to obtain the correct answer," or
stated another way; no single evaluation procedure would have detected
the majority of the economically important chemical compounds presently
available and registered for use as herbicides and growth regulators in
the United States. Some of the most widely used herbicides on the market
today were tested and discarded by other firms employing different eval-
uation screening procedures for physiological activity in plants.
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2.2 MECHANISMS OF TOXICANT ACTION '
The world-wide searches by pesticide manufacturers for chemical con-
figurations that exhibit physiological activities towards the Tracheophyta,
more specifically the Spermatophyta, have elicited hundreds of substances,
perhaps thousands, with such effects. Some obviously are herbicidal,
others herbistatic, and still others growth regulants in various ways.
The overwhelming majority of these configurations are never developed
commercially. In the search for this activity most of the firms' research
and development divisions immediately evaluate through various screening
procedures those chemicals closest at hand. These would include all
chemicals manufactured, chemical process intermediates, chemical process
by- or waste products, and stock chemicals on the shelves for one reason
or another. Through the empirical process many major breakthroughs in
herbicide technology have occurred. Once a certain configuration is
determined active, then organic specialists rapidly synthesize as many
analogues and related compounds as possible seeking the most appropriate
specific compound.
In this manner, we believe that in the last 30-year period since the
introduction of herbicides into world-wide use, many of the common
mechanisms for phytotoxic effects in plants have been elucidated.
Thus a review of the herbicide literature dealing with mechanisms of
action will shed light on probable mechanisms in the total range of
toxic substances. Furthermore, this body of literature
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points out the fallacy in attempting to use a single screening or eval-
uation procedure against all kinds of potential toxicants with any hope
of success. Hilton et al. (1) points out this multiplicity of mechanisms
all of which influence the kinds of testing required to determine effects.
The chemical categories discussed in the following pages illustrate
the diversity of principles involved in mechanisms of phytotoxic action.
The inclusion or exclusion of a group of toxicants does not necessarily
imply greater or lesser significance.
In reference to plants the term growth has been given a variety of
meanings, often being used synonymously with yield. Whereas the end
product sought by the practical agriculturalist is yield, yield is not
growth per se. Instead yield is a result of growth. Growth can be de-
fined better in terms of production of more protoplasm and cellular de-
velopment, namely: cell division, elongation, and differentiation. These
processes.bring about an irreversible increase in volume.
Growth rates do not continue at a constant velocity because of
hormone inactivation or inhibition. Growth rates may also be affected
by the inactivation of phosphate transferring enzyme systems. Shifts in
growth from production of vegetative parts to the production of reproduc-
tive parts usually results in the reduction or cessation of the former.
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Patterns of growth in plants are accompanied by certain gross
changes in the proportions of the main chemical constituents of the plant
parts. These gross changes, expressed as percentage of dry weight, are
primarily shifts in protoplasmic constituents from a high level in rapidly
growing tissues to lower levels as these tissues cease growth and their
cells differentiate secondary wall materials.
Growth of higher plants from seed to maturity and the production of
more seed is the intimate involvement of a number of complex processes.
Some of the more readily identifiable processes include imbibition, dif-
fusion, osmosis, active and passive absorption, mineral function, trans-
piration, chlorophyll and other pigment synthesis, photosynthesis, syn-
thesis of carbohydrates, fats, proteins, vitamins, hormones, etc., digestion,
assimilation, translocation, respiration, and reproduction. The processes
are interdependent and influenced by environmental, genetic and pathological
factors, enzymes, hormones, and vitamins.
Chemicals can affect the growth and development of plants in many
diverse ways, ranging from subtle alteration of the normal development
of some plants to outright death of others. The means by which chemicals
affect the growth and development of plants are diverse, in many instances
unknown, and theoretically as numerous as the processes essential to
plant life.
It is not necessarily true that all chemicals affect plant growth,
or, that those that do affect plant growth do so only in an abnormal or
detrimental manner. The agricultural scientist in cooperation with the
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chemists have learned to produce many chemicals for selective control of
certain types of plants in crop production and for the control of other
plants for industrial, health, and recreational purposes. Other types
of chemicals have been developed that alter plant growth in specific
ways, thus are useful for the economic culturing of such species of plants.
The Committee on Plant and Animal Pests of the National Research
Council's Agricultural Board considered the question of what effects
chemicals that are used for the control of pests may have on host plants
(2). Attention was accorded in their study to any secondary effect pro-
duced at all stages of plant growth, i.e., seed germination* vegetative
development, sexual reproduction, development of storage organs, matura-
tion, harvest and post-harvest behavior, nutritional value, and market
quality of fresh and processed food products by such chemicals when used
within their normally prescribed dosages for various pest control pur-
poses. Their careful sampling of the vast number of literature references
to pesticide trial reports resulted in the substantial listing of over
600 citations that made reference to secondary effects produced by 110
different chemicals used for agricultural purposes. The major secondary
effects noted for these chemicals are listed in Table 2.1.
Superficial observations and published literature, stating that
there are no effects to the crop plants following application of certain
chemical pesticides have led to the widely held assumption that such
chemicals do not cause significant effects on plants exposed to them.
Thus the information in Table 2.1, in addition to demonstrating the. erron-
eousness of this belief, also emphasizes that not all of the secondary
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Table 2.1 Different Types of Secondary Effects Observed in Plants After
to a Variety of Agricultural Chemicals
phytotoxicity
decreased and increased phytosynthetic activity
altered chlorophyll content
induced chlorosis due to interaction with minerals or other chemicals
decreased and increased respiration
reduced transpiration
altered carbon dioxide assimilation
enhancement of vegetative growth
stimulated or delayed root development
proliferation and suppression of vegetative growth
affected setting of fruit
affected pollen germination
malformation or'atrophy of various plant parts
potentiation of other chemicals
decreased or increased harvestable yield
reduction or increases in size and/or number of fruit
delayed or accelerated fruit maturity
improved appearance of fruit and vegetables
russetting of fruit
discoloration of leaves
absicission of leaves and fruit
hardening and stunting of plants
altered thickness of leaves
altered senescence
resistance and susceptibility to drought
resistance and susceptibility to freezing
susceptibility to scorching or burning of leaves
increases or decreases in plant composition (including carbohydrates,
protein, amino acids, lipids, and vitamins)
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Table 2.1 Different Types of Secondary Effects Observed in Plants After
Exposure to a Variety of Agricultural Chemicals (Cont'd)
increased or decreased specific gravity of fruit
increased or decreased soluble content of fruit
accumulation of chemicals from the soil
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effects produced by some chemicals in plants are detrimental. Indeed,
some of the effects that have been noted can be considered as desirable,
and the chemical might be exploited to induce these effects for
economic purposes. In this regard, the testing of chemicals for effects
on plant growth should not necessarily be considered as another
obstacle or objectionable feature in its commercial development; but
rather should create some excitement in anticipation of serendipitous
discoveries of other possible uses for the chemical, since it is rarely
possible to predict what effect most chemicals might have on plant life.
Our greatest source of information about the ways in which chemicals
affect plants resides in the literature pertaining to those chemicals that
are used for pest control purposes, and most specifically those chemicals
that are used as herbicides. In spite of much experimental work, part-
icularly with the auxin and photosynthetic inhibiting groups of chemicals,
our knowledge about the relationship between molecular structure and
herbicidal activity is restricted.
The relationship between chemical constitution and physiological
activity has been the subject of intensive study and speculation since
the earliest days of organic chemistry. This is not surprising since
the initial stimulus to that science came from medicine and from the
need to find a wide range of drugs for the cure of diseases. This
stimulus remains, but with passage of time and because of rising world
population the control of pests in agriculture has also become an urgent
problem that has resulted in the development of a massive chemical in-
dustry in endeavoring to provide solutions to these problems. The
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triumphs achieved in some areas of pest control have been many and strik-
ing, yet a precise theoretical basis for the design of pesticides still
eludes us just as it does in the search for chemotherapeutic agents.
An understanding of enzyme and other systems involved in vital processes,
and their vulnerability in different organisms is necessary for a more
rational development of specific biologically active chemicals. Given
such an understanding it would be possible to more readily design chemicals
that would interfere with vital systems and if problems of stability
and transport in the organism can be surmounted, would provide effec-
tive agents needed in the control of diseases and pests.,
Almost all commercial herbicides were first developed empirically,
and studies of their mode of action were undertaken only after their
economic benefits were recognized. The results of these investigations
are of considerable interest to biology and agriculture, since they furnish
new insights into the physiological processes of plants and at the
same time provide some information that is useful toward the rational
development of new chemicals to satisfy specific needs.
Although the contrary view is often stated, the current level of
biochemical knowledge provides considerable understanding of the primary
site of action of many of the herbicides (and other pesticides) in use
today. However, information is lacking about the detailed mode of action
of phytotoxicants that are thought to act in areas where the fundamental
biochemistry of the particular process is obscure, e.g., chemicals that
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are thought to interfere with cell division, axonal transmission, or
growth processes in plants.
The biophysical properties of phytotoxic chemicals have so far
attracted much less attention than their biochemical properties. With
the exception of those chemicals related to indoleacetic acid (auxins),
little is known about their effect on biophysical systems in plants. A
proper biophysical organization is necessary for the efficient biochemical
functioning of the plant.
In view of the importance of biophysical processes in the economy
of plants, it would be very helpful to know to what extent interferences
by phytotoxicants with these processes may be responsible for the death
of plants. An adverse effect of phytotoxic chemicals on biophysical
systems operating in the plant may have a profound effect on its growth
and development. Thus, more knowledge is necessary to determine how .
chemicals affect (1) the osmotic pressure and water uptake of cells, (2) the
properties of the cell wall, (3) transpiration and stomatal opening,
(4) cell permeability, (5) the uptake of mineral ions, and (6) proto-
plasmic streaming and the viscosity of the protoplasm.
In a discipline concerned with the physical properties of a living
system, particular attention needs to be given to factors of physical
environment such as temperature, humidity, light intensity and quality,
before and after the experiment. Much of the value of some past invest-
igations is lost because of a failure either to control the environment
adequately or to have defined the conditions clearly in the published
work. Nonetheless, from the evidence found in the literature, it is
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unlikely that death of plants caused by interferences of phytotoxic
chemicals is due to any large extent to effects on biophysical processes.
For this reason subsequent emphasis in this report will be placed on
the biochemical effects of chemicals as they relate to phytotoxicity.
It must be realized that the effects of chemicals on plants, whether
phytotoxic or stimulatory, are not due to.a simple physiological pro-
perty but may result from many different processes, each physiologically
distinct, being affected. For example, one group of chemicals may act
in high concentration by osmotic action on living cells; another group
may interact at low concentration with the photosynthetic system within
green cells; yet a third class of chemicals might by their interaction
with the plant's own hormonal system disturb cell elongation or multipli-
cation or their component processes. That there are various physiological
processes, either beneficial or detrimental, affected by chemicals in
plants is readily emphasized in Table 2.1.
The diversity of principles involved in the action of chemicals on
plants precludes unqualified generalizations. For most 'chemicals, multiple
sites and mechanisms of action must be considered a probability along
with the possibility that the most sensitive sites differ among species.
Many possible sites of action have been described for chemicals used as
herbicides, however, their contribution to lethal action may still be
uncertain in some cases and unknown in other cases.
Table 2.2 has been included to list the most likely primary mode of
action and major function that is modified or disrupted in plants by
2-11
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Table 2.2 Mode of Action by Which Various Chemicals Disrupt Plant Growth
1. Inhibit Lipid Synthesis; structural organization disrupted.
Thiocarbamates
S-ethyl diisobutylthiocarbamate butylate
S-ethyl tf-ethylthiocyclohexanecarbamate , cycloate
S- (2,3~dichloroa1lyl) diisopropylthiocarbamate diallate
S-ethyl dipropylthiocarbamate EPIC
S-ethyl hexahydro-15-azepine-l-carbothioate molinate
S-propyl butylethylthiocarbamate pebulate
S-(2,3,3"trichloroallyl) diisopropylthiocarbamate triallate
S-propyl dipropyl thiocarbamate vernolate
2. Disrupt Cell Membranes.
Petroleum oi1s
Bi pyri dy1i urns
6,7-dihydrodipyrido [1,2-a:2',1'-C] pyrazinediium ion diquat
1,1' -dimethyl-4,V -bipyridinium ion paraquat
3. Divert Photosynthetic Electron Transport; energy supply disrupted.
Bipyridyliums
6,7~dihydrodipyrido [1,2-a:2',1'-c] pyrazinediium ion diquat
1,1 '-dimethyl-k,k'-bipyridinium ion
paraquat
4. Inhibit Enzyme Systems; energy supply disrupted..'.
Arsenicals
hydroxydimethylarsine oxide
disodium methanearsonate
monosodium methanearsonate
2-12
cacodylic acid
DSMA, (DMA)
MSMA
-------
Table 2.2 Mode of Action by Which Various Chemicals Disrupt Plant Growth-
(Cont'd)
Inorganic salts
ammonium sulphamate
borax
calcium cyanamide
copper sulfate
sodium chlorate
sodium metaborate
potassium cyanate
Miscellaneous compounds
allyl alcohol
acrolein
5. Uncouple Oxidative. Phosphorylation.
Di ni trophenol s
2-sec-butyl-^,5~dini trophenol
2- (1-methylbuty l)-^,6-dini trophenol
4,6-dini tro-o-cresol
Hydroxybenzoni tri les
3,5~di bromo-^-hydroxybenzoni tr i le
-S.S-di iodobenzoni tr i le
2-propen-l-ol,
2-propenal
Mi seel laneous
pentachlorophenol
dini trobuty1 phenol
dini trocresol
bromoxyni1
• ioxynil
PCP
2-13
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Table 2.2 Mode of Action by Which Various Chemicals Disrupt Plant Growth
(Cont'd)
6. Affect Photosynthetic Electron Transport ( Hill Reaction Inhibited)
and Chlorophyll Destruction via Inhibition of Carotenoid Synthesis;
Ureas
3~ (p-chlorophenyl)-l-methy1-l-(l-methyl-2-propyny1) urea buturon
3- (^-bromo-S-chlorophenyO-l-methoxy-l-methylurea chlorbromuron
3~ [p~ (p-chlorophenoxy)phenyl ]-l , 1-dimethylurea chloroxuron
3-cyclooctyl-l,1-dimethyl urea eye 11 uron
3-(3,^-dichlorophenyl)-!,1-dimethyl urea diuron
1,l-dimethyl-3~phenylurea fenuron
1,1-dimethyl-3-(a,a,a-trif1uoro-m-tolyl) urea fluometuron
tert-butylcarbamic acid ester with 3"(m-hydroxyphenyl)-l,1-
dimethylurea karbutilate
3-(3,^-dichlorophenyl)-l-methoxy-l-methylurea 1inuron
3-(p-bromophenyl)-1-methoxy-l-methyl urea metobromuron
3~(p-chlorophenyl)-1-metboxy-1-methyl urea monolinuron
3-(p-chlorophenyl)-l,1-dimethyl urea monuron
3-(hexahydro-^.y-methanoindan-S-yl)-l»1-dimethylurea norea
1-(2-methylcyclohexyl)-3-phenylurea siduron
s-Triazines
2-(ethyl ami no)-k-(isopropy 1 ami no)-6-(methylthio)
-s-triazine ametryne
2-chloro-^-(ethylamino)-6-(isopropy lamino)-s-triazine atrazine
2-(ethylamino)-^-(isopropy 1 ami no)-6-methoxy-s-triazine atratone
2- [[^-chloro-6-(ethylamino)-s-triazin-2-yl amino]-2-methyl-
propionitrile cyanazine
2-chloro-4-(cyclopropylamino)-6-(isopropy 1 ami no)
-s-triazine cyprozine
2-(i sopropy 1 ami no)-4-(methyl ami no)-6-(methylthio
-s-triazine desmetryne
2-14
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Table 2.2 Mode of Action by Which Various Chemicals Disrupt Plant Growth
(Cont'd}-
2-(ethylthio)-it,6-bis(isopropylamino)-s-triazine d.ipropetryn
2-chloro-A-(diethyl ami no)-6-(isopropylamino)-s-triazine ipazine
2,4-bis(isopropylamino)-6-methoxy-s-triazine prometone
2,A-bis(isopropylamino)-6-(methylthio)-s-triazine prometryne
2-chloro-*f,6-bis(isop ropy 1 ami no)-s-triazine propazine
2-chloro-'*,6-bis(ethylamino)-s-triazine simazine
2,^-bis(ethyl ami no)-6-(methylthio)-s-triazine simetryne
2,4-bis(ethy1 ami no)-6-methoxy-s-triazine si metone
2-(tert-butylamino)-4-(ethylamino)-6-(methylthio)
-s-triazine terbutryne
2-chloro-Jf- (di ethyl ami no)-6- (ethyl ami no)-s-tr iazine trietazine
Acylani1 ides
3',V-dichlorocyclopropanecarboxani1ide cypromid
fl-[5-(2-chloro-l,l-dimethylethyl)-l,3,^-thiadiazol-2-yl]-
cyclopropanecarboxamide
3',^'-dichloropropionani1ide propani1
2-chloro-2' ,6'-diethyl-/l/- (methoxymethyl) ace tan i 1 ide alachlor
Hydroxybenzoni triles
S.S'dibromo-^t-hydroxybenzoni tr i le bromoxyni 1
4-hydroxy-3,5-di iodobenzoni trile ioxyni1
Uraci1s
5-bromo-3~see-butyl-6-methyluraci1 bromaci1
3-eye 1ohexy1-6,7~d ihyd ro-1H-cyc1 open tapy r i mi d i ne
-2,M3#,5#)-dione lenacil
3-tert-butyl-5-chloro-6-methyluraci1 terbaci1
Ethers
p-nitrophenyl a,a,a-trifluoro-2-nitro-p-tolyl ether fluorodifen
2,4-dichlorophenyl-p-nitrophenyl ether nitrofen
2-15
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Table 2.2 Mode of Action by Which Various Chemicals Disrupt Plant Growth
(Cont'd)
Pyridasinones
l»-chloro-5-(methylamino)-2-(a,a,a-trlf 1uoro-m-tolyl)-3
(2#)-py r i daz i none monometf1urazon
5-am i no-A-ch1o ro-2-p heny1 - 3(2ff)-py r i da z i none
N-phenyIcarbamates
methyl m-hydroxycarbanilate m-methylcarbanilate
methyl 3»^~dichlorocarbanilate
7. Inhibit Carotenoid Synthesis; destroy chlorophyll.1
pyrazon
phenmepipham
swep
3-amino-l,2,^-triazole
2,3,5-trichloro-A-pyridinol
8. Inhibit Cellular or Nuclear Division.
ami trole
pyriclor
/V-pheny Icarbamates
^-chloro-2-butyny 1 m-chlorocarbani late
D-#-ethylacetamide carbanilate (ester)
isopropyl m-chlorocarbanilate
isopropyl carbanilate
methyl 3»^"dichlorocarbanilate
Other carbamates
methyl sulfanilylcarbamate
2,6-d i-tevt-buty1-p-to1y1 me thy 1ca rbama te
Dini troani1i nes
N- bu ty 1 -N- e t hy 1 -a, a., a-1 r i f 1 uo ro- 2,6-d i n i t ro-p- to 1 u i d i ne
ff^^-diethyl-a.a.a-trif 1 uoro-3,5-dini trotoluene-2,4-
diami ne
2,6-d i n i t ro-N^N-di propy 1 cum i d i ne
barban
desmed i pham
chlorpropham
propham
swep
asulam
terbutol
benefin
di ni trami ne
i sopropali n
2-16
-------
Table 2.2 Mode of Action by Which Various Chemicals Disrupt Plant Growth
(Cont'd)
k- (methylsulfonyl )-2,6-dini tro-tf,/l/-dipropylani 1 ine ni tral in
3,5~dini tro-A^jA^-dipropylsulfani1 amide oryzalin
a,a,a-1 r i f1uoro-2,6-d i n i t ro-N3N-di p ropy 1-p-toluidine trifluralin
Miscellaneous compounds
6-hydroxy-3(2ff)-pyridazinone MH
2-chloro-ff-isopropylacetani1ide propachlor
9. Mimic Indolacetic Acids.
' Phenoxyalkonic acids
2,^-dichlorophenoxyacetic acid 2,^-D
k-(2,A-dichlorophenoxy)butyric acid 2,4-DB
tris-(2,4-dichlorophenoxyethyl) phosphite and
bis (2,4-dichlorophenoxyethyl) phosphite 2,Jj-DEP
2-(2,A-dichlorophenoxy)propionic acid dichlorprop
2-(2,A,5~trichlorophenoxy)ethyl 2,2-dichloropropionate erbon
(2,3,6-trichlorophenyl)acetic acid fenac
2-(2,4,5~trichlorophenoxy)propionic acid silvex
[(4-chloro-o-tolyl)oxy]acetic acid MCPA
4-[(A-chloro-o-tolyl)oxy]butyric acid MCPB
2-[4-chloro-o-tolyl)oxy]propionic acid mecoprop
1-naphthaleneacetic acid NAA
2,^,5'trichlorophenoxyacetic acid 2,4,5~T
Benzole acids
3-amino-2,5~dichlorobenzoic acid amiben
3,6-dichloro-o-anisic acid dicamba
2,3,6-trichlorobenzoic acid 2,3,6-TBA
2-17
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Table 2.2 Mode of Action by Which Various Chemcals Disrupt Plant Growth
(Cont'd)
Pico]inic aci d
i»-amino-3,5,6-trichloropicol inic acid picloram
10. Interfere with Transport of Indolacetic Acid.
W-1-naphthylphthalamic acid NPA
2,3,5-triiodobenzoic acid TIBA
11. Inhibit Gibberellins; growth and reproduction disrupted.
2-(chloroethyl)trimethylammonium chloride chloromequat chloride, CCC
N-(dimethylamino)succinamic acid succinic acid; DMSA
(2,lt-dichlorobenzyl) tributylphosphonium chloride phosphon
12. Affect Ethylene Levels; growth and reproduction disrupted.
2-chloroethylphosphonic acid ethephon
13. May Combine with Proteins.
Chlorinated aliphatics
2,2-dichloropropionic acid dalapon
trichloroacetic acid TCA
ethylene glycol bis(trichloroacetate) glytac
.14. Mimic Nitrate Ion; disrupt metabolism of nitrogen compounds.
sodium chlorate
.15. Mode of Action not Known.
2,6-dichlorobenzonitrile dichlobenil
0,0-diethyl di thiobi s [thioformate] bisethylxanthogen
1,1,1,3,3,3-hexachloro-2-propanone HCA
potassium hexaf1uoroarsenate hexaflurate
2-18
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Table 2.2 Mode of Action by Which Various Chemicals Disrupt Plant Growth
(Cont'd)
0,0-diisopropy 1 phosphorodithloate 5-ester with
N-(2-mercaptoethyl)benzenesulfonamide bensulide
3-i sop ropy 1 -1H-2,1,3-benzoth i ad i az i n-(k)3#-one 2,2-
dioxide bentazon
(benzamidooxy)acetic acid benzadox
^ff-dimethyl-2,2-diphenylacetamide diphenamid
2-(a-naphthoxy)-W,#-diethylpropionamide napropamide-
2-tert-butyl-A-(2,4-dichloro-S-isopropoxyphenyl)-A2-l, oxadiazon
3,^-oxadiazolin-5~one
2-19
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chemicals that are used to control plant life. Over 125 chemical sub-
stances ranging from the relatively simple inorganic salts to the more
intricate synthetic organic chemicals and complex materials such as
petroleum oil are included in this survey. The synthetic organic chem-
icals are represented by diverse chemical structures consisting of de-
rivatives of acylanilides, alchols, arsenicals, benzoic acid, benzoni-
triles, bipyridinyls, carbamates, chlorinated aliphatics, chlorophenols,
dinitroanilines, dinitrophenols, ethers, methylcarbamates, pyridazionones,
sulfonamides, s-triazines, triazole, uracil, and ureas.
The available information reveals that death of plants from such
chemicals results after they affect such major functions as structural
organization, energy supply, and growth and reproduction through at
least 12 identified mechanisms of action. Such mechanisms of action
listed in Table 2.2 include (1) disruption of lipid synthesis, (2) disturb-
ances of cell membranes, (3) interference with electron transport, (4)
inhibition of enzymes, (5) uncoupling of oxidative phosphorylation, (6)
inhibition of photosynthesis, (7) inhibition of pigment synthesis
and the destruction of chlorophyll, (8) mimicry of plant auxins,
(9) interference with plant hormone metabolism, (10) affect level of
ethylene, (11) combination with proteins, and (12) interference with
mineral metabolism.
The phytotoxic properties of petroleum oils are in direct relation-
ship to the proportion of aromatic constituents they contain. Oils per
se probably are not metabolic inhibitors, but act by physically plugging
the tissue vessels during transport.
/
2-20
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A number of substances such as inorganic salts, chloropicrin, methyl
bromide, phenylmercuric acetate, and the substituted chlorophenols are
metabolic inhibitors; thus, general poisons that are not specific for
plants only. Their effects on plants may be through their established
roles of blocking or uncoupling specific enzyme systems, although other
unknown toxic actions may be involved.
The best evidence is that arsenic and copper containing chemicals
act at least partially by combination or interference with vital thiol
groups in the pyruvate dehydrogenase or the a-ketoglutarate dehydrogenase
system, or both of them. Pentavalent arsenic may act by mimicking the
phosphate ion and be incorporated into key energy containing inter-
mediates that degrade rapidly if they contain arsenate in place of
the normal phosphate. In spite of the long usage of ammonium sul-
famate, borax, potassium cyanate, and sodium chlorate no satisfactory
explanation of their phytotoxic action has been advanced. The general
toxic action of calcium cyanamide and sulfuric acid to plants may be
ascribed to tissue corrosion on physical contact; although, some as yet
unrecognized metabolic action may also be involved.
There is no generally accepted demonstration of any effect of the
five types of plant hormones, auxins, gibberellins, ethylene, cytokinins,
and abscisic acid, at physiologically realistic concentration in a cell
free system. Neither has the mode of action of the synthetic compounds,
which affect plant growth, and which are used either as herbicides or as
plant growth regulators, been demonstrated with cell free systems.
2-21
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Corbett (3) postulates that the synthetic growth-regulating chemicals
probably act by interfering with the action of the known natural hor-
mones unless there are yet undiscovered natural plant growth hormones.
that are interfered with by synthetic compounds or if some of the syn-
thetic compounds act at quite different levels than the natural hormones.
A likely exception, however, would be those compounds that inhibit cell
division. Corbett (3) concludes then that there are only seven ways by
which synthetic compounds can interfere with the action of a known natural
hormone. It can (1) release or (2) combine with it, (3) interfere with
its synthesis or (4) degradation, (5) combine at the site of action, (6)
modify its transport or (7) its deposition at an inert site.
The phenoxyalkanoic acids derivatives and related compounds listed
under 9 in Table 2.2 acts as auxins. They produce morphological effects
on plants that indicate exaggeration of normal auxin action. Hanson
and Slife (4) conclude that plants treated with 2,4-dichlorophenoxyacetic
acid die due to the aberrant growth produced by its persistent auxin
effect. The roots and stem of the treated plant proliferate rapidly, thus
appropriating most of the available food, which results in senescence and
physiological malfunction of leaves and secondary roots, so that the
plant is unable to feed itself, and death ensues. Existing reports in
the literature offer ample evidence to rule out both interference with
oxidative phosphorylation in mitochondria and inhibition of the Hill
reaction in chloroplasts as mechanisms to explain the phytotoxic action
of the phenoxyalkanoic acid type of chemicals.
2-22
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The phenoxyalkanoic acid chemicals are eminently more phytotoxic
to dicotyledons than to monocotyledons, generally killing the former but
not the latter. There are striking exceptions, however since onions
(monocotyledons) are susceptible to these chemicals, where as chickweed
and cleavers (Goose Grass-dicotyledons) are resistant.
Although 4-amino-3,5,6-trichloropicolinic acid (picloram) is struct-
urally distinct from the other auxins, it has been shown to possess the
typical properties of an auxin. Thus, it causes cell elongation in a
variety of plant stems, stem proliferation, leaf and stem bending, loss
of chlorophyll, and adventitious rooting.
Derivatives of benzoic acid act in the same way as the phenoxyalkanoic
acids, i.e., they are persistent auxins, with the exception of 2,3,5-
triiodobenzoic acid (TIBA) which is not an active auxin. TIBA is known
to exert its phytotoxic effects by way of interfering with auxin trans-
port, and possibly by modifying auxin action in some other manner. A
similar mode of action has been identified for the naphthyl-phthalamic
acid and chloroflurecol derivatives.
The chemicals listed under 9 and 12 in Table 2.2 affect plants by
reducing growth without permanently stunting or malforming the plant, and
their action is distinguished from that of such growth inhibitors as
maleic hydrazide. These chemicals are in contrast with almost all of
the others listed in Table 2.2. Whereas the latter group are primarily
used for their phytotoxic properties to kill plants, the naphthyl-ph.th-
alamic acids and chloroflurecols produce compact plants with shortened
2-23
-------
internodes and leaf stems and are used commercially for such purposes
to prevent lodging of cereal stems, increase resistance to insect and
fungal attack, control vegetative growth of fruit trees, and to modify
the shape and height of ornamental plants.
2-Chloroethylphosphonic acid (ethephon) acts in plants by decomposing to
ethylene, thus produce all of the characteristic responses in plants
attributed to ethylene. The chemical, therefore, finds practical appli-
cation to induce flowering, promote fruit ripening, and fruit abscission.
The W-phenyl carbamic acid derivatives (Table 2.2,8) affect cell ,
division in plants. They are not used as growth regulators, as are the
chemicals mentioned in the preceding paragraph, but rather find extensive
application as herbicides, and in this regard are referred to as "mitotic
poisons." The inhibitory effect of the A/-phenylcarbamates on cell divi-
sion appears to be due to disorientation of the microtubules while other sub-
cellular structures are unaffected. Microtubules are small tube-like
structures made of sub-units of globular protein, which may function
to aid in the separation and alignment of chromosomes during nuclear
division. The action of these chemicals is in contrast to colchicine,
a compound known for its ability to inhibit cell division which actually
disrupts the microtubules, probably by binding to a component protein.
Isopropyl carbanilate (propham), an A'-phenylcarbamate, has been shown
to have no deleterious effect on the tubules themselves, but only on
their organization. Morphological and cytological investigations indicate
a similar mode of action for all carbamates.
2-24
-------
Moreland and Hill (5) concluded that carbamates interfered with
more than one system in plants, and produced evidence to show that photo-
synthesis might be such a system. It is uncertain whether the inhibition
of photosynthesis is a primary effect of this class of chemicals. In
vitro results strongly suggest that the inhibition of photosynthesis can
only be a side effect for most of these chemicals. The W-phenylcarbamates
that inhibit photosynthesis are structurally similar to the phenylurea
and acylanilide derivatives (Table 2.2, 6), chemicals are known as potent in-
hibitors of photosynthesis, but their toxic symptoms differ and the im-
plication on inhibited photosynthesis to overall herbicidal action is
uncertain. Most of them inhibit cell division in both susceptible and
resistant plant species.
The benzenesulfonyl and W-methyl carbamate derivatives appear to
exert their phytotoxic activity also by inhibiting cell division. The
dinitroaniline compounds (Table 2.2,8) are analogous in their mode of '
action to the carbamate class of chemicals. It is known that the dini-
troanilines inhibit mitosis, their primary mode of phytotoxic activity.
The exact mechanism of how they .disrupt nuclear and cell division
is not known. The most typical symptoms of plants treated with dini-
troaniline chemicals is the inhibition of lateral root formation.
They also cause growth reduction in roots and shoots, and swelling and
irregularities in various tissues. This class of chemicals is more toxic
to monocotyledons than to dicotyledons.
2-25
-------
The effect of maleic hydrazide on plants is due to its ability to
inhibit cell division, but not cell enlargement. It is an isomer of
uracil, a pyrimidine found in ribonucleic acid. It may interfere
with mitosis by becoming incorporated into ribonucleic acid, as recent
studies have demonstrated..
Herbicides that are toxic to plants by interfering with photosynthesis
do so by two distinct modes of action. The majority of the chemicals,
for which there is experimental evidence, do so by inhibiting electron
transfer, but other compounds such as the bipyridylium derivatives short
circuit electron transfer and generate toxic molecular species.
A wide variety of chemicals (Table 2.2, 6) will inhibit photosynthetic
electron transfer, and many of them are used as herbicides. The urea
and s-triazine family have more such herbicides than any other class of
chemicals. More is known about the mode of action of 3-(3,4-dichloro-
phenyl)-l,l-dimethylurea (diuron) than for any other inhibitor of the
Hill reaction. The studies with diuron have contributed significantly .
to our understanding of the mechamisms of photosynthesis and herbicidal
properties of such chemicals. Only a limited amount of biochemical re-
search has been reported for ureas other than diuron and 3-(4-chloro-
phenyl)-l,l-dimethylurea (monuron); however, the available data suggest
that all urea herbicides inhibit the Hill reaction and thus appear to
have similar modes of phytotoxic action.
2-26
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The phototoxic action of the substituted urea chemicals appears to
be due to other factors as well as the inhibition of photosynthesis.
Diuron, for example, affects some non-photosynthetic processes, but only
at very high concentrations. Death of plants caused by this class of
chemicals is probably due to the direct interruption of the food supply
caused by inhibition of photosynthesis, and by irreversible damage to
the photosynthetic system, resulting in permanent inhibition of food
supply.
The s-triazine class of chemicals are also potent inhibitors of
the Hill reaction, thus their primary mode of action is due to the inhibi-
tion of photosynthesis. The available information suggests that they
act in an essentially similar way to the substituted urea compounds and
kill plants in a similar but not necessarily identical manner as the
ureas.
The acylanailides have been studied much less than the ureas and the
s-triazines, however, they also have proved to be powerful inhibitors of
the Hill reaction. The available evidence suggests that they act in a
similar manner as the ureas and s-triazines. The hydroxybenzonitriles
are both uncouplers of oxidative phosphorylation and inhibitors of photo-
synthesis. It is not possible to be conclusive about which of these
effects is most important in the phytotoxic action. The fact that light
is necessary to achieve rapid and complete killing of plants with some
of the hydroxybenzonitrile compounds indicates that the herbicidal action
of these chemicals is mainly due to inhibition of photosynthesis.
2-27
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The uracil derivatives cited in Table 2.2 inhibit the Hill reaction
and are regarded to exert phytotoxic effects by their inhibitory action
on photosynthesis. They appear to have little effect on non-photosyn-
thetic processes in plants. The available information about the uracil
compounds reveals that they act in a similar fashion to the ureas. Some
of the uracil derivatives have been shown to inhibit nitrate reductase
activity in excised plant tissue, but the significance of these observa-
tions to the phytotoxic action of these chemicals is obscure.
Some of the nitrophenyl ethers have been found to inhibit the Hill
reaction and to be active uncouplers of electron transport. Either of
these inhibitory effects might be sufficient to explain their phytotoxic
action, but there is not sufficient information in the literature that
permits one to decide whether the inhibitory effect on photosynthesis or
respiration is the primary mode of action in whole plants. The pyridaz-
inones and pyrimidones are also strong inhibitors of the Hill reaction
and exert their phytotoxic activity by inhibiting the photosynthetic pro-
cesses in plants. Other classes of organic chemicals that have been
found to be effective inhibitors of the Hill reaction include substituted
benzimidazoles, imidazopyridines, imidazoles, imidazoquinoxalines, and
thiadiazoles. The benzimidazoles and the imidazopyridines are also un-
couplers of oxidative and photophosphorylation. None of these chemicals,
however, has been developed for weed control purposes.
The mode of action of the chlorinated aliphatic acids is not known,
but it has been suggested that they act by combination with proteins.
2-28
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Both trichloroacetic acid (TCA) and 2,2-dichloropropionic acid (dalapon)
have been shown to inhibit the synthesis of the surface layer of wax on
plant leaves, but it is not known whether or not the inhibition of lipid
synthesis contributes to the lethal effect of these chemicals, after they
are applied to plants. Lisner (6) states that dalapon is a specific
blocking agent in the biosynthesis of pantothenic acid in plants. Thus,
plants with a low requirement for this vitamin might be expected to be
resistant to dalapon.
The extensive literature on the possible mode of action of 3-amino-
1,2,4-triazole (amitrole) suggests that it might exert its phytotoxic
effect by inhibiting more than one reaction in plants. This evidence
suggests that amitrole prevents the synthesis of a component that is
required for the production of chlorophyll, but does not affect the
synthesis of chlorophyll itself. Since, as it has been stated pre-
viously, there is reasonable evidence that carotenoids are necessary
to prevent the photooxidation of chlorophyll and the disruption of
chloroplast structure it seems likely that the inhibition of carotenoid
biosynthesis is the primary site of action of amitrole.
On the preceding pages many possible sites and mechanisms of phy-
totoxic action have been cited for a wide array of chemicals. These find-
ings are a result of the great advances in the development of many new
methods to test biochemical responses in in vitro and in vivo systems.
Consequently, it is logical to question whether or not such tests could
be utilized to evaluate chemicals for potential effects to plant life.
2-29
-------
Photosynthesis is the ultimate source of all energy used in plants
(and animals) and the toxic action of the largest number of chemicals
listed in Table 2.2 have been attributed to their effects on this biochem-
ical process. Therefore, it is obvious that this might be the first
useful evaluation to be performed to screen a chemical for possible phy-
totoxic effects.
There are a number of relatively simple tests for determining the
effects of a chemical on photosynthesis. Most of these are described in
detail by Wilkinson (7). The Hill reaction, which was mentioned most
often in the preceding pages, is a rapid and sensitive spectrophotometric
assay by which the photolysis of water by a cell free preparation of
photosynthetic tissue can be studied. The usefulness of this test, how-
ever, is greater in studying the mechanism of action of a chemical more
so than in predicting whether or not a chemical will be toxic to a plant.
This is because not all chemicals that effectively inhibit the Hill
reaction are phytotoxic when tested on whole plant systems. The converse
of this, however, has also been demonstrated, i.e., all chemicals whose
mechanism of action has been determined to be due to inhibition of photo-
synthesis are also inhibitors of the Hill reaction. By way of illustra-
tion, all of the herbicidally active s-triazines yield a 50% inhibition
response of the Hill reaction in concentrations of ICT^to 10~7M. Although
there is generally good agreement between the herbicidal activity and
the concentration of the chemical necessary to inhibit the Hill reaction,
this relationship is not always consistent. For example, 2-chloro-4-
ethylamino-6-n-butylamino-s-triazine is one of the most active members
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of this class of chemicals as measured by the Hill reaction, yet it is
virtually devoid of herbicidal properties on growing plants in a field.
From the preceding example it is apparent that the interference of
a fundamental physiological process, as determined by in vitro testing,
does not automatically lead to a similar effect in whole organisms.
The reason for this is due to some additional and specific morphological,
physiological or biochemical properties of plants and edaphic factors
/
that may influence the stability, fate and behavior of the chemical in
soil; hence its availability to the plant.
The diversity of principles involved in phytotoxic activity pre-
cludes unqualified generalizations about mechanisms. For most chemicals
multiple sites and mechanisms of action must be considered a probability
along with the possibility that the most sensitive sites differ among
plant species. Many possible sites of action have been described, but
their contribution to lethal action is uncertain. The most difficult
aspect of mechanism research on phytotoxicity is substantiating the phys-
iological significance of in vitro results under field conditions. At
the present state of knowledge in the field of plant physiology this
appears to be equally true for whatever biochemical or in vitro parameter
that is being considered, i.e., effects on respiration (C0_ production,
0? consumption, or high energy phosphate, ATP production); photosynthesis
(0- release, CO incorporation, chlorophyll content, photophosphoryla-
14
tion, NADP reduction, CO- fixation, or the Hill reaction); amino acid
and protein metabolism; plant enzyme systems; lipid synthesis; mineral
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metabolism; and plant nucleic acids. The same would also be applicable
to morphological, anatomical, and cytological studies.
The susceptibility of a plant to a particular chemical is influenced
by morphological, physiological, biochemical, and genetic factors. These
factors influence the concentration of material that reaches the site or
sites of action at any one time. The site(s) of action in different
species of plants may also be differentially sensitive to the chemical.
The quantity of a chemical accumulated per unit weight of plant
may be important, since as a rule the higher the concentration of chem-
ical in the plant the more likely it is to damage it. The difference
in the degree of absorption can be caused by the morphological character-
istics of the plant in question. After a chemical is absorbed by a
plant it must be translocated to the site of action and not remain at
the site of its application. While a study of the morphological attributes
of plants often allows one to predict the characteristics necessary for
penetration of a chemical, similar predictions cannot always be made
with regard to the physiological response of the plant. Experiments
alone can decide whether a particular chemical is more toxic to one
species than to another.
The differences in the responses of the same dose of chemicals to
different plant species has been the basis of much research. A number
of factors may operate in determining the selectivity of this response,
amongst the important ones are (1) the extent to which the applied chem-
ical is absorbed by the plant, (2) the ease with which the chemical
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moves in the plant tissue, (3) the stability of the chemical within the
plant, and (4) its potential activity at the site of action. A chemical
may be applied in one form, translocated in a metabolized form, and
express its inhibitory effect at the site of action in still another form;
whereas, another chemical may be absorbed, translocated, and accumulated
at the site of action in its native state. Almost all organic chemicals
are metabolized by plants, many rapidly and extensively.
Metabolism of a chemical is not necessarily tantamount to detoxifica-
tion, although this is usually the case. There are a number of instances
in which a relatively non-toxic chemical is converted by plant metabolism
to phytotoxic material, e.g., reduction of dipyridyl compounds and the
B-oxidation or ester hydrolysis of some of the phenoxy compounds.
The problem of extrapolating in vitro tests to in vivo conditions
is confounded also by the problem of secondary effects. Despite our
reasonably good knowledge of the primary sites of action of many chemicals,
generally very little is known about the secondary reactions resulting
from the initial action of the chemical.
The complexity of methodology and high cost are also additional
deterrents to the use of biochemical and other laboratory tests for
studying the effects of a chemical on plant life. A number of biochemical
tests such as the Hill reaction and other tests for photosynthesis and
respiratory activity are relatively simple and can be conducted by most
competent laboratory technicians with equipment available in any adequately
equipped chemical laboratory.
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However, many other biochemical testing procedures require a considerable
amount of costly and specialized equipment and supplies as well as highly
skilled personnel not usually found in laboratories that are not normally
involved with biochemical operations. The degree of specialization and
expertise becomes even increasingly greater for cytological investiga-
tions.
It is therefore concluded that for the present it is not feasible
to employ biochemical test methods to obtain quantitative estimations
of the phytotoxic properties of chemicals that could be correlated in
any meaningful way with the actual behavior of the chemical under field
conditions. In view of our present knowledge of primary biochemical
disturbances that can be caused by chemicals in plants, the complexity,
high cost, and requirements for highly specialized laboratory equipment
and personnel, the phytotoxic properties of chemicals can best be com-
pared on relatively simple criteria based on numbers of plant individuals
damaged or destroyed in bioassay type tests.
A very large amount of time was spent during an intensive effort to
consider the practicality of using biochemical test methods to elucidate
the potential phytotoxic properties of chemicals. The results of this
effort have been negative in respect to adapting such procedures on a
practical basis at the present. However, the effort expended revealed that
the present state-of-the-art is somewhat incoherent, in disarray, but
excitingly evolutionary. The writer would be remiss, therefore, if he
neglected to recommend that this specific phase of the present contract
be considered for further in depth consideration. There appears to be
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a need for an intensive study to compile and to correlate the vast amount
of available information regarding the biochemical responses of plants
to chemicals, with the expected resultant effect that a number of exist-
ing procedures might already be available to establish a better relation-
ship between chemical structure and physiological activity, and to cite
new areas of research that should be undertaken to provide presently
missing answers for more practical utilization of biochemical test methods
in the field of phytotoxicity.
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2.2.1 References
1. Hilton, J. L., L. L. Jansen, and H. M. Hull. Mechanisms of
Herbicide Action. Ann. Rev. of Plant Phys. 14:353-384 ; 1963.
2. National Academy of Sciences. Principles of Plant and Animal
Pest Control, Vol. 6: Effects of Pesticides on Fruit and Veget-
able Physiology. National Academy of Sciences, Washington,
D.C., 90 pp.,1968.
3. Corbett, J. R. The Biochemical Mode of Action of Pesticides^
Academic Press, New York, 330 pp.,1974.
4. Hanson, J. B. and F. W. Slife. Role of metabolism in the
action of auxin herbicides. Residue Reviews. 25:59-67 ; 1969.
I
5. Moreland, D. E. and K. L. Hill. Interference of herbicides
with the Hill reaction of isolated chloroplasts. Weeds 10:
229-236 51962.
6. Linser, H. The Physiology and Biochemistry of Herbicides. Ed.
L. J. Audus, Academic Press, New York, 550 pp.,1964.
7. Wilkinson, R. E. Methods in Weed Science. Southern Weed Science
Society, Experiment, Georgia, 198 pp.,1971.
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2.3 FATE OF TOXICANTS IN OR ON PLANTS
The fate of any compound contacting a plant may follow several path-
ways. In some instances this may result in degradation and finally the
material's passing from existence. In other instances the compound may
remain intact and merely recycle in the environment. The alternatives
have been outlined by Norris in a paper presented at the Environmental
and Physiologic Chemodynamics Symposium at Oregon State University in
1969. The following discussion is based on that paper.
The initial point of chemical-plant contact depends on the method
of release. In some instances the foliage and stems may be the primary
intercepting organs. In other instances roots or rhizomes may be the
initial contact.
2.3.1 Aerial Portions of the Plant
Materials intercepted by aerial portions of the plant may under-
go several processes:
2.3.1.1 Absorption. The degree of absorption will dictate the severity
of effects with systemic chemicals. The amount of absorption
depends on the nature of the chemical, the plant species and
the chemical residue characteristics. Absorption must take
place if toxicity to the plant is to occur.
2.3.1.2 Surface adsorption. The extent of adsorption depends on the
physical and chemical properties of both the chemical and
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the leaf surface. Surface adsorption may inactivate
chemicals since it prevents absorption and reduces contact
action. It is important to realize, however, that surface
adsorption is not final; it is an equilibrium reaction.
Environmental factors define the equilibrium between adsorb-
jici and free chemicals and a change in environmental conditions
will alter the point of equilibrium. Any reduction in the
amount of free chemical leads to a release of adsorbed chem-
ical until equilibrium is reestablished.
2.3.1.3 Volatilization. This process is not important for chemicals
with a low vapor pressure or a high heat of vaporization. On
the other hand, losses may be appreciable for pesticide com-
pounds like ethyl ff,tf-dipropylthiolcarbamate (EPIC) or the
isopropyl ester of 2,4-D. Although volatilization reduces
chemical residues on the plant, it adds to the total load of
atmospheric pollutants.
2.3.1.4 Washoff. The amount of chemical not absorbed, adsorbed, de-
graded, or lost through volatilization may be subjected to
washoff. Washoff may carry materials in solution or suspen-
sion depending on their water solubilities. Chemicals washed
to the soil may be leached to the root zone and absorbed by
the plant.
2.3.1.5 Degradation. Degradation of surface residues may reduce
activity by removing the chemical from the site of action.
On the other hand, degradation is the only mechanism which
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can reduce the total load of environmental pollutants.
Absorption, adsorption, volatilization, and washoff only
store or transport hazardous substances to other parts of
the environment.
2.3.2 Underground Portions of the Plant
Chemicals in the root zone are subjected to the same processes
as chemicals intercepted by aerial portions of the plant. How-
ever, the degree to which a particular process operates may be
quite different.
Water-soluble materials are readily absorbed by the roots and
may be transported to other parts of the plant. Surface adsorp-
tion also occurs. Volatilization is relatively unimportant from
root surfaces, but may occur from the soil surface. Washoff does
not occur, but leaching of chemicals from the root zone is an
analogous process. Photochemical degradation does not occur on
roots, but chemical and biological degradation in the root zone
is important.
2.3.3 Behavior of Substances Inside the Plant
The action of chemicals inside the plant depends on absorption
and activity. If significant amounts are absorbed, the substance
may have profound physiologic effects on plant growth. Chemicals
inside plants may undergo several processes:
2.3.3.1 Translocation. Translocation is important because the fate
of chemicals may vary in different plant parts. Materials
absorbed by foliage but not translocated to other plant parts
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may be lost in leaf fall, while those transported to the
roots may be exuded into the soil. Generally, mobility and
water solubility are positively correlated.
2.3.3.2 Storage. The chemical or physical binding of substances to
plant constituents, may occur in any part of the plant.
Largest amounts are frequently found close to the point of
absorption, in storage cells adjacent to the paths of trans-
location, and in areas of intense metabolic activity.
Storage may be active or passive. Active storage is accumulat-
ed against a concentration gradient and requires expenditure
of metabolic energy. Chemicals may be passively adsorbed to
structural components of plants. Both active and passive
storage are reversible, and materials may be released and
translocated to other parts of the plant as conditions in
the plant change.
2.3.3.3. Metabolism. Alteration in chemical structure may result in
detoxication or activation and may occur anywhere in the
plant. Metabolism of most chemicals is nearly always a de-
toxication process for the plant, but the products may be
biologically active in other systems and, therefore, still
important as residues. The phenoxybutyric herbicides are
an exception. They are inactive as herbicides, but their
herbicidally active acetic acid derivatives are produced
through 6-oxidation of the butyric side chain.
2.3.3.4 Exudation. Volatile substances and metabolites may leave
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the plant as vapors through the stomates (pores in the
leaves). Some chemical herbicides like 2,4-D, 2-methoxy-
3,6-dichlorobenzoic acid (dicamba) or 4-amino-3,5,6-
trichloropicolinic acid (picloram) are exuded from the roots.
In contrast with animals, fish, and birds, however, exuda-
tion of chemicals from plants is not extensive.
2.3.4 Conclusions
The chemical characteristics of a substance determines its fate in
all parts of the environment including plants. Chemicals in plants may
be absorbed, stored, metabolized, and/or recycled to the environment.
These processes determine both the substance's impact on the plant and
its residue characteristics.
The behavior of a chemical results from the interaction between the
properties of the compound and the environment. The environment has
many components, and a chemical may interact with any or all of them.
The chemical behavior we observe in a pollutant in nature is an inte-
gration of many single interaction.
Physiologists can accurately measure both the chemical properties
and the environmental factors which interact to produce behavior. The
results of some simple interactions can be predicted. However, the field
of chemodynamics has not yet attained the sophistication necessary to
quantify the multiple interactions which may occur between chemicals and
their environment.
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It will be impossible to derive a single predictive test which in-
cludes the important primary and secondary interactions which produce
chemical behavior and effects-within the plant.
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2.4 CRITERIA FOR SELECTION OF TEST METHODS
Choice of protocols for evaluation of substances for physiologic
activity depends on the following factors:
1. Chemical configuration of compound under study
2. Geographic location of entry site(s) into environment
3. Type of entry
a. Unintentional direct emission - from manufacturing or pro-
cessing facilities
b. Intentional release - for Intended use within natural
environment
c. Indirect release - from various products, endproducts or
components containing the questionable substance
4. Concentration of compound at entry
a. Weight of compound per weight of transport or contact medium
b. Total quantity of release
5. Phase of compound at entry
a. Solid
b. Vapor
c. Liquid
(1) Solution
(2) Suspension
6. Site of plant interception
a. Aerial portion
(1) Foliage
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(2) Support structure
(3) Fruit
b. Underground portion
(1) Roots
(2) Rhizomes, tubers, etc.
Selection of a test procedure can be made on a much sounder basis
if information about the preceding factors is available at the time of
testing. Direct emission of a particulate substance through air control
system of a manufacturing facility near intensive vegetable crop acreages
represents a completely different situation from liquid effluent released
into a waste water stream used for irrigation of forest land.
Knowledge of chemical stability or persistance is important. Will
the material hydrolyze rapidly and degrade after initial entry to the
environment? In this instance the potential for plant damage may be
present only at the time of release and in the vicinity of first contact.
On the other hand a stable molecule may remain active in soils and waters
and even recycle in the plant-to-plant system, affecting future genera-
tions of crops and/or native vegetation or plant communities.
Ideally, a test procedure should evaluate a complete life cycle or
generation, i.e. seed to seed or spore to spore. Unfortunately, for most
materials, this is not possible. Resources are hot available to carry
out such procedures. In the case of woody perennials, the time involved,
especially in community population studies, could span a human lifetime
or more.
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Chemical evaluation and screening procedures have been used for over
twenty-five years with the goal of seeking physiologic activity for re-
finement herbicides and/or plant growth regulators. Much has been learned
I
from this activity within the plant1 science research disciplines. The
procedures may be grouped in the following way:
2.4.1 Scope or Extent of Procedure
1. Seed germination (petri dishes)
a. incubator
b. growth chamber
2. Seedling evaluation
a. incubator
b. growth chamber
3. Growth effects (seedling or transplants)
a. liquid medium
b. solid medium
4. Growth effects (complete life cycle)
a. pots
b. field plots
5. Species shifts
a. synthesized communities
b. natural communities
6. Population shifts
a. synthesized communities
b. natural communities
2.4.2 Facilities for Test Procedures
1. Laboratory
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2. Incubator
3. Growth chamber
4. Greenhouse
5. Field plots
6. Native vegetation areas
2.4.3 Treatment Methods for Test Substance
1. Seed treatment—(soak, dust, etc.)
2. Other germination substrate, e.g. filter paper,
(dip, soak, etc.)
3. Seedling foliage spray or growth medium amendment
4. Soil amendment, pre-plant
5. Soil drench - post plant
6. Soil vapor transfer, (volatilization chamber)
7. Mature plant foliage spray
8. Field soil spray
a. pre-emergence
b. post-emergence
9. Field soil granular incorporation (pre-plant)
10. Exposure to' atmospheric gas or aerosal
11. Field spray or granules (native plant communities)
2.4.4 Evaluation of Factors Influencing Plant Response and Chemical
Activity
1. Chemical treatment procedure
a. Spray or drench
(1) spray droplet size and pressure
(2) chemical dilution and/or dosage
(3) wetting agents
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(4) spray volume per plant or unit of growth
b. Soil amendment
(1) granule or dust particle size
c. Germination substrate, e.g. filter paper
(1) incorporation method
(2) solvent choice
2. Prertreatment plant environment
a. Above ground
(1) temperature
(2) humidity
(3) light intensity
(4) photoperiod
b, Below ground
(1) soil moisture
(2) soil temperature
(3) pH of soil solution
(4) soil texture
(5) organic matter
(6) nutrient element balance
c. Chemical residue persistance and mobility in soil
3. Treatment plant environment
a. Above ground
(1) temperature
(2) humidity
(3) light intensity
(4) photoperiod.
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b. Below ground
(1) soil moisture
(2) soil temperature
(3) pH of soil solution
(4) soil texture
(5) organic matter
(6) nutrient element balance
c. Chemical residue persistance and mobility in soil
4. Post-treatment plant environment
a. Above ground
(1) temperature
(2) humidity
(3) light intensity
(4) photoperiod
b. Below ground
(1) soil moisture
(2) soil temperature
(3) pH of soil solution
(4) soil texture
(5) organic matter
(6) nutrient element balance
c. Chemical residue persistance and mobility in soil
2.4.5 Plant Age at Treatment and Exposure Duration
1. Seed germination
2. Seedling
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3. Mature plant
4. Complete life cycle
5. Complete life cycle in ecosystem-community situation
Evaluation Criteria for Chemical Treatment
(Most tissues and organs have been employed)
1. Size
2. Weight
3. Morphology
4, Crop yield
5. Reproduction
6. Competition and survival in natural communities
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3. RECOMMENDED TEST PROTOCOLS
The difficulty in predicting toxicity from a single or even a
limited number of test procedures has been repeatedly emphasized.
However, any evaluation program must have a beginning. In view of this
situation a sequence of three levels of testing beginning with laboratory-
growth chamber (seed germination-seedling'effects), greenhouse
and extending to open plot has been recommended. These procedures
are not intended to be adhered to rigidly but rather to serve as
framework for further development and procedural modification as
the needs may arise. In addition, a procedure is provided for bacterial
nitrogen-cycle and fungal cellulose decomposition inhibition testing.
These procedures have been derived from the relevant procedures
cited as well as from interviews and the knowledge of the consultants.
In the later stages of testing, special attention should be given to
the listing of references involving environmental effects and inter-
actions, such as soil temperature, pH, moisture, texture, air environment,
and chemical interactions. The need to move to greenhouse and field
testing depends on the anticipated amount of environmental release
of the substance.
3-1
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The fate and effects of many chemicals or their degradation pro-
ducts are of concern because such chemicals enter the soil through the
various manners by which they are used. Many are applied as pesticides
directly to the soil to selectively control soil insects, fungi, nema-
todes or weeds, or, to be taken up from the soil by plant roots and
translocated into their aerial portions to protect them by means of
systemic action from bacteria, fungi, insects and mites, or, to unselec-
tively kill vegetation on industrial sites. Still other pesticides
reach the soil as "fallout" or drift after spray applications to the
aerial parts of plants or find their way into the soil as a result of
the decay of plants that have been treated with them.
Apart from the desired toxic effect of the pesticide on the target
organism there are a number of possible secondary effects that they may
have on non-target organisms. Some of these chemicals, especially those
used as herbicides, may persist in the soil for a long period, thus
damaging or reducing the yield of sensitive crops subsequently grown in
treated soils. The'chemicals may also leach into drainage water, ulti-
mately resulting in a potential danger to man, animals, and plants
through the contamination of potable and irrigational water. Yet
another very important possibility is the direct action of such chemi-
cals on one or more of the components of the complex microbial popula-
tion in the soil. Soil fertility depends on a very delicate equilibrium
3-2
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being maintained in this population. Its disturbance by incident
chemicals might indeed affect fertility adversely.
It is of concern, therefore, to determine whether or not chemicals
in the soil may cause undesirable changes in the microbial populations
or in the metabolism of the microbial community. Such changes could
affect the development and vigor of plants through nutritional distur-
bances, a shift in biological equilibrium, or the appearance of micro-
bial inhibitors for plant growth. Chemicals entering the soil in large
amounts, at frequent intervals, or those that do not detoxify rapidly
could conceivably eliminate or suppress microbial groups with a sub-
sequent reduction in crop yield or quality.
Considerable investigative attention has been directed toward
studying the influence of pesticides on biological process or on the
development of specific microorganisms in the soil. Excellent reviews
by Audus (1, 2), Alexander (3), Martin (4), Domsch (5), Bollen (6), and
Fletcher (7) have covered the subject extensively.
Bioassay tests have been developed to assess the toxicity of
pesticides to microorganisms by observing their effects on the rate of
growth of selected species, by comparing the numbers of soil micro-
organisms capable of growing in media containing various rates of the
chemical, and by noting the rates of changes in specific biochemical
processes in enrichment cultures fortified with various rates of the
pesticide. Respiration, nitrogen mineralization, rate of nitrification,
and cellulose decomposition are the biochemical processes most commonly
considered for this purpose. No one of these techniques is entirely
3-3
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suitable to characterize the influence of the added chemical and some
provide little information of ecological significance. However, an
insight into the possible alterations of the soil microflora can be
obtained by judicious use of several of the procedures.
Herbicides are applied to soil at very low rates (several parts
per million) and at these rates most of them appear not to affect the
dominant microbial groups. Tenfold or higher concentrations of herbi-
cides than those employed for recommended weed control purposes are
usually required before inhibitions are noted. A few of the herbicides
studied, however, have been observed to cause extended depressions of
microbial numbers. Dinoseb, at low rates, can reduce the overall bac-
terial population for as long as three months (8). Dalapon and EPIC,
at normal field rates, reduced total bacterial and actinomycete numbers
for approximately two months, although nitrifying organisms were un-
affected (9). Fungal populations were depressed at normal or just above
normal field rates by dinoseb and pyrazon in calcareous soil (10).
Fitzgerald (11) reported that algae are sensitive to low levels of
monuron.
Insecticides are often applied to or reach the soil as "fallout"
in appreciably higher concentrations than herbicides. However, reason-
ably large quantities are required before significant inhibitory effects
are observed. Partial inhibition of one or another microbial group has
been reported in certain soils with many insecticides, but these changes
are rarely dramatic (12).
Fungicides and soil fumigants have a much greater, effect on micro-
3-4
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bial populations than herbicides or insecticides. In general, fungi-
cides at normal rates augment bacterial numbers, presumably in an in-
direct manner by killing fungi, thus increasing the organic substrates
for bacterial growth, and reducing the level of antibiotic substances
produced by these fungi (2).
Many fungicides are broad spectrum toxicants and can serve as
partial soil sterilants, killing large segments of the saprophytic
population. The microbial response to these fungicides and fumigants
can be divided into four phases (3). In the initial phase, populations
of microorganisms commonly decrease and may remain low for long periods.
Fewer fungi have been found in some soils fumigated with D-D than in
parallel untreated plots even three years after treatment (13). The
second stage, lasting for shorter periods of time of up to eight
months or so, includes the multiplication of a variety of microbial
types which often attain cell densities in excess of those in the
original unamended soil. In the third phase, a new biological equili-
brium frequently is established as a result of the pesticide applica-
tion. The composition of the microflora is less diverse as a result of
treatment and often one, two or more species assume dominance. The
final phase is essentially the climax community characteristic of the
original soil. These oscillations in microbial numbers probably follow
the rise and fall of available nutrients from autolysing organisms that
have been killed by the fungicide or from the added fungicide itself.
Antagonistic effects between specific groups of microorganisms probably
play a role in these oscillations.
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The organisms which decompose the various forms of cellulose are
some of the most important microorganisms contributing to the humifi-
cation processes in soils. The effects of pesticides on these pro-
cesses have been studied mainly by direct observations of changes in
populations of cellulolytic organisms, and also by following the decom-
position of cellulosic substrates buried in treated soils (2). A few
herbicides when applied at normal field rates have shown significant
inhibition of cellulolytic bacteria for relatively short intervals,
e.g., fenuron, atrazine, simazine, and propazine (2). Most of the in-
secticides tested have no effects on the numbers of activities of cellu-
lolytic organisms in soil at normal field rates. Knowledge is frag-
mentary on the effects of fungicides and fumigants on cellulolysis in
soils. Methyl bromide and D-D at normal rates completely suppress
activity for days while ethylene dibromide is only inhibitory at very
high rates (2).
Most herbicides fail to inhibit the rates of respiration, organic
matter turnover, and nitrification in the soil at normal field rates.
Nonsymbiotic nitrogen fixation, nitrogen mineralization, and ammonifica-
tion usually show no response to soil treatment with herbicides. Nitri-
fication is one of the most sensitive conversions in the soil being
inhibited by concentrations of chemicals not inhibiting other important
biochemical reactions. Quantities of herbicides required for a signifi-
cant decline in the rate of nitrate formation are often appreciable,
e.g., 12 ppm CDAA, CDEC, or CIPC, 10 ppm DNEP or 25 ppm of monuron.
In general, insecticides have no effects on the C02 production,
3-6
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cellulose decomposition, ammonification, and nitrification process in
the soil except at relatively high dose rates (14, 15). Nitrification
and nodulation of leguminous plants have been shown to be inhibited by
some insecticides, but the effects are not consistent from soil and
rarely is the toxicity complete (3).
Fungicides and fumigants, when applied at normal field rates, are
the compounds that most often dramatically alter the biological activity
in soil. Nitrification has been found to be inhibited by a number of
fungicides and fumigants. Following treatment, the typical pattern
consists of a strong suppression of the nitrifying microorganisms fol-
lowed by a slow recovery as shown by methyl bromide (recovery several
weeks or more), D-D (recovery 50 days or more), chloropicrin (recovery
50 to 120 days), nabam (recovery 60 days), thiram (recovery 60 days),
ferbam (recovery 28 days), maneb (recovery 25 days), zineb (recovery 17
days), mylon (recovery more than 60 days), and CS2 (recovery over five
months) (2). Temporary suppression of nitrate biosynthesis can be bene-
ficial in as much as the nitrogen is not lost from the plant's rooting
zone by leaching or denitrification; however, ammonium accumulation
arising from the lack of nitrifying activity in treated soils may lead
to serious root injury (3). Also destruction of the nitrifying bac-
teria may be responsible for a reduction in plant growth or crop yield
because of lack of nitrate availability. Morris and Gibbons (16)
reported that nitrate fertilizers were especially beneficial to tobacco
growing in a soil in which the nitrifiers had been killed as a conse-
quence of fumigation.
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Differential effects of the microscopic species in the soil is
part of the general pattern of pesticide action. Toxicity varies with
the individual microbial species, the morphological and physiological
stage of the organism (endospore, conidia, sclerotia, vegetative cells,
hyphae, etc.), the chemical and physical composition of the environ-
ment, and the time of exposure of the organism to the toxicant (e.g.,
the age of the organism) (3).
Kreutzer (17) reviewed many instances of selective toxicity of
chemicals to soil microorganisms. In general, the sensitive species are
reduced in abundance, and their processes retarded. The tolerant
species, and their processes, assume dominance as a result of the
elimination of competing species. Major emphasis has been placed on the
selective effects of pesticides on soil fungi, especially the plant
pathogenic fungi. Alexander (3) states that chemicals can alter the
populations of a plant pathogen in three ways: (1) they may exert a
direct destructive action upon the pathogen; (2) they may have no direct
eradicating influence upon the pathogen, but rather the chemical might
destroy or reduce the number of the pathogen's natural antagonists,
resulting in a rise in the abundance of the pathogen and/or an increase
in the severity of the disease it incites; and (3) they might destroy
the organisms which serve to control the pathogen's antagonists so that
the antagonists become more numerous and suppress the pathogen.
The selection of antagonistic species following the treatment of a
soil with a specific chemical may occur because of a selective destruc-
tion of components of the indigenous population or because of the rapid
3-8
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rate of recolonization of the antagonists following partial soil steri-
lization. Strains of the genus Trichoderma are considered important
antagonists since they suppress Armillaria, Pythium, Rhizoatonia,
Phytophthora, and other soil-borne pathogens (3). Of special signifi-
cance is the fact that T. viride is frequently found to be the dominant
fungus following the application of toxic chemicals to soil (18,19).
Its dominance appears to be due to its high rate of growth as much as
to its tolerance of the chemical (6).
These same selective phenomena could also account for the abundance
of increased activity of soil-borne pathogens following the treatment
of soil with a specific chemical. For example, the incidence of tomato
wilt caused by Fusarium oxysporium f. lycopersici was increased after
the soil was treated with yellow oxide of mercury (20) and PCNB in-
creased damping-off caused by a Pythium species (21). The enhancement
of pathogenic activity could be caused by (1) a decline in the popula-
tions of organisms functioning to check the abundance or activity of the
pathogen while the pathogen remains largely unharmed, or (2) a rapid
reinfestation by the pathogen before its antagonists become sufficiently
well-established.
3-9
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REFERENCES
1. Audus, L. J. Herbicide behavior in the soil. .The Physiology and
Biochemistry of Herbicides. Academic Press, pp. 168-206; 1969.
2. Audus, L. J. The action of herbicides and pesticides on the
microflora. Meded. Rifksfak. Landbouw. Gent. 35: 465-92; 1970.
3. Alexander, M. Microbial degradation and biological effects of
pesticides in soil. Soil biology: reviews of research. Paris,
UNESCO, 209-40; 1969.
4. Martin, J. P. Influence of pesticide residues on soil microbiolo-
gical and chemical properties. Residue Rev. 4: 96-129; 1963.
5. Domsch, K. H. Einflus von Pflanzenschutzmitteln auf die Boden-
mikroflora. Mitt. Biol. Bundesanst. Land-Forstuirtsch. Berlin-
Dahlem 107: 5-53; 1963.
6. Bollen, W. B. Interactions between pesticides and soil micro-
flora. Annu. Rev. Microbiol. 15: 69-92; 1961.
7. Fletcher, W. W. The effects of herbicides on soil microorganisms.
Herbicides and the Soil3 Ed. Woodford, E. K. and G. R. Sager,
Oxford, pp. 1-41; 1960.
8. Gamble, S. J. R., C. J. Meyhew and W. E. Chappel. Respiration
rates and plate counts for determining effects of herbicides on
heterotrophic soil microorganisms. Soil Sci. 74: 347-50; 1952.
9. Rakhimov, A. A. and V. F. Rybina. Effects of some herbicides on
soil microflora. Usbeksk. Biol. Zhur. 7: 74-6; 1963.
10. Mezharaupe, V. A. Effects of Phenazone and Other Herbicides on
Development of Soil Microorganisms. Mikroog. Radt. Trudy. Inst.
Mikrobiol. Akad. Nauk. S.S.S.R., pp. 65-83, 1967.
11. Fitzgerald, G. P. The control of the growth of algae with CMU.
Wise. Acad. Sci.3 Arts and Letters 46: 281-94; 1962.
12. Eno, C. F. Insecticides and the soil. J. Agr. Food Chem. 6: 348-
51; 1958.
13 Martin, J. P., R. C. Baines and J. 0. Ervin. Influence of soil
fumigation for citrus replants on the fungus population of the
soil. Proc. Soil Soi. Soc. Amer. 21: 163-66; 1957.
14. Duda, J. The effect of hexachlorocyclohexane and chlordane on
soil microflora. Acta. Microbiol. Polonica 7: 237-44; 1958.
3-10
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15. Pathak, A. N., H. Shanter and K. S. Awasthi. Effect of some
pesticides on available nutrients and soil microflora. J. Indian
Soo. Soil Soi. 9: 197-200; 1961.
16. Morris, H. D. and J. E. Giddens. Response of several crops to
ammonium and nitrate forms of nitrogen as influenced by soil
fumigation and liming. Agron. J. 55: 372-74; 1963.
17. Kreutzer, W. A. Selective toxicity of chemicals to soil micro-
organisms. ' Annu. Rev. Phytopath. 1" 101-26; 1963.
18. Mofe, W., J. P. Martin and R. C. Baines. Structural effects of
some organic compounds on soil organisms and citrus seedlings
grown in an old citrus soil. J. Agr. Food Chem. 5: 32-6; 1957.
19. Bliss, D. E. The destruction of Armillaria mellea in citrus
soils. Phytopathology 41: 665-83; 1951.
20. Tobolsky, I. and I. Wahl. Effectiveness of various soil treat-
ments for the control of Fusarium wilt on tomatoes in the green-
house. Plant Dis. Reptr. 47: 301-5; 1963.
21. Gibson, A. S. A., M. Ledger and E. Boehm. An anomalous effect of
pentachloronitrobenzene on the incidence of dampening-off caused
by a Pythium species. Phytopathology 51: 531-33; 1961.
3-11
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Recommended and suggested test protocols
are presented in the style issued by the
American Society for Testing and Materials,
3-12
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3.1 PROCEDURE FOR THE EVALUATION OF A CHEMICAL'S EFFECT ON THE SOIL
MICROBE POPULATIONS.
1. SCOPE
1.1 This procedure provides guidelines for determining the effect
of a chemical on the populations of bacteria, fungi, and
act.inomycetes in soil. The number of colonies that appear on
the growth medium provides an estimate of the number of
viable propagules (spores, bacterial cells, and mycelial
fragments) in the sample. All motions of the operator should
be kept as standard as possible to minimize variability from
human error.
2. SIGNIFICANCE
2.1 The equilibrium of the very delicate balance that exists
( between various types of microorganisms in the soil may be
altered by chemicals in a variety of ways. First, there is
the possibility that a chemical may have a toxic effect on a
broad spectrum of microorganisms by inhibiting some essential
metabolic activity, e.g., respiration. Second, the chemical
may be selectively toxic to a certain group of microorganisms.
Specific metabolic effects of this kind may alter population
equilibria in an indirect manner by changing the competitive
efficiency of one group or another. The. third possibility is
that the chemical may promote the growth of one or more types
of soil organisms. The types favored could be either benefi-
cial or harmful to soil fertility.
3-13
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3.1 Standard Laboratory facilities for sterile culture.
3.1.1 Growtl> medium and glassware sterilizer
3.1.2 Glassware washing facilities
3.1.3 Incubators
3.1.4 Standard complement of glassware including: pipettes,
petri dishes, flasks, balances
3.1.5 Medium preparation facilities
3.1.6 Sterile transfer facilities
3.1.7 Culture maintenance refrigerators
3.2 Materials for preparation of various media.
3.2.1 Agar
3.2.2 Mono-basic potassium phosphate
3.2.3 Di-basic potassium phosphate
3.2.4 Magnesium sulfate (MgS04-7H20)
3.2.5 Calcium chloride
3.2.6 Sodium chloride
3.2.7 Ferric chloride
3.2.8 Potassium nitrate
3.2.9 Calcium carbonate
3.2.10 Peptone
3.2.11 Dextrose
3.2.12 Rose bengal
3.2.13 Streptomycin
3.2.14 Asparagine
3.2.15 Mannitol
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3.2.16 Glycerol
3.2.17 Sodium asparaginate
4. PROCEDURES
4.1 Dilution and plate count method.
4.1.1 A quantity of soil is serially diluted through water
blanks and a suitable end dilution is plated on appro-
priate culture media. The number of colonies that
appear provides an estimate of the number of viable
propagules in the sample.
4.2 Growth medium
4.2.1 Thornton's standardized medium for bacterial culturing
4.2.2 Rose bengal agar for culturing fungi
4.2.3 Glycerol asparaginate medium for culturing actinomy-
cetes
4.3 Soil preparation
4.3.1 Soil should be representative of the region(s) of
concern.
4.3.2 Source of the soil is preferably from an agricultural
field .
4.3.3 The characteristics of the soil should be defined as to
soil type, composition of organic matter, sand, silt
and clay, cation exchange capacity, pH, and field
moisture holding capacity .
4.3.4 A composite bulk sample is obtained by collecting a
3-15
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number of random sub-samples, using a clean metal soil
tube or soil auger, from the upper six inches of the
field .
4.3.5 The composite soil mass is mixed thoroughly and then
screened through a 2-mm sieve (U. S. Standard Sieve
No. 10).
4.3.6 The soil should be processed immediately after collec-
tion and employed without delay .
4.3.7 Storage, if necessary, should be in a fresh state (not
air-dried) at approximately 5°C and should not exceed
1 week.
4.4 Treatment of soil
4.4.1 Determine the average moisutre content prior to making
aerial dilutions of the soil on three portions of 10
to 25 g dried for 24 hr at 100°C±5°.
4.4.2 All soil sample weights are subsequently based on
oven-dry weight (o.d.).
4.4.3 Transfer at least 300 g of soil (o.d.) into a 600-ml
beaker or other type of vessel suitable for mixing
purposes .
4.4.4 Distribute evenly on the surface of the soil contained
in separate mixing vessels a uniform but minimum amount
of solution, made with the most appropriate solvent, to
treat the soil with 0, 5, 25, 125, and 625 g of the
test chemical per g of soil (o.d.), respectively.
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4.4.5 Allow the solvent to evaporate from the soil, this may
facilitated through the use of a gentle stream of
filtered air taking precautions into account for vola-
tile characteristics of the test chemical .
4.4.6 Mix the soil thoroughly with a heavy duty glass rod to
insure that the chemical is distributed evenly through-
out the soil sample. Use a standard technique that
will yield mixtures of uniform distribution of the
chemical and no great variability in organic solvent
content .
4.4.7 Transfer equal quantities of the treated sample into
each of three 250-ml Erlenmeyer flasks, properly
labeled as to treatment and replication.
4.4.8 Remove approximately 10 g of the soil sample from each
flask for moisture determination .
4.4.9 Transfer a weighed aliquot of each sample in the range
of 1 g (o.d.) into a 99-ml sterile distilled water
blank, to be used for propagule enumeration at 0-hr.
4.4.10 Add a sufficient amount of sterile distilled water to
the soil in the Erlenmeyer flasks to adjust its mois-
ture content to 75% of field moisture capacity
4.4.11 Close flask with Morton steel closures, plastic foam
or cotton plugs.
4.4.12 Incubate soil samples at 28°C at high relative humidity
4.4.13 Agitate the prepared milk dilution bottles on a a
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mechanical shaker for 30 minutes.
4.4.14 From the original dilution (1:100) make additional
serial dilutions of 1:1,000, 1:10,000, and 1:100,000
using a uniform shaking period of 30 sec. and settling
period of 30 sec. between each subsequent dilution.
4.4.15 Aliquots of 0.1 ml of the desired end dilution is
transferred to each of a minimum of 5 sterile petri
dishes of the appropriate agar medium .
4.4.16 Inoculate the surface of the plates with a flamed glass
rod while the plate is being rotated.
4.4.17 Incubate the plates at 28°C at high relative humidity.
4.4.18 At the end of 2, 4, and 7 days of incubation count the
number of fungal, bacterial, and actinomycetes colo-
nies, with the aid of a magnifier such as the Quebec
colony counter, on the Thronton's, rose bengal, and
glycerol-asparaginate agar media, respectively.
4.4.19 Repeat 4.4.8, 4.4.9, and 4.4.13 through 4.4.18 after
intervals of 7, 14, 28 and 56 days of incubation.
5. DATA REPORTING
5.1 Express the number of progagules per gram of soil (o.d.) and
analyze the results statistically for treatment and sampling
times, factorily accounting for the variations due to repli-
cate vs. treatments, plates x sampling times vs. treatments,
and among plates .
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6. PROCEDURAL MODIFICATION
6.1 The above procedures are suggested with the assumption that
the worker who uses them is familiar with the fundamentals of
microbial physiology and culturing of microorganisms. Aseptic
techniques should be used wherever appropriate to avoid inad-
vertent contamination.
6.2 Variations of the basic procedures are conceivable, however,
shortcuts to reduce the amount of glassware and number of
steps may also reduce precision. Improved precision can be
gained by employing a large sample, ca. 1,000 g but not to
exceed a depth of 1 to 1 1/2 in., and using a soil aliquot
for analysis of 25 g for the first dilution made with a total
volume of 250 ml of 1% carboxymethyl cellulose or 0.2% agar.
(7.4)
7. REFERENCES
7.1 Thornton, J. G. On the development of a standardized agar
medium for counting soil bacteria, with especial regard to the
repression of spreading colonies. Ann. Appl. Biol. 9: 241-
274; 1922.
7.2 Martin, J. P. Use of acid, rose bengal and streptomycin in
the plate count method for estimating soil fungi. Soil Sci.
69: 215-232; 1950.
7.3 Tuite, J. Plant Pathological Methods: Fungi and Bacteria.
Burgess Publ. Co., Minneapolis, p. 35; 1969.
7.4 Curl, E. A. and Rodriguez-Kubana, R. Research Methods in Wood
Science. Ed. R. E. Wilkinson. Creative Printers, Griffin,
Georgia, pp. 161-194; 1972.
3-19
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3.2 EVALUATION OF A CHEMICAL'S POTENTIAL TOXICITY TO THE MAJOR SOIL
NITROGEN CYCLE BACTERIA
1. SCOPE
1.1 This suggested procedure provides guidelines for the evalua-
tion of the potential toxicity of a chemical to the two major
soil nitrification bacterial genera Nitrosomonas and Nitro-
bacter. This procedure is not intended to be a final authorita-
tive and restrictive protocol but rather a guideline for
studies from which further refinement and sophistication
may be developed.
\
2. SIGNIFICANCE
2.1 The conversion of protein and other organic nitrogen compounds
to nitrates for plant utilization , as well as the loss of
nitrogen through nitrate leaching or denitrification, depends
on this bacterial conversion. Substances which exhibit a
high level of toxicity to Nitrobaater and Nitrosomonas
represent a potential threat in many natural plant community
soils as well as crop soils. Where materials exhibit signifi-
cant toxicity at concentrations that would approach those
occurring in soils through environmental release of the
material, further evaluation is indicated. However, in
certain agricultural soils nitrification inhibition is
desirable and is done intentionally through various chemical
compounds marketed for this purpose.
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3. EQUIPMENT AND FACILITIES
3.1 General chemistry laboratory equipment and supplies.
3.1.1 Soil perfusion apparatus i.e. the Audus apparatus (7.2)
3.1.2 Sandy loam soil.
3.1.3 Appropriate reagents for nitrite and nitrate analysis
in liquid solutions.
4. TEST PROCEDURE
4.1 Select source of sandy loam soil which is free from known
chemical use or chemical contamination.
4.1.1 Sieve soil to remove coarse and fine particles. Collect
aggregates of 2-4 mm size and air dry (in laboratory).
4.2 Soil bacterial enrichment.
4.2.1 Using an Audus apparatus, or other appropriate technique,
_3
continuously perfuse soil with 6.2 x 10 M ammonium
sulfate solution (starting concentration) for 20-25 days.
4.2.2 Check nitrification through chemical analysis.
4.2.3 After 25 days, drain soil columns and wash with
distilled water. Perfuse with fresh ammonium sulfate
(starting concentration) until chemical analysis indicates
a constant maximum rate of nitrification.
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4.2.4 Wash soil samples with successive changes of distilled
water and prepare for use.
4.3 Assessment of toxicant effects on nitrification process.
4.3.1 Prepare a series of perfusion columns each containing
50g of bacterially enriched soil (from the same enrich-
ment procedure).
4.3.2 Replicate design treatments appropriately for
statistical analysis of the data.
4.3.3 Perfuse columns at^room temperature with 200 ml of
_3
6.2 x 10 M (NH,)~ SO containing concentrations of
the test chemical ranging from .01. yg/ml to 1000
Ug/ml in a log,., progression.
4.3.4 Various formulation procedures may be needed to keep
the test substance in solution or in suspension. In
these instances appropriate checks should be introduced
to evaluate adjuvant effects.
4.3.5 Conduct perfusion for 10 days; withdrawing 1 ml
samples daily for nitrate and nitrite analysis.
4.3.5.1 Nitrite detection may be performed by the
Lees & Quastel method using the Illosvay
reagent or other suitable technique (7.2).
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4.3.5.2 Nitrate analysis may be done using the
phenoldisulphonic acid method of Lees &
Quatsel or other suitable technique (7.2).
4.3.6 In the absence of a toxicant, nitrite buildup is
normally linear during the 10 day period.
5. PROCEDURAL MODIFICATION
5.1 The general procedures outlined here may be modified as
needed to increase accuracy and effectiveness of the measure-
ment technique. This general procedure is based on Debona
and Audus (7.3).
6. DATA REPORTING
6.1 If the test substance will be introduced into soils (under
manufacturing or use situations) in quantities approaching
0.01 the level that results in a 50 percent reduction in
nitrification after 10 days of perfusion, then further
detailed testing is suggested prior to environmental release.
7. REFERENCES
7.1 Audus, L. J. A new soil perfusion apparatus. Nature. London
158:419; 1946.
7.2 Lees, H. and J. H. Quastel. Biochemistry of soil nitrification.
I.kinetics and effects of poisons on nitrification as studied
by soil perfusion technique. Bioehem. J. 40: 803-815; 1946.
7.3 Debona, A. C. and L. J. Audus. Studies on the effects of her-
bicides on soil nitrification. 'Weeds Res. 10: 250-263; 1970.
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3.3 EVALUATION OF CHEMICAL PHYTOTOXICITY TO SOIL FUNGI OF IMPORTANCE
IN CELLULOSE DECOMPOSITION
1. SCOPE
1.1 Guidelines are given to evaluate the potential toxicity of
a compound to cellulose decomposing fungi in soil. It is
not intended as a definitive or all-inclusive procedure but
it will indicate the level of toxicity exhibited by a material
under laboratory as well as simulated soil conditions.
Testing may be conducted by persons competent in mycological/
microbiological laboratory procedures in a general microbiological
laboratory.
2. SIGNIFICANCE
2.1 Cellulose decomposition is an essential portion of the earth's
carbon cycle. High levels of toxicity exhibited by a material
in this procedure indicates the need for further testing in
those instances where release of the material to soil through
air, water, or direct placement is anticipated.
3. EQUIPMENT AND FACILITIES
3.1 Laboratory facilities for the sterile culture of fungi.
3.1.1 Growth media and glassware sterlizer.
3.1.2 Glassware washing facilities.
3.1.3 Incubators.
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3.1.4 Normal complement of glassware including pipettes,
petri dished, flasks, balances, etc.
3.1.5 Medium preparation facilities.
3.1.6 Sterile transfer facilities.
3.1.7 Culture maintenance refrigerators.
4. TEST ORGANISMS-SOURCE AND IDENTIFICATION
4.1 American Type Culture Collection
4.1.1 A.T.C. #9095 Myrotheeium verrucaria Ditmar ex Fries
maintenance medium, A.T.C. malt extract #325 .
4.1.2 A.T.C. #24687 Trichoderma viride Persoon ex Fries
maintenance medium, A.T.C. potato dextrose agar #336.
4.1.3 A.T.C. #6205 Chaetonrium globosum Kunz ex Fries Main-
tenance medium, A.T.C. mineral salts agar #329 and
filter paper.
4.2 Maintain test species under conditions that will insure
stability and similarity of the isolate over time. This
should prevent changes in response pattern to chemical exposure.
5. TEST PROCEDURE (8.1 and 8.2 with modifications)
5.1 Agar culture "poisoned food" technique.
5.1.1 Growth medium should be malt extract agar (ATC #325)
amended with the test chemical and poured into petri
dishes.
3-25
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5.1.2 Dissolve or suspend test material in sterile water
or appropriate sterile solvent prior to blending.
5.1.3 Pour plate technique should take into account test
material solubility, solvent toxicity, or other
problems associated with achieving a uniform mixture
of test substance with the malt agar.
5.1.4 Agar should be partially cooled prior to toxicant
addition to prevent change in test material. Pour
and cool agar immediately after amendment.
5.1.5 Test material dosage should be calculated on the
basis of chemical wt. to agar volume relationship
i.e. ug/ml of agar. Initial dosages should be on
a log-ip. progression beginning at 0.01 yg/ml and
progressing to 1000 yg/ml.
5.1.6 Seed plates with uniform discs of fungal mycelium
cut from equi-age (same radius) mycelium of colonies
of the fungus grown in malt agar in petri dishes.
5.1.7 Replicate and design treatments appropriately for
statistical analysis of the data.
5.1.8 Incubate "seeded" plates near the optimum temperature
for vegetative growth of the fungi (24 C).
3-26
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5.1.9 Record growth data by measuring the diameter of the
fungal colonies when the growth leaves the original
disc and prior to reaching the outer edge of the
petri plate. Fungal growth is linear with time
across agar. Radial growth may be calculated in the
following manner:
radial growth = colony diameter day Y-colony diameter day X
in mm/day 2 x no. days growth (Y - X)
5.2 Soil amendment technique (8.12 with modification)
5.2.1 Fine sandy loam(pH 7.0-7.5)should be air dried, sieved
through a 20 mesh screen and sterilized fro 45 min.
at 15 psi in an autoclave.
5.2.2 The test material in a volatile solvent or in a finely
divided state should be mixed with the dry sterile
soil and then diluted with soil further so that a
series of soil concentrations of 0.01 yg per ml air
dry soil to 1000 yg per ml soil in a login progression
can be established.
5.2.3 Place the soils containing the test dosages in
sterile 25 ml shell vials (20 mm diam x 85 mm deep)
to a depth of 25 mm.
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5.2.4 Place A 10-mm disc from the rapidly growing margin
of a fungal colony on malt extract agar on the soil
surface. Cover the disc with 25 mm of soil containing
the same concentration of chemical.
5.2.5 Add five (5) ml of sterile distilled water; plug
the vials with cotton and incubate for 24 hours at
24°C.
5.2.6 Empty each vial onto a wire screen. Wash the disc
with sterile distilled water and blot on sterile
paper with towels. Place discs on malt extract agar
to determine viability by visible growth from the disc
after 48-72 hours.
5.2.7 Record data in terms of growth (+) or no growth (-).
Estimate an ED _ for each test chemical.
5.2.8 Experimental design should allow for statistical analysis
of the data.
6. MODIFICATION OF PROCEDURES
6.1 The preceding "poisoned food" and soil amendment procedures
have been widely used by various researchers. Variations
among the procedures are common. Modifications may be made
by utilizing good experimental practice and design.
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7. DATA INTERPRETATION
7.1 If the test substance is introducted into the soil in
quantities equivalent to 0.01 ED . for inactivation of
fungal discs in soil, then further testing of the material
is in order prior to intentional or unintentional soil
introduction. This also applies to the "poisoned food"
agar plate if there is complete growth inhibition at 0.01
yg/ml of agar (or less).
8. REFERENCES
8.1 Bateman, E. The effect of concentration on the toxicity of
chemicals to living organisms USDA Tech. Bull 346 1-53; 1933.
8.2 Brancato, F. P. and N. S. Golding. The diameter of the mold
colony as a reliable measure of growth Myoologia 45: 848-854;
1953.
8.3 Zentmeyer, G. A. A laboratory method for testing soil fung-
icides with Phylopthora cinnamomi as test organism. Phytopath-
ology 45: 398-404; 1955.
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3.4 ADDITIONAL LISTINGS TEST PROCEDURES FOR SOIL MICROFLORA
Soil forms the basis for terrestrial plant ecosystems. The
nutrients released as a result of the microbiological interaction with
inorganic and organic soil constituents are essential for terrestrial
plant growth.
Substances toxic to soil microflora components including nitrifica-
tion bacteria, cellulose decomposing fungi and soil algae may reach
soils through water or air transport mechanisms. The research con-
ducted in this area and the resultant publication of assay methods
has not been directed towards pollution effects. Rather the work
has revolved around pesticide effects on these processes or, in the
case of soil fungi, the procedures have been developed to screen
and evaluate potential chemical compounds toxic to fungi to be used
as fungicides against soil-borne and air-borne plant pathogens. The
procedures listed in most instances have not been used for evaluation
of pollutant effects and thus should be modified and adapted to the
purpose at hand, recognizing that the specific chemical and exposure
route may modify the test procedure.
3.4.1 nitrification
Nitrosomonas and Nitrobacter - major most significant bacteria in
nitrogen cycle.
1. Effect on Nitrosomonas NH.+ N0~
Nitrobaoter NO" NO"
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Soil perfusion to achieve soil saturated with nitrifying
organisms .
Debona, A. C. and L. J. Audus. Studies on the effects of her-
bicides in soil nitrification. Weeds Res. 10: 250-263; 1970,
2. Effect on Nitrosomonas NH.+
Use of specific Nitrosomonas inhibitor in comparison with non-
treated checks and test substances.
Thorneburg, R. P. and J. A. Tweedy. A rapid procedure to eval-
uate the effect of pesticides on nitrification. Weed Soi. 21:
397-399; 1973.
3.4.2 Soil Alqal Growth
1. Green algae Chlamydomonas and Chlorella.
Autotrophic and heterotrophic growth. Liquid culture.
Loeppky, Carol and B. G. Tweedy. Effects of selected herbicides
upon growth of soil algae. Weed Soi. 17: 110-113; 1969.
2. Bluegreen algae Cylindrosporiwn
Green algae Chlorella^ Chloroooooum.
Solid and liquid culture. Autotrophic growth.
Arvik, J. H., D. L. Wilson, and L. C. Darlington. Response
of soil algae to picloram 2,4-D mixtures. Weed Soi. 19:
276-278; 1971.
Above procedures based on Jansen et al.
3. Green algae Chlorella pyrenoidosa
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Chlorella pyrenoidosa.—great deal of research knowledge as
well as important soil alga.
Simple quick method. Semi-solid media. Probably applicable
to bluegreen algae.
Thomas, V. M. Jr., L. J. Buckley, J. D. Sullivan, and Miyoshi
Ikawa. Effect of herbicides on the growth of Chlorella and
Bacillus using the paper disc method. Weed Soi. 21:449-451;1973.
Jansen, L. L., W. A. Centner, and J. L. Hilson. A new method
for evaluation of potential algicides and determination of
algicidal properties of several substituted urea and s-triazine
compounds. Weeds 6: 390-398; 1958.
3.4.3 Soil Fungi Grov.'th
The procedures listed below are suitable for testing the reaction
of a variety of fungi to various chemicals; several of which determine
the volatile toxic action of the test substance. These were
developed for fungitoxicity procedures against plant pathogens; hence,
a modification of the test organisms to soil fungi important in
saprophytic cellulose or organic matter decay or normal soil fungal
constituents. The suggested test fungi should be representatives of
the following genera with the species selection at the option of the
investigator:
*1. Myrotheciiun sp. (Myrothecium verrucariae)
2. Fusarium sp.
3. Aspergillus sp.
*4. Trichoderma sp. (Trichoderma viride)
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. *5. Chaetomium sp. (Chaetomium globoswn)
1. Simulates soil incorporation of pollutant. Measures diffusi-
bility.
Munnecke, D. E. A biological assay of non-volatile diffusible
fungicides in soil. Phytopathology 48: 61-63; 1958.
2. Measures kill of fungi and complete eradication of organisms
from soil.
Corden, M. E. and R. A. Young. Evaluation of eradicant soil
fungicides in the laboratory. Phytopathology 52: 503-509;
1962.
3. Can be used with above designated fungi as well as the
Phytophthora plant pathogen. A very comprehensive procedure.
Zentmeyer, G. A. A laboratory method for testing soil fungi-
cides with Phylopthora cinrcononi as test organism. Phytopath-
ology 45: 398-404; 1955.
4. Requires diffusibility. Simple, easy to do.
Thornberry, H. H. A paper-disk plate method for the quantita-
tive evaluation of fungicides and bacteriacides. Phytopath-
ology 40: 419-429; 1949.
*Myrotheaium3 Trichoderma, & CTiaetomiwm-important genera in cellulose
decomposition in soil.
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5. Measures fungal spore toxicity.
Chinn, S. H. F. and R. J. Ledingham. A laboratory method for
testing the fungicidal effect of chemicals on fungal spores in
soil. Phytopathology 52: 1041-1044; 1962.
6. Separates the fungistatic from fungicidal toxicants.
Neely, D. and E. B. Himelick. Simultaneous determination of
fungistatic and fungicidal properties of chemicals. Phytopath-
ology 56: 203-209; 1965.
7. These two procedures allow measurement of the toxicity of
materials by volatile vapor phase action.
Richardson, L. T. and D. E. Munnecke. A bioassay for volatile
toxicants from fungicides in soil. Phytopathology 54: 836-839;
1964.
Latham, A. J. and M. B. Linn. An evaluation of certain fungi-
cides for volatility, toxicity and specificity using a double
petri dish diffusion chamber. Plant Dist. Peptr. 49: 398-400;
1965.
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3.5 PRELIMINARY EVALUATION OF TOXICITY TO TERRESTRIAL PLANTS THROUGH
LABORATORY SEED GERMINATION AND SEEDLING GROWTH TESTING
1. SCOPE
1.1 These procedures will allow evaluation of toxicity of chemical
configurations which are inhibitory to seed germination or
seedling growth in the first three weeks after emergence. The
procedures are not suitable for measurement of long term growth
effects or for viewing maturational or reproductive effects.
2. SIGNIFICANCE
2.1 Many plant species are most sensitive to toxicants in the seed
germination or seedling stage. Seeds or seedlings occupy a
minimum of space and do not create the logistic problems of
full scale field experiments. High levels of toxicities in
seed or seedling tests strongly suggest the need for further
greenhouse or field tests.
3. EQUIPMENT AND FACILITIES
3.1 Glassware and supplies associated with general horticultural
and plant research laboratory.
3.2 Seed germinator or dark cabinet capable of maintaining a
reasonably uniform temperature in the 21-27° C. range.
3.3 Plant growth facility-alternatives.
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3.3.1 Plant growth chambers. Many types of controlled environ-
. ment systems for raising plants are available commer-
cially and their use in research is increasing. The
systems vary considerably in size and capability, rang-
ing from simple lighted cabinets to those providing
.rigorous control of several environmental parameters.
Growth chambers are used primarily to (1) obtain uni-
form plant material, (2) permit selection of environ-
mental conditions appropriate for a given plant species
without regard to season, and (3) permit environmental
manipulation as an experimental variable. If more than
one type of growth chamber is available, the selection
of the most appropriate one will depend on its intended
use.
In general, all growth chambers contain mechanical,
electrical, and perhaps electronic components and con-
trols, all of which are subject to breakdown and require
maintenance. The more sophisticated chambers generally
provide greater flexibility of operation but often re-
quire more frequent or more complicated maintenance.
Usually the simplest chamber that will meet the re-
quired environmental conditions is the best and often
the least expensive. However, satisfactory control of
a few major parameters will result in uniform and re-
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producible plant growth.
Temperature control is usually achieved through ther-
mostatically controlled refrigeration and heating
systems. It is useful to have two thermostats, one
to control day temperature and the other for night
temperature, with a timeclock to switch from one to
the other. Switching need not coincide with the light-
dark cycle. Temperature control of i 2.0° C. is
adequate for many purposes. Continuous temperature
programming is provided in some growth chambers, but
it is not needed for most experimental work. The degree
of temperature control provided in an ordinary labora-
tory or workroom may be adequate for many purposes, and
the on-off cycling of adequately ventilated banks of
lights will provide some day-night temperature differ-
ential.
The lighting for growth chambers is commonly provided
by a combination of fluorescent and incandescent illum-
ination, banks of which commonly occupy the entire ceil-
ing. The quality of the light from artificial lamps is
not equal to sunlight. However, a satisfactory light
quality may be achieved by using approximately 4 watts
of cool-white fluorescent illumination per watt of in-
candescent illumination. The incandescent lamps need
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not be larger than 60 watts, although 100-watt lamps
are sometimes used.
Control of day length is an essential part of environ-
mental regulation. Timeclocks that can be set to
turn the lights on or off at any 15-minute interval
are satisfactory for most purposes. Regulation of light
intensity is most commonly achieved by a combination of
varying the distance between the plant bed and the light
bank and by controlling the number of lamps lighted at
any given time. Most growth chambers contain a mechan-
ism for adjusting the plant bed height and several time-
clocks, each controlling a part of the lights. Although
illumination as low as 400 ft-c may be desirable
for some purposes, most plants will grow satisfactorily
in a white-walled chamber under a measured illumination
of 1000 to 2500 ft-c.
Many growth chambers can provide illumination consider-
ably in excess of 2500 ft-c; however, the literature
reveals that 1600 to 2000 ft-c is usually sufficient
for vigorous growth. Achieving maximum growth rates
may require higher illumination.
The lamp bank and associated electrical ballasts gen-
erate a considerable amount of heat and this must be
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dissipated if temperature control is to be acheived.
One of the better growth chambers has the ballasts in
a compartment insulated from the plant growth space and
a transparent barrier isolating the lamp bank.
Some chambers have a humidity sensing apparatus, which
controls the operation of a refrigeration coil to trap
unwanted water vapor, and a steam generator, wet pad,
or aerosol generator to increase moisture in the air.
In closed growth chambers without humidity control,
precautions must be taken to insure adequate ventilation
and thereby prevent the excessive moisture buildup that
results from an enclosed space. Increased humidity may
promote mildew development. If plants are grown under
a light bank in a laboratory or workroom, humidity con-
trol is not practical and normal watering of the plants
should be sufficient. A fresh-air change every 2 hours
in the chamber is desirable to prevent excessive carbon
dioxide buildup at night and a depletion during the day.
3.3.2 Laboratory tables or benches may be used with supple-
mental lighting by fluorescent plant growth tubes to
1600 ft-c on time clock control, and room temperature
range from 21-27° C. These may be used in place of
growth chambers.
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3.4 Balances, scales, rules, and other plant measurement equipment.
4. TEST PROCEDURES
4.1 Test plant species.
Monocotyledons
Oats—Avena sativa L. 'Cllntford1
Ryegrass—Loliwn perenne L. 'Manhattan'
Corn—lea mays L. 'Butter and sugar1
Dicotyledons
Cucumber—Cucwnis sativus L. 'Marketer'
Bean—Phaseolus vulgaris L. 'Pinto'
Tomato—Lycopersioon esculentum Mill. 'Rutgers'
4.1.1 All six species should be tested with all procedures.
4.2 Seed germination.
4.2.1 Test materials should be prepared as solutions, sus-
pensions, or emulsions in distilled water in concen-
trations ranging from 0.01 yg/ml to 1000 yg/ml in Iog1f)
progression including the control.
4.2.2 Pour 100 ml of the solution in a 250-ml Erlenmeyer
flask containing 50 seeds. Place on a shaker for 24
hours at room temperature.
4.2.3 Remove seeds and wash in distilled water over a screen
or Buchner funnel.
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4.2.4 Place the washed seed on 3 layers of clean moist filter
paper in a large petri dish (approx. 200 mm x 20 mm)
and maintain in the dark in an incubator or germinator
at 21-27° C. for 10 days. Determine the percent ger-
mination at the end of 5 and 10 days. A seed with any
degree of sprouting is considered germinated.
4.2.5 The treatments should be replicated and the experimental
design suitable for statistical analysis of data ob-
tained. Appropriate controls and blanks should be in-
cluded where emulsifiers or solvents were used to ob-
tain solution or suspension of the material.
4.2.6 For each substance and test species an ED<-n should be
estimated. In this case the ED n is that dosage in
yg/ml solution which results in inhibition of 50 percent
of the germinable (viable) seeds in the test population
when compared with the germination percentage follow-
ing distilled water treatment.
4.3 Seedling growth effects.
4.3.1 This method consists of transplanting seedlings into a
vermiculite medium previously amended with a test sub-
stance and then measuring growth effects after a 3-week
period or transplanted seedling foliage may be sprayed
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with the test substance in aqueous solution and growth
effects measured after a 3-week period.
4.3.2 Wet two paper towels and place on waxed paper. Fold
back 5 cm. of the upper paper and place seed on the
second paper along the fold. Fold back upper paper
over seed. Loosely roll absorbent paper towels inside
the waxed paper. Place in a beaker with 5 cm. of water
and incubate 4-5 days at the desired temperature, in
the dark. Remove seedlings from the incubator and
expose to room light (100-200 ft-c) for a day. Trans-
plant hardened seedlings and place in an environment
with required illumination.
Seed germination by the "paper roll" method may require
more time and care than direct seeding in flats. How-
ever, distinct advantages make this method worthwhile.
Seedlings germinated by this method may be selected for
uniformity of root and shoot development and may be
transferred to vermiculite growth medium with minimum
root damage and without particulate matter adhering to
them.
4.3.3 Culture techniques.
4.3.3.1 Containers for the brief 3 week period utilized
this evaluation should be disposable. Plants may
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Magnesium sulfate MgSO ' 7H 0 246.5
Iron solution- The iron chelates of ethylenediaminetetraacetic acid
(EDTA) are commercially available, often as Versene or Sequestrene.
Dilute the commercial product to obtain a 5 percent w/v solution.
Dilute 200 ml of this solution in distilled water and make to 1
liter. This stock solution contains 10,000 ppm of iron chelate.
One ml of stock solution will give an iron concentration of 1 ppm
when diluted to 1 liter.
Micronutrients- The microelements or tract elements are dissolved
together in 1 liter of deionized water to make a stock solution.
The microelements should be added to the water in the order listed
and each dissolved before the next is added to avoid precipitation.
Chemical Formula Grams per liter
Boric acid H3B°3 2-5°
Zinc chloride ZnCl 0.50
Cuprous chloride CuCl -H 0
0.05
Molybdenum oxide MoO 0.05
Manganese chloride MnCl '4H 0 0.50
Preparation of nutrient solution from stock solutions-The follow-
ing volumes of the six stock solutions are then individually added
to about 500 ml of deionized water with stirring. After all stock
solutions have been added, the nutrient solution is made to 1 liter
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with deionized water to obtain full-strength solution. The solu
tion is usually diluted to one-third, one-half, or three-fourths
strength.
Chemical Formula Milliliters
Potassium dihydrogen
phosphate. . .. .......
.. ....... ,
Potassium nitrate ...... KNO 5
Calcium nitrate ....... Ca(NO ) 5
Magnesium sulfate ...... MgSO. 2
Iron ...................... 1
Micronutrients ................. 1
4.4 Experimental design-The test procedures should be planned in
such a manner that replications, pot arrangements, experimental
and control organisms are suitable for appropriate statistical
analysis. Special precautions in the design should allow
measurement of the independent effects of chemical adjuvants,
i. e. solvents, emulsifiers, etc. that are used in making the
test substance suitable for soil amendment, spraying, or
seed treatment.
5. PROCEDURAL MODIFICATIONS
5.1 The general procedures outlined here may be modified as needed
to increase accuracy and effectiveness of the evaluation
technique. The general plant cultural procedures recommended
are taken from the report cited in reference No. 8.1
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6. DATA REPORTING
6.1 Seed germination
6.1.1 Estimation of the ED n for seed germination inhibition
should be made for 5 and 10 days.
6.2 Seedling growth — soil treatment and foliar spray
Estimation of the LD n dosage which results in the complete
death of 50 percent of the test plants through root or foliage
exposure should be made for each exposure method. In add-
ition detailed records should be kept of plant heights, dry
and fresh weights of top growth, and morphologic growth changes
at the end of the 3-week period.
7. DATA INTERPRETATION
7.1 The germination and plant growth data should be reviewed against
the anticipated dosages and concentrations that are likely to
occur through environmental release of the test substance. If
the environmental accumulation will be 0.01 times that con-
centration resulting in significant seed germination or .
seedling growth effects in the test procedures, then further
greenhouse and field testing is suggested.
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8. REFERENCES
8.1 Anonymous. Growing plants without soil for experimental use.
U.S.D.A. Miscellaneous Pub. No. 1251. Superintendent of doc-
uments, U.S. Govt. Printing Office. Washington, D. C. 17 p. 1972.
8.2 Ashton, F. M., Elizabeth G. Cutter, and Donna Huffstutter.
Growth and structural modifications of oats induced by Bromacil.
Weed Res. 9: 198-204; 1969.
8.3 Chang. In-kook, and C. L. Foy. Effect of Picloram on germina-
tion and seedling development of four species. Weed Soi.19:
58-64; 1971.
8.4 Cutter, Elizabeth, F. M. Ashton and Donna Huffstutter., The
effects of bensulide on the growth, morphology, and anatomy
of oat roots. Weed Res. 8: 346-352; 1968.
8.5 Centner, W. A., A technique to assay herbicide translocation
and its effect on root growth. Weed Soi. 18: 715-716; 1970.
8.6 Cowing, D. P., A method of comparing herbicides and assessing
herbicide mixtures at the screening level. Weeds 7: 66-76;
1959.
8.7 Horowitz, M. and Nira Hulin., A rapid bioassay for diphenamid
and its application in soil studies. Weed Res. 11: 143-149;
1971.
8.8 Horowitz, M., A rapid bioassay for PEBC and its application
in volatilization and adsorption studies. Weed Res. 6: 22-36;
1966.
8.9 Kratky, B. A. and G. F. Warren., The use of three simple rapid
bio-assays on forty-two herbicides. Weed Res. 11: 257-262;
1971.
8.10 Leasure, J. K. Bioassay methods for 4 amino-3,5,6-trichloro-
picolinic acid. Weed Soi. 12: 232-234; 1964.
8.11 Parker, C., A rapid bio-assay method for the detection of her-
bicides which inhibit photosynthesis. Weed Res. 5: 181-184;
1965.
8.12 Santelmann, P. W., J. B. Weber, and A. F. Wiese., A study of
soil bioassay technique using prometryne. Weed Soi. 19: 170-
174 ; 1970.
8.13 Sund, K. A. and N. Nomura., Laboratory evaluation of several
herbicides. Weed Res. 3: 35-43 ; 1963.
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3.6 GREENHOUSE EVALUATION OF TOXICITY TO TERRESTRIAL PLANTS THROUGH
FOLIAR SPRAY AND SOIL AMENDMENT TESTING
1. SCOPE
1.1 These procedures represent a continuing expansion of the initial
growth chamber laboratory bench seedling procedures. Green-
house plant cultures can be adjusted with appropriate container
sizes and plant species to examine the effects of a test sub-
stance on the complete growth cycle of a plant from seedling
to mature plant and may in some circumstances include flower-
ing and fruit production. In addition, interactions between
potentially antagonistic or synergistic toxic substances may
be examined.
2. SIGNIFICANCE
2.1 In some instances greenhouse testing allows interpretation of
results as being representative of anticipated field results.
The cost of greenhouse testing may be less than growth chambers.
However, the greenhouse environment differs from the outdoors
in light quality and temperatures. Therefore, greenhouse
testing should not be considered a substitute for in situ field
testing under actual toxicant release conditions. However,
i
a long term greenhouse test provides a greater in depth evalu-
ation of the toxicity hazard than a short term laboratory
bench or growth chamber tests.
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3. EQUIPMENT
3.1 Greenhouse with temperature and light control, headhouse facil-
ities for potting, transplanting, etc.
3.1.1 Greenhouses
3.2 Growth media blending and mixing equipment
3.3 Pot or flat spraying equipment for uniform chemical applica-
tions.
3.4 Scales, balances, and plant measurement supplies.
3.5 Soil nutrient supplies.
4. TEST PROCEDURES-PLANT CULTURE AND PRODUCTION (n.l)
4.1 Greenhouse selection and maintenance. Many types of green-
houses are available commercially, and vary considerably in
size, shape and construction materials. The covering may be
glass or translucent plastic. Glass is more transparent, but
it is susceptible to breakage and increases heat retention.
Regardless of the type of greenhouse available, certain
features are necessary to insure its usefulness for cultivating
plants throughout the year. Provisions must be made for heat-
ting and supplemental lighting during winter. A reduction in
temperature can be provided by a combination of shading, cool-
ing, and ventilation.
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Shading of greenhouses in the summer is essential to prevent
excessive heat buildup. This may be accomplished either by
painting the houses with a lime-linseed oil shading compound
or by using a shade screen. The shading compound will weather
during the summer, often providing inadequate shading during
the. last part of the season. Eventually it will etch the
glass and reduce the transparency of the greenhouse during the
winter.
Temperature may be controlled by using ridge or side vents.
These are often used in conjunction with forced-air evapor-
ative coolers, which bring air into the greenhouse. A high
degree of summer temperature control can be achieved if both
the evaporative coolers and motorized vents are thermostat-
ically activated. The covering of vent openings with a fine
screen will exclude birds and most insects, thereby reducing
plant damage and the frequency of fumigation. Without cool-
ing, the temperature in a greenhouse during the summer may
range between 20-45° C.
Supplementary light controlled by timeclocks is best supplied
by fluorescent lamps without reflectors. These provide minimum
shading of plant benches when the sun is shining and can be
left in place permanently. Four 244-cm fluorescent lamps over
each 122 x 244 cm table will supply from 300 to 600 foot-
candles (ft-c) of illumination, which controls day length and
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is sufficient to maintain many species above the compensation
point during the winter. These lamps have an average life of
approximately 7500 hours and can be used in the greenhouse
for at least two winters.
With an overcast sky during the day, the natural illumination
in the greenhouse may be 900 to 1100 ft-c, and on a bright
day it may reach 5000 to 7000 ft-c.
The ideal situation is to cultivate each species under its
specific optimum conditions. Because of limitations in cost,
space, and time, it is often necessary to grow several species
in the same greenhouse section or growth chamber. Therefore,
compromises may have to be planned. When two species are
grown together, neither will respond optimally to compromise
of growth conditions. Every attempt should be made to grow
plants together that have similar growth requirements.
Under varying environments the water and nutritional require-
ments of plants will differ. During the summer it may be nec-
essary to water the plants twice daily, whereas once a day may
be satisfactory in the winter. The same is true when nutrient
solution is applied two or three times a week, alternating
with water only. If the growth rate of the plants increases,
additional nutrients are necessary. As plants approach ma-
turity and growth rate decreases, their nutrient requirements
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are reduced. For certain species of plants, the flowering
stage may be delayed or hastened according to the photoperiod
they receive.
4.2 Cultural conditions. Illumination and quality of light, photo-
period, temperature, relative humidity, and nutrition all In-
teract to affect plant growth. Less than optimum conditions
can result in poor plant growth.
4.2.1 Light. The illumination, light quality, and photoperiod
requirements vary among species. A foot-candle meter
is often used to measure illumination. Although foot-
candle meters can be obtained, photographic light meters
are also practical for light measurement. Photographic
light-meter readings can be converted into foot-candles
by the following formula:
B = 20 (f)2
TS
Where
B = illumination in foot-candles
f = aperture in f stop
T = shutter speed in seconds
S = film speed in ASA units
To measure illumination with a photographic light meter,
reflected rather than incident light must be measured.
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To do this, place a large sheet of white paper on the
surface to be measured, set an appropriate ASA film
speed on the meter, and read the shutter speed re-
quired for proper exposure at a given f stop.
Ft-c values for meter settings may be obtained by sol-
ving the equation. The results will be approximate
depending on the accuracy of the meter and the cone of
light it accepts. For example, false low readings may
result if the meter accepts light from an area greater
than that of the white paper at which it is directed.
Nevertheless the readings can suffice to determine
whether illumination is adequate for good plant growth.
About 1200 ft-c of light in a growth chamber is satis-
factory for many plants such as cabbage, carrots, peas,
and tobacco, whereas other plants such as corn, cotton,
rice and sorghum grow better when supplied with 1600
to 1800 ft-c.
Different plant species vary in their photoperiod re-
quirements. For example, barley requires a photoperiod
greater than 12 hours for good flowering, whereas soy-
beans can mature under a 12-hour day.
During the winter a 12-hour photoperiod is generally
used as a compromise day length under which reasonable
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vegetative growth can be maintained. The short natural
photoperiod in the winter is supplemented with fluor-
escent light.
4.2.2 Temperature. During the summer it is often difficult
to maintain sufficiently cool temperatures in the green-
house for certain species of plants. For example, >
head lettuce and peas will often grow poorly in mid-
summer. It is not recommended to grow these species
until conditions are more favorable.
4.2.3 Relative humidity. An average of 50-percent relative
humidity is satisfactory for many species. A relative
humidity of 100 percent is often needed to germinate
very small seeds such as those of tobacco, bluegrass
or root sugarcane stem sections. The required humidity
can be obtained by covering the plant containers with
plastic bags.
4.2.4 Growth media. Vermiculite of a medium fine texture is
readily obtained from local dealers. Local tapwater
may be too contaminated, saline, or alkaline for use
in nutrient culture. It may be deionized by passage
through a commercial mixed-bed deionizer.
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4.2.5 Nutrition. Environmental conditions affect the amount
and rate of nutrient uptake by individual plants. If
many different plants are to be grown, considerable
variation in nutrient requirements can be expected. By
careful observation, early deficiency symptoms can be
diagnosed and readily cured. If fairly exact nutrient
requirements are not known for a given species, it is
best to use a standard solution, taking care not to
overfertilize, for it is easier to correct a nutrient
deficiency than an excess. Many plants seem to tolerate
wide variations in nutrient supply. It is desirable to
supply nutrients at the optimum level.
4.3 Seed germination. A temperature-controlled incubator may be
used for seed germination. Most seeds are germinated in the
. dark, although some require light. If a dark incubator is not
available, any dark cabinet in which the temperature remains
fairly constant (21-27° C) can be used. Reasonably uniform
and reproducible seed germination will be obtained.
4.3.1 Method 1. Fill a small flat, 30 x 20 x 10 cm,
with 5 cm of vermiculite. Place a seed on the sur-
face and cover with 1.3 cm of vermiculite for alfalfa
seeds and 2,5 cm for pea seeds. Very small seeds,
i.e-itobacco, are mixed with sand to increase the volume
for better distribution. These seeds are not covered
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after seeding. The vermiculite is wetted from the bot-
tom. An enamel tray is placed under the flat to main-
tain the moisture level. The flat with tray is placed
in an incubator at the desired temperature for the
required length of time. If the seedlings are kept in
flat for an extended period of time, a dilute nutrient
solution can be used in place of water. Metal flats
should be of stainless steel, or if galvanized they
should be coated with an asphaltic material to prevent
toxic levels of zinc from leaching into the germinating
med ium.
This method is generally the easiest and most success-
ful for many types of seeds. The advantages of using
vermiculite for germinating seed are as follows: (1)
adequate moisture-holding capacity and aeration, (2)
lack of toxic materials and nutrients, (3) root dev-
elopment encouraged (4) light weight and easy to handle.
Although peat may be mixed with vermiculite for some
purposes, its high absorptivity may interfere with
subsequent"chemical treatments applied to the roots.
4.3.2 Method 2. Wet two paper towels and place on waxed
paper. Fold back 5 cm. of the upper paper and place
seed on the second paper along the fold. Fold back
upper paper over seed. Loosely roll absorbent paper
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towels inside the waxed paper. Place in a beaker with
5 cm of water and incubate 4r5 days at.the desired
temperature, in the dark. Remove seedlings from the
incubator and expose to room light (100-200 ft-c) for
a day. Transplant .hardened seedlings and place in an
environment with required illumination.
Seed germination by the "paper roll" method may require .•... i
more time and .care than direct seeding in flats. How-
ever, distinct advantages, make the method worthwhile.
Seedlings germinated., by the paper roll may be selected
for uniformity of..root and shoot development and may
be transferred to nutrient or other solutions with
minimum root damage and without particulate matter
adhering to them..
4.4 Culture techniques
4.4.1 Containers. The plant container must be of sufficient
size to permit adequate root growth. For example, a
pea plant can be grown to maturity in a 10-cm plastic
pot filled with vermiculite or in a 500 ml jar filled
with nutrient solution. A corn plant grown in a 10-cm
pot or 500-ml jar becomes rootbound after 5 to 6 weeks
i
of growth and abnormalities will occur. Corn plants
require an 18 cm pot or 2 liter jar to grow to maturity.
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For this reason various types and sizes of containers
are used, such as jars, stainless-steel troughs with
lids, and plastic pots with saucers. Milk cartons can
also be used as disposable containers. They will often
last for 6 to 8 weeks. Glass jars are covered with a
coat of black paint followed by a coat of aluminum
paint. The black paint excludes light, inhibits growth
of algae in the nutrient solution, and prevents abnormal
root pigmentation. Since the aluminum paint reflects
sunlight, the jars remain cool.
When many plants are to be grown together in the same
container in nutrient solution, use a stainless-steel
trough, 66 x 16 x 10 cm, with a stainless-steel lid.
The plants are placed in holes in the lid, which serves
as a support and keeps the light out. Soft plastic
collars may be used for additional stem support. The
size of the holes should be adequate to prevent stem
girdling. Alternatively the flat steel trough lids
may be replaced by wood or plastic frames with nylon
screen bottoms. These frames are supported within the
troughs, above the bottom. The screen is filled with
vermlculite. The plants can be grown directly from
seed or transplanted. Nutrient solution is added to
wet the vermiculite. The plant roots grow through the
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screen and into the nutrient solution. Many plants
grow well in this manner. The screen gives good root
support, and most of the roots can be harvested.
Plexiglass framing is preferred to wood to avoid leach-
ing of chemicals from the frame. If the experiment
requires root treatment by the addition of a chemical
to the nutrient solution, a wooden frame may become
contaminated and should be discarded.
If plants are grown in jars, then jar lids, tinfoil,
waxed cork, Masonite or other materials can be used for
plant support. Many people have found paper cups most
satisfactory for support. They can be used in various
ways depending on the species of plant to be grown.
For example, when pea plants are grown, two cups are
glued together on the bottom. The lower cup slips
over the outside of the jar; the upper cup is used for
plant support. One small hole is punched through the
bottom of each cup for the seedling and another small
hole through the lower cup for the aeration tube.
The rationale for this technique is: (1) the cups are
chemically unreactive, (2) they may be discarded after
use, (3) sharp cutting edges are eliminated, (4) gird-
ling can be prevented by gradually enlarging the hole
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as the plant stem increases in diameter, (5) cups can'
be easily lifted so that nutrient solution can be ad-
ded, (6) plants can be easily transferred to other
jars, (7) there is more stem support for certain plants
than with many other types of lids.
4.4.2 Aeration. When plants are grown directly in a liquid
culture, air must be supplied to the nutrient solution.
A small electric pump ,i.e., a fish tank aerator, can
force air through rubber or plastic tubing to the jar
or troughs. Glass capillary tubing is inserted in the
solution. A well-regulated uniform flow rate can be
obtained for several aerators on the same line by in-
serting a 2.5-cm length of 0.25-mm bore capillary tub-
ing into the air line for each aerator tube. Aerating
can be done continuously or at regular intervals, (two
hours on, two hours off) by using a timeclock.
If compressed air is available, this may be more
economical than using many small pumps, which require
frequent maintenance. Oil vapors from a rotary-type
compressor must be trapped out with a charcoal filter
before the air reaches the plants.
4.4.3 Subirrigation. Plants grown in vermiculite-filled pots
or flats should receive water and nutrient solution by
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subirrigation. Saucers or shallow pans are used under
the pots and enamel trays under the flats. The amount
of water or nutrient solution added is dependent on the
plant and growing conditions. It is not as easy to
overwater plants grown in vermiculite as it is in soil.
Plants that require well-aerated media can become water-
logged if the vermiculite is watered excessively. Care
must be taken to avoid this. When in doubt, consult
those who have grown these plants, or refer to liter-
ature on the specific plant.
Some salt accumulation may occur on the surface of the
vermiculite after a period of time, and this should be
leached out once a month. This is done by applying
excessive water at the top and letting it drain through
the container. Subirrigation may then be resumed on the
usual schedule. Salts are leached away very rapidly
from vermiculite by this method. The pots and flats are
watered from the top only at seeding time. When very
small seeds are placed in flats and left uncovered, sub-
irrigation is used. If they are watered from the top,
a fine spray must be used to prevent the seed from being
carried too deep into the vermiculite to emerge.
The following methods can be used to apply nutrients to
plants: (1) mix the nutrients directly into the water-
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ing system (2) add nutrient solution two or three times
per week and water at other times (3) supply a nutrient
solution every day without mixing it in the watering
system; the concentration can then be varied as needed.
When using method 2, tapwater can often be substituted
for distilled or deionized water if the plants are not
going to be used for a critical experiment in which
nutrition should be carefully controlled. The use of
tapwater several times per week reduces the volume of
deionized water required. However, it must be employed
with caution, since some domestic water supplies may
contain levels of salts that will damage plants. Soft
water is not necessarily low in salt content since the
softening process exchanges highly soluble salts for
less soluble ones.
The acidity or alkalinity of the water used may also
affect plant growth adversely. Some injurious pH
effects result from changes in the availability of
nutrient salts. For example, excessive watering of
corn with alkaline (pH9) tapwater can cause the leaf
margins to become chlorotic and torn. These symptoms
are reminiscent of certain nutrient deficiencies. The
addition of dilute acids, bases, or buffering salts to
the tapwater or nutrient solution can help overcome
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these difficulties. Care ;must be taken, however, to
insure that neither the nutrient .solution nor the tap-
water used between additions contains too much salt.
It is necessary to experiment in order to determine the
optimum amounts of nutrient for individual species .
4.4.4 Nutrient solution. The following modified formula for
Hoagland and Arnon should be used. Preparation is as
follows:
Major elements. — Individual 1 M stock solutions of
each major element are made with deioriized water.
Chemical . Formula Grams per liter
Potassium dihydrogen
phosphate .......... KH2P04 131.1
Potassium nitrate ...... KNO~ 101.1
Calcium nitrate ....... Ca (NO ) . 4H 0 236.2
Magnesium sulfate ...... MgSOyO ' 246.5
Iron solution. The iron chelates of ethylenediaminetetraacetic
acid (EDTA) are commercially available, often as Versene or Seques
trene. Dilute the commercial product to obtain a 5 percent w/v
solution. Dilute 200 ml of this solution in distilled water and
make to 1 liter. This stock solution contains 10,000 ppm of iron
chelate. One ml of stock solution will give an iron concentration
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of 1 ppm, when diluted to 1 liter
Micronutrients. The microelements or trace elements are dissolved
together in 1 liter of deionized water to make a stock solution.
The microelements should be added to the water in the order listed
and each dissolved before the next is added. This prevents pre-
cipitates from forming.
Chemical Formula Grams per liter
Boric acid H3B°3 ?'50
Zinc chloride ZnCl 0.50
Cuprous chloride CuCl.-H.O 0.05
Molybdenum oxide MoO_ 0.05
Manganese chloride. .... MnCl -4H 0 0.50
Preparation of nutrient solution from stock solutions. The follow-
ing volumes of the six stock solutions are then individually added
to about 500 ml of deionized water with stirring. After all stock
solutions have been added, the nutrient solution is made to 1 liter
with deionized water to obtain a full-strength solution. This
solution is usually diluted to one-third, one-half, or three-
fourths strength.
Chemical Formula Milliliters
Potassium dihydrogen
phosphate
Potassium nitrate. . . .
. . KH PO
lvl 2 4
. . KNO,,
\
1
5
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Calcium nitrate Ca(N03)2 -5
Magnesium sulfate MgSO, 2
Iron 1
1 i
Micronutrients 1
5. TEST PROCEDURES.--CHEMICAL EXPOSURE METHODS
Test substances may be applied to plants either through foliar spray
or root contact through growth media amendment. A liquid or ver-
miculite culture is suitable for the foliar spr,ay technique. A
vermiculite culture is most appropriate for growth media amendment.
Both foliar spray and soil amendment exposure methods ^hould be
tested.
5.1 Foliar spray techniques may be performed by one of two tech-
niques. The first is through foliar contact. The test
solution is sprayed with an atomizer to the point of run-off.
Tip the pot or cover the medium surface to avoid chemical con-
tact. The alternative method is to spray pots or flats from
overhead with both foliage and vermiculite surface receiving
chemical contact with a known surface area dosage. In this
manner, a known surface area dosage of chemical contacts both
the foliage and vermiculite surface.
5.2 Growth media amendment should be done prior to transplanting.
The test substance is mixed with water to form a solution,
suspension, or emulsion. This is combined with th^ vermic-
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ulite using enough diluent to achieve thorough blending.
Dosages should be calculated on a weight/volume relationship
with the test dosages ranging from 0.01 yg/ml growth media
(vermiculite) to 1000 yg/ml on a log,n progression. Uniform
amounts of the amended media should be placed in each con-
tainer and the seedlings transplanted. The seedling and con-
tainer should be subirrigated with an individual pan or petri
dish "half" beneath each container. Nutrient concentrations
should be weak to minimize the need for periodic leaching from
above to reduce salt concentrations, which may leach out the
test substance.
6. TEST PLANT SPECIES
6.1 Monocotyledons
Oats- Avena sativa L. 'Clintford'
Ryegrass- Loliwn perenne L. 'Manhattan'
Corn- Zea mays L. 'Butter and sugar'
6.2 Dicotyledons
Cucumber- Cuawnis sativus L. 'Marketer'
Bean- Phaseolus vulgaris L. 'Pinto'
Tomato- Lyoopersicon esculentum Mill. 'Rutgers'
6.3 All six species should be tested with all procedures.
6.4 In addition to these six basic species other additional species
and cultivars may be used as appropriate.
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7. DURATION OF TESTING
7.1 The basic test period varies depending on the needs of the
evaluation. Foliar tests may involve seedling sprays, re-
peated sprays, or mature plant sprays, depending on the test
objective. The duration of the experiment may proceed through
plant maturity, including flowering and reproduction.
8. PROCEDURAL MODIFICATION
8.1 The general procedures outlined here may be modified as needed
to increase accuracy and effectiveness of the evaluation. For
certain purposes, natural soils may be used instead of ver-
miculite. (11.3)
9. DATA REPORTING
9.1 Potential effects of test substances are listed below. The
effects denoted by an asterisk should be determined in all
instances.
9.1.1 Visual changes in morphology.
9.1.1.1 Above ground.
a. foliage*- leaves, petioles, foliar arrangements
size, shape, distortion, color.
b. support structure*- stems, limbs, stolons, tillers
size, shape, distortion, color
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c. reproductive organs*- flowers, fruits, seeds
size, shape, distortion, color.
9.1.1.2 Below ground.
a. rhizomes, tubers, corms, bulbs—-size, shape,
distortion, color.
b. roots*- size, shape, distortion, color.
9.1.2 Changes not visually detectable.
9.1.2.1 Anatomical—tissue and cellular arrangement.
9.1.2.2 Physiological.
a. Chemical constituents.
1. nutrients, vitamins, etc.
2. metallic and non-metallic elements.
3. other organic and inorganic constituents.
b. Crop plant yields or other subtle changes in
morphology which are not visually detectable but
only discernible through quantitative measurements*,
i. e. yield, height*, weight*, etc.
c. Changes in reproduction.
1. mutations
2. progeny abnormalities in FI populations.
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10. DATA INTERPRETATION
10.1 Interpretation in comprehensive greenhouse testing will be
complex. At this stage in testing a great deal may be learn-
ed about the potential physiologic effects of a substance.
The major problem is to determine if such greenhouse-demon-
strated effects are likely to occur under natural conditions.
10.2 If a given substance does exert profound physiologic effects
at low levels and if significant amounts reach the environ-
ment in soils and waters or will be deposited directly on
plants, then the rule of 0.01 (11.5) should apply. That is,
if the anticipated environmental concentration and exposure
route will reach 0.01 the dosage required to exert physiologic
effects in the greenhouse tests, then field testing should
be initiated. This includes in situ testing with species
occurring in the anticipated release areas as well as the six
standard species.
11. REFERENCES
11.1 Anonymous. Growing plants without soil for experimental use.
USDA Miscellaneous Pub. No. 1251. U.S. Govt. Printing Office.
Washington D. C. 17 pp. 1972.
11.2 Mason, E. B. B. and R. M. Adamson. A sprayer for applying
herbicides to pots or flats. Weeds 10: 330-332; 1962.
11.3 Shaw, W. C. and C. R. Swanson. Techniques and equipment used
in evaluating chemicals for their herbicidal properties. Weeds
1: 352-365; 1951.
11.4 Wilcox, M. A. sprayer for application of small amounts of
herbicides to flats. Weed Sci. 16: 263-264; 1968.
11.5 Lehman, A. J., FDA, Federal Register p. 1493. March 11, 1955.
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3.7 EVALUATION OF PLANT TOXICITY THROUGH FIELD TESTING
1. SCOPE
1.1 These guidelines will allow development of field plot or field-
scale testing of potential toxicants. From these procedures
the scale may vary from small/centare size individual plots
to hectare size whole field plots. Testing may be conducted
on the site of the actual anticipated release or simulated
areas. Although the basic six species used for growth
chamber and greenhouse testing are suggested for inclusion,
additional species may also be added as appropriate.
2. SIGNIFICANCE
2.1 The underlying premise for field evaluation is that such ex-
perimentation will be representative of the effects of toxic
substances under actual commercial manufacture and release
situations. How well such experiments simulate reality de-
pends on the soundness of the experimental design. Properly
conceived experiments will predict accurately the actual
effects of toxicants and may be used as the final criteria
in manufacturing and environmental release situations.
3. EQUIPMENT AND FACILITIES
3.1 Suitable experimental land areas, i.e., field plot sites,
must be free from chemical treatment history. These sites
should be level and uniform in all directions.
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3.2 Soil tillage and planting equipment should provide uniform
results with minimal variation. The capability of the equip-
ment depends on the extensiveness of the plot.
3.3 Test substance application equipment.
3.3.1 Granule applicators and incorporation blenders.
3.3.2 Even-dosage plot sprayers.-gasoline, electric, com-
pressed gas and hand-powered. (7.3)
3.3.3 Logarithmic sprayers.-electric, compressed-gas and
hand-powered. (7.3)
3.4 Surveying, measuring and weighing equipment.
3.4.1 Tapes, transits, stakes, rulers, etc.
3.4.2 Scales, balances, etc.
4. TEST PROCEDURES
4.1 Test plant species
Monocotyledons
Oats—Avena sativa 'Clintford1
Ryegrass—Loliwn perenne 'Manhattan1
Corn—Zea mays L. 'Butter and sugar'
Dicotyledons
Cucumber—Cuownis sativus L.'Marketer'
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Bean—Phaseolus vulgaris L. 'Pinto'
Tomato—Lycopers-icon esculentum Mill. 'Rutgers'
4.1.1 All six species should be tested with all procedures.
4.1.2 In addition to the six species above, other species
should be included, depending on the vegetation and
crops of the exposure area(s).
4.2 Field plot experimental design
4.2.1 Certain principles involved in the use of field plots
must be considered. Variability in the test plot area
soil is an outstanding problem. Soil is universally
heterogenous. Consequently, any two plots taken at
random in a field and sown with the same kind of seed
will almost always fail to give the same plant growth.
Error is reduced if conspicuous variations in soil are
avoided which makes the choice of land a major considera-
tion.
Placing two plots close together ordinarily will make
them more alike than if they are some distance apart.
Hence, there is a positive correlation between the
response of plots placed close together. For example,
in comparing two soil toxicant treatments, it follows
from the nature of soil variability that they should be
near one another. Similarly, for more that two treatments
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it is essential that all the plots be placed in a com-
pact group.
4.2.2 Plot shape, plot size- The shape of long narrow plots
makes it possible to bring them closer together than
square plots. Severe practical restrictions on the
narrowness of plots are certain considerations, such
as border effects, diffusion of treatments, and the
mechanics of seeding, cultivation, and evaluation. The
ideal plot provides a proper balance between these con-
siderations.
The size of the plot is more difficult to decide. For
example, small seeded cereal grain plots are commonly
3-4 rows wide and 5 m long for preliminary tests and in
1/100 hectare plots for final tests. To avoid margin
effects only the center rows are harvested.
4.2.3 Replications.- Replications are essential because of the
variables present in field experiments. The usual plan
is to have a single plot of each treatment in a compact
group or block and to replicate the blocks as often
as necessary to secure the desired accuracy. This
gives some degree of control over the error of the
experiment, and furnishes the mechanism for determin-
ing the experimental error, which cannot be obtained
otherwise.
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4.2.4 Plot plan (7.1.1). Many plot plans are possible; but
two are commonly used: the randomized block and the
Latin square.
4.2.4.1 Randomized Block. This method consists of
replicate blocks, containing a number of rows
equal to the number of treatments. Treatments
are assigned at random. The shapes of the
entire planting of the replication blocks, and
of the individual treatment plots within the
replication blocks are important for error
control. The smallest units, square treat-
ment plots, are less likely to respond similar-
ly than long narrow plots placed side by side.
In many situations a long narrow arrangement
(within limits) may be preferable. Similarly,
the differences that exist in the soil selected,
should be as great as possible between repli-
cation blocks. Thus, the shape of these blocks
should be as nearly square as possible. The
most compact and suitable arrangement approaches
a square. However, local conditions may in-
dicate a modification.
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4.2.4.2 Latin Square. This plan is accurate when the
number of treatments is eight or less. The
field is rectangular and divided into an equal
number of rows and columns. Each treatment
will occur once in each row and column. The
advantage of the Latin Square is that it
controls variability of soil, etc., in two
directions across the field. This is part-
icularly valuable when the direction of the
important fertility trends cannot be predicted,
Although plots may be rectangular, the effi-
ciency of the Latin Square to the randomized
block is less obvious with an increase in
length and width.
4.2.5 Miscellaneous problems. The effects of competition
between a plant and its neighbor are sometimes mani-
fested at the edge of experimental plots unless pre-
cautions are taken to prevent them. It is desirable
to arrange for extra plants on the ends and sides of
an experimental plot to equalize the competition be-
tween the plants under examination. This is consider-
ed in cases where groups of plants in the trials are
either killed or conspicuously stunted by the test
substance treatment.
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Also, the rate of seeding must be taken into considera-
tion. Ordinarily, the optimum rate of slightly above
is employed. This factor is usually not of consequence
except where the size of seed in different varieties
varies greatly and the competition between plants, and
between rows, influences the result.
4.3 Test plant establishment.
4.3.1 Test plants may be established through seeding by
hand or with mechanical seeders.
4.3.2 Test plants may be established through hand transplant-
ing or through mechanical transplanters.
4.3.3 In the case where the test substance is employed as a
soil amendment, seeding or transplanting may be done
after the soil amendment has taken place.
4.4 Test substance application techniques.- Test substances may
be applied to plants and soils in the field in the following
ways:
4.4.1 Spraying or spreading the substance on the soil surface
then "rototilling" or discing into soil profile, follow-
ed by planting.
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4.4.2 Spraying of spreading on soil surface either before or
after planting, i.e., pre-plant-pre-emergence; post-
plant-post-emergence .
4.4.3 Spraying or spreading on foliage post-emergence.
4.5 Treatment dosages.- Greenhouse and growth chamber testing
usually provides a basis for field test dosage selection. In
all instances a control should be included. The dosages should
range from the "no effect" level to rather severe drastic plant
damage so that a full range of plant responses may be
observed.
4.5.1 Field plot soil amendment may be calculated on a volumet-
ric basis with either overall amendment of the test
plot area to a 15-cm depth or band amendment in a band
50 cm wide by 15 cm deep. Normally the test substance,
in a finely divided state, is evenly deposited on the
soil surface and then "rototilled" into the profile
followed by planting in the center of the band. Depend-
ing on previous experience, the test dosages may range
from 0.1 ng/ml to 1000 yg/ml soil volume to log,n pro-
gression volumetric basis.
4.5.2 Spray or granule treatments may be made to the soil
surface post-seeding but pre-emergence of seedlings.
Treatment may be made overall to emerged foliage as well
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as exposed soil, or only to foliage. Dosages in this
instance are normally calculated on the basis of sur-
face area treated. Applications may be made to seed-
lings, mature plants, singly or repetitively depending
on the purpose of the test. A possible dosage range
would be from 0.1 kg per hectare to 1000 kg per hectare
equivalent. The logarithmic dosage technique is one of
the best ways of achieving a wide range of response.
(7.3.4)
4.6 Application equipment.- The major criterion for application
equipment is that the substance is applied uniformly and re-
producibly at the dosage or range of dosages desired. Sprayers,
dusters, or granule spreaders may be used.
4.6.1 Granule spreaders of normal commercial use are satis-
factory if properly calibrated.
4.6.2 Many types of sprayers have been developed for herbicide
evaluation. (7.3.1 to 7.3.12)
5. DATA REPORTING—EFFECTS EVALUATION.
5.1 Potential effects are listed below. An asterisk denotes those
effects that should be determined in all instances.
5.1.1 Visual changes in morphology.
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5.1.1.1 Above ground
a. foliage*- leaves, petioles, foliar arrangements-
size, shape, distortion, color.
b. support structure*- stems, limbs, stolons, tillers,
-size, shape, distortion, color.
c. reproductive organs*- flowers, fruits, seeds-
shape, distortion, color.
5.1.1.2 Changes not visually detectable
a. rhizomes, tubers, corms, bulbs- size, shape dis-
tortion, color.
b. roots*- size, shape, distortion, color.
5.1.2 Below ground.
5.1.2.1 Anatomical— tissue and cellular arrangement.
5.1.2.2 Physiological.
a. Chemical constituents.
1. nutrients, vitamins, etc.
2. metallic and non-metallic elements.
3. other organic and inorganic constituents.
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b. Crop plant yields or other subtle changes in
morphology which are not visually detectable
but only discernible through quantitative measure-
ments* ,i.e., yield*, height*, weight*, etc.
c. Changes in reproduction.
1. mutations
2. progeny abnormalities in F.. populations
6. DATA INTERPRETATION.
Appropriate statistical analysis or quantitative information obtained
should be made.
6.1 Perhaps the statistical method best adapted to the study of
results from field plots is the analysis of variance. (7.11,
7.12) Certain aspects of the analysis of variance should be
considered:
6.1.1 The analysis of variance enables a researcher to test
the significance of the observed results of the experi-
ment. The objective is to obtain a variance for the
observed effects, such as treatment differences, and
a variance for error. The ratio of these variances
will be equal to one if there are no effects owing to
the treatments. If the ratio is greater than one, no
significance can be attached to the results. When the
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ratio is equal to or greater than its 5 percent point
it can be concluded that varietal differences are real.
6.1.2 Replicates increase the precision of an experiment by
controlling error. The error variance results from
differences between plots that are alike. When re-
plicates are used, each treatment appears in each
replicate. In such experiments the differences between
the plots of any one variety are due to experimental
error, the average differences between the replicates.
Therefore, the replicate variance is removed from the
error. The larger the proportion of the total varia-
bility that is removed, the more accurate the experiment,
6.2 If the anticipated exposure level of the test substance in
situ will reach 0.01 of the levels that result in significant
effects during field testing, then the release level of the
substance in actual manufacture should either be reduced to
a satisfactory level or the release prevented from taking place.
Long-term effects from low-level chronic doses presents a
problem which requires long term testing with multi-exposure
or continuous exposure techniques. The procedures are beyond
the scope of this outline.
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7. REFERENCES "
7.1 Experimental statistics.
7.1.1 Snedecor, G. W. and G. W. Cochran. Statistical methods,
6th Ed. Iowa State Univ. Press, Ames, Iowa. 623 pp.
1969.
7.1.2 Sokol, R. R. and F. J. Rohlf. Biometry. W. H. Freeman
& Co., San Francisco, Cal. 776 pp. 1969.
7.2 General evaluation—field plot procedures.
7.2.1 Shaw, W. C. and C. R. Swanson. Techniques and equip-
ment used in evaluating chemicals for their herbicidal
properties. Weeds 1: 352-356.
7.3 Field plot sprayers.
7.3.1 Derscheid, L. A. Sprayers for use on experimental plots.
Weeds 1: 329-337 ; 1951.
7.3.2 Ries, S. K. and C. W. Terry. The design and evaluation
of a small-plot sprayer. Weeds 1: 160-173 ; 1951.
7.3.3 Leasure, J. K. A logarithmic-concentration sprayer for
small plot use. Weeds 7: 91-97; 1959.
7.3.4 Webster, D. H. and J. S. Leefe. A small plot sprayer
using disposable spray containers. Weeds 9: 323-324;
1961.
7.3.5 Lillie, D. T. A carbon dioxide pressured portable field
sprayer. Weeds 9: 491-492 ; 1961.
7.3.6 Leefe, J. S. A variable dosage sprayer for treating
small plots. Weeds 9: 325-327; 1961.
7.3.7 Kerr, H. D. and W. C. Robocker. A portable compressed
air sprayer for experimental plots. Weeds 9: 660-663;
1961.
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7.3.8 Grover, R. and H. Clarke. A precision sprayer for small
experimental field plots. Weed Res. 3: 246-249; 1963.
7.3.9 Clarke, G. S. and A. A. Ross. A small-scale variable
dosage (logarithmic) sprayer. Weed Res. 4: 249-255;
1964.
7.3.10 Kasasian, L. An easily-made, inexpensive, multi-purpose
experimental sprayer. Weed Res. 4: 256-260; 1964.
7.3.11 Turgeon, A. J. and W. F. Meggit. A small plot sprayer.
Weed Sci. 19: 245-247; 1971.
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3.8 ADDITIONAL LISTINGS OF TEST PROCEDURES FOR TERRESTRIAL TRACHEOPHYTA
FOR LABORATORY AND GREENHOUSE
Terrestrial vascular plants form the basis for the earth's
crops for human and animal food and for support of a multitude
of other organisms. In addition, through the photosynthetic
processes conducted by these plants a major portion of the
earth's organic molecules are produced and transformed either
directly or indirectly. However, the effect of soil and water
pollutants is almost unknown. The available knowledge is
is restricted to a few crop plants with little information
about native plant communities.
Our search of the world's literature produced no information
regarding test procedures or investigations with the Bryophyta.
Among the Tracheophyta no research has been conducted with the
Lycopsida, Pteropsida, or Sphenopsida. The bulk of the avail-
able information pertained to crop plants and "weed" plants
within the Angiospermae and Coniferophyta.
As noted previously, test procedures may be performed in the
laboratory, growth chambers, greenhouses, fields, or with entire
plant communities in nature. Laboratory incubator, growth
chamber and greenhouse procedures can be performed at any time
of the year with results obtained in the shortest interval.
However, the correlation between small pot, petri dish, tube,
sand, or liquid culture test results and field or natural plant
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communities results may be very poor, especially if the effects
are exerted primarily or exclusively on the mature plant or its
reproduction. The soil and air environment exert major effects
on test results. Some substances may be toxic only under
environmental stress.
Plant species are extremely variable in response to different
toxicants. Due to the lack of knowledge of chemical effects
(except for the Spermatophyta), it is strongly recommended that
initial testing be restricted to the monocotyledoneae and di-
cotyledoneae. In later secondary test procedures Coniferophyta
may be appropriate under circumstances where substances will
come in direct contact with coniferous vegetation. Crop plants
should be employed because the information available concerning
species and cultivar variability, environmental and toxic re-
sponses, and overall physiology is far greater than for any
wild plants regardless of genera or species. The significance
and importance of crop plant response is much easier to grasp
than with unknown wild species.
Laboratory, incubator, growth chamber, and greenhouse procedures
1. This procedure represents a general sequence of testing
beginning with seed germination and growth chamber studies
of seeds surrounded by toxicant soaked filter paper pro-
ceeding to the germination and growth of seeds in toxicant
treated soil. It can employ chemicals with various modes
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of action and a number of crop species.
Chang, In-kook, and C. L. Foy Effect of picloram on germination
and seedling development of four species. Weed Sci. 19: 58-64;
1971.
2. A sequence of three bioassays is proposed which will pro-
vide a broad spectrum of sensitivity to varying modes of
toxic action. These procedures consist of a seedling root
elongation, shoot elongation, and a Chlorella chlorophyll
extraction. These can be conducted in the growth chamber.
Kratky, B. A. and G. F. Warren. The use of three simple rapid
bio-assays on fourth-two herbicides. Need Res. 11: 257-262; 1971.
3. This is a rapid method which should detect toxicants that
inhibit photosynthesis.
Parker, C. A rapid bio-assay method for the detection of herbicides
which inhibit photosynthesis. Weed Res. 5: 181-184; 1965.
4. Growth chamber procedures involving seed treatment, sprout-
ed seeds and seedling exposure in nutrient solutions are
presented. Injury is expressed in terms of MD,-n (a mo-
larity dosage which causes 50% kill or inhibition of the
organism tested).
Sund, K. A. and N. Nomura. Laboratory evaluation of several her-
bicides. Weed Res. 3: 35-43; 1963.
5. This technique provides a measure of toxicant injury to
root growth as well as evaluating translocation potential,
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Centner, W. A. A technique to assay herbicide translocation and
its effect on root growth. Weed Soi. 18: 715-716; 1970.
6. This procedure, although primarily developed to quantify her-
bicide residues by bioassay, may be used to measure toxicant
effects through soil exposure routes. This method stresses
uniformity of procedure especially soil and air environments.
Santelmann, P. W., J. B. Weber, and A. F. Wiese. A study of soil
bioassay technique using prometryne. Need Sci. 19: 170-174; 1970.
7. Rapid techniques are described for a foliage and three com-
plementary soil bioassays to measure the presence of toxicants
from various exposure routes. This series should be effective
in measuring phytotoxicity from exposure to a wide range of
toxicants as well as the pichloram employed in the report.
Leasure, J. K. Bioassay methods for 4-amino-3,4,4-trichloropicolinic
acid. Weed Sci. 12: 232-234; 1964.
8-9 These papers describe a growth chamber procedure to evaluate
the effects of toxicants on seed germination and seedling
growth including morphological and anatomical modifications
of shoots and roots. Very little space is required for these
tests.
Cutter, Elizabeth, F. M. Ashton, and Donna Huffstutter. The effects
of bensulide on the growth, morphology, and anatomy of oat roots.
Weed Res. 8: 346-352; 1968.
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Ashton, F. M., Elizabeth G, Cutter, and Donna Huffstutter. Growth
and structural modifications of oats induced by Bromacil. Weed Res,
9: 198-204; 1969.
10. This unique approach to bioassay involves soil-toxicant mix-
tures in petri dishes in which seeds are germinated. In add-
ition, standard soil cup-seedling growth procedures are des-
cribed for toxicant incorporation into soil. Although the petri
dish technique required only an incubator, the short duration
would not detect photosynthesis inhibiting compounds.
Horowitz, M. and Nira Hulin. A rapid bioassay for diphenamid and
its application in soil studies.. Weed Res. 11:143-149; 1971.
11. This petri dish technique employs pre-germinated seeds with
dishes placed in vertical position during incubation. Root
and shoot elongation inhibition may be determined. Variables
may be incorporated into the experiment to include such things
as soil moisture levels, adsorption, soil texture, etc. The
short duration would not be effective in detecting photosyn-
thesis inhibitors.
Horowitz M. A rapid bioassay for PEBC and its application in volatil-
ization and adsorption studies. Weed Res. 6: 22-36; 1966.
12. Although twenty-five years old, the series of procedures de-
tailed in this paper still provide a good basis for detection
of phytotoxicity. Growth chamber or greenhouse techniques with
preemergence soil surface sprays as well as pre-and post
emergence field plot sprays are included.
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Shaw, W. D. and C. R. Swanson. Techniques and equipment used
evaluating chemicals for their herbicidal properties. Weeds 1:
352-365; 1951
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3.9 ADDITIONAL LISTINGS OF TEST PROCEDURES TO DETERMINE INFLUENCE OF
ENVIRONMENT ON PHYTOTOXICITY IN LABORATORY, GREENHOUSE AND FIELD.
As evident from previous discussions, many environmental and
treatment factors may influence toxicity by test substances.
When any initial screening procedure in laboratory incubator,
greenhouse or growth chamber indicates potential phytotoxicity
the next essential step is elucidation of the influence, both
exposure factors and the environment, on symptom expression.
The following must be evaluated:
1. Exposure factors-spray droplets size.
2. Environmental factors.
a. Climatic
(1) temperature
(2) humidity
(3) light
b. Edaphic
(1) soil pH
(2) nutrients
(3) texture
(4) temperature
(5) organic matter.
The acceptable procedures available to determine these factors
are listed below by categories.
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3.9.1 Influence of Spray Droplet Size
Douglas, G. The influence of size of droplets on the herbicidal
activity of diquat and paraquat. Weed Res. 8:205-212; 1968.
McKinlay, K. S., R. Ashford, and R. J. Ford. Effects of drop
size, spray volume, and dosage or paraquat toxicity. Weed
Sci. 22:31-34; 1974.
McKinlay, K. S., S. A. Brandt, P. Morse, and R. Ashford.
Droplet size and phytotoxicity of herbicides. Weed Sci. 20:
450-452; 1971.
3.9.2 j Air and Soil Environment
Bovey, R. W. and J. D. Diaz-Colon. Effect of simulated rain-
fall on herbicide performance. Weed Sci. 17: 154-157; 1969.
Buchholtz, K. P. Factors influencing oat injury from triazine
residues in soil. Weeds 13:362-367; 1965.
Burnside, 0. C., C. R. Fenster, G. A. Wicks, and J. V. Drew.
Effect of soil and climate on herbicides dissipation. Weed
Sci. 17:241-244; 1969.
Corbin, F. T., R. P. Upchurch, and F. L. Selman. Influence
of pH on the phytotoxicity of herbicides in soil. Weed Sci.
19:233-239; 1971.
Day, B. E., L-. S. Jordan and V. A. Jolliffe. The influence of
soil characteristics on the adsorption and phytotoxicity of
simazine. Weed Sci. 17:209-213; 1969.
Grover, R. Influence of organic matter, texture, and available
water on the toxicity of simazine in soil. Weeds 14:148-151;
1966.
Hance, R. J., S. D. Hocombe, and J. Holroyd. The phytotoxicity
of some herbicides in field and pot experiments in relation
to soil properties. Weed Res. 8:136-144; 1968.
Rahman, A. Effects of temperature and soil type on the phytotox-
icity of trifluralin. Weed Res. 13:267-272; 1973.
Stickler, R. L., E. L. Knake, and T. D. Hinesly. Soil moisture
and effectiveness of preemergence herbicides. Weed Sci. 17:
257-259; 1969.
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Upchurch, R. P. and D. D. Mason. The influence of soil organic
matter on the phytotoxicity of herbicides . Weeds 10:9-14 ; 1962,
3.9.3 Exposure Site
Appleby, A. P. and W. R. Furtick. A technique for controlled
exposure of emerging grass seedlings to soil active herbicides.
Weeds 13:172-173; 1965.
Eshel, Y., and G. N. Prendeville, A technique for studying
root vs. shoot uptake of soil-applied herbicides. Weed Res.
7:242-245; 1967.
Geronimo, J., L. L. Smith Jr., and G. D. Stockdale. Effect of
site of exposure to nitrapyrin and 6-chloropicolinic acid on
growth of cotton and wheat seedlings. Agronomy J. 65:692-693;
3.9.4 Pretreatment Environment
Darwent, A. L. and R. Behrens. Effect of pretreatment environ-
ment on 2,4-D phytotoxicity. Weed Si. 20:540-544; 1972.
3.9.5 Vapor Transfer from Soil
Swann, Charles, W. and Richard Behrens. Phytotoxicity of
trifluralin vapors. Weed Soi. 20:143-146; 1972.
3.9.6 Persistence and Movement in Soil
Eshel, Y. and G. F. Warren. A simplified method for determ-
ining phytoxicity, leaching and adsorption of herbicides in
soils. Weeds 15:115-118; 1967.
Fink, Rodney J. Phytotoxicity of herbicide residues in soils.
Agron. J. 64:804-805 ; 1972.
Harris. C. I., Adsorption, movement, and phytoxicity of monuron
and s-triazine herbicides in soil. Weeds 14:6-10; 1966.
u in™ rharles S. D. D. Kaufman and Charles T. Dieter.
flgaebioasTay Lotion of pesticide .ability in soils.
Soi. 19:685-690 ; 1971.
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Lambert, S. M., P. E. Porter and R. H. Schifferstein. Move-
ment and sorption of chemicals applied to the soil. Weeds 13:
185-190 ; 1965.
Phillips, W. M. and K. C. Fletner. Persistence and movement
of picloram in two Kansas soils. Weed Sai. 20:110-116; 1972.
3.9.7 Phytotoxic Interactions between Chemicals in Soil
Colby, S. R. Calculating synergistic and antagonistic responses
of herbicide combinations. Weeds 15:20-22; 1967.
Cowing, D. P. A method of comparing herbicides and assessing
herbicide mixtures at the screening level. Weeds 7:66-76;
1959.
Nash, R. G. Phytotoxic pesticide interactions in soil.
Agronomy J. 59:227-230. 1967.
Nash, R. G. and W. G. Harris. Screening for phytotoxic pest-
cide interactions. J of Environ. Qual. 2:493-497. 1973.
Nash, R. G. and L. L. Jansen. Determining phytotoxic pesticide
interaction in soil. J of Environ. Qual. 2:503-310. 1973.
Tammes, P.M.L. Isoboles, a graphic representation of
synergism in pesticides. Neth. J. Plant Path. 70:73-80 ; 1964.
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3 10 ADDITIONAL LISTINGS OF SPRAY APPLICATION EQUIPMENT FOR PHYTOTOXICANTS
IN GROWTH CHAMBERS, GREENHOUSE AND FIELD
3.10.1 Growth Chamber and Greenhouse
Bouse, L. F. and R. W. Bovey. A laboratory sprayer for potted
plants. Weeds 15:89-91; 1967.
Day, B. E., L. S. Jordan, and R. T. Hendrixson. A pendulum
sprayer for pot cultures. Weeds 11:174-176; 1963.
Mason E. B. B., and R. M. Adamson. A sprayer for applying
herbicides to pots or flats. Weeds 10: 330-332; 1962.
Wilcox, Merill. A sprayer for application of small amounts
of herbicides to flats.
3.10.2 Field Plots
Clarke, G. S. and A. A. Ross. A small-scale variable dosage
(logarithmic) sprayer. Weed Res. 4:249-255 ; 1964.
Danielson, L. L. and R. E. Wister. Logarithmic evaluations
of herbicides in horticultural crops. Weeds 7:324-331; 1959.
Derscheid, L. A. Sprayers for use on experimental plots. Weeds
1:329-337; 1951.
Grover, R. and H. Clarke. A precision sprayer for small
experimental field plots. Weed Res. 3:246-249; 1963.
Kasasian, L. An easily-made, inexpensive, multipurpose
experimental sprayer. Weed Res. 4:256-260; 1964.
Kerr, H. D. and W. C. Robocker. A portable compressed air
sprayer for experimental plots. Weeds 9:660-663; 1961.
Leasure, J. K. A logarithmic-concentration sprayer for small
plot use. Weeds 7:91-97 ; 1959.
Leefe, J. S. A variable dosage sprayer for testing small
plots. Weeds 9:325-327; 1961.
Lillie, D. T. A carbon dioxide pressured protable field
sprayer. Weeds 9:491-492; 1961.
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Ries, S. K., and C. W. Terry. The design and evaluation of a
small-plot sprayer. Weeds 1:160-17351951.
Turgeon, A. J. and W. F. Meggit. A small plot sprayer. Weed
Sci. 19:245-247 ; 1971.
Webster, D. H. and J. S. Leefe. A small plot sprayer using
disposable spray containers. Weeds 9:323-324; 1961.
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a..11 MEASUREMENT OF PHYTOTOXICITY USING AQUATIC PLANTS
3.11.1 Introduction
Stephan A. Forbes, in his now classic 1887 paper titled, "The Lake
as a Microcosm," noted the great interdependence within aquatic
ecosystems. The freedom of one organism from the influence of
another is often much more restricted than in many terrestrial
systems. As an example, metabolites released by terrestrial organ-
isms are generally more spatially limited in effect than are the
readily diffusible materials within the aquatic medium. However,
since aquatic zones have not been utilized for agricultural pur-
poses, as have terrestrial regions, the aquatic community had his-
torically been treated as a source of fish and other readily
apparent members of the top of the fiid chain, rather than as a
highly integrated 'microcosm.'
In recent years, however, a wise shift has been taking place as the
primary producers of aquatic systems have received increased
attention. Part of the emphasis has been due to the adverse effects
of nuisance plant growth on fish life, but an increasing amount of
it has hopefully been undertaken with a view of the basic importance
of aquatic plants to both aquatic and terrestrial life.
Numerous efforts have been put forth to establish standardized
testing procedures for aquatic plants, but no one protocol has been
found to be applicable to all situations. As noted for terrestrial
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plant testing, plant species, culture age, dosage, chemical formula-
tion, and other factors greatly influence test results. Specific
influences in the aquatic environment include solubility, degrada-
tion and deactivation potential, method of release into the eco-
system, dilution, and chemical characteristics of the receiving
water.
Certainly a screening test using either algae or aquatic vascular
plants will not produce a complete answer to material toxicity with-
in the aquatic environment. Such tests will, however, demonstrate
some effects of specified materials upon some basic members or
representatives of the aquatic ecosystem. The extent of testing and
the species used would necessarily be dependent on a variety of
factors relative to the nature of the material in question and the
receiving water system.
3.11.2 Algae
The use of algae as test organisms is well established in plant
physiology. Their use as bioassay organisms has been much more
limited, however, and thus techniques have not been generally
standardized. Except for relatively recent attempts coordinated by
T. E. Maloney (Algal Assay Procedure, 1971), few efforts to dev-
elop a sizeable data base upon which to build a standard protocol
have obtained any broad acceptance. Many of these attempts have
viewed algae principally as potential nuisance organisms in
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eutrophication rather than as bioassay test organisms in their own
right.
Methods of measuring effects of chemicals on algae:
A wide variety of procedures are available to measure alterations
in algal activities. The following list includes some of the re-
ported approaches:
1) oxygen evolution rate
2) carbon-14 uptake rate
3) increase or decrease in cell numbers
4) increase or decrease in cell size
5) chlorophyll -a content
6) chlorophyll-carotenoid ratio
7) nitrogenase activity
8) heterocyst frequency
9) akinete development
10) biomass changes
11) ATP content
12) cell division rate
13) plant composition variations
14) optical density of cultures
15) community structure
16) diversity estimates
The choice of approach reported in much of the literature has
obviously been strongly influenced by instrumentation availability,
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taxonomic capability, and interest of the investigator. Only in a
relatively few instances have algal bioassay investigators reported
adequate preliminary research to determine the method or methods
most applicable to the situation. As a result, workers have chosen
their own light quality and quantity, evaluation of effect pro-
cedure, duration of run, specie of organism, growth medium, and any
of numerous other design features.
No single species of aquatic plant (vascular or algae) and no single
test procedure can fully evaluate the toxic capabilities of any
given material. The addition of an aquatic plant early in any
overall test regime will, however, greatly enhance the potential
to evaluate possible phytotoxicity more completely. Since most
bioassay procedures use single species cultures, great care must be
exercised in extrapolating information derived from such tests to
the environment in general.
The first factor in determining the extent of algal testing is the
expected quantity and area of environmental release. The lack of
direct aquatic release does not diminish the efficacy of algal bio-
assays. With the proper choice of organisms, the test results are
equally applicable to the evaluation of effects on soil algae. Due
to the flowing nature of many potential receiving waters, no direct
aquatic discharge can be considered as a strictly local application.
Therefore, even relatively small quantities of materials to be re-
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leased into the aquatic environment should have some algal bioassay
testing.
The chemical nature of the material to be released is an important
consideration. Information concerning biodegradibility, nutrient
potential, or other possible effects may be determined ahead of time.
In each case, it must be recognized that different environments
react differently to a given effluent.
The actual method of waste handling, product disposal, or other
method by which material may reach the aquatic system will also
require consideration when determining the most appropriate testing
program. To paraphase a common cliche, the dilution factor should
not be considered as a solution to pollution. Nevertheless, such
factors are important in establishing a basis for appropriate test-
ing. As an example, in the absence of biological concentration
mechanisms, materials should be worked wit^h in concentrati9ns
approximating those expected in discharges rather than at concen-
trations several orders of magnitude higher.
Two major categories of test materials emerge from these consid-
erations: 1) pure compounds and 2) mixed pr composite effluents.
Bioassays using materials from the first category are relatively
easy to work with. Tests are repeatable and analytical work is
simplified. Materials in the second category, however, impose
a different set of considerations. It is at this point that the
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bottomless pit opens, as daily variations in effluents, synergistic
reactions, season, photochemical processes, and innumerable other
factors enter in. Obviously, to examine the effects of all such
possible combinations of factors becomes prohibitive in both time
and resources. Therefore, rather than test for all possible effects,
a feedback system is recommended that allows retesting of materials
using different regimes as evidence points to such a need.
In general, algal bioassays are divided into two major approaches,
laboratory (in vitro) and field (in situ). Laboratory bioassays
are further subdivided into static and flow-through designs. Field
bioassays may be subdivided into open-system designs, such as ponds,
and closed system bottle designs.
Static in vitro tests are the most commonly used form of algal bio-
assay, but continuous flow designs applicable to certain analysis
have recently been further developed. To be generally applicable
for the purposes of this report, however, the key requirements for
test fitness listed in the PAAP must be considered
1) They should be so designed that technician-level personnel
can do them .
2) Equipment and instrumentation requirements should be relatively
modest and readily attainable .
3) The procedures should be so standardized that results are
acceptably reproducible .
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4) Geographic location should not affect the test results .
5) The results can be applied with judgment to field conditions.
A central point in acheiving reproducible algal bioassays is the
use of proper media. Available formulae range from highly defined
chemical mixtures to indeterminate soil extracts. The use of an
applicable defined medium is considered to be a necessary
beginning point, even though, undefined media, such as those
using prepared effluent receiving water, may be more appropriate
during final testing stages.
The selection of test species for either static or continuous flow
bioassays should be based on a variety of criteria, among them the
following: (1) general availability of standard cultures; (2)
similarity of test species to locally known flora; and (3) knowl-
edge of the organism's physiology',and (4) suitability of the or-
ganism for routine culturing in defined media.
The selection of species for flow-through apparatus is further re-
stricted to organisms that grow attached to a substrate or are
otherwise suitable for such applications. Greater technical
capabilities are probably needed for continuous-flow operations than
for static testing.
Large scale pond type in situ testing requires fairly large areas,
extensive equipment, and considerable expertise for proper eval-
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uation. As a result, this type of algal testing is more research
oriented than bioassay directed. In situ bottle tests, on the
other hand, are well established as an evaluation method and are
highly applicable to some testing situations. Carbon uptake rates,
using carbon-14 or other physiological rate measurements, are
frequently used to assess a variety of effects of environmental
releases of materials.
Acute phytotoxicity tests, using algae, can be completed in rel-
atively short time periods, ranging from a few hours to a few days.
Methods of determining chronic effects are, however, poorly devel-
oped for general application and interpretation of results is, at
at this time, questionable.
The appropriate applications of algal bioassays are many and varied.
Inclusion of one or two algae in the earliest screening seems
appropriate both from an information and a cost point of view. A
static algal bioassay during screening is fairly inexpensive and
straight forward interpretation is possible with applications to
both terrestrial and aquatic systems. As the need to test a given
material increases so can the complexity and effectiveness of algal
testing increase.
3.11.3 Vascular Plants
According to the literature and interviews conducted for this
project , the major use of aquatic vascular plants has been to
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evaluate herbicidal qualities of chemicals. This type of activity,
though informative, has not resulted in a published information
based wholly applicable to routine bioassay of low-level phytotoxic
effects.
Methods are appended for use of aquatic vascular plants in bio-
assay, but considerably more work needs to be published and stand-
ardized before wholesale adoption of such tests in routine practice.
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3.12. PROCEDURE FOR PRELIMINARY EVALUATION OF POTENTIAL TOXICITY TO ALGAE
THROUGH LABORATORY BIOASSAY TESTING. (Adapted from the Provisional
Algal Assay Procedure -EPA), (PAAP)
1. SCOPE
1.1 These procedures are intended to provide a basis for evalua-
ting the effects of a variety of chemical compounds and/or mix-
tures on the growth and death rate of cultured algae. The pro-
cedures are suitable for determining gross effects and are not
intended for use in detailed physiological or biochemical studies.
2. SIGNIFICANCE
2.1 Significant effects observed in these tests point to the need
for more refined approaches to ellucidate probable modes of
action, since coagulation, nutrient binding, and other indirect
causes may result in apparent toxicity.
3. EQUIPMENT AND FACILITIES
3.1 General laboratory facilities suitable for algal, culturing and
growth measurements.
3.1.1 Culturing and incubation facilities - either a temperature
stable room or an incubator.
3.1.2 Culture vessels - good quality Erlenmeyer flasks such
as Pyrex or Kimax. For uniform light transmission, etc.,
the same brand should be used within a laboratory. To
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achieve optimum surface to volume ratios for adequate
carbon dioxide transfer, the following ratios are sug-
gested:
(a) 40-50 ml liquid in 125 ml flasks
(b) 60-80 ml liquid in 250 ml flasks
(c) 100-130 ml liquid in 500 ml flasks
3.1.3 Culture closures of foam, gauze, or other material that
permits adequate gas exchange but prevents contamina-
tion.
3.1.4 Lighting facilities to provide equal illumination to
all flasks. "Cool White" or similar type fluorescent
illumination should be able to provide between 200 and
800 ft-c.
3.1.5 Light meter to measure foot-candles or use photographic
light meter and convert to ft-c according to formula
given elsewhere in this paper.
3.1.6 Microscope of good quality and up to 400x magnification.
3.1.7 Counting chamber - Palmer cell, hemacytometer, or sim-
ilar type cell. The Sedgewick-Rafter chamber is gener-
^
ally unacceptable for algal identification and ennumera-
tion.
3.1.8 pH meter accurate within +0.1 units.
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3.1.9 Spectrophotometer for use between 600 and 750 nm
(optional).
3.1.10 Coulter electronic cell counter (optional).
3.1.11 Fluorometer (optional).
3.1.12 Shaker capable of approximately 100 cycles/minute
(optional).
4. TEST ORGANISMS
4.1 Fresh water (for source cultures see 7.7)
4.1.1 Selenastrum capriaornutwn Printz. (green alga)
4.1.2 Anaoystis cyanea Drouet and Dailey. (blue-green alga)
(formerly Microcystis aeruginosa Kutz.- emend Elenkin)
4.1.3 Anabaena flos-aquae (Lyngb.) DeBrebisson (blue-green
alga)
4.1.4 Other fresh water algae with similar background in bio-
assay or physiological studies may be considered equally
appropriate.
4.2 Marine
4.2.1 DunoH-ella teTrtioleota Butcher (green flagellate alga)
4.2.2 Thalassiosira pseudonana Hasle and Heimdal (diatom alga)
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5.
4.2.3 Other marine or brackish water algae with similar
background in bioassay of physiological studies may be
considered equally appropriate.
4.3 Test species, if maintained in the laboratory, should be kept
under conditions that will insure stability and similarity of
the isolate over time to prevent changes in response pattern
to chemical exposure.
TEST PROCEDURES
5.1 Culture medium (fresh water)
5.1.1 Composition (macronutrients)
Compound
Final Concentration
' (mg/1)
Quantity to make
1 liter stock
solution at lOOOx
NaN03
K2HP04
MgCl2
MgS04.7H20
Cad2.2H20
NaHC03
(micronutrients)
MnCl2
ZnCl2
CoCl0
25.500
1.055
5.700
14.700
4.410
15.000
(yg/D
185.520
264.264
32.709
0.780
25.500 g
1.044 g
5.700 g
14.700 g
4.410 g
15.000 g
0.185520 g
0.264264 g
0.032709 g
0.000780 g
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CuCl2
Na2Mo04
FeCl3
NaEDTA-
5.1.2
Element
N
P
Mg
S
C
Ca
Na
K
0.009
•2H20 7.260
96.000
2H20 300.000
Element concentrations
Concentration Element
(mg/D
4 . 200 B
0.186 Mn
2.904 Zn
1.911 Co
2.143 Cu
1.202 Mo
11.001 Fe
0.469
0.000009
0.007260
0.096000
0.30000
g
g
g
g
Concentration
(yg/ml)
32.460
115.374
15.691
0.354
0.004
2.878
33.051
5.1.3 Stock solutions of 1000 times final concentration are
used by combining 1 ml of each solution into a final
volume of 1 liter distilled water (glass-distilled is
preferred).
5.1.4 Autoclaving of final solutions should be at 15 psi
o
(1.1 kg/cm ) at 121°C for 10-15 minutes. Adjust volume
following autoclaving. if an electric particle counter
is to be used for counting cells, filter and autoclave
solution through a 0.45y membrane filter.
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5.1.5 If the test organism is a diatom, add the following to
the culture medium:
Compound Concentration g™A in stock Element Concentration
_ (mg/1) (mg/1)
Na2Si03- 101.214 101.214 gm Si 9.980
5.2 Culture medium (marine)
5.2.1 Seawater media vary accoring to the organism and other
factors. Proper selection of ingredients, including
micronutrients and vitamins will need to be made in
accord with test requirements. The media of Guillard
and Ryther (1962), Woods Hole modifications of Guillard
and Ryther, the Marine Algal Assay Procedure: Bottle
Test, and numerous others are available.
5.3 Algal inoculum
5.3.1 Cultures, up to three weeks old may be used as a source
of inoculum. For Selenastrum and most other green
algae and diatoms, one-week incubation is often suffi-
icient to provide enough cells. Two to three weeks may
be required to provide inocula for assays with blue-
green species.
5.3.2 The inoculum may be used directly from stock cultures or
the cells may be centrifuged. The sedimented cells should
then be resuspended in an appropriate volume of distilled
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water containing 15 mg NaHCO-/l and again centrifuged.
The sedimented algae should again be resuspended in the
water-bicarbonate solution and used as the inoculum.
Centrifugation should not exceed either 1000 rpm or
10 minutes.
5.4 Incubation
5.4.1 Incubation should be light as described, with the illumina-
tion measured adjacent to the flask at liquid level.
Temperature should remain constant (± 2°C).
5.4.2 To prevent excessive cell death due to settling, the
cultures should be incubated on a shaker at approximately
100 cycles per minute or swirled by hand, on a regular
schedule, at least twice a day. Shaker incubation has
the added advantage of increasing CO- exchange.
6. TEST EVALUATION
6.1 Growth parameters
6.1.1 Maximum specific growth rate
6.1.1.1 The maximum growth rate (y ) for an individual
III 3.X
flask is the largest specific growth rate (y)
occurring at any time during incubation. The
y for a set of replicate flasks is determin-
max
ed by averaging y of the individual flasks.
nicix
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The specific growth rate, y, is defined by
ln(X2/X1) _1
y = — days
where X« =biomass concentration at end of
selected time interval
X. =biomass concentration at beginning
of selected time interval
t_ 1 =elapsed time (in days) between
selected determinations of bio-
mass
NOTE: IF BIOMASS (DRY WEIGHT) IS DETERMINED
INDIRECTLY, E.G., BY CELL COUNTS, THE
SPECIFIC GROWTH RATE MAY BE COMPUTED
DIRECTLY FROM THESE DETERMINATIONS WITH-
OUT CONVERSION TO BIOMASS, PROVIDED THE
FACTOR RELATING THE INDIRECT DETERMINA-
TION TO BIOMASS REMAINS CONSTANT FOR THE
PERIOD CONSIDERED.
6.1.1.2 Laboratory measurements - The specific growth
rate occurs during the logarithmic phase of
growth - usually between day 0 and day 5 - and
therefore it is necessary that measurements of
biomass be made at least during the first 5 days
of incubation to determine this maximum rate.
Indirect measurements of biomass, such as cell
counts, will normally be required because of
the difficulty in making accurate gravimetric
3-111
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measurements are made should be recorded for
use in the compoutations.
6.1.1.3 Computation of maximum specific growth rate -
The maximum specific growth rate (y ) can- be
1113. X
determined by calculation using the equation
in 6.1.1.1 to determine the daily specific
growth rate (y) for each replicate flask and
averaging the largest value for each flask.
It may also be determined by preparing a semi-
log plot of biomass concentration versus time
for each replicate flask. Ideally, the ex-
ponential growth phase can be identified by 3
or 4 points which lie on a straight line on
this plot. However, the data often deviate
somewhat from a straight line, so a line judged
to approximate most closely the exponential
growth phase is drawn on the plot. If it
appears that the data described two straight
lines, the line of steepest slope should be
used. A linear regression analysis of the
data may also be used to determine the best fit
straight line. Two data points that most
closely fit the line are selected and the
specific growth rate (y) is determined accord-
ing to the equation given in 6.1.1.1. The
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largest specific growth rates for the replicate
flasks are averaged to obtain p
max.
6.1.2 Maximum standing crop
6.1.2.1 Definition - The maximum standing crop in any
flask is defined as the maximum algal biomass
achieved during incubation. For practical
purposes, it may be assumed that the maximum
standing crop has been achieved when the in-
crease in biomass is less than 5 percent per
day.
6.1.2.2 Laboratory measurement - After the maximum
standing crop has been achieved, the dry weight
of algal biomass may be determined gravimetrically
using either the aluminum-dish or filteration
technique. If biomass is determined indirectly,
the results should be converted to an equival-
- ent dry weight using appropriate conversion
factors.
6.1.3 Biomass monitoring - several methods may be used, but
they must always be related to dry weight.
6.1.3.1 Dry Weight - gravimetrically
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6.1.3.2 By direct microscopic counting (appropriate
counting cell) or the use of an electronic part-
icle counter. Anabaena flos-aquae, or other
filamentous forms, are not amendable to count-
ing with an electronic particle.counter. Micro-
scopic counting can be facilitated by breaking
up the algal filaments with a high speed blender
or by sonication.
6.1.3.3 Absorbance - with a spectrophotometer or color-
imeter at a wavelength of 600-750 nm . In re-
porting the results, the instrument make or
model, the geometry and path length of the cuvette,
the wave length used, and the equivalence to
biomass should be reported.
6.1.3.4 Chlorophyll - after extraction or by direct
fluorometric determination. The equivalence
between chlorophyll content and biomass should
be reported.
6.1.3.5 Total cell carbon - by carbon analyzer. Equival-
ence between total cell carbon and biomass
should be reported.
6.2 Data analysis
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6.2.1 The principal measures of the culture activity are:dry
weight, cell numbers, pigment content, or other biomass
indicators as previously noted. It is usually appropri-'
ate to include experimentally determined conversion
factors between the indicator used and the dry weight.
More than one growth or biomass indicator should be used
whenever possible.
6.2.2 The overall evaluation of algal bioassay results con-
sists of two parts. The first is the determination of
whether a given set of results is significant when con-
sidered as a laboratory measurement. Several methods
are available such as Student's t- test and analysis of
variance. (A sufficient number or replicates is there-
fore necessary for statistical analysis.) It must be
emphasized, however, that no set criteria presently
exist to determine what level response is significant.
Each evaluation must be conducted on the basis of specific
test objectives using valid statistical procedures.
(Some laboratories do not use set evaluative routines,
such as an equivalent to an LC ) The second part of
the overall evaluation is the correlation of laboratory
bioassay results with those observed or predicted in the
field. No specific guidelines are yet available for
this purpose.
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7. REFERENCES
7.1 Glass, Gary E. Bioassay techniques and environmental chemistry.
Ann Arbor Science Publ., Inc. Ann Arbor, Michigan. 1973.
7.2 Johnson, J.M., T.O. Odlaug, T.A. Olson, and O.R. Ruschmeyer.
The potential productivity of freshwater environments as de-
termined by an algal bioassay technique. U. Minn. Water Re-
sources Research Center. Bulletin #20 ;1970.
7.3 Joint Industry - Government Task Force on Eutrophication. Pro-
visional algal assay procedure. N.Y., N.Y. 1969.
7.4 Martin, D.M. Freshwater laboratory bioassays - a tool in
environmental decisions. Contributions from the Dept. of
Limnology. ANSP. #3 ; 1973.
7.5 Murry, S., J. Scherfig, and P.S. Dixon. Evaluation of algal
assay procedures - PAAP batch tests. J. Water Poll. Cont. Fed.
43:1991-2003; 1971.
7.6 National Environmental Research Center. Marine algal assay
procedure - bottle test. EPA; 1974.
7.7 National Eutrophication Research Program. Algal assay procedure
- bottle test. EPA; 1971.
7.8 Weiss, C.M. and R.W. Helms. The interlaboratory precision test:
an eight laboratory evaluation of the provisional algal assay
procedure bottle test. Dept. of Env. Sci. and Eng. U.North
Carolina. 1971.
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3.13- APPROACH FOR THE EVALUATION OF TOXICITY TO AQUATIC VASCULAR PLANTS
1. SCOPE
1.1 This procedure is intended to provide guidelines for those who
wish to use aquatic vascular plants in the screening of poten-
tially phytotoxic materials. It is not intended as a definitive
test, but will provide an indication of gross effects on vascular
plants in an aquatic environment.
2. SIGNIFICANCE
2.1 High levels of toxicity exhibited by a material in this procedure
indicates the need for further testing in those-instances where
aquatic or run-off release is anticipated.
3. EQUIPMENT AND FACILITIES
3.1 General laboratory facilities suitable for culturing and main-
taining small aquatic vascular plants.
3.1.1 Temperature stable room or incubator.
3.1.2 Culture vessels - Erlenmeyer flasks or other suitable
containers. For uniform light transmission, the same
brand should be used within any given experimental run.
Flask sizes up to 1500 ml, test tubes 175 x 20 mm, etc.
3.1.3 Culture flask closures of foam, gauze, or other material
that permits adequate gas exchange but prevents contamina-
tion.
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3.1.4 Lighting of the "Cool White" fluorescent type to provide
equal illumination to all flasks of between 200 and 400
ft-c.
3.1.5 Light meter to measure ft-c or use photographic light
meter and convert to ft-c according to formula given
elsewhere in this paper.
4. TEST ORGANISMS
4.1 Lemna gibba L., Lerrma minor L., or others.
4.2 Cabomba oaroliniana Gray.
4.3 Elodea oanadensis Rich, in Michx.
4.4 Other small aquatic vascular plants may be equally suitable.
5. TEST PROCEDURES
5.1 Culture medium (modified Hoagland's solution)
Compound Stock Solution cc of stock to
(g/1) make one liter of
nutrient solution
(cc/1)
Ca(»03)2.4H20
KNO-
3
MgSO -7H 0
118.08 10
50.25 10
24.08 10
KH0PO. 13.61 10
2 4
the following (except EDTA and Fe) to be combined into one stock
H-BO 2.860
Z'4H20 1.810
or MnS04«H20 1.540
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Compound Stock Solution cc of stock to
(g/1) make one liter of
nutrient solution
(cc/1)
ZnS04-7H20 0.220
•5H,0 0.080
0.090
or MoCyH20 0.075
EDTA (potassium salt) 2.500
•7H^O 2.500
(It may be desirable to add 10 grams per liter sucrose to the
nutrient medium of some plants.)
5.2 Nutrient solutions should be autoclaved in stoppered containers
3
at 15 psi (1. 1 kg-cm ) at 121°C for 15 minutes.
5.3 Sterile cultures (if needed to avoid interferences)
5.3.1 Immerse in 0.1% HgCl2 for 45 seconds and rinse in sterile
water.
5.3.2 Immerse in 50% ethyl alcohol for 30 seconds and rinse
twice in sterile nutrient.
5.3.3 Place sterile plants in sterile culture flasks where
regrowth will develop sterile cultures.
5.3.4 Transfer plants every week to maintain appropriate
growth conditions and to prevent crowding.
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5.4 Three to ten plants or plant tips, depending on species,
are used in testing materials. At least duplicate tests at
appropriate concentrations should be run for 1-3 weeks.
6. TEST EVALUATION
6.1 The effect is evaluated by comparing appearance of test plants
with control plants grown in nutrient solution.
7. REFERENCES
7.1 Feichtmeir, E.F. Shell Development Company Agricultural
Chemicals and consumer products division. 1974.
7.2 Dow Chemical Co. Submersed and floating aquatic phytotoxins.
1974. .
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3.14 EVALUATION OF THE PHYTOTOXICITY OF AIRBORNE SUBSTANCES
One can formulate a possible strategy for the regulation of air-
borne emissions of potential toxic substances. The strategy out-
lined below takes into account only direct effects on plants, but
indirect effects such as the alteration by a toxicant of disease
susceptibility; the plant as an accumulator of a toxicant and the
means by which it can be transferred to other components of the
biotic environment; or subtle effects of a toxicant on plant growth
and form, yield or quality are not considered. The strategy also
includes two phases: (1) an initial screening for compounds that
will have limited production and (2) a secondary screening that will
require establishment of the threshold for plant injury.
1. Initial Screening
The purpose of the initial screening is to establish the rela-
tive toxicity to selected plant species of the chemical to be
released.
1.1 Plant Materials - Because it will be necessary to conduct
tests on phytotoxicity of chemicals in different parts of
the United States, reliance solely on dominant species of
native vegetation especially for initial screening may be
impractical for several reasons: 1) they may be difficult
to transplant or grow, 2) they may be slow growing,
3-121
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3) they may be difficult to evaluate with respect to injury.
It would be simpler and more practical to select plants that
grow rapidly and uniformly, inexpensive to produce in large
quantities, relatively susceptible to some known toxicants,
and are representative of large groups of plants. These
criteria for initial screening can be met by the use of a
cultivar of the common bean (Phaseolus vulgaris L.) and corn
(Zea mays L.). Bean, a dicotyledonous plant, is representa-
tive of the broad-leaved plants and corn, a monocotyledonous
plant, of the grains and grasses. For purposes of uniformity
and simplicity, plants could be grown in 4-inch pots in a
synthetic medium which can be reproduced easily. Any of the
peat-perlite, peat-vermiculite, or other available mixes can
be used. Six seeds should be sown in each pot and, after
emergence, the seedlings should be thinned to three per pot.
Each fumigation should consist of at least 6 pots of each
species (18 plants).
1.2 Fumigation Equipment The fumigation chambers used should
have the following characteristics: 1) they should be of a
size to accommodate a sufficient number of replicates of
the species under test; 2) they should have an air delivery
system capable of exchanging the chamber air about once
each minute; 3) there should be an inlet into the air de-
livery system for introduction of the chemical being tested;
4) the air delivery system should be designed in such a way
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as to give even distribution through the chamber of the
chemical being tested. The air supply for the chambers
should pass through a particulate filter and through another
filter to remove ambient phytotoxicants other than that be-
ing tested. The most common and universally distributed
ambient phytotoxicant is ozone and it can be removed by
passing air through an activated charcoal filter. A sat-
isfactory chamber for the purpose has been described by Heck
et al. (1,2).
Appropriate devices must be adapted or developed to meter
the test chemical into the treatment chamber, to maintain
desired concentrations, and to monitor the concentration of
the chemical during the exposure period. No single device
can be recommended which is satisfactory for all substances
under all conditions; but a commonly used apparatus is the
Greenburg-Smith impinger (3,4). The Greenburg-Smith impinger
is based upon the impingement of a gas or particle-contain-
ing stream in an appropriate liquid medium. After the sampl-
ing period, an analysis is made for the substance of interest.
1-3 Fumigation Procedures
1.3.1. Concentration - There is no completely satisfactory
method to estimate the concentration of the test sub-
stance to be used, but it would seem to be an unnecessary
requirement at this stage of testing to attempt to
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establish the threshold dose for plant injury. There-
fore, for purposes of initial screening, one concentra-
tion might be used and it might be established as five
times the highest predicted ambient concentration (at
the property line) based upon estimated losses to the
atmosphere and local meteorological and typographical
conditions.
1.3.2. Duration - .A four-hour exposure period is recommended.
1.3.3. Test Conditions - No specific parameters can be required
except that tests should be carried out at temperatures
and relative humidities as near as possible to those
found under ambient conditions during midsummer. Fumiga-
tions should always be made under light conditions of
at least 1500 ft-c .
1.3.4. Plant Evaluation - Any foliar lesion produced by ex-
posure to the test substance should be considered as a
positive effect.
1.4 Results
1.4.1. No Effect - If no visible change has occurred on the
plant 48 hours after exposure, the assumption is made
that the test substance is safe for limited production.
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1.4.2. Effect - If visible lesions have been induced on the
plant 48 hours after exposure, manufacture is restricted
or a more adequate atmospheric control system will be
required.
2 SECONDARY SCREENING
The purpose of the secondary screening is to establish the
threshold of the chemical for plant injury when (1) the chemical
is to be produced in large quantities or (2) if the chemical
fails to pass the initial screening.
2.1. Plant Materials - The same species recommended for initial
screening should be used also for secondary screening.
Where practicable, consideration should also be given for
testing of easily cultivated native species, both woody and
herbaceous. Adequate replication should be included in each
fumigation.
2.2 Fumigation Equipment ~ Tne same chambers recommended for
initial screening are suitable for these tests.
2.3 Fumigation Procedures
2.3.1. Concentration - One approach to the determination of
the threshold for plant injury is estimation by up-and-
down techniques, one of which is the staircase method
of Finney (5). By this method, the EDS_ (effective
dose at which 50% of the subjects respond) is estimated
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from the data acquired. In the staircase method, a
series of equally spaced log doses is chosen, e.g.,
. . . . , x^~, x^,j, x , x~, x.. , x_ , . . . . , where x_
is believed to be near the log ED . The initial screen-
ing may be useful for this estimation.. The first group
of plants is tested at X_. Thereafter, the result of any
test determines the dose for the next test: if an effect
is produced, the next test is conducted at a dose one
step lower; if an effect is not produced, the next test
is at a dose one step higher. Whether or not the 'first
dose is successfully chosen, later doses tend to con-
centrate about the ED . The advantages and disadvantages
of the staircase method are discussed by Finney and some
modifications are given.
2.3.2. Duration - A four-hour exposure period is recommended.
2.3.3. Test Conditions - The same recommendations as given for
the initial screening should be used.
2.3.4. Plant Evaluation - Any foliar lesion produced by ex-
posure to the test substance should be considered as.
a positive effect.
2.4 Results - When the tests .are completed, the threshold con-
centration for a four-hour exposure should be estimated for
the species most susceptible to the test substance. If the
concentration that, is predicted to occur in the ambient air
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(for a four-hour period) is less than the threshold con-
centration determined experimentally, the substance may be
deemed acceptable and cleared for further production. If
the threshold for injury is less than that expected in the
ambient air, the substance may be deemed unacceptable and
increased production would require more effective atmospheric
controls. In evaluating the results of these tests, a number
of compromises have been made which may prove later to be
unsatisfactory. One of these is the conditions under which
the tests are to be carried out. Relatively small differ-
ences in temperature or relative humidity can have drastic
effects on the response of plants to a phytotoxicant. Both
relative humidity and the frequency of precipitation will
be significant factors in phytotoxic effects of particulate
materials. The tests do not consider that plants respond
differently to phytotoxicants at different ages or stages
of development. The screening tests also assume that ambient
exposures of only four hours will occur in the field or that
the effects induced will be based upon total dose (time x
concentration). In all probability, this is not true; thus,
3 3-1
a concentration of 100 yg/m for 4 hours (400 yg/m -hr )
will probably be of a different degree of phytotoxicity than
4 yg/m for 100 hours (400 yg/m "hr"1).
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3. REFERENCES
1. Heck, W. W., J.A. Dunning and H. Johnson. Design of a simple
plant exposure chamber. 60th Annual Meeting of APCA, Cleveland,
Ohio. 1967.
2. Heck, W. W., J.A. Dunning, and H. Johnson. Design of a simple
plant exposure chamber, Nat. Air Poll. Control Adm. Publ. APTD
68-6: 24; 1968.
3. ASTM, Standard Method of Test for Inorganic Fluoride in the
Atmosphere, D1606-60, Book of ASTM Standards, Part 23, pp. 639-
649; 1964.
4. Greenburg, L., and G. W. Smith, A new instrument for sampling
aerial dust, U.S. Bureau of Mines3 Report of Investigation 3392.
1922.
5. Finney, D. J., Probit Analysis. 3rd Edition. Cambridge Univer-
sity Press, pp. 211-219; 1971.
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3.15 PROBLEMS IN THE DEVELOPMENT OF SCREENING PROTOCOLS FOR AIR-BORNE
POLLUTANTS TEST CHAMBERS
Two types of chambers have been used for studies on the effects of
air pollution on plants: (1) the portable or fixed field chamber used
to study the effects of pollution on field-grown plants, and (2) con-
trolled-environment chamber for use in the laboratory or greenhouse,
which allowed from minimal environmental control to highly sophisticated
chambers with elaborate controls. Perhaps the first of these chambers
was used by Schroeder and Schmitz-Dumont in Germany in 1896 (1). Haywood
(2) described a portable chamber in 1908, and Wislicenus (1) used a
sophisticated attached greenhouse. Many other chambers have been deve-
loped and used since the first ones 79 years ago (3,4,5,6,7,8,9,10,11,12,
13,14,15,16,17). Open-top field fumigation chambers have been introduced
recently (16,17).
The first problem is that most of these chambers have not been built
under sufficiently rigid specifications to be acceptable for studies on the
effects of air pollution on plants (18,19). The commonly used chambers
are deficient with respect to many parameters, including good environ-
mental control, uniform air distribution, a one-pass circulation system
which can provide filtered air to the chamber, and types of materials
used in construction. Commercially-available plant growth chambers
have not been designed for use in air pollution studies. Although they
generally provide acceptable control of temperature, relative humidity,
and photoperiod, they do not usually provide control over light in-
tensity. This problem is common to studies with water- or soil-borne
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as well as air-borne pollutants, so it. does not present a serious obstacle.
Because most chambers have not been designed for air pollution studies, the
means of introduction and uniform dispersion of gaseous or particulate
substances are less than adequate. Not only must the concentration of
a pollutant be uniform throughout the chamber, but the air flow must .
be uniform because pollutant uptake (and injury) of plants is related
to both concentration and the amount of the pollutant which passes
over the foliar surface ( 19,20,21) . A second problem is that it is
inescapable that some substances to be tested will have corrosive
properties, and/or may absorb or react with the inner chamber
surfaces. These potential problems emphasize the need for a chamber with
inner surfaces as chemically inert as possible. Corrosion of chamber parts
is obviously unsatisfactory, but even absorption onto surfaces can cause
problems of monitoring and contamination.
Thirdly, because the most satisfactory air distribution system is
a non-recirculating arrangement, proper venting of the chamber exhaust
gases must be provided. Finally, the presence of ambient pollutants in
the incoming air-stream can be unsatisfactory for two reasons: (1) ambient
pollutants, such as ozone, are highly phytotoxic; and (2) the presence
of ambient pollutants may potentiate or antagonize the phytotoxic action
of a test chemical, resulting in invalid test results. This necessitates
the introduction of an appropriate filter (such as activated charcoal) in
the incoming air stream to remove ambient pollutants.
3.15.1 Introduction of Pollutant into the Chamber
These protocols will require the testing of two physical forms of
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air-borne chemicals: gas and particle. The introduction and maintenance
of desired atmospheric concentrations of either type of pollutant present
a number of problems which will be discussed in this and in the subsequent
section.
A number of methods have been used to generate or introduce gaseous
pollutants into chambers. The simplest method is represented by metering
commercially-available bottled gas into the chamber through an appropriate
valve. The gas is often diluted with an inert gas such as nitrogen when
bottled. This method has been used successfully for sulfur dioxide, ox-
ides of nitrogen, chlorine, ethylene, and other less common air pollutants.
A second method involves the generation and bottling of the pollutant,
followed by appropriate dilution and metering into the chamber. Per-
oxyacetyl nitrate is handled in this manner. A third method involves the
generation of the pollutant as it is introduced into the chamber. This
is best represented by the generation of ozone by passing oxygen or air
across an electrical discharge. The generated ozone is then metered into
the chamber at an appropriate rate. Another example of this approach might
be the generation of nitrogen dioxide by reacting copper with.nitric acid
or of potassium bisulfite with a strong acid, but neither is used com-
monly because of the poor control over concentration. A fourth method
involves the volatilization of an aqueous solution of a substance to produce
a gas, and is used commonly for fumigating with HF. (22). Introduction
of test substances as gases might be accomplished by adapting one of these
methods or may require new technology.
Generation of aerosols (air-borne particles) is more difficult than
gases. Information on methods of introduction and design of appropriate
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chambers is limited because there is no large body of literature
(23). The Bacho Microparticle Classifier was recommended by Barley et
al. (23) for the introduction of dry particles but it is no longer avai-
lable and the Wright Dust Food Mechanism (L. Adams Ltd., London, England)
is a satisfactory substitute. Chemicals soluble in water can be introduced
as aerosols into chambers by the use of pneumatic nozzles (such as Sprayco
Model //686C/RXH 11 AM assembly). The particle size and degree of hydra-
tion can be controlled by the rate of supply of the test liquid, by the
air pressure to the nozzle, and by use of other appropriate nozzles. One
serious problem with aerosol fumigations often is poor distribution to all
parts of the chamber. This can be improved to a large extent by the use of
a turntable to equalize the aerosol dosage and environmental conditions over
the plants used in an exposure.
3.15.2 Monitoring of Pollutant
The methods of introduction and control of any fumigant are only as
good as the method used to monitor the concentration in the chamber.
Monitoring is composed of two important steps: Collection and analysis.
Many methods of collection of gaseous pollutants have been used, depending
upon the chemical and physical characteristics of the gas, available
equipment, and desired accuracy and reproducibility of the methods. The
gases have been collected in impingers and bubblers containing water, acid,
alkali, or other solvent; or in dry collectors with treated filter paper,
membrane filters, charcoal, or other media. In some cases, the gas has been
passed directly through an appropriate cell where its concentration is
measured by a direct physical means (e.g., infrared or ultraviolet spec-
troscopy).
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Particles are also collected in impingers or bubblers containing water
or other solvents. But they are most commonly collected with a high-
volume sampler or filter holder on glass, membrane, or paper filters or
with a paper sampler. The particle size distribution can be determined
by use of cascade impactors such as the Andersen or a Lundgren Rotating
Drum Impactor.
The use of proper equipment does not guarantee a proper result, however.
Improper placement of the sampling probe (for gases) or device can give
fallacious results, and for this reason, tests should be made to insure
uniform mixing or dispersion of the pollutant. In the case of gases, the
composition of the sampling probe can be very significant. Certain materials
such as polyvinyl chloride should be avoided; other materials such as
Teflon, glass, stainless steel, polyethylene, or polypropylene may be
satisfactory for some materials but not for others. Some sampling problems can
be solved by the use of a heated probe.
When lengthy exposures are required, the frequency of sampling is
important. For example, a single 10-minute sample in a 24-hour fumigation
would not be sufficient to determine whether the proper concentration had
been attained or maintained. Obviously, the more frequent the sampling the
better the control can be, assuming that a rapid analytical method is
available.
It is difficult to perform exposures at a desired concentration if
a rapid analytical method is not available that will allow adjustments
in the rate of introduction of the pollutant periodically. The analytical
method should also have a high order of accuracy and precision.
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3.15.3 Dose-Response
What response should be measured? The response measured could be
the production of any foliar lesion, and this would be the simplest app-
roach. The response measured could also be an effect on the fresh or
dry mass of the top of the plant. There are innumerable parameters that
could be measured. But can one be certain that the response measured is
a significant one with respect to the native or cultivated flora of the
area? For example, the presence of a foliar lesion may have no measur-
able effect upon the growth or vitality of the plant, or on its intended
use, unless, of course, the leaves of the plant are eaten or it is an
ornamental. A reduction in the mass of the plant may be unimportant if
it is a potato, carrot, or beet and the tuber or root yield has not been
affected. On the other hand, exposure to a chemical during the testing
period may produce no foliar lesions or no affect on the mass of the plant,
and produce an important effect. For example, a chemical may alter the
nutritional composition of the plant; induce sterility by affecting pollen
viability or fertilization; or through direct absorption or metabolism,
be toxic to foraging animals, insects, birds, etc.
What duration of exposure should be used? It is probably impractical
to require long-term exposures with test chemicals for many reasons:
(1) it may be economically unfeasible; (2) the longer the exposure, the
more sophisticated the equipment required to maintain good control; (3)
most controlled environment chambers will not support normal growth of
many species for a long duration.
Should a dose-response curve be established for each chemical to be
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tested? The difficulties in the establishment of dose-response curves
should be apparent from the preceding discussions, but there are other
problems not yet discussed. One of these is that a given "dose" (a meas-
ured concentration of a toxicant for a known duration of time) of an air-
borne pollutant may produce different effects on the plant, depending
upon how it is applied. For example, the phytotoxicity of, say, 1 ppm
of a compound for 168 hours (7 days) may be completely different from
168 ppm for 1 hour, but the dose is the same (168 ppm hrs.). The lack
of reciprocity of a given dose applied in different ways has been shown
experimentally for SO (24), ozone (25), and HF (26). The difference
between the two types of exposures is the difference between acute and
chronic injury. In the former case (high concentration-short duration),
the toxicant may essentially "swamp" the metabolic systems of the plant
which have no opportunity to accommodate to this insult and major injury
may occur. In the latter case (low concentration-long duration), the
plant may detoxify the pollutant by metabolic change (e.g., sulfur di-
oxide to sulfate), insolubilization (e.g., HF to CaF), changes in the
metabolic pathways of the plant cell, or by other means (27), (28) ,and
injury may be slight and difficult to measure.
Because fumigations in the field are more likely to result in low
concentrations of pollutant for long durations than the more extreme
insult, one must question the value of establishing screening procedures
based upon a type of exposure that is least likely to occur. On the
other hand, long-term, low-level, fumigations are prohibitive economically
and pose technical problems that have not yet been satisfactorily resolved.
3-135
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3.15.4 Factors Which Affect Response of the Plant to a Pollutant"
The response of the plant to air-borne, as well as water- and soil-
borne pollutants, is affected by many factors. Although most of these
are important for the three types of pollutant, some are more important
to the air-borne pollutants.
First, there are a number of climatic factors which influence plant
response. Generally, the phytotoxicity of the pollutant increases with
temperature (29), (30), and light intensity (33). The photoperiod in
relation to the time of exposure of the plant to ozone or peroxyacetyl
nitrate is also important (33). In the case of air-borne pollutants , pre-
cipitation or the presence of free water on foliar surfaces can be a
determining factor in whether or not injury will occur from a given ex-
posure. This is especially important in the case of particulate materials
which reside on the plant surfaces, but can also be important with gaseous
pollutants. In the case of particulate materials, the occurrence of light
precipitation or dew can solubilize the particles and aid in foliar
penetration, thus increasing the potential for injury. Heavy precipitation
can remove the particles from the plant surfaces and, in some cases,ses,
remove phytotoxic materials that were adsorbed to the surface or absorbed
as a gas and excreted (34).
Second, are several edaphic factors which affect the response of
the plant to pollutants introduced through the air, water, or soil.
These include the nutrient status of the plant, a subject about which
there is only limited information, and soil moisture. The presence of
adequate soil moisture generally favors the injury of plants by air-borne
pollutants (35).
3-136
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Third, factors associated with the pollutant itself are important
in determining the production of plant injury. These factors include
concentration, duration of exposure, recurrency of exposure, physical
and chemical properties of the pollutant, and the presence of other
pollutants during the exposure period. The importance of concentration
and duration of exposure have been discussed earlier. The recurrency
(or frequency) of exposure is also a significant factor with respect to
the response of the plant. Obviously, the more recurrent the exposure,
the greater the probability of producing injury to the receptor. But
recurrent exposures can be less phytotoxic than continuous ones because
the plant has time to accommodate to this intrusion by detoxification,
excretion, or metabolism. The types and recurrency of exposures that
will occur near any manufacturing facility will be determined by the
nature of the manufacturing processes, and by meteorological and topo-
graphical characteristics of the area. The importance of the physical
and chemical nature of the pollutant has been discussed briefly in an
earlier section, but several important features should be mentioned.
With respect to particulate substances, phytotoxicity is closely assoc-
iated with particle size and degree of hydration of the particle. Very
small particles (ca. <1 y) may not deposit on vegetation but may act as
a gas. Larger particles will impact on the plant but will not be trans-
ported over distances as great as smaller particles. Small particles of
relatively insoluble materials will be more soluble than large particles.
Hydrated particles are more immediately phytotoxic than dry particles, but
the presence of surface moisture as dew or after a rainfall would elim-
inate this difference. Finally, the presence of other pollutants before,
3-137
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during, or after exposure to an air pollutant may alter its phytotoxicity,
and the occurrence of synergistic and antagonistic effects are known
I
(36,37,38,39,40,41,42). There is a high probability that other pollutants
i • .*
will also occur in the atmosphere near any chemical manufacturing facility,
either from other .chemical processes, nearby industries, long-range tran-
sport, etc., and the possibility of interactive effects should be considered
in the evaluation of potential effects on plants.
Fourth, the response of a plant to a toxicant will be influenced by
several biological factors, including the stage of plant development,
the age of the leaf, and heredity. During the ontogeny of a plant, its
susceptibility to environmental stresses, including air pollution, will
change. The direction and extent of this change will depend upon .the
plant and the pollutant.
Young plants are usually more susceptible to air-borne pollutants
than older plants, and the flowering stage of plants can be a particularly
susceptible period. As the leaf ages, its susceptibility to pollutants
changes, decreasing for some, increasing for others. There is a wide
disparity between plants in their susceptibility to any pollutant, but
the order of susceptibility among species is different for each pollutant.
Thus, a test plant may be a susceptible receptor for one chemical and a
resistant receptor for another. Even within a field population of plants,
such as pines, wide differences will be found in the susceptibility of
the various genotypes to any one pollutant (35).
3.15.5 Indirect Effects on the Pollutant-Plant Interaction
Although the possible indirect effects of air-borne pollutants are
3-138
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not a necessary component in the preparation of the screening methods,
the contractor and user of these methods should recognize the fact that
these effects occur, and may produce effects that are more important
ecologically than the direct effect of the pollutant alone. The uptake
and accumulation of some pollutants may have little or no direct effect
on the plant, but may have serious consequences on other components of
the biotic environment. For example, although accumulation of fluoride,
lead, mercury, cadmium, other heavy metals, and nuclides can affect many
plants, depending upon species, stage of development, environmental
factors, and other circumstances, ingestion of the plant by foraging
animals (cattle and other herbivores), birds, or insects can cause dis-
ease, such as fluorosis in the case of fluoride (43). The loss of leaves
through natural processes can transfer accumulated toxicants to the soil,
where an effect may be produced in the soil microbial population. Changes
Changes that occur in metabolism and certain volatile constituents
may affect the suitability of the plant as a habitat for destructive
insects or plant diseases. Although little is known of this area, the
plant may metabolize a substance to a form more toxic to the plant or the
biotoc environment. The metabolism of inorganic fluoride ion to mono-
fluoroacetic acid in a number of African, Australian, and Brazilian
species has been responsible for the innumerable cattle fatalities (44).
Finally, the pollutant may affect the intended use of the plant, whether
for food, fiber, or for aesthetic purposes. Thus, a subtle effect, such
as small difference in plant form may make the plant unsuitable for mec-
hanical harvesting and decrease its value (45).
3-13?
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3.15.6 References
1. Wislicenus, H. Experimentelle Rauschschaden. Versuche liber die ausseren
und inneren VorgMnge der Einwirkung von Russ, sauren Nebeln and stark
verdunnten sauren gasen auf die Pflanze. Samml. Abh. Abga.se Rauchsch'dden,
Paul Parey, Berlin. No. 10: 1-168; 1914.
2. Haywood, J. K. Injury to vegetation and animal life by smelter wastes.
U.S, Dep. Agr.3 Bur. of Chem., Bull. No. 113: 40 ; 1908.
3. Adams, D. F., H. G. Applegate, and J. W. Hendrix. Relationship among
exposure periods, foliar burn, and fluorine content of plants exposed
to hydrogen fluoride, j. Agric. Food Chem.. 5: 108-116; 1957.
4. Guderian, R. 1966. Reaktionen von Pflanzengemeinschaften des Feldfutter-
baues of Schwefeldioxideinwirkungen. Schriftenr. Landesanstalt Immissions-
und Bodennut-zungsschutz. Landes NW 4: 80-100; 1966.
5. Haselhoff, E. and G. Lindau. Die Beschadigung der Vegetation durch Rauch.
Gebr. Borntraeger-Verl. Berlin ; 1903.
6. Heck, W. W., J. A. Dunning, and H. Johnson. Design of a simple plant
exposure chamber. Natl. Air Pollut. Control Adm. Publ. APTD 68-6 ; 24,
1968.
7. Hill, G. R., Jr. and M. D. Thomas. Influence of leaf destruction by
sulphur dioxide and by clipping on yield of alfalfa. Plant Physiol.
8: 223-245 ; 1933.
8. Hindawi, I. J. Injury by sulfur dioxide, hydrogen fluoride, and chlorine
as observed and reflected on vegetation in the field. J. Air Pollut.
Control Asso_c. 18: 307-312 ; 1968..
9. Hitchcock, A. E., P. W. Zimmerman, and R. R. Coe. Results of ten years'
work (1951-1960) on the effect of fluorides on gladiolus. Contrib.
Boyoe Thompson Inst. 21: 303-344 ;1962.
10. Juhren, M., W. Noble, and F. W. Went. The standardization of Poa annua
as an indicator of smog concentrations. 1. Effects of temperature,
photoperiod, and light intensity during growth of the test-plants.
-Plant Physiol. 32: 576-586 ; 1957.
11. Katz, M., A. W. McCallum, G. A. Ledingham, and A. E. Harris. Description
of plots and apparatus used in experimental fumigations. In Effect of
Sulphur Dioxide on Vegetation. 'Natl. Res. Counc. Can. Ottawa, Ontario,
p. 207-217 ; 1939.
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12. Leonard, C. D. and H. B. Graves, Jr. Effect of air-borne fluorides on
"Valencia" orange yields. Proa. Ft. State Hort. Soo. 79: 79-86 ; 1966.
13. Thompson, C. R., and 0. C. Taylor. Plastic-covered greenhouses supply
controlled atmospheres to citrus trees. Trans. Am. Soo. Agr. Eng. 9:
338-339 ; 1966.
14. Zhan, R. Wirkungen von Schwefeldioxyd auf die Vegetation, Ergebnisse aus
Begasungsversuchen. Staub. 21: 56-60 ; 1961.
15. Zimmerman, P. W. and A. E. Hitchcock. Susceptibility of plants to hydro-
fluoric acid and sulfur dioxide gases. Contrib. Boyoe Thompson Jnsf. 18:
^63-279; 1956.
16. Mandl, R. H., L. H. Weinstein, D. C. McCune, and M. Keveny. A cylindrical
open-top chamber for the exposure of plants to air pollutants in the
field. J. Environ. Qual., 2: 371-376 ; 1973.
17. Heagle, A. S., D. E. Body, and W. W. Heck. An open-top field chamber
to assess the impact of air pollution on plants. J. Environ. Qual. 2:
365-368 ; 1973.
18. Wood, F. A., D. B. Drummond, R. G. Wilhour, and D. D. David. An exposure
chamber for studying the effects of air pollutants on plants. Progress
Report 335. The Pennsylvania State University, p. 7 ; 1972.
19. Hill, A. C. A special purpose plant environmental chamber for air pol-
lution studies. J. Air Pollut. Control Assoo. 17: 743-748 ; 1967.
20. Brennan, E. and I. A. Leone. The response of plants to sulfur dioxide or
ozone-polluted air supplied at varying flow rates. Phytopathology 58:
1661-1664 ; 1968.
21. MacDowell, F. D. H., E. J. Mukammul, and A. F. W. Cole. A direct
correlation of air-polluting ozone and tobacco weather fleck. Can. J.
Plant Sci/44: 410-417 ; _1964.
22- Mandl, R. H., L. H. Weinstein, G. J. Weiskopf, and J. L. Major. The
separation and collection of gaseous and particulate fluorides. In H. M.
Englund and W. T. Beery (ed.) Proa. Second Int.Clean Air Congr. , Academic
Press, New York. p. 450-458 ; 1971.
23. Barley, E. F., S. Lerman, and R. J. Oshima. Plant exposure chambers for
dust studies. J. Air Pollut. Control Assoa. 18: 28-29; 1968.
24. Temple, P. J. Dose-response of urban trees to sulfur dioxide. J. Air Pollut.
Control Assoa. 22: 271-274; 1972.
25. Heck, W. W., J. A. Dunning, and J. J. Hindawi. Ozone: nonlinear relation
of does and injury in plants. Science 151: 577-578; 1966.
3-141
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26. MacLean, D. C., R. E. Schneider, and L.'H. Weinstein. Accumulation of
fluoride by forage crops. Contrib. Boyoe Thompson Inst.. 24: 165-166 ;
1969.
27.McCune, D. C. and L. H. Weinstein. Metabolic effects of atmospheric
fluorides on plants. Environ. Pollut. 1: 169-174 ; 1971.
28. Mudd, J. B. Biochemical effects of some air pollutants on plants. In:
"Air Pollution Damage to Vegetation", John A. Naegele (ed.) Adv. Chem.
Ser. 122. -Amer. Chem. Soc. Washington, D. C. p. 31-47 ; 1973.
MacLean, D. C. and R. E. Schneider. Fluoride phytotoxicity: its altera-
tion by temperature, pp. 292-295. Proc. Second Int. Clean Air Congr-
Washington, D. C., Academic Press ; 1971.
30. Thomas, M. D., R. H. Hendricks, and G. R.Hill. The action of sulfur di-
oxide on vegetation. Proc. Natl. Air Pollut. Symp. , 1st, Pasadena, Calif.,
p. 142-147 ; 1949.
31. Benedict, H. M. and W. H. Breen. The use of weeds as a means of evaluat-
ing vegetation damage caused by air pollution. Proc. Natl. Air Pollut.
Symp., 3rd, Pasadena, Calif., pp. 177-190; 1955.
32. MacLean, D. C., R. E. Schneider, and D. C. McCune. Fluoride phytotoxicity
as affected by relative humidity. Proc. Third Int. Clean Air Congr.
Dusseldorf, p. A143-A145; 1973.
33. Dugger, W. M. and I. P. Ting. Air pollution oxidants — their effects
on metabolic processes in plants. Annu. Rev. Plant Physiol. 21: 215-
234; 1970.
34. Jacobson, J. S., L. H. Weinstein, D. C. McCune, and A. E. Hitchcock.
The accumulation of fluorine by plants. J. Air Pollut. Control Assoc.
16: 412-417 ; 1966.
35. Weinstein, L. H. and D. C. McCune. Effects of air pollution on vegetation.
Air Pollution Manual, Part 1 - Evaluation, 2nd Ed., Am. Ind. Hyg. Assoc.
p. 60-74 ; 1972.
36. Banfield, W. Comparative response of eastern white pine to low level
fumigation with respectively ozone, sulfur dioxide, and mixtures of these
two air pollutants. Abst. Northeast. For. Pathol. Workshop. University of
New Hampshire, March 30-31; 1971.
37. Applegate, H. G. and L. C. Durant. Synergistic action of ozone-sulfur
dioxide on peanuts. Environ. Sci. Technol_. 3: 759-760 ; 1969.
38. Dochinger, L. S., F. W. Bender, F. L. Fox, and W. W. Heck. Chlorotic
dwarf of eastern white pine caused by an ozone and sulfur dioxide inter-
action. Nature. 225: 476 ;1970.
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39. Grosso, J. J., H. A. Menser, G. H. Godges, and H. H. McKinney.
Effects of air pollutants on Nicotiana cultivars and species used
for virus studies. Phytopathology. 61: 945-950; 1971.
40. Mandl, R. H., L. H. Weinstein, and M. Keveny. Effects of hydrogen
fluoride and sulfur dioxide alone and in combination on several
species of plants. Environ. Pollut. In press; 1975.
41. Menser, H. A. and H. E. Heggerstad. Ozone and sulfur dioxide
synergism: Injury to tobacco plants. Science. 153: 424-425; 1966.
42. Tingey, D. T., R. A. Reinert, J. A. Dunning, and W. W. Heck.
Foliar injury responses of eleven plant species to ozone/sulfur
dioxide mixtures. Atmos. Environ. 7: 201-208; 1973.
43. National Academy of Sciences. Biological Effects of Atmospheric
Pollutants. Fluorides, Washington, D. C., 1971.
44. Peters, R. A. Lethal synthesis. Proc. Roy. Soc. B139: 143-170;
1952.
45. Weinstein, Leonard H. and Delbert C. McCune. Effects of fluoride
on agriculture. J. Air Pollut. Control Assoc. 21: 410-413; 1971.
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4. SUMMARY
The herbicide literature has provided .information on the mode of
action of chemicals from a variety of structural classes on numerous
types of plants. The majority of available information on the effects
of chemicals on plants has come from research on herbicides and other
pesticides. Although research on air pollution effects on plants has
provided additional insight into the problem, these studies have been
restricted to a relatively small group of pollutants and their effects
on specific receptor species.
The important crop plants provide most of the data base of test
species. Studies on naturally growing plants in the ecosystem have
been limited to forest management techniques and the like and have not
focused on toxicity per se. Although methods for determining chemical
effects on trees are reported, the time involved in testing from seed
to seed precludes their use as test plants.
Because it would be impossible as well as impractical to test every
significant species and strain against even a single chemical, a limited
group of preliminary test species has been recommended. These were
selected on the basis of their importance as crops, their susceptibility
to chemicals, and certain anatomical and physiological characteristics.
Bioassay methods have been selected as the procedures, since they more
closely reflect natural conditions than do techniques involving bio-
chemical or cytological examination. It is recommended that the extent
of plant toxicity testing be directly proportional to the quantity of a
4-1
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chemical which is to be manufactured and the resulting hazard to the
environments.
4-2
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5. CONCLUSIONS
5.1 STATE OF THE ART
5.1.1 Herbicide research, air pollution studies on
plants, and investigations of the aquatic algae and
their environments provided substantial quantities of
data for this report. The results of herbicide re-
search have provided significant information pertaining
to chemical effects on crop plants. These studies
focused on the differential toxicity between wanted and
unwanted species. The bulk of industrial, academic,
and government research has been oriented toward crop
protection by chemicals rather from chemicals.
5.1.2 Air pollution studies have provided data on the
effects of some pollutants on specific species. These
receptor plants were selected because of their known
responses and are often non-representative of either
naturally-growing or commercial crop plants as in the
case of Bel-Ws tobacco. Ecological studies of chemical
effects on plants have been limited mainly to aquatic
environments and have been performed principally with
algae both individually and in communities. The under-
standing of the ecological significance of interrela-
tionships among aquatic algae and diatoms in the food
chain is greater than that achieved in the case of
aquatic vascular plants.
5-1
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5.1.3 Terrestrial plant communities present several
complex problems. There are hundreds of major com-
munities throughout the United States and the problem
is complicated by the number of species involved.
Therefore, emphasis should be placed on species of
ecological importance. When studying a particular
ecosystem, species whould be selected from the study
site.
5.2 Test Methods Selected
5.2.1 Bioassay type determination of chemical effects
on plants should be performed by bioassay techniques
during the critical stages of the plant's life cycle.
These tests were chosen because they assess the effects
of toxic chemicals rather than study the mechanism of
action. «
5.2.2 Preliminary sequence testing should be performed
in a laboratory incubator/growth chamber prior to
greenhouse and field testing. For incubator, growth
chamber, and greenhouse testing, terrestrial plants
should be grown in sand or vermiculite. Temperautre,
humidity, light intensity, nutrients, and duration of
exposure should be standardized. Application of
chemicals may be pre-plant, pre-emergence, post-plant,
or post-emergence.
5-2
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5.2.3 Aquatic plant testing has been limited to algae.
Under controlled conditions both static and flow
through techniques are recommended.
5.2.4 When the hazardous substance is airborne, air
pollution type testing should be used. These tests
should be conducted under controlled conditions in
fumigation chambers. Development of a routine bio-
assay for effects of typical air pollutants and other
airborne chemicals on plants does not seem feasible at
present due to the inadequacy of existing methodology.
5.3 Species Selection
Criteria for species selection included: knowledge of the
plant's physiology, commercial and/or ecological significance,
sensitivity, resistivity, and availability of uniform strains.
No single strain or species can be considered to be the most
susceptible or the most resistant to chemical insult in
general.
5-3
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6. RECOMMENDATIONS
Pilot studies should be performed to test the validity of the procedures
recommended in this report. To determine their reproducibility, these
should be conducted simultaneously in several laboratories. Chemical
standards should include, but not be limited to, a representative of
each major category of herbicides and plant growth regulators listed
in Table 2.2,p. 2-12, Mode of Action by Various Chemicals Disrupt
Plant Growth.
1. Lipid Synthesis Inhibition; structural organization disrupted.
EPTC
2 & 3. Cell Membrane Disruption/Electron Transport Inhibition
diquat
paraquat
4. Enzyme System Inhibition
DSMA
5. Oxidative Phosphorylation Uncoupling
PCP
6. Photosynthetic Electron Transport Inhibition
diuron
atrazine
7. Carotenoid Synthesis Inhibition
amitrole
6-1
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8. Cellular or Nuclear Division Inhibition
chlorpropham
9. Indoleacetic Acid Mimicking
2,4-D
picloram
10. Indoleacetic Acid Transport Interference
naptalam
11. Gibberlin Inhibition
phosphon
12. Affect Ethylene Production
ethephon
13. Combination With Proteins
dalapon
14. Nitrogen Metabolism Disrupter
sodium chlorate
15. Mode of Action Unknown
dichlobenil
diphenamid
bensulide
6-2
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Should any of these effects fail to be reflected by the species suggested,
additional species may be studied. Following successful pilot testing,
a list of industrial (non-agricultural) chemicals should be subjected to
testing based on the quantity manufactured, the environmental release,
and predicted hazards to the environment.
Throughout this project, two major deficiencies in current knowledge
have become apparent. These are the inadequate utilization of infor-
mation and research results tangentially related to pollutant effects on
plant growth, i. e., herbicidal research, and the lack of information
pertaining to chemical effects on natural plant communities. There-
fore, new research efforts.are encouraged with respect to these factors.
The following are recommendations:
The development of research procedures to evaluate the
effects of chemical toxicants on natural plant communities and
ecosystems. These would cover single growing seasons as well
as long term studies that encompass the complete life cycles
of perennial herbaceous and woody plants. Exposure routes
should include soil, water, aerosols, and vapor.
* The development of correlative procedures for elucidating
relationships of growth chamber, greenhouse, and field plot
tests to natural ecosystems. This would provide a basis for
extrapolation of effects found in small scale systems to
natural plant communities.
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* The development of compressed life cycle procedures so that
potential effects may evolve in short time spans. This would
be applicable to forests and perennial vegetation communities.
6-4
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APPENDIX A
-------
APPENDIX A
INTERVIEWS
In the course of gathering information for this report, leading
members of the scientific community were interviewed for first-hand in-
formation. Not all of those contacted were available for personal con-
sultation; several were interviewed solely by telephone. A number of
those interviewed personally requested that they not be quoted or iden-
tified. In deference to their wishes, a listing of only the -organizations
visited is presented. The figure in parentheses indicates the number of
individuals interviewed.
Ag-Organics Dept. (4)
Dow Chemical Co.
Walnut Creek, CA 94598
Agricultural Chemical Div. (1)
Amchem Products Inc.
Ambler, PA 19002
Argicultural Div. (2)
CIBA-GEIGY Corp.
Greensboro, NC 27409
Agricultural Division (2)
Shell Chemical Co.
Modesto, CA 94598
Agricultural Research Service (7)
U.S. Dept. of Agriculture
Beltsville, MD 20705
Biological Research Center
ICI-America Inc.
Goldsboro, NC 27530
(1)
Dept. of Crop Science, Botany (1)
& Forestry
North Carolina State University
Raleigh, NC 27607
Dept of Entomology (1)
Pesticide Research Laboratory
Pennsylvania State University
University Park, PA 16802
Dept. of Forestry (1)
Oregon State University
Corvallis, OR 97331
Dept. of Limnology (1)
Academy of Natural Sciences
19 & Benjamin Franklin Parkway
Philadelphia, PA 19103
Dept. of Plant Biology (2)
Cook College
Rutgers University
New Brunswick, NJ 08903
A-l
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APPENDIX B
-------
TAXONOMIC LISTING OF SUGGESTED TEST SPECIES
SOIL BACTERIA
Schizomycophyta; Schizomycetes; -Pseudomondales; Pseudomonadinaea;
Nitrobacteraceae; Nitrosomonas and Nitrobacter.
BLUE-GREEN ALAGE
Myxophyta (Cyanophyta); Cyanophyceae; Nostocales; Nostocaceae;
Andbaena flor-aquae.
Myxophyta (Cyanophyta); Cyanophyceae; Chroocooccales; Chroococcaceae;
Anacystis Cyanea.
SOIL FUNGI
Eumycophyta (Myxomycophyta); Deuteromycetes; Moniliales; Moniliaceae
Triooderna vi-ride.
GREEN ALAGE
Chlorophyta; Chlorococcales; Oocystaceae; Selenastrwn capvioornutwn.
AQUATIC VASCULAR PLANTS
Magnoliophyta; Magnoliatae; Ronales; Nymphaeceae; Cabomba oarol'in'iana.
Duckweed
Magnoliophyta; Liliatae; Arales; Lemnaceae; Lerrtna minor.
Magnoliophyta; Liliatae; Arales; Lemnaceae; Lenrna gibba.
Waterweed
Magnoliophyta; Liliatae; Hydrocharitales; Hydrocharitaceae;
Elodea canadensis.
B-l
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TERRESTRIAL VASCULAR PLANTS
CORN
Magnoliophyta (Angiospermae); Liliatae (Monocotyledonae); Cyperales;
Gramineae; Panicoideae; Tripsaceae; Zea mays; 'Butter1 and 'Sugar1.
OATS
Magnoliophyta (Angiospermae); Liliatae (Monocotyledonae); Cyperales;
Gramineae; Poacoideae; Avena sativas 'Clintford1.
RYEGRASS
Magnoliophyta (Angiospermae); Liliatae (Monocotyledoneae); Cyperales;
Gramineae; Poacoideae; Hordeae; LoHum perenne; 'Manhattan'.
BEAN
Magnoliophyta (Angiospermae); Magnoliatae (Dicotyledonae); Resales;
Legumiosae; Fabacea; Phaseolus vulgaris 'Pinto'.
CUCUMBER
Magnoliophyta ( Angiospermae); Magnoliatae (Dicotyledonae); Violales;
Cucurbitaceae; Cuownis sativus; 'Marketer'.
TOMATO
Magnoliophyta ( Angiospermae); Magnoliatae (Dicotyledonae); Polemoniales;
Solanaceae; Lycopersicon esculentwn; 'Rutgers'.
B-2
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Phylogenetic origins of terrestrial plants.
KINGDOM PLANTAE (EUPHYTA)
Dicotyledoneae Monocotyledoneae
Coniferophyta
(conifers)'
Ginkophyta
Pteropsida
(ferns)
Lycopsida
(club mosses
Angiospermae
(flower plants)
.Spermatophyta
(seed plants)
Hepaticae
(1iverworts]
Sphenopsida
(horsetails)
Musca
(mosses]
, Tracheophyta
(vascular plants)
Psilopsida
Bryophyta
Chlorophyta
(green algae)
Chrysophta
(yellow-green and golden brown algae,
and diatoms)
Pyrrophyta
(dinoflagellates
Myxomycophta
(siime molds)
Eumycophyta
(true fungi
Euglenophyta
(euglenoids)
Phyaeophyt?.
'(brown-algae)
,Rhodophyta
(red algae)
Schizomycete
(bateria)
Single celled ancestors
KINGDOM MONERA
•Cyanophyceae
(blue-green algae)
Figure 2. Phylogenetic Origins of Terrestrial Plants
B-3
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA 560/5-75-008
2.
3. RECIPIENT'S ACCESSION-NO.
4. TITLE AND SUBTITLE
Test Methods for Assessing the Effects of Chemicals on
Plants
5. REPORT DATE
June 30, 1975
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(s)Richard Rubinstein, Eunice Cuirle, Herbert Cole
and Charles Ercegovich (Penn State U.)> Leonard Weinste
(Boyce Thompson), and Jerry Smith (Academy of Natural S
8. PERFORMING ORGANIZATION REPORT NO.
.n
iences)
9. PERFORMING ORGANIZATION NAME AND ADDRESS
The Franklin Institute Research Laboratories
The Benjamin Franklin Parkway
Philadelphia, Pennsylvania 19103
10. PROGRAM ELEMENT NO.
2LA328
11. CONTRACT/GRANT NO.
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Final
14. SPONSORING AGENCY CODE
15. SUPPLEMENTARY NOTES
16. ABSTRACT
This report is the result of a survey undertaken to develop a series of accept-
able test protocols for assessing the effects of chemicals on plants. Plant exposure
to air, soil, and water contaminants were considered. Test species were selected on
the basis of physiology, anatomy, importance as crops, and their susceptibility to
chemicals. Bioassay methods were chosen because they are most representative of
natural conditions. The recommended sequence of test procedures includes:, growth
chamber or laboratory testing, greenhouse testing, field plot testing, progeny testing
and, finally, population studies in the ecosystem. It is suggested that the extent
of testing should depend upon the quantity of the chemical to be manufactured and its
potential hazard to the environment.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b. IDENTIFIERS/OPEN ENDED TERMS
c. COSATI Field/Group
Airborne contaminants, bioassay, indicator
species, pesticides, plant ecology, plant
physiology, soil microbiology, tests, toxi-
city and water pollution.
52Agriculture
36 01 Bioassay
36 03 Botany
)6 06 Pesticides
)6 13 Microbiology
)8 01 Aquatic Org.
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