ORNL/EPA-1
State-of-the-Art and Proposed Testing
for Environmental Transport
of Toxic Substances
Environmental Sciences Division Publication No. 893
Prepared for
Environmental Protection Agency
Office of Toxic Substances
4th and M Streets, S.W.
Washington, D.C. 20460
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ORNl/EPA-1
Contract No. W-7405-eng-26
STATE-OF-THE-ART AND PROPOSED TESTING FOR ENVIRONMENTAL
TRANSPORT OF TOXIC SUBSTANCES
Project Officer
MICHAEL J. PRIVAL
Prepared by
J. P. Witherspoon, E. A. Bondietti, S. Draggan,
F. P. Taub,* N. Pearson,* and J. R. Trabalka
Report No. EPA-560/5-76-001
prepared for
Environmental Protection Agency
Office of Toxic Substances
Washington, D.C. 20460
under Contract No. 40-404-73
AUGUST 1976
Environmental Sciences Division Publication No. 893
*University of Washington, Seattle, Washington.
OAK RIDGE NATIONAL LABORATORY
Oak Ridge, Tennessee 37830
operated by
UNION CARBIDE CORPORATION
for the
ENERGY RESEARCH AND DEVELOPMENT ADMINISTRATION
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ABSTRACT
WITHERSPOON, J. P., E. A. BONDIETTI, S, DRAGGAN, F. B. TAUB,
N. PEARSON, and J. R. TRABALKA. 1976. State-of-the-
art and proposed testing for environmental transport
of toxic substances. ORNL/EPA-1. Oak Ridge National
Laboratory, Oak Ridge, Tennessee.
This study is a review and evaluation of the use of soil, laboratory
microcosm, and field tests to determine transport of chemicals. The soil
thin-layer chromatography test appears to be the best method for eval-
uating mobility of chemicals in soil. Laboratory results with this method
have been verified in a number of field studies.. Review of studies on
sediment-water interactions indicated that batch equilibrium techniques
may be used to test mobility of chemicals in sediments. Review of lab-
oratory microcosm studies revealed that microcosms have served as useful
tools in the study of movement of nutrients, toxic substances, and energy.
Although experimental microcosms have employed a wide variety of organisms
and levels of complexity, all of them omit large-scale natural processes.
Microcosms of reduced complexity are suitable for measuring rate proc-
esses over a short time range, but more complex microcosms present diffi-
culties in measurement of some rate processes due to the importance of
mutualistic and competing processes. It is proposed that, until research
on microcosms progresses, simple-food chain tests should be used.to de-
termine whether toxicants are likely to bioaccumulate or biomagnify.
Field test systems contain a large (frequently unknown) number of species;
and the number of toxic effects, chemical transfers, and species inter-
actions that can be studied is limited only by the time and resources
available to the experimenter.
iii
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SUMMARY
This study is a survey (review and evaluation) of the use of soil,
laboratory microcosm, and field tests to determine transport of chemi-
cals. It also contains some proposals for tests that may be^ed to
predict the nature and extent of chemical transport in the environment.
It is expected that the results of this effort will assist The Environ-
mental Protection Agency in its responsibilities related to the regula-
tion of toxic substances.
Review of literature on the transport of chemicals in soils revealed
that it is dominated by studies on pesticides. This excludes, of course,
the vast literature on soil and chemica-ls of nutritional significance to
plants. Soil column methods, batch equilibrium techniques, and soil
thin-layer chromatography all have been applied to the study of mobility
of chemicals in soils. In some cases, results of laboratory tests have
been compared to field results on pesticides. Although a sizable liter-
ature on absorption of pesticides by soils exists, there are no standard-
ized test methods which are widely accepted. There is also no recognized
standard soil(s).
The soil thin-layer chromatography test appears to be the best
method for evaluating mobility of chemicals in soil. Laboratory results
with this method have been verified in a number of field studies with
pesticides. Moreover, the method has great value as a routine procedure
because of its reproducibility and because chemicals of known mobility
can be tested simultaneously with unknowns.
Review of studies on sediment-water interactions indicated that
batch equilibrium techniques may be used to test mobility of chemicals
in sediments. These tests determine the degree of adsorption of chemi-
cals to sediments and are relatively economical in terms of money and
time.
The loss of volatile chemicals from soil or sediment-water systems
has been estimated by determining the potential of low solubility com-
pounds to evaporate with water and by measuring either gas-phase diffusion
losses from or residence times of volatile chemicals in soil or water.
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All three of these techniques are useful for estimating the rate of
volatilization of chemicals from land or water to the atmosphere.
Review of laboratory microcosm studies revealed that microcosms
have served as useful tools in the study of movement of nutrients, toxic
substances, and energy. Although experimental microcosms have employed
a wide variety of species of organisms and levels of complexity, all of
them onrit large-scale processes such as alluvial deposition or long
distance migration. Microcosms of reduced complexity are suitable for
measuring one or a few rate processes over a short time range. The more
complex microcosms, while being more like natural systems, present dif-
ficulties in measurement of some rate processes due to the importance of
mutualistic and competing processes.
Three critical problems arise in using microcosm results for pre-
dicting actual environmental results. There is the question of applying
results from an isolated process to a more complex system. There is not
yet enough field verification of microcosm results to assume that this
interpolation is valid. Secondly, the comparability of similar, but not
identical, model systems has not been evaluated. And, perhaps most
important, there is a general lack of estimates of confidence limits on
parameter measurements made in microcosm studies. It is unusual to find
replicated experiments in the literature except for the simplest micro-
cosms.
No standardized microcosm was found to exist, although the seven
species system devised by Metcalf has been used to test a number of
toxicants. This model terrestrial-aquatic ecosystem, however, still
excludes some functionally important groups such as soil and benthic
organisms.
It is proposed that, until research on microcosms progresses and
until there is more field validation of microcosm results, simple food
chain tests should be used to determine whether toxicants are likely to
bioaccumulate or biomagnify. The food chain tests which were selected
(one for terrestrial transfers, two for aquatic freshwater transfers,
and one for marine transfers) offer a maximum chance for a chemical to
bioaccumulate. Moreover, enough trophic levels are included to give a
reasonable prediction of the extent of biomagnification.
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A final category of experimental ecosystem is the field test plot
in which an enclosed portion of a natural ecosystem is treated with a
chemical under otherwise uncontrolled conditions. Review of literature
on the use of field tests for studying chemical transport revealed that
grassland plots, portions of forest, and experimental ponds and streams
all have been used. These systems contain a large (frequently unknown)
number of species; and the number of toxic effects, chemical transfers,
and species interactions that can be studied is limited only by the time
and resources available to the experimenter.
It is proposed that field tests be used for toxic chemicals which
have been shown to bioaccumulate in laboratory food chain tests. The
estimated cost of a field test ($10,000 to $15,000) is about ten times
higher than that for a 30-day laboratory test. This additional cost is
justified in the case of toxic, persistent chemicals which will have
widespread dispersal or local, high-level dispersal.
A hierarchy of testing is suggested in which results from tests on
chemicals for toxicity, persistence, and solubility in water and lipid
are coupled with information on dispersal characteristics to determine
the necessity for laboratory food chain tests. The laboratory tests are
selected on the basis of expected entry routes (atmospheric, landfill,
aquatic) and tests on mobility of the chemical in soil.
Use of the proposed testing scheme should result in enough informa-
tion to prevent the release of a toxicant which, through chemical trans-
port in the environment, has the potential to damage man or other biota.
vii
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ACKNOWLEDGMENT
A number of people contributed to this study as active participants
or as "sounding boards" for ideas. Initially, the study was expedited
by an excellent literature search conducted by the Toxic Materials Infor-
mation Center of Oak Ridge National Laboratory. A bibliography was pre-
pared by this group under Emily D. Copenhaver. About 1300 references
were compiled and copies of over 700 literature documents were procured.
Both copies of the bibliography (hard cover) and procured documents were
furnished to the Office of Toxic Substances, U.S. Environmental Protec-
tion Agency. This bibliography also has been published as an Oak Ridge
National Laboratory report (B. K. Wilkinson, L. S. Corrill and E. D.
Copenhaver. 1974. Environmental transport of chemicals bibliography.
ORNL/EIS-74/68.
Several consultants contributed to the study. Dr. Gordon Chester,
of the Water Resources Center, University of Wisconsin furnished useful
ideas in the area of soil and sediment reactions with chemicals. Dr.
Charles R. Malone, Environmental Studies Board, National Research
Council (formerly with the Environmental Sciences Division), initiated
the literature search and identified institutions involved in chemical
transport work. Visits to other laboratories and information exchanges
were also helpful.
Finally a number of our colleagues in the Environmental Sciences
Division contributed both to the literature search and formulation of
the study. Drs. S. V. Kaye, D. L. Eyman, J. W. Huckabee, G. S. Henderson,
C. W. Gehrs, and R. I. Van Hook lent their expertise in this regard.
viii
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TABLE OF CONTENTS
Page
INTRODUCTION , 1
I. GENERAL .' 1
A. Description of Transport Pathways ... 1
B. Purpose of Study 4
II. GLOSSARY 5
"STATE-OF-THE-ART" OF THE USE OF LABORATORY TESTS TO DETERMINE
MOBILITY OF CHEMICALS IN PHYSICAL ENVIRONMENTAL COMPARTMENTS . 8
I. GENERAL CONSIDERATION OF SOIL TESTS 8
II. SURVEY OF SOIL TEST STUDIES 9
A. Chemical-Soil Interactions 9
The Organic Chemical 9
Soil Water 10
Soil Properties 11
B. Test Methods for Evaluating Mobility Adsorption . . 11
Batch Equilibration Studies 12
Column Leaching Studies 13
Soil Thin-Layer Chromatography 14
C. Sediments 18
D. Concluding Comments on "State-of-the-Art" for
Mobility 20
III. VOLATILITY 22
A. General 22
B. Predictive and Experimental Methods 23
IV. LITERATURE CITED - 29
"STATE-OF-THE-ART" OF THE USE OF LABORATORY MICROCO.SMS TO
DETERMINE ENVIRONMENTAL FATE OF CHEMICALS 33
I. MOVEMENT OF CHEMICALS INTO BIOTA 33
A. General 33
B. Chemical Transport in Food Chains 34
II. GENERAL CONSIDERATIONS OF LABORATORY MICROCOSMS .... 36
A. Advantages of Microcosms . 36
B. Predictions of Actual Environmental Events 37
C. Types 39
ix
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Page
D. Parameters Measured 43
E. Reproducibility 43
F. Applicability • 44
G. Duration 44
H. Recovery 44
I. Environmental Control 45
J. Equipment Costs 45
III. SURVEY OF LABORATORY MICROCOSM STUDIES 46
A. Terrestrial Microcosms 46
B. Terrestrial-Aquatic Microcosms 51
Farm-Pond ("Metcalf Microcosms") 51
Rail-Streambank-Pond 59
Soil-Stream-Lake 59
C. Laboratory Stream Microcosms 60
D. Aquatic-Batch Microcosms 62
Nutrient Cycles 69
Continuous Monitoring; Diurnal Cycles 71
Equilibrium 72
Route of Entry . . 73
Duration of Zooplankton-Fish Studies 73
Single vs Continuous Input 73
Equivalency of Similar Organisms 73
Detritus Feeders 74
Volatile Chemicals 74
Accumulation of Organic Pesticides by
Components of Aquatic Microcosms 75
Degradation or Chemical Modification 75
Microorganisms Uptake (Including
Solvent Effects) 76
Reproducibility 77
E. Aquatic-Continuous Cultures 79
Introduction 79
Apparatus 83
Principles of Operation 83
Parameters Measured—Applicability 84
Cases from the Literature 86
Reproducibility 88
F. Special Types 88
Species Defined (Gnotobiotic Microcosms) 88
In Situ Bioassay 88
Naturally-Occurring Microcosms 90
Closed Ecological Systems (Bioregenerative
Life Support Systems) 90
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IV. LITERATURE CITED
V. APPENDIX . . 102
A. Comparable Data on Microcosms Adaptable to
Chemical Transport Studies 103
"STATE-OF-THE-ART" OF THE USE OF FIELD TESTS TO DETERMINE
ENVIRONMENTAL FATE OF CHEMICALS 109
I. GENERAL CONSIDERATIONS OF FIELD TESTS 109
II. SURVEY OF FIELD TEST STUDIES ..... 110
A. Terrestrial Studies 110
B. Aquatic Studies 110
C. Substance Categorization and Analysis 117
D. Size and Its Relation to Field Variability 118
E. Mode of Entry 119
F. Processes Observed 121
G. Duration . . . . 122
III. LITERATURE CITED ..... 123
RECOMMENDATIONS FOR TESTING ENVIRONMENTAL FATE OF CHEMICALS . 128
I. INTRODUCTION 128
II. TEST PROTOCOLS 129
A. Value of Short-term Tests 129
B. Cost Effectiveness of Tests ..... 129
C. General Testing Hierarchy 130
D. Specific Testing Hierarchy 133
1. Tests for mobility in soil and sediments .... 135
Objectives and general comments 135
Soil test protocol 136
Cost and time of soil tests 137
Degree of predictability of soil tests 138
Required soil and sediment information 138
Sediment test protocol 139
Cost and time of sediment tests 140
Degree of predictability of sediment tests ... 140
Volatility test protocol 140
Cost and time of volatility test 141
Degree of predictability of volatility tests . . 142
XI
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Page
2. Laboratory tests for terrestrial bioaccumulation
and biomagnification 142
Objectives and general cormients 142
Terrestrial test protoco] 143
Cost and time 144
Degree of predictability 144
3. Laboratory tests for aquatic bioaccumulation
and biomagnification 145
General comments 145
Freshwater test protocol 147
Cost and time 148
Estuarine-marine test protocol 148
4. Field tests for transport of toxicants ..... 149
General comments 149
Terrestrial field test protocol 153
Cost and time 156
Terrestrial-aquatic field test protocol .... 157
Aquatic-estuarine-roarine field test protocol . . 157
Personnel required 159
III. LITERATURE CITED 160
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INTRODUCTION
I. GENERAL
An evaluation of the potential hazard of releasing chemicals to
the environment should include determination of their toxicity, persist-
ence and transport in the environment. Understanding the pathways and
processes of transport is especially important in terms of determining
the potential effect of an environmental contaminant. Given that a
chemical is toxic and does not degrade to innocuous material, the extent
to which it disperses in the physical environment and becomes available
to biota, including man, governs its hazard. Of course, transport proc-
esses also may be related to persistence of a substance in the environ-
ment if chemical and biological degradation processes occur during
transport.
Figure 1 shows the major pathways of transport of chemicals in the
environment. Some of the pathways include several transfer mechanisms.
From the various sources of entry of chemicals into environmental com-
partments (boxes), they may remain immobile or be transported at various
rates to other environmental compartments along pathways (arrows) of
physical or biological transfer mechanisms.
A. Description of Transport Pathways
Most planned or accidental releases of chemicals into the environ-
ment are initially to the atmosphere, soil, or water. One significant
exception may be the application of herbicides or pesticides directly
to terrestrial biota.
The Atmosphere
Chemicals may initially enter the atmosphere as gasses or small
particulates dispersed via stacks, vents, or open incineration. In
addition to this direct mode of entry, many substances (organic solvents,
for example) may be transported from soil or water to the atmosphere via
volatilization. Once in the atmosphere, these chemicals will diffuse
from their point of entrance at rates and distances governed by their
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physico-chemical properties and meteorological conditions of the atmos-
phere. Chemicals may be removed from the atmosphere via deposition
which includes both dryfall and wetfall processes. Deposition is the
major pathway of transport from the atmosphere to soil and water.
Direct ^cosition upon terrestrial biota.&lso may occur.
*V9*i I£A&^^
The Soil
Chemicals may directly enter the soil as a result of intended ap-
plication, burial of wastes, or spills. Indirectly, chemicals are trans-
ported to soil by deposition from the atmosphere and by irrigation.
The transport of chemicals (mobility) in soil is related to physico-
chemical properties of the chemical and the soil and the amount of water
present. Chemicals which are not strongly adsorbed to soil will be
transported to terrestrial biota via uptake by plants. They also will
be transported by surface runoff to aquatic ecosystems and by leaching
into ground water. Erosion is another pathway by which transport from
soil to aquatic ecosystems may occur. This is especially true of agri-
cultural soils which may not have as much vegetation cover as natural
soils.
The Water
Saline and fresh waters may receive direct input of chemicals as
wastes or spills. Transports from other environmental compartments
include deposition from the atmosphere and erosion and runoff from soil.
Chemicals entering a water mass may stay in solution or become sorbed
to particles of suspended and bottom sediments at a rate of quantity
dependent upon physico-chemical properties of the chemical, the water,
and the sediment. Chemicals which are strongly sorbed may be transported
to biota by direct uptake (absorption) from water or by use of the water
for drinking or irrigation. Volatile chemicals may be released to the
atmosphere for dispersion and possible uptake by terrestrial biota.
The Biota
The biota, or living environmental component, represents the focal
point for evaluating the potential hazard of a chemical released into
the environment. Chemicals transported to biota by the pathways of
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uptake mentioned above may accumulate in organisms which take up the
chemical from physical components or in consumer organisms via food
chains. The food chain serves as a transport pathway between organisms
and as a mechanism by which substances from the physical environment can
be obtained by organisms which are unable to take up the substances di-
rectly. As in the case of organisms taking up chemicals from the phys-
ical components, higher level organisms may or may not accumulate
transported substances. Thus, the causation of adverse effects from
chemicals transported to biota is dependent upon whether the chemicals
accumulate to levels which will affect the organisms.
B. Purpose of Study
It is the purpose of this study to review and evaluate methods
which have been used to measure or predict transport pathways of chemi-
cals in the environment. Special attention is devoted to laboratory
microcosms and field studies of chemical transport. On the basis of
this review and evaluation, several laboratory tests are proposed for
determing the potential of toxic chemicals to move along key transport
pathways and to accumulate in biota. We believe these tests to be
simple, economical, reproducible and reasonably similar to "real world"
transport. Methods for field testing of chemical transport also are
suggested for evaluation of chemicals which have been determined to be
highly mobile and of widespread initial dispersal into the environment.
Evaluation of the potential hazard of new toxic chemicals will
require both information on chemical transport and persistence of chem-
icals in the environment. It is hoped that information contained in
this study can be used with that furnished by the Syracuse University
Research Corporation on techniques for determining persistence of chem-
icals in the environment (Howard, P. H., J. Saxena, P. R. Durkin and
L. T. Ou. 1975. Review and evaluation of available techniques for
determining persistence and routes of degradation of chemical substances
in the environment. Report No. PB243825/AS, Nat1!. Tech. Info. Ser.,
Springfield, Va.).
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II. GLOSSARY
The following definitions of terms may be useful to the reader who is
not familiar with the several disciplines from which this study was
drawn.
adsorption - The gathering of a gas, liquid or dissolved substance on
a surface in a condensed layer.
bioaccumulation or accumulation - Uptake which results in a residue of
the chemical in an organism.
bioconcentration - Bioaccumulation in which the resulting concentration
of the chemical in the organism exceeds that of the chemical in the
food or surrounding medium.
biodegradation - A reduction of the complexity of a chemical compound
accomplished by the biological action of living organisms.
biomagnification - Bioconcentration in two or more successive trophic
levels with that in the higher level due to consumption of organisms
in the lower level.
biomass - Mass of living matter per unit area or volume of habitat.
biota - The animal and plant life of an area or habitat.
chromatography - The separation of mixtures into their constituents by
preferential adsorption by a solid such as silica or paper.
desorption - The unbinding of a substance which had been adsorbed.
detritus - The dead and fragmented organic matter at the bottom of a
body of water or on the forest floor.
dissociation - The reversible decomposition of a complex substance into
simpler constituents caused by variation in physical conditions.
diversity - The number of species per unit area, volume or time.
dryfall - A deposition of matter from the atmosphere during periods of
no rain.
ecosystem - A system formed by the interaction of a community of organ-
isms with their physical environment.
environmental control - The regulation of environmental factors in
laboratory studies.
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environmental fate - Refers collectively to the transport, accumulation
and transformation of a substance released to the environment.
exchange capacity (soils) - The capacity of a soil to adsorb cations or
anions in an exchangeable form. Units are milliequivalents per 100
grams of soil.
food chain - A series of organisms interrelated in their feeding habits.
litter fall - The loss of foliage from plants.
microcosm - A confined ecosystem that may be subjected to laboratory
controls due to its reduction in size or complexity. Such systems
may be created artificially or they may represent portions of
natural ecosystems which can be brought in the laboratory for study.
persistence - Refers to the ability of a chemical compound to degrade
slowly or not at all in the environment.
root exudation - The release of chemical substances from living plant
roots.
runoff - Water, suspended soil and chemicals that drain horizontally
over soil.
solubility - The mass of a substance contained in a solution which is
in equilibrium with an excess of the substance.
solubility of a gas - The ratio, at equilibrium, of concentration of the
gas in solution to concentration of the gas above the solution.
solubility of a substance in air - A method of expressing vapor pressure
(saturated). Given the molecular weight of a chemical compound, its
concentration in air at saturation can be calculated from the ideal
gas law and the vapor pressure of the compound.
throughfall - The rain, suspended solids, and dissolved substances that
fall through forest foliage to the ground.
trophic level - A level in a food chain. Plants (producers) occupy the
first level, plant eaters (herbivores) the second level, animals that
eat herbivores (carnivores) the upper level.
uptake - The movement of a chemical into an organism from its food or
directly from the surrounding medium.
vapor pressure - The pressure of a confined body of vapor. At saturation,
the pressure of a vapor is dependent on temperature only.
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volatilization - The evaporative loss of a chemical compound. Volatil-
ization rates are dependent on the vapor pressure of the compound
and the environmental factors influencing diffusion from the evapora-
tive surface.
wetfall - A deposition of chemicals which are washed out or scavenged
from the atmosphere by rain.
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8
"STATE-OF-THE-ART" OF THE USE OF LABORATORY TESTS
TO DETERMINE MOBILITY OF CHEMICALS IN PHYSICAL ENVIRONMENTAL
COMPARTMENTS
I. GENERAL CONSIDERATION OF SOIL TESTS
Review of a large number of studies on absorption and mobility of
chemicals in soils revealed that pesticide sorption studies dominate the
literature. Such studies include sorbates like referenced clay minerals,
purified humic substances, and other simplified substrates. Experiments
with soils or sediments included primarily soil column studies, batch
equilibration work, and soil thin layer chromatography studies. Where
possible, field studies were reviewed to determine if laboratory findings
agreed with field observations. The bulk of pesticide adsorption papers
in the open literature are not directly suitable for the purpose of this
review because of special considerations designed into each experiment.
There are no standardized test methods widely accepted in the area of
mobility. However, it is evident from the literature that methods
exist for evaluating relative mobility in soil. A distinction should be
drawn between mobility and adsorption because, while the two concepts
are very closely related, adsorption is only part of mobility. Desorption,
a phenomenon directly related to mobility, has been largely ignored in
the literature (Hamaker, 1971). Thus a batch equilibration study which
measures the affinity of a substance for soil (i.e., a partition between
aqueous and solid phases) tells little about the desorption behavior of
a substance during leaching.
The most significant approach to studying relative mobility appears
to be that of soil thin layer chromotography. It is a new approach (1968)
but promises to be ideally suited for screening-type experiments. As
a method, it has been used to evaluate over 40 pesticides for grouping
into mobility classes. Further work on establishing mobility classes
are needed if this method is accepted as a standard test.
Volatility is much more difficult to evaluate than mobility. As
will be illustrated, knowing a compound's tendency to partition between
air and water can aid in evaluating its potential for vapor phase losses.
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II. SURVEY OF SOIL TEST STUDIES
A. Chemical-Soil Interactions
The adsorption, desorption and movement of organic compounds in
soils are related to the physiochemical nature of the organic substance,
the amount of water present in the soil or moving through it and certain
physical and chemical properties of the soil itself.
The Organic Chemical
Extensive research on the adsorption of natural and synthetic organic
substances to soil has delinated physiochemical properties important in
adsorption. Bailey and White (1970) discuss the following properties:
1. Chemical character, shape and configuration of the adsorbate.
This broad property encompasses the nature of the molecule itself; the
functional groups present; the nature of substituting groups on the
molecule, aromaticity, length and position of alkyl groups, etc. These
characteristics will determine the extent of adsorption. Molecules
with large numbers of nonpolar substituents will be largely hydrophobic
and, consequently, due to low water solubility, will adsorb strongly
to organic matter in soils. The halogenated hydrocarbons are examples.
The herbicide 2,4-0 is sold in many forms, from the free acid to complex
esterified forms. Each alteration strongly affects its behavior in
water and soil and its subsequent biological toxicity.
2. Dissociation constants of functional groups. Soil colloids
are negatively charged at environmental acidities. Consequently, acidic
compounds exhibit various degrees of affinity for soils since anion ex-
clusion will affect adsorption. For example, the weak acid herbicide,
4-amino-3,5,6-trichloropicolinic acid (picloram) shows an adsorbability
to soils closely related to the amount of ionized acid present (Hamaker
and Thompson, 1972). At pH 2, almost all of the picloram added will be
adsorbed to soils. At pH 5, virtually all the herbicide is ionized,
adsorption falls to less than 20% of the amount added. As will be
discussed under testing methods, picloram is a fairly mobfle compound.
The low adsorption is due to anion repulsion forces between the negatively
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10
charged soil and herbicide. Weakly basic (cationic) compounds behave
somewhat similarly in that adsorption is pH-dependent. However, the
reasoning is opposite that for the weak acids. As pH rises, strongly
adsorbed protonated forms of organic cations dissociate to yield non-
ionic or more neutrally charged molecules with less affinity for soil
colloids.
3. Water solubility. Regardless of the mechanism(s) in influenc-
ing adsorption, movement of organic chemicals in soils is linked to
water solubility of the chemical. However, while water solubility is
not necessarily the mechanism controlling soil solution concentrations,
highly water-soluble compounds are more mobile than less soluble
substances.
4. Charge distribution on organic cations. Positively charged
organic molecules can vary in adsorption characteristics due to the
nature or distribution of .the electrostatic charge and surface charge
density. This phenomenon has been noted for the dipyridyl herbicides,
diquat and paraquat.
5. Molecular size. The molecule's size affects adsorption since
large molecules present more functional groups which can interact with
adsorption sites on soil colloids.
6. Polarity. Dipole moment and dielectric constants can be used to
understand potential adsorption to solid surfaces. Basically, the mole-
cule is partitioning between water and the solid phase in soils. The
stronger the affinity or solvation in water, the more difficult adsorp-
tion is to less polar substances like clay minerals, organic matter, etc.
Soil Water
Precipitation and irrigation affect mobility. A fundamental con-
sideration required for determining potential mobility of chemicals is
the net downward movement of water in soils. In areas of low to inter-
mediate rainfall, organic compounds may move up and down. The soil
profile in the upper soil horizons responds to evapotranspiration losses
and precipitation/irrigation inputs. Only that fraction of incipient
water which moves past this zone downward or semi-laterally due to
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11
gravitational and topographical influences could carry chemical sub-
stances into drainage water, groundwater or lateral seepages. Sanitary
landfills present ideal situations for toxicant movement by water if the
land surface conditions allow maximum movement of water to underlying
strata.
Soil Properties
A complete description of the relationship between soil properties
and organic molecule adsorption is not within the scope of this report.
Some general comments should be made, however. The experience of pesti-
cide research has revealed that adsorption is related to pH, organic
Carbon content, and clay content. Organic matter seems to be the single
most identifiable soil parameter which affects organic substance adsorp-
tion, especially when the contaminant is present in trace amounts. The
soil pH is extremely important in considering the movement of weak acidic
or basic substances. The soil thin-layer method of Helling (1971c)
illustrates this quite well. Picloram, as mentioned, is an acidic herbi-
cide whose affinity for soil surfaces is a function of pH. Helling
determined picloram's mobility on 14 soils ranging in textural class
from sandy loam to clay. Movement of picloram was correlated to pH at
the 10% level of significance. Movement was not correlated to organic
matter contents. Helling studied a total of 13 different pesticides on
the 14 soils and concluded that acidic pesticides behave distinctly
differently from non-ionic compounds in that acidic compound mobility is
directly related to pH and water flux. The most mobile organic compounds
studied thus far appear to be the acidic compounds.
B. Test Methods for Evaluating Mobility and Adsorption
The literature is rather voluminous in this area. Helling (1970)
reviewed the literature on pesticide mobility and discussed the meth-
odology for studying mobility. No standardized test methods were
discussed because the requirements for such were not evident at the
time. It should be noted that much research in the area of pesticides
has been devoted to studying adsorption processes to evaluate the
-------
12
extent to which applied substances are not immobilized and consequently
reach the target organisms.
The methods for evaluating or predicting potential movement of a
substance in soils can be generally grouped into four categories:
batch equilibration studies; column leaching studies; soil thin layer
chromotography; and field-scale experiments.
Batch Equilibration Studies
This method measures adsorption, and not mobility per se. A given
soil is shaken with a aqueous solution containing the compound of inter-
est. After centrifugation or filtration, the concentration in solution
is determined by radiotracer or chromatographic methods. Hamaker and
Thompson (1972) recently reviewed over 177 adsorption studies on pesti-
cides using this approach. This review represents the best available
compilation of data in this area. The magnitude of this type of study
in the open literature necessitates a discussion of the merits of this
approach. Adsorption is typically expressed in three forms:
1. £ = K C 1/N ; the Freundlich equation
2. - = KDC ; the KQ expression
3< m = K1K2 'W^1 + K2 *W ' the Lan9muir equation
The term X/m is the concentration (ug/g) of adsorbate in the soil; K is
the equilibrium constant, and C is the concentration of adsorbate
remaining in solution (ug/ml). The K- or K term therefore becomes the
ratio of the fraction adsorbed to the fraction in solution (ug/g). In
many soil adsorption studies, the 1/N term of the Freundlich equation
has been found to approach unity; thus the equation becomes the same as
K_. The Langmuir equation has not found widespread use in soil adsorp-
tion studies because of the heterogenous nature of the soil surface.
It has not proven to be as useful in predicting adsorption in soils
(Hamaker and Thompson, 1972). The simplified Freundlich or KQ equation
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13
assumes no limiting value in surface concentration. A low constant means
poor adsorption and a high value represents high adsorption. For example,
if equal weights of water and soil are equilibrated and 50 percent of the
tested substance adsorbed, the K- would be 1. If 90 percent adsorbed,
KD would be 9; and if 99 percent adsorbed, KQ would be 99. An examina-
tion of Hamaker and Thompson's compililation of 1C values for 50 chemi-
cals reported in over 30 studies shows wide variations among individual
chemicals because of differences in soils and experimental conditions.
In some cases KQ values vary by a factor of over 100 for a particular
compound, depending on experimental conditions. This illustrates the
necessity of standardized procedures. If all experiments are conducted
at the same soil/water rates, the KQ expression can be substituted by
the simpler expression of percent adsorbed. As a qualitative tool, sim-
ple adsorption experiments can be of value in determining the affinity
of a compound for soil or sediment. Harris and Sheets (1966), Hance
•
(1965), Helling (1971 c), Huggenburger e_t £]_. (1973) and Damanakis et al.
(1970) and Lotse e_t al_. (1968) describe experimental systems to measure
adsorption of organic compounds to soils by batch equilibration techniques.
Batch equilibration methods, however, do not measure the influence of
water movement (flux) on toxicant movement. This is a disadvantage of
such tests because they do not evaluate mobility per se, but only adsorp-
tion. However, for screening purposes, adsorption studies which evaluate
the tendency of a compound to partition between soil, or sediment, and
water are very useful.
Column Leaching Studies
Leaching a substance through a column filled with soil is a much
more direct method of evaluating mobility than adsorption equilibrium.
Harris (1967) described a column leaching technique by which he evalu-
ated the mobility of 28 herbicides. The two soils used were a silty
clay loam and a sandy loam. Aluminum columns contained the soils which
were packed uniformly to a known bulk density (gms soil/cm of column).
Water and herbicide were applied at the bottom. Thus, herbicide movement
occurred with capillary movement of water up the column. Bioassays using
oats were carried out on sections of the columns after the leaching study.
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14
From the results of the bioassay, Harris developed mobility factors for
the herbicides which integrated distance moved as well as the concentra-
tion profile within the column. To minimize the effects of differences
in experimental conditions over time or soils, a relative mobility factor
was also proposed, in which individual herbicide mobility factors were
related to the mobility factor of monuron, a herbicide which gave rela-
tively constant results from one experiment to another. Lambert et^ a_1.
(1965) proposed a system which they tested with only one herbicide.
Soil was packed into a tube which was slotted on one side to allow plant-
ing of seeds. The degree of movement was evaluated by growth inhibition
of the plants. In this study system mobility and_ sorption are evaluated
since phytotoxicity is related to the amount remaining in the soil solu-
tion and not adsorbed; By application of chromatographic theory and
evaluations of the "active" adsorption sites, Lambert e_t al_. attempted
to develop predictive equations for evaluating pesticide movement in
soil. Many other workers have done column leaching experiments, but none
serve as an example which can be used to develop a mobility testing scheme.
The disadvantages of soil columns are their large soil requirement, ne-
cessity for uniform packing, and the difficulty in determining where a
compound is in the soil column. With mobile compounds, column effluent
can be monitored, but this becomes rather elaborate and difficult to
relate to an absolute mobility scheme.
Soil Thin-Layer Chromatography
Helling and Turner (1968) reported on the use of soil thin-layer
Chromatography (soil TLC) to evaluate pesticide movement by comparing
R- values (Rf = distance moved by the test compound/total distance of
water movement). They observed that this technique correlated well with
existing information on movement of the tested compounds. In the origi-
nal paper, 16 pesticides were evaluated and a mobility classification
scheme was proposed. In a later series of papers (Helling'et^ al_., 1968;
1971a,b,c) a total of 40 pesticides were evaluated and grouped according
to mobility class. Table 1 gives the five mobility classes chosen by
Helling and the pesticides grouped with each class. Mobility class 1
covers relatively immobile pesticides with Rf values 0.0-0,09; the last
-------
Table 1. Mobility classification scheme for pesticides
proposed by Helling (1971b) using soil TLC1
Mobility class
1
Chloroxuron
Diquat
Chlorphenamidlne
Fluorodifen
Morestan
Oleldrln
Paraquat
Trlfluralln
Endrin
DDT
(0.09)
(0.06)
(0.04)
(0.04)
(0.02)
(0.00)
(0.00)
(0.00)
(0.00)
(0.00)
2
Slduron
Prometryne
Propanll
Dluron
D1chloben1l
Chlorpropham
Azinphosmethyl
(0.
(0.
(0.
(0.
(0.
(0.
(0.
30)
25
24)
24)
22)
18)
15)
3
Propachlor
Prometone
2,4,5-T
Propham
Fluometuron
Dlphenamid
Monuron
Atrazlne
S1maz1ne
Ametryne
Propazlne
Trletazlne
4
(0
(0
(0
(0
(0
(0
(0
(0
(0
(0
(0
(0
.63)
.60)
.54)
.51)
.50)
.49)
.48)
.47)
.45)
.44)
.41)
.36)
Plcloram
Fenac
MCPA
Amltrole
2,4-D
Bromacil
Nortron
(0.
(0.
(0.
(0.
(0.
(0.
(0.
84)
84)
78)
73)
69)
69)
65)
5
TCA (0.96)
Dalapon (0.96)
Dlcamba (0.96)
Anil ben (0.91)
ch
values 1n parentheses determined using Hagerstown silt soil.
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16
class, 5, covers the very mobile compounds, Rf 0.90-1.00. The mobility
classification was developed using Hagerstown silty clay loam, a soil
from Frederick County, Md. The advantages of soil TLC as delineated by
the authors include rapidity, reproducibility, and low equipment costs.
The procedure for preparing soil TL£olates is basically the same
as for conventional TLC. A soil-water slurry is applied to clean glass
plates using a variable thickness applicator. After the soil plates are
air dried, the compound to be tested is applied to the soil near the
bottom of the plate. Amounts between 1-10 ug have proved useful for
radioisotope-labeled pesticides. The plate is placed in a solvent tank
containing water. Water leaches through the application zone (spot)
while ascending the plate. When the water front reaches the top of the
plate, the plate is removed and allowed to air-dry. The location of the
test spot can be most readily determined using auto-radiography or radio-
chromatogram scanners. Alternatively, bioassay techniques have been
used. Extraction and analysis of successive vertical zones of the plates
can be used if radio!abeled or biologically active substances are tested.
Helling (1968; 1971a,b,c) has compared relative movement data derived
from soil TLC tests with soil column leaching studies and found good
agreement. The column work of Harris (1966, 1967), for example, consid-
ered many of the same pesticides and used two of the same soils that were
treated by Helling. Results of both methods revealed that adsorptive
differences in the two soils were apparent and that commonly tested pes-
ticides followed the same order of mobility.
Inch et^lL- (1972) used soil TLC to study the movement of relatively
immobile insecticides. Using repeated ascending and descending Teachings,
they found that descending chromatogram development caused more movement
of certain test compounds than ascending techniques. The authors con-
cluded that downward chromatogram development contained elements of
saturated water flow, as well as unsaturated flow, while ascending
chromatography represent unsaturated flow. Helling (1971 a) observed
that water flux (i.e., rate of water movement) affected mobility to a
small extent. Descending leaching caused a slight increase in pesticide
mobility expressed as R. values, especially for mobile, acid compounds
like 2,4-0. Chapman e_t aj_. (1970) used soil TLC to investigate triazine
-------
17
(a family of herbicides) movement in soil. Abernathy and Wax (1973) used
soil TLC to evaluate movement of three pesticides on a sandy loam soil.
They found that bentazon, a postemergence herbicide, is very mobile on
TLC plates (Rf 1.0).
Because soil TLC seems to offer a simple, yet reliable method of
evaluating relative mobility in soils, an effort was made to compare the
literature data on field mobility studies with laboratory soil TLC eval-
uations. These comparisons came in part from Hell ing's own comparisons
(Helling and Turner, 1968; Helling, 1970; Helling, 1971b,c; Helling et
al., 1971b) and from literature reviewed for this study. In a 15-year
field experiment (Nash and Wool son, 1968) the chlorinated hydrocarbon
insecticides lindane, endrin, and dieldrin were found to be very immobile
in a Maryland soil, penetrating, over a 13-year period, to a depth of
15-30 on. The field site was near Beltsville, MD on a sandy loam soil.
The chlorinated hydrocarbon and organo-phosphorous insecticides exhibit
very low mobilities on soil TLC, as illustrated in Table 1. Dieldrin
and endrin both show R^. values of 0.00. Lichtenstein (1958) showed that
lindane, aldrin and DDE stayed in the surface 22 cm of a Wisconsin field
plot after 3 years.
Trifluralin, a toluidine herbicide, was immobile in soil TLC (Rf
0.00, see table) and column studies (Harris, 1967). Smith (1972) showed
that 11 to 22% of soil-applied trifluralin remained in the top 0-5 cm of
soil and 22% was found in the 5-10 cm depth after a 5 month test in
Regina, Saskatchewan, Canada. Degradation and volatilization presumably
accounted for the bulk of herbicide loss. Picloram is a relatively
mobile herbicide (Rf 0.84). Field studies have shown that indeed picloram
does leach to some extent in soils. Dowler e_t afl_. (1968) found picloram
at depths of 90-120 cm three months after application under high rainfall
conditions in Puerto Rico. Hunter and Stobbe (1972) reported movement
of picloram in field experiments (Canada) to 30-60 cm depths over a
four-year period in a clay soil. Lutz ejb al_. (1973) compared movement
of picloram and 2,4,5-T (R, 0.54) on a western North Carolina watershed
and found that higher concentrations of picloram had moved to depths of
30-40 cm than 2,4,5-T 100 days after application. (This is predicted
by the differences in R. values determined by Helling.) In general,
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18
picloram was found to be more evenly distributed through the soil profile
(0-40 cm) than 2,4,5-T. Highest concentrations of both compounds were
observed at the 0-7.5 cm depth. Scifes e£ a1_, (1971), found picloram
moving to 45 cm depths five months after application to a semiarid
rangeland soil. ^
*"*Sfe
La Fleur e£.al.. (1973) studied the movement of toxaphene, an organo-
chlon'ne insecticide, and fluometuron, a substituted phenylurea herbicide,
applied (rates were 100 and 40 kg/ha, respectively) to a field plot in
South Carolina. The water table was usually less than 3 m from the sur-
face. After one year, about 20 times more fluometuron was found in the
ground water below the plots than toxaphene. Helling (1971b) showed
that fluometuron had an R- of 0.50. Toxaphene was not tested by Helling
but as a chlorinated, non-acidic compound, it is expected to be immobile.
Swoboda et. aK (1971) found that toxaphene did not move as far as
DDT in a clay soil over a ten-year period. Almost all of the recovered
pesticides were in the <1 ft depth. Mansell and Hammond (1971) studied
the movement of 2,4-0 and paraquat in sterilized soil columns. The
leaching of these two compounds through peat and sand resulted in more
2,4-0 movement than paraquat. Paraquat is a cationic herbicide which
normally shows no mobility on soil TLC (R,; 0.00), while 2,4-D is an
acidic herbicide which Helling found to be mobile (Rf 0.69). Damanakis
et al. (1970) found paraquat to be very immobile. Deli and Warren (1971)
studied the Teachability of diphenamid, 2,4-0, and chlorpropham from
soil columns. Leachability from a silt loam soil followed the order
2,4-D > diphenamid > chlorpropham. This is in agreement with Hell ing's
work on soil TLC (see Table 1). The above comparisons between labora-
tory, column field and soil TLC work were chosen to illustrate the
usefulness of soil TLC in measuring relative mobility of a compound in
soil.
C. Sediments
Most of the principles applicable to adsorption-desorption inter-
actions in soil are applicable to sediments. Sediments generally are
of finer texture than soils and they contain more amorphous organic
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19
matter and clay. This is due to the erodability of finer soil compo-
nents and the biotic activity in water. Because of the finer texture
and higher organic content, sediments generally show higher sorption
tendencies than soils (Poinke and Chesters, 1973; Lotse e_t _al_. , 1968).
However, Poinke and Chesters (1973) point out that little information
exists on sediment-organic contaminant interactions to indicate differ-
ences from typical soil -contaminant interactions.
The Freundlich equation (see Section B) has been applied to
pesticide-sediment interactions (Poinke and Chesters, 1973). The
equation
m eq
describes the adsorption of a chemical compound as a function of concen-
V
tration. The — term is the ratio of test substance to sediment mass
m
(ug/g) . C is the concentration of compound in solution at equilibrium
and K and N are constants. At low concentrations (parts per million)
V
of contaminant, a plot of log -^versus log C is linear and 1/N approaches
unity. The K, or K- if 1/N is unity, thus becomes the constant describing
the affinity of the sediment for the contaminant. Hance (1965) empha-
sizes the necessity of conducting experiments such that K can be compared
for various substrates when testing a given compound. For any given
soil or sediment, K values for different compounds also are comparable.
Hance (1965) used a reference concentration of C = 1 ug/ml for calcu-
lating K values.
One characteristic of sediments that is distinctly different from
most soils is the tendency for sediments to become anaerobiotic. This
condition results from a lack of oxygen in the sediment and it affects
the chemical characteristics of the sediment-water interface. For ex-
ample, iron and manganese can be solubilized due to reduction of iron
and manganese oxides. The soluble iron and manganese may affect organic-
sediment interactions. Such effects, however, have not been extensively
studied (Poinke and Chesters, 1973).
Patrick e_t al_. (1973) described a simple system for controlling the
reduction-oxidation potential of soil and sediments. They reported that
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20
the reduction-oxidation potential can be controlled within ± 3 mV and
the pH within ± 0.05 pH units. Their system could be utilized for eval-
uating the effect of reducing and oxidizing conditions on the interactions
of organic compounds with sediments. However, further testing would be
required to validate the general utility of this method.
Sediment interactions with inorganic chemicals (stable and radio-
active elements) have been reviewed by Booth (1975). He formulated a
system analysis model for estimating the exchange of chemical elements
between water (fresh and saline) and sediments. The most important
parameter of this model is the 1C value. Compartments of this model are
the receiving water, interstitial water in bottom sediments, bottom
sediments that undergo sorption-desorption reactions with water and
bottom sediments (sinks) that undergo only sorption reactions with inter-
stitial water. This model has been used to generate equilibrium concen-
trations and turnover times of elements in water and sediments.
D. Summary on "State-of-the-Art" for Mobility
The state of predicting mobility in soil or sediments of substances
introduced into ecosystems is not well developed as a routine procedure.
The soil TLC approach of Helling comes closest to a developed screening
test for evaluating mobility. It appears to be the best method for
further development because mobility can be expressed as an R, value for
a particular soil. Although it has not been done, a standard compound of
intermediate mobility could be selected to which all other compounds are
related. This would allow comparisons of compounds while minimizing
their behavior on different soils. Harris (1967) attempted this by
using monuron as a reference compound. Relating movement to one compound
minimized variances in experimental results and techniques. Helling
(1968) concluded that soil TLC would be a useful tool for evaluating
mobility if a single, nationally recognized soil standard were used.
A mobility classification scheme could then be developed that would have
widespread applications. A standard soil is not available at this time.
The American Society of Agronomy and the Soil Science Society of American
are currently assembling a soil bank which at present includes 12 soils
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21
representative of distinct characteristics found in soils of the U.S.
Or. Robert Holmgren, USDA, SCS, Soil Survey Laboratory, 1325 N St.,
4th Floor, Lincoln, NE 68508, is chairman of the committee overseeing
this project. An agreement has been reached with the National Bureau of
Standards for dispersal of samples. This is one existing soil collection
program which could be tapped for the collection, classification, and
storage of national soil sample(s) for use in any testing procedures
developed.
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22
III. VOLATILITY
A. General
The volatilization of organic compounds from soils and natural
waters is more difficult to assess than adsorption or mobility. Environ-
mental factors (temperature, humidity, soil type, soil moisture content,
evaporation, mixing, air movement) complicate the evaluation of the rates
of loss from soil or water surfaces.
In general, volatilization rates from soil or water are related to
the vapor pressure of the substance and the characteristics of the soil-
water system in which the substance resides. Volatilization increases
with higher temperatures as well as with decreasing soil organic matter
and clay contents (i.e., the adsorptive sites). Losses are greater from
moist soil than dry soil. Contaminant transport to the soil surface
("wick effect") results from mass flow movement of water during evapora-
tion, and affects the rate of loss from the soil surface. Compounds of
intermediate water solubility, adsorbability, and volatility are most
susceptible to losses by evaporation-induced mass flow. For example,
Spencer and Cliath (1973) reported on pesticide volatilization as related
to water loss from soil. They attempted to relate pesticide loss to soil
water conditions at the soil surface and the relative humidity of the
air at the soil surface. In comparing the influence of humidity on the
volatilization of lindane and dieldrin (lindane is more water soluble
and more mobile in soils than dieldrin), they observed that lindane
volatilization was greater than dieldrin when water loss by evaporation
occurred at the soil surface. This was attributed to mass flow movement
of the more soluble insecticide to the soil surface and concurrent evap-
oration. At low relative humidities, the less soluble dieldrin accumu-
lated at the drying soil surface. When the relative humidity was changed
to 100%, the remoistening of the soil surface caused the volatilization
of this accumulated dieldrin. The authors concluded that the magnitude
of the "wick effect" on pesticide volatilization is related to water
evaporation rate, pesticide vapor pressure, and concentration of pesticide
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23
in the soil solution. The latter property is a function of adsorption
to the soil and water solubility.
The difference in volatility due to wet and dry soil is a result
of the displacement of the pesticide from the soil colloid surface by
water molecules. Spencer and Cliath pointed out that in moist soils,
volatilization of pesticide and water will occur at relative rates
approximating the ratio of pesticide/water, and that expected volatili-
zation rates due to mass flow can be calculated reasonably well when
transfer rates from adsorbed to solution phases of the pesticide are
known.
B. Predictive and Experimental Methods
Since volatilization of environmental contaminents is from dilute
solutions, an important property of a organic compound is its distri-
bution coefficient between water and air (Hartley, 1969). This coeffi-
cient is calculated from the vapor pressure of the chemical and its
solubility in water (mg/liter). For convenience, vapor pressure is con-
verted to mg of compound per liter of air by application of the ideal
gas law, given the molecular weight of the compound and a reference
temperature. Hamaker (1972) reports distribution coefficients for a
number of pesticides. An illustration of his data which gives values
of the distribution coefficient (microgram compound/liter water at
saturation divided by microgram/liter air at saturation) is a comparison
of the organo-phosphorous insecticide dimethoate (vapor pressure, 8.5
x 10 mm Hg at 20°C; water solubility, 30,000 ppm) and the organo-
chlorine insecticide aldrin (vapor pressure, 6.0 x 10" mm Hg at 25°C;
water solubility, 0.2 ppm). Both substances have similar vapor pressures
which would indicate similiar concentrations in air at the same temper-
ature. However, because of the large difference in water solubility,
Q
dimethoate has a distribution coefficient of 2.8 x 10 , while aldrin is
only 2.3 x 10 . Thus for equal concentrations to exist in solution,
there would have to be about 10 times more aldrin in the vapor phase
at equilibrium.
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24
Goring (1972) used the distribution coefficients calculated by
Hamaker (1972) to predict the percentage of eleven agricultural chemicals
which would be in the soil air (vapor phase) of a soil containing three
of organic matter (main sorption site). Even though the compounds
varied widely in sorption affinity for soil, the relative, calculated
concentrations in soil air were decreased as the relative tendency of
the compound to partition into water increased. The higher the distri-
bution coefficient (water/air), the lower the amount of chemical in the
soil air, usually irrespective of sorption affinity. An example of this
calculation is a comparison of disulfoton and lindane. Both chemicals
sorb to soil organic material quite differently. The soil sorption (K.)
value for disulfoton is about 15 times that of lindane. Lindane has
a water/air partition coefficient of 19,600 as compared to 5560 for
difulfoton (i.e., 3,5 times greater). Therefore, even though lindane
does not sorb as well as disulfoton, a larger fraction of the non-sorbed
lindane will be associated with the soil water than the soil air when
compared to disulfoton. Using the partition value for the chemical
between soil and water (lO. the water/air partition coefficient, and
the classical 50% solid, 25% air, and 25% water, volume distribution
for a typical soil, Goring calculated the percentage of the chemical
which could be in the soil air under the specified conditions. Ten
times more disulfoton was estimated to be in the soil air than lindane,
even though disulfoton is much more strongly sorbed to the solid phase.
The point to be made here is that it is not sorption affinity of the
chemical to soil but the partition coefficient between water and air
which will control soil air concentrations under non-evaporative
conditions.
Goring (1972) also grouped agricultural chemicals into three cate-
gories in characterizing them relative to diffusion-mediated transport.
When the water/air partition coefficient is 10,000 or below, diffusion
of the chemical in soils is primarily through the vapor phase. When
the coefficient is above 30,000, Goring believes that diffusion will
be predominantly through the soil water. Between 10,000 and 30,000,
the movement of the chemical in the soil by diffusion will be divided
-------
25
between both phases.' Therefore, substances with high partition coeffi-
cients will be lost by volatilization mainly during water loss (evapo-
ration) while chemicals with very low water/air partition coefficients
will be lost both during water loss, but more importantly, by diffusion
in the gas phase.
The same concept has also been applied for volatilization from
natural waters, Mackay and Wolkoff (1972) derived equations to evaluate
the rate of evaporation from water of compounds like hydrocarbons and
chlorinated hydrocarbons which have low water solubilities. Their cal-
culations appear to have merit in evaluating the relative tendencies of
compounds to evaporate from a water column. The equation used,
T = 12.48 L Pw C1$/(E P 1$ M.) ,
predicts the evaporation half-life (T) of a chemical present at less
2
than saturation concentrations in 1 m of water of depth L meters under-
going constant mixing. P is the partial pressure of water at the ex-
w
perimental temperature; C. is the solubility of the compound in water
in mg/i; E is the evaporation rate of water from the column in g/day;
P. is the vapor pressure of the compound; and M. is the compound's
molecular weight. Table 2 presents some examples of substances chosen
to illustrate the predictive equation. For L = 1 meter (i.e., a total
3
volume of 1 m ) and 25°C, T was calculated assuming that E, the evapora-
2
tion rate, was 2740 g/m /day (arbitrarily chosen). The partial pressure
of water at 25°C is 23.76 mm Hg. The pesticides DDT and aldrin have
very short half lives (less than 2 weeks). This is in accord with their
high volatilization rates from water. Aromatic hydrocarbons all have
very short calculated residence times, as do the PCB's. The equation
for predicting residence time is based on the assumptions that the water
column undergoes continuous mixing and that the compound is present in
true solution and not adsorbed, complexed, etc.
Mackay and Leinonen (1975) modified the initial calculations to
include a better estimate of liquid phase mass transfer rates. The re-
sulting changes in the values for evaporation half-life for the chemicals
in Table 2 were small and not significantly different from these estimates.
-------
26
Table 2. Calculated evaporation half-life, T, for selected compounds
in water at 25°C (Mackay and Wolkoff, 1973)
Compounds ' '
Alkanes
n-Octane 0.66 14.1 3.8 sec
2,2,4-Trimethylpentane 2.44 49.3 4.1 sec
Aromatics
Benzene 1780 95.2 37.3 min
Toluene 515 28.4 30.6 min
o.-Xylene 175 6.6 38.8 min
Cumene 50 4.6 14.2 min
Naphthalene 33 0.23 2.9 hr
Biphenyl 7.48 0.057 2.2 hr
Pesticides
DOT 1.2 x 1Q-3 1 x 10-7 3.7 days
Lindane 7.3 9.4 x 1Q-6 289 days
Dieldrin 0.25 1 x 10'7 723 days
Aldrin 0.2 6 x l(r6 10.1 days
PCB's
Aroclor
Aroclor
Aroclor
Aroclor
1242
1248
1254
1260
0
5
1
2
.24
A x
• "T "
O v
'.7 x
10-
10-
10-
2
2
3
4
4
7
4
.06 x
.94 x
.71 x
.05 x
10-
10-
10-
10-
i*
i*
s
5
5.96
58.3
1.2
28.8
hr
min
min
min
= 1 meter, E = 2740 g/m2/day.
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27
Dilling etaK (1975) experimentally measured the evaporation
half-lives, T, of 22 low molecular weight chlorinated hydrocarbons from
water. Measured values were compared to values calculated with the
Mackay and Wolkoff (1973) equation. Although the measured values were
larger (the half-lives in water were longer), Dill ing e_t al_. concluded
that the Mackay and Wolkoff equation adequately predicted that the half-
lives would be in the order of several minutes. Measured values were
obtained in this study by adding the hydrocarbons to water in beakers
and sealed flasks, stirring the resultant suspensions and measuring the
concentration of hydrocarbons in the air phase by mass spectrometry.
Environmental studies of volatilization have progressed from use
of wide-mouthed jars open to the laboratory atmosphere to use of con-
trolled environment techniques (Farmer et ajL, 1972 and Igue et aJL,
1972). Bowman et_ al_. (1959), Lichtenstein and Schulz (1970), Buescher
el^aK (1964), Bowman (1964) and Acree et ajL (1963) studied volatili-
zation of DOT and other chlorinated hydrocarbons from aqueous solutions.
These studies were relatively simple, but effective. The chemical com-
pounds were added to jars or beakers and either incubated under static
conditions, mechanically agitated or subjected to air blown across the
liquid surface. Losses of pesticide were determined by analysis of that
remaining in the aqueous phase. Lloyd-Jones (1971) and Anderson et al.
(1952) placed test chemicals on glass or aluminum surfaces and evaluated
losses by evaporation. Foy (1964) and Jordan et a_L (1965) reported
the use of nickel or aluminum-coated planchets as test surfaces for
evaluating the volatility of S-triazine herbicides.
Burt (1974) evaluated the volatility of radio!abeled (carbon-14)
atrazine herbicide from plant and glass surfaces. A trap containing
activated magnesium silicate was placed in flasks or adapted to cover
live plant material. Air was swept over the surfaces containing the
herbicide and into the trapping matrix. Adsorbed atrazine was subse-
quently eluted from the magnesium silicate and the amount of volatilized
herbicide was determined by radiometric analysis. Hylin and Chin (1968)
described a simple plant incubation chamber in which volatile metabolites
of dimethyl dithiocarbamate fungicide were studied. Radiolabeling with
carbon-14 and sulphur-35 facilitated detection of the volatile compounds.
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28
In addition to evaluating abiotic parameters such as temperature,
windspeed and humidity, the influence of various substrates on the
volatilization of pesticides in water has been studied by Dilling et aK
(1975) and Lichtenstein and Schulz (1970). The addition of peat moss,
clays and other sublimates had the general effect of reducing volatili-
zation of pesticides but had little effect on highly volatile chemicals
such as chloroform.
In summary, the calculational techniques of Mackay and Wolkoff
(1973) and the experimental methods of Spencer and Cliath (1973) and
Dill ing e_t al_. (1975) seem useful for estimating loss of volatile chemi-
cals from soil and sediment-water systems. They appear to be relatively
simple and economic and they offer a reasonable degree of accuracy.
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29
IV. LITERATURE CITED
Abernathy, J. R. and L. M. Wax. 1973. Bentazon mobility and adsorption
in twelve Illinois soils. Weed Sci. 21(3):224-227.
Acree, F. Jr., M.JBeroza, and $LC. Bowman. 1963. J. Ari. Food. Chem.
11:278-280.^ ****
Anderson, W. P., B. J. Linder, and J. W. Mitchell. 1952. Evaporation
of some plant growth regulators and its possible effect on their
acti vi ty. Sci en ce' 116:502.
Bailey, G. W. and J. L. White. 1970. Factors influencing the adsorption,
desorption and movement of pesticides in soil. pp. 29-92. In Residue
Reviews, Francis A. Gunther (ed.). Vol. 32, Triazine HerbicTcfes.
Springer-Verlag, New York-Berlin-Heidelberg. 413 pp.
Booth, R. S. 1975. A systems analysis model for calculating radio-
nuclide transport between receiving waters and bottom sediments.
ORNL/TM-4751.
Bowman, M. C., F. Acree, Jr., C. H. Schmidt, and M. Beroza. 1959. fate
of DDT in larvicide suspensions. J. Econ. Entomol. 52:1038-1042.
Bowman, M. C. 1964. Chlorinated insecticides: fate in aqueous suspen-
sions containing mosquito larvae. Science 146:1480.
Buescher, C. A., J. H. Doughterty, and R. T. Skrinde. 1964. Chemical
oxidation of selected organic pesticides. J. Water Pollution
Centr. Fed. 36:1005.
Burt, G. W. 1974. Volatility of atrazine from plant, soil, and glass
surfaces. J. Environ. Quality. 3(2):114-117.
Caseley, J. C. 1968. The loss of three chloronitrobenzene fungicides
from soil. Bull. Env. Contam. Tox. 3(6):180.
Chapman, T., D. Jordan, D. H. Payne, W. J. Hughes, and R. H.
Schieferstein. 1968. A new triazine herbicide. Proc. 9th Brit.
Weed Control Conf. 9:1018.
Damanakis, M., D. S. H. Drennan, J. D. Fryer, and K. Holly. 1970. The
adsorption and mobility of paraquat on different soils and soil
constituents. Weed Res. 10(3):264-277.
Deli, J. and G. F. Warren. 1971. Adsorption, desorption, and leaching
of diphenamid in soils. Weed Sci. 19(l):67-69.
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30
Oil ling, W. L., N. B. Tefertiller, and G. J. Kallos. 1975. Evaporation
rates and reactivities of methylene chloride, chloroform, 1,1,1,-
trichloroethane, trichoroethylene, tetrachloroethylene, and other
chlorinated compounds in dilute aqueous solutions. Environ. Sci.
Technol. 9(9):833-838.
Dowler, C. C., W. Forestier, and F. H. Tschirley. 1968. Effect and
persistence of herbicides applied to soil in Puerto Rican forests.
Meed Sci. 16:45.
Farmer, W. J., K. Igue, W. F. Spencer and J. P. Martin. 1972. Volatility
of organochlorine insecticides from soil: I. Effect of concentration,
temperature, air flow rate, and vapor pressure. Soil Sci. Soc.
Amer. Proc. 36(3):443-447.
Foy, C. L. 1964. Volatility and tracer studies with alkylamino-s-
triazines. Weeds 12:103-108.
Goring, C. A. 1972. Agricultural chemicals in the environment., _In_
C. A. Goring and J. W. Hamaker (eds.), Organic chemicals in the
soil environment, Vol 2. Marcel Dekker, Inc., New York. 440 pp.
Hamaker, 0. W. 1972. Diffusion and volatilization, pp 341-397. J,n_
C. A. I. Goring and J. W. Hamaker (eds.), Organic chemicals in the
soil environment. Vol. 1. Marcel Dekker, Inc., New York. 440 pp.
Hamaker, J. W., and J. M. Thompson. 1972. Adsorption., pp. 49-144.
In C. A. I. Goring and J. W. Hamaker (eds.), Organic chemicals in
tfie soil environment. Vol. 1. Marcel Dekker, Inc., New York.
440 pp.
Hance, R. J. 1965. The adsorption of urea and some of its derivatives
by a variety of soils. Weed. Res. 5:98-107.
Harris, C. I. 1967. Movement of herbicides in soil. Weeds. 15(3)214-
216.
Hartley, G. S. 1969. Advances in chemistry, Series 86. pp 115-134.
Helling, C. S. 1971a. Pesticide mobility in soils. I. Parameters of
soil thin-layer chromatography. Soil Sci. Soc. Amer. Proc. 35(5):
732-737.
Helling, C. S. 1971b. Pesticide mobility in soils. II. Applications
of soil thin-layer chromatography. Soil Sci. Soc. Amer. Proc.
35(5):737-743.
Helling, C. S. 1971c. Pesticide mobility in soils. III. Influence of
soil properties. Soil Sci. Soc. Amer. Proc. 35(5):743-748.
Helling, C. S., and B. C. Turner. 1968. Pesticide mobility:
Determination by soil thin-layer chromatography. Science 162:562-
563.
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31
Helling, C. S. 1970. Movement of s-triazine herbicides in soils.
pp. 175-210 In, F. A. Gunther (ed.), Residue Reviews. Vol. 32,
Triazine Herbicides. Springer-Verlag, New York-Berlin-Heidelberg.
413 p.
Helling, C. S., D. D. Kaufman, and C. T. Dieter. 1971. Algae bioassay
detection of pesticide mobility in soils. Weed Sci. 19(6):685-690.
Helling, C. S., P. C. Kearney, and M. Alexander. 1971. Behavior of
pesticides in soil. pp. 147-240. _In_ N. C. Brady (ed.), Advances
in agronomy, Vol. 23. Academic Press, New York. 407 pp.
Huggenberger, F., J. Letey, and W. J. Farmer. 1973. Effect of two
nonionic surfactants on adsorption and mobility of selected
pesticides in a soil system. Soil Sci. Soc. Amer. Proc. 37(2):215-
219.
Hunter, J. H., and E. H. Stobbe. 1972. Movement and persistance of
picloram in soil. Weed Sci. 20(5):486-489.
Hylin, J. W., and Byong Hn Chin. 1968. Volatile metabolites from
dime thy! dithio-carbamate fungicide residues. Bull. Environ. Contain.
Toxicol. 3(6):322-332.
Igue, K., W. J. Farmer, W. F. Spencer, and J. P. Martin. 1972. Vola-
tility of organochlorine insecticides from soil: II. Effect of
relative humidity and soil water content on dieldrin volatility.
Soil Sci. Soc. Amer. Proc. 36(3):447-450.
Inch, T. D., R. V. Ley, and D. Utley. 1972. Mobility of some organo-
phosphorus sheep dip insecticides in soil. Pestic. Sci. 3(3):243-
253.
Jordan, L. S., J. D. Mann, and B. E. Day. 1965. Effects of ultraviolet
light on herbicides. Weeds 13:43-46.
La Fleur, K. S., G. A. Wojeck, and W. R. McCaskill. 1973. Movement
of toxaphene and fluometuron through Dunbar soil to underlying
ground water. J. Environ. Qua!. 2(4):515-518.
Lambert, S. M., P. E. Porter, and R. H. Schieferstein. 1965. Movement
and sorption of chemicals applied to the soil. Weeds 13:185-190.
Lichtenstein, E. P. 1959. Movement of insecticides in soils under
leaching and non-leaching conditions. J. Econ. Entomol. 51:380-383.
Lichtenstein, E. P., and K. R. Schulz. 1970. Volatilization of insec-
ticides from van'ous substrates. J. Agr. Food Chem. 18(5):814-818.
Lloyd-Jones, C. P. 1971. Evaporation of DDT. Nature, London. 229:65.
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32
Lotse, E. G., D. A. Graetz, G. Chesters, G. B. Lee, and L. W. Newland.
1968. Undane adsorption by lake sediments. Environ. Sci.
Techno!. 5:353-357.
Lutz, J. F., G. E. Byers, and T. J. Sheets. 1973. The persistence and
movement of picloram and 2,4,5-T in soils. J. Environ. Qual.
2(4):485-488.
Mackay, D., and A. W, Wolkoff. 1973. Rate of evaporation of low-
solubility contaminants from water bodies to atmosphere. Environ.
Sci. Technol. 7(7):611-614.
Mackay, D., and P. J. Leinonen. 1975. Rate of evaporation of low-
solubility contaminants from water bodies to atmosphere. Environ.
Sci. Technol. 9(13):1178-1180.
Mansell, R. S., and L. C. Hammond. 1971. Movement and absorption of
pesticides in sterilized soil columns. Rept. No. WRRC-PUB-16,
Water Resources Research Center, Univ. Florida, Gainesville. 68 pp,
Nash, R. G., and E. A. Woolson. 1968. Distribution of chlorinated
insecticides in cultivated soil. Soil Sci. Soc. Amer. Proc.
32:525-527.
Patrick, W. H., Jr., B. G. Williams, and J. T. Moraghan. 1973. A
simple system for controlling redox potential and pH in soil
suspensions. Soil Sci. Soc. Amer. Proc. 37(2):331.
Pionke, H. B., and G. Chesters. 1973. Pesticide-sediment-water
interactions. J. Environ. Quality. 2(l):29-45.
Scifres, C. J., R. R. Hahn, J. Diaz-Colon, and M. G. Merkle. 1971.
Picloram persistence in semiarid rangeland soils and water.
Weed Sci. 19(4)-.381-384.
Smith, A. E. 1972. Persistence of trifluralin in small field plots as
analyzed by a rapid gas chromatographic method. J. Agric. Food
Chem. 20(4):829-831.
Spencer, W. F., and M. M. Cliath. 1973. Pesticide volatilization as
related to water loss from soil. J. Environ. Qual. 2(2):284-289.
Swoboda, A. R., G. W. Thomas, F. B. Cady, R. W. Baird, and W. G. Knisel.
1971. Distribution of DDT and toxaphene in Houston black clay on
three watersheds. Environ. Sci. Technol. 5(2):141-145.
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33
"STATE-OF-THE-ART" OF THE USE OF LABORATORY MICROCOSMS
TO DETERMINE ENVIRONMENTAL FATE OF CHEMICALS
I. MOVEMENT OF CHEMICALS INTO BIOTA
A. General
There are literally thousands of papers in the literature dealing
with uptake and concentration of chemicals in organisms and the physical
environment. There also is a voluminous literature on uptake of stable
and radioactive elements by organisms and the factors that govern rates
of uptake. Many food chains, both natural and those leading to man, have
been identified in a variety of different ecosystems. It is not within
the scope of this study to review and evaluate this literature which
consists of studies on single species, groups of unrelated species or
simple food chains. However, most of our knowledge on uptake and bio-
accumulation of chemical substances is derived from this literature.
Two recent reviews (Thomas, 1972 and Thomas e£ aj_., 1973) on the use of
organisms to indicate environmental quality have drawn heavily from
this literature. Moreover, the experimental design of many laboratory
microcosm studies is based on information derived from single organism
or trophic level studies.
The transport of energy, as well as chemical substances, is accom-
plished in all ecological systems by food chains. All food chains con-
tain the same functional groups or links known as producers (plants),
primary consumers (herbivores), secondary consumers (carnivores or
omnivores), perhaps tertiary consumers (higher carnivores) and decom-
posers (microbes) which reduce dead organic matter back into useable
components. The individual or species composition of food chains differs
depending on climate, evolutionary history, and the chemical and physical
nature of the supporting substrate which is soil or water (Auerbach et_
al., 1974). A food chain should have at least the first three links to
be complete and they are rarely longer than five links (Kendeigh, 1961).
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34
B. Chemical Transport in Food Chains
Many field'and laboratory studies on the movement of chemicals into.
biota have been conducted on single species or with incomplete (two-link)
food chains. Such studies have usually focused on the metabolic behavior
of a particular chemical, or the environmental factors effecting trans-
port, or the bioaccumulation tendencies of a particular species or
trophic level. These studies are on uptake and bioaccumulation from
physical components (soil, air or water) by plants, or movement of chem-
icals from plants to herbivores or from herbivore to carnivore, etc.
Much of our knowledge of the transport of chemicals in food chains has
been "pieced" together from studies on transport between two trophic
levels — incomplete food chains. Examples of such studies are given
below.
The two major paths of entry of chemicals into food chains of plants
are 1) absorption of substances deposited on foliage and 2) uptake from
soil via roots. In both paths the biological availability of the chem-
ical depends on its solubility in the aqueous media that is in contact
with plant tissues. Examples of transfers of toxic or radioactive sub-
stances into plants have been given in reviews by Copenhaver et al.,
1973 (arsenic); Fulkerson and Goeller, 1973 (cadmium); Sartor e_t al.,
1966 (radioisotopes) and Adams e_t al_., 1957 (fluorides).
Studies on uptake and bioaccumulation by herbivores are illustrated
by the work of Van Hook and Crossley (1969) who determined the biological
turnover rates of several radionuclides in the brown cricket, Acheta
domesticus. This common, herbivorous species feeds on many kinds of
vegetation and is considered a good example of a terrestrial herbivore.
Daphnia, the water flea, is a good example of an aquatic herbivore.
Food transfers to this common organism have been studied by Richman (1958)
and Christensen (1973) who determined types (algae) and uptake rates of
food into Daphnia.
The literature on effects of toxic substances often contains studies
on uptake and bioaccumulation by single organisms. For example, Brungs
(1969) studied transfers of zinc to fathead minnows; Pringle e_t a]_. (1968)
studied trace element uptake by molluscs; Biesinger and Christensen (1972)
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35
studied uptake of metals by Daphm'a; and Arther and Leonard (1970)
studied uptake of copper by isopods and snails.
The literature on culturing techniques for organisms contains both
information on food preferences and food uptake. Examples are culture
studies by Frank e£al_. (1957) on Daphm'a, Robertson et al_. (1974) on
ostracods and Loosanoff (1965) and Loosanoff and Davis (1963) on oysters.
Studies on simple uptake and bioaccumulation of radionuclides by
algae, invertebrates and fish are reviewed by Vanderploeg et ah (1975)
for freshwater species. This review treats both the methods of deter-
mining bioaccumulation in aquatic organisms and the factors that in-
fluence bioaccumulation in natural habitats. A similar review for insect
food chains is given by Reich!e and Van Hook (1970) and for terrestrial
and aquatic food chains by Reichle e£ al_. (1970).
While only a brief treatment of the voluminous literature on food
chains has been given here, the examples indicate ways in which general-
ized food chains can be constructed for laboratory study of transport
of chemicals.
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II. GENERAL CONSIDERATIONS OF LABORATORY MICROCOSMS
A. Advantages of Microcosms
Microcosms are among the useful techniques of studying the behavior
of chemicals of interest, be they potential nutrients or toxicants. In
comparison to field techniques laboratory microcosms offer the advantages
of: control of system complexity, replication and environmental condi-
tions.
Within laboratory microcosms, the level of complexity may be varied
to suit experimental need. All laboratory microcosms omit large-scale
processes, such as alluvial deposition and long-distance migration.
Nevertheless, complex laboratory microcosms are suitable for identifying
toxic chemical accumulation and for assessing the importance of mutual-
istic as well as competing processes. A significant shortcoming is that
complex microcosm studies are inadequate to measure rates of these proc-
esses. Microcosms of reduced complexity are suitable for measuring
rates of the one or few processes over a short time range.
The prime advantage of complex microcosms over simple ones is the
more faithful duplication of heterogeneous environmental conditions and
diversity of organisms. "Mixed culture phenomena are not merely compos-
ites of the pure culture behavior of the organisms present; the perform-
ance of a complex microbial process depends upon interactions between
the species and strains" (Bungay and Bungay, 1968). For example, DDT
may degrade only if subjected to different bacteria in a cycle of
anaerobic-aerobic conditions in the presence of a utilizable energy
source (Pfaender and Alexander, 1972); or, accumulations of microbial
waste products having inhibitory effects may be removed by other microbes,
In this way, complex microcosms serve as a practical device for the inte-
grating myriad biotic-abiotic interactions, and hence may be used as a
screening tool for identifying the potential fate of hazardous chemicals
for further study.
Reasonably simplified microcosms may be used for the verification
of the pathways of chemical transport and the measurement of the corre-
sponding transfer rates. Batch culture techniques are widely used to
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37
measure maximum growth rates, maximum chemical uptake rates, and bio-
accumulation factors. They have the advantage of simplicity of operation
and low cost, but they disallow control of nutrient, toxicant,.metabolite
concentrations, and pH as well and other key parameters. Continuous
culture techniques have been extensively used to measure growth rate as
a function of nutrient level, and to measure a number of cel.l properties,
such as protein content, ONA and RNA content, as a function of growth
rate. They have the advantage over batch culture when controlled sub-
maximal growth rate or controlled chemical introduction are desired,
while suffering the disadvantage of equipment complexity and cost of
operation. Occasional large disparities between batch and continuous
culture growth rate measurements (Button et_al_., 1973) and biotransfor-
mation effects (Vosjan and van der Hook, 1972) have invited unresolved
speculation as to which more faithfully represents a natural system of
interest.
B. Predictions of Actual Environmental Events
Three critical problems arise in using microcosm results for predict-
ing actual environmental results:
1. Applying results of an isolated process to a more complex system
2. Evaluating the comparability of similar but not identical
model systems
3. Estimating confidence intervals of the parameter measurements.
Until these have been evaluated, the "validation" of microcosm
studies cannot be discussed in great depth.
1. Care must be taken when applying results from a lower to a
higher level of complexity, such as from microcosm studies to the real
environment. Parameters measured in isolation may well be strongly
dependent on a factor missing in the microcosm, but existing in the more
complex environment. For example, in a microcosm one may measure an
organism's uptake rate of phosphate as a function of the limiting phos-
phate concentration. However, certain chemicals in a given lake might
inhibit phosphate uptake rate in a manner which could not have been
foreseen from the simple microcosm experiment. This does not negate the
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38
results of the particular experiment, which might well be relevant to a
great many other lakes. Moreover, that particular microcosm experiment
would normally precede more detailed work on the effects of phosphate
inhibitors. Thus one should beware of naive applications of microcosm
results to natural environments, yet should not let obvious environmental
complexity deter mechanism studies which are best performed at a much
simpler level.
2. Is a given microcosm an adequate model of an actual environment?
Authors generally justify their selection of sediment, water, and organ-
isms as typical food chain representatives, readily available, easily
reproducible, and not interfering with chemical analyses. Yet many
studies report differences due to such variables as types of clay, pH of
water, nutrient level, and species of organisms. How similar are two
"model" freshwater microcosms, say one using Chlamydomonas-Daphnia-
Gambusia. and another using Chiorel1a-Cyclops-Lebistes. How much differ-
ence does light cycle or temperature make? The use of standardized
microcosms has advantages of comparability between trials and experiment-
ers; the use of locally-derived components has advantages of local pre-
diction.
3. The lack of precise agreement between replicates where they
have been done, and the general practice of doing only 3 simultaneous
replicates, seriously limits the confidence that can be placed on many
parameter estimates (see E. Reproducibility). In making wide-ranging
decisions, there should be some estimate of the probability that the
results would be similar if the experiment were redone at a later time,
or in a different laboratory. In justification of most microcosm studies,
most researchers made the best use of the resources available to them.
The lack of replicates is often related to the complexity, size, and
intensive labor and cost requirements of the study. The purpose of
most studies is to analyze or simulate, not to validate. Most values
are accepted as being within an "order of magnitude" and they are prob-
ably at least as good as this* However, for practical decision making,
a more precise estimate may be needed.
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39
C. Types
Studies of fate are not simple because they include the many proc-
esses shown in Table 1. Most of the parameters needed to construct
biosphere models (Cramer, 1973; Woodwell et al_., 1971) could be studied
in microcosms. -Identifying the controlling or limiting processes for a
given chemical can make the prediction of its fate more amenable to
analysis.
A review of studies, comparison of techniques and evaluation of.
applicability is not simple because of the diversity of methods and
parameters used (Table 2). No standard laboratory microcosm design
exists; specific types have been developed by various researchers to
simulate each of several habitats:
Terrestrial
Terrestrial - Aquatic
River
Aquatic - Batch
Aquatic - Continuous Culture
Chemostat and Turbidostat
Special Microcosms
Species Defined (Gnotobiotic)
In-situ Bioassay
Closed (Bioregenerative Life Support)
Naturally Occurring Microcosms
Most microcosms are not used for determining chemical fate, but for
studying the properties of community metabolism, photosynthesis, respi-
ration and biomass. Such microcosms adaptable to chemical fate studies
are listed in a supplementary bibliography, Appendix A.
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40
Table 1. Fates of Chemicals in the Environment
I. Transport (measured by "rates")
A. Nonbiological, e.g., complexed or sorbed onto particles
1. Diffusion
2. Convection
3. Settling
4. Evaporation
5. Leaching
6. Long-range effects and cataclysmic events
B. Biological
1. Across cell boundary
2. Migration (vertical or horizontal)
II. Accumulation (measured by concentration levels)
A. Nonbiological
1. Adsorption onto transportable particulate material
2. Held in bottom sediments
B. Biological
1. On nonliving cuticle
2. On cell membrane
a. At protein "recognitive sites"
b. In lipid superstructure
3. In cytoplasm
4. In nucleus
5. In specific tissues of higher organisms
III. Transformation
A. Nonbiological, e.g., photo-oxidative
B. Biological, e.g., metabolic
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41
Table 2. Methods Commonly Used In Microcosm Studies
I. Types of Inputs
A. Solution
1. Single entry, point source
2. Single entry, mixed into system
3. Multiple entry, point or mixed
4. Continuous entry
B. Biological entity
1. Live, e.g., a labelled prey
2. Dead, e.g., tagged leaf litter
C. Nonbiological entity
1. Solid, e.g., labelled fly ash
2. Liquid, e.g., labelled rain
3. Gas, e.g., lSN
II. Timing of Measurements
A. Initial sampling (validation of initial input)
B. Periodic sampling, using subsamples of replicate (s)
C. Terminal sampling on a portion of the replicates (e.g., destructive
sampling of 1/3 of the replicates at 3 different durations)
D. Terminal sampling of all replicates simultaneously
III. Compartments Measured
A. One (i.e., species of interest [fish] or system output [leachate])
B. Special
C. All compartments except container surfaces and atmospheric gases
D. All compartments including container surfaces and atmospheric gases
IV. Entity Measured per Compartment
A. Chemical of interest (e.g., radionuclide or DDT)
B. Carrier (or stable isotope) mass
C. Degradation products (e.g., ODD, DDE)
D. Compartment mass (e.g., dry weight)
V. Calculated Values
A. Concentration of entity in a compartment
B. Transfer rates between compartments (first order, function of mass
in donor compartment; second order, function of mass in donor and
recipient compartments) requiring sequence of measurements and
model of interactions
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42
Table 2. Methods Commonly Used in Microcosm Studies (continued)
C. Specific activity
D. Concentration factor, also called biological or ecological magni-
fication, bioaccumulation factor or index (ratio of concentration
in recipient compartment/donor compartment—donor may be water or
food)
E. Biodegradability index (ratio of concentration of breakdown
productions/parent compounds, e.g., polar/nonpolar labelled com-
pounds, Metcalf et al. 1971b)
F. Distribution among major compartments, e.g., % in soil, water,
algae, snails
G. Total budget studies
VI. Special Types
A. Species defined (gnotofaiotic)
B. In situ bioassay
C. Naturally occurring microcosms
D. Closed (bioregenerative life support)
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43
D. Parameters Measured
The parameters most frequently measured are either (1) transfer rates
and compartment size, which together can also be expressed as turnover
rate or (2) bioaccumulation factor and (3) biodegradation index. If the
relationships controlling transfer rates were known, bioaccumulatton could
be calculated. Bioaccumulation factor is frequently considered very ap-
proximate, probably within an order of magnitude, as it is an- integration
of many complex processes and has been shown to be quite variable. Bio-
degradation can be measured only if the products can be identified, e.g.,
by the presence of a radioactive label.
It is not known if the parameter estimates are markedly affected by
the relative masses of various components, e.g., sediment, water, algae,
etc. The relative masses have been arbitrarily determined by the inves-
tigators, possibly with a consideration of the sample size needed. Most
microcosms did not allow control of the relative densities of organisms.
In particular, fish are often stocked at higher densities than the micro-
cosm can support. "Balanced aquaria" are not generally in use anywhere,
although contrary to Atz (1949a and b) they probably can be developed
(Odum and Johnson, 1955; Richardson, 1930).
Though chemical fate is the principal concern of this report, con-
siderations of chemical fate cannot entirely ignore chemical effects.
Physical and chemical properties and toxicological, effects should be
known for appropriate design of experiments as well as for the protection
of the researchers. Chemical effects may also alter the fate of a chem-
ical by eliminating potential transport or degradation pathways. The
effects of one chemical may alter the fate of many others: terrestrial
pollutants may reduce the inventory of soil nutrients and release them
to streams (Woodwell, 1970).
E. Reproducibility
Few investigators perform repetitions of their studies, and even
fewer perform enough repetitions to make statistical inferences (Table
3). Where investigators have sought to measure microcosm reproducibility
-------
44
in mixed communities, they have found satisfactory reproducibility in
community properties such as productivity and respiration. Much less
reproducibility has been observed when counting individuals of a partic-
ular species (see III. D. Aquatic-Batch Reproducibility for details).
In continuous culture, we have seen no data where the experiment was
repeated more than once (see III. E. Aquatic-Continuous Reproducibility).
F. Applicability
The fate of elements, radioactive or stable, and of various organic
or inorganic compounds can be studied in any of the various types of
microcosms. The use of a radioactive tracer confers the advantages of
greater sensitivity of analyses and the ability to identify degradation
products of complex organic compounds. Since laboratory microcosms tend
to be small, sample size is limited, which, along with the low concen-
tration of chemicals being studied, frequently necessitates radionuclide
tracers. Their use occurred for practical reasons since many of the
early studies (Patten and Witkamp, 1967; Whittaker, 1961) were designed
to study the fate of nuclear reactor releases of radionuclides.
G. Duration
Duration of studies ranged from several hours to approximately one
year. Most are ended when steady state has been approached and the
researchers telieve no further change will occur. Other time periods
are determined by practical considerations; in zooplankton-fish studies
within aquaria, all of the zooplankton are likely to have been eaten by
the second day. Sampling the fish is done on day 3'in the farm-pond
microcosm (Kapoor e_t a\_., 1970). Death of organisms is more likely to
occur in prolonged experiments.
H. Recovery
Although laboratory microcosms present optimal opportunity for re-
covering the test chemical, often complete recovery is not obtained even
-------
45
when container surfaces are eluted for sorted materials. Evaporation,
co-distillation, and degradation are often postulated. More attention
should be given to this matter. .Some attempts to recover all of the
chemical under study in microcosms have been made and these are discussed
later.
I. Environmental Control
Environmental control is expensive, and while an advantage when
equipment is working, becomes a risk when experiments are prolonged.
It is also a form of simplification. For example, the researcher may
find it easier to conduct and analyze a constant temperature and light
experiment, but he cannot use its results to predict the natural environ-
ment unless he knows the natural ranges of light and temperatures, and
has information on temperature and light effects on the microcosm or the
processes within it. To be more specific, how does the researcher ex-
trapolate from a Daphnia-algae microcosm which was run at 20°C, 12 hours
light, to explain the behavior of biota in an arctic pond which in the
summer is 24°C with 24 hours of light and 2 months later is 5°C with 4
hours of light?
J. Equipment Costs
Microcosms have been housed in everything from small vials, jars,
bottles, and aquaria, to sophisticated continuous culture apparatus.
Many of the ingenious arrangements were designed and constructed by the
researchers. However, chemostats, turbidostats, and biooxidation sys-
tems are also commercially available.
Cost is difficult to estimate. Other than continuous culture and
large stream microcosms, the cost of the microcosm equipment is usually
insignificant relative to the costs of labor and equipment necessary for
the accompanying chemical analyses. On a maintenance basis, the major
equipment cost will probably be for environmental temperature control
and monitoring. Most of these expenses are common to any laboratory
experiment.
-------
46
III. SURVEY OF LABORATORY MICROCOSM STUDIES
A. Terrestrial Microcosms.
The most pertinent transport studies originated at Oak Ridge National
Laboratory (Table 3). They involved selected components of the leaf
litter-soil habitat and used simulated rain and leachate collection to
evaluate binding. All involved radionuclides. Mathematical compartment
models aided in calculating transfer kinetics. It is recognized thai-
three studies involved transport of elements or inorganic compounds and
that environmental persistence was not a variable. With many organic
compounds, transport to biota may affect persistence.
In the first study (Patten and Witkamp, 1967), laboratory experiments
134
were conducted to determine patterns and rates of Cs exchange in
microecosysterns composed of different combinations of radioactive leaf
litter, soil, microflora, millipedes, and aqueous leachate. Rate con-
stants for a five-compartment model were determined by fitting models
to data with an analog computer. Simulations with the models permitted
examination of the parameters of radiocesium equilibrium, steady-state
concentrations, concentration factors, input and output fluxes, turnover
rates, and stability.
Of extreme importance, they found that the rate of transfer between
two compartments was altered by the addition of new compartments: in
the absence of soil, the transfer rate from litter to leachate was rapid
(0.037 day ) whereas in the presence of soil (the transfer from litter
to leachate) was reduced (0.001 day ). In that case, the loss rate of
134
Cs from the litter was unchanged, but the fate of the Cs was altered.
However, the loss rate of Cs from the litter was changed by the presence
of microflora and millipedes (from 0.037 to 0.199 day" ). Therefore,
caution must be exercised in using rate measurements from isolated proc-
esses in more complete models. Patten and Witkamp were able to use
their transfer rates in a simulation model of soil communities because
they had appropriate rate constants for all processes alone and in
combination.
-------
Table 3. Terrestrial Microcosms Used for Chemical Fate Studies
Size
Reference Transport of (liters
Bordeleau and
Bartha (1972)
Goswaml and
Green (1971)
Patten and Ult-
kamp (1967)
Vlsser (1971)
Vlsser et al.
(1973)
Ultkamp (1972)
"C labelled
l-chloroanlllne
ioll micro-
irganisns
"•Cs
»N
'N
"Cs
.1
.2
.06
.05
.05
1.7
Duration Parameters Experinental
Equipment Mode of entry Replicates (days) Complexity measured variables Comments
Covered beakers
tater circula-
tion between
soil-filled
tube and
reservoir jar
Slass funnel
lespiroroeter
Mask
lesplrometer
Mask
Transparent
ilastlc box
Single applica-
tion In ethanol
.eaves from
:runk-labelled
trees
i-day exposure
to "Hi
1-day exposure
to liN2
.eaves from
:runk-labelled
trees
actorial, 3
ilcrocosns per
:ond1tion
'actorial, 2
:ultures per
:ondition
;actoria1. 2
:ultures per
rendition
:actor1al. 3
ilcrocosms per
:ond1tton
14
18
27
27
149
Soil
Soil, micro-
organisms.
*ater reservoli
Combinations
of leaves.
nicroflora.
nillipedes.
soil; Simula. tec
rainfall
Clay, sterile
nedlum,
N-fixlng bac-
terium, other
>acteria, duck-
weed
Clay, sterile
nedlum. duck-
teed, H-fixing
lacteriuo.
other bacteria
Combination of
leaf litter.
nlcroftora.
nilltpedes.
snails, plants.
soil; slmulatec
•alnfall
lelative con-
:entrat1ons of
i-chloroanlltnc
transformatloor
>roducts
Distribution o<
"»Cs In leach
ate and micro-
cosm components
rate constants
Total N and
"N:1^ ratio
in duckweed,
liquid, clay '
Total N and
N: N ratio
In duckweed,
liquid, clay;
transfer
coefficients
Xstributlon 01
"'Cs In leach-
ite and micro-
:osra components,
rate constants
Soil, several
nodel enzyme
systems
Combinations
of leaves.
nicroflora.
nillipedes.
soil
Tine. N-fixlng
>acterlun
alone.
N-fixlng plus
other bacteria
Tine. N-flxing
lacterium
alone,
M-flxIng plus
other bacteria
Combinations ot
nllllpedes.
snails, plants
Compares degra-
dation of
anilines In
soil to that in
nodel enzyme
systems
Apparatus to
culture soil
nicroorganisas
-------
Table 3. Terrestrial Hlcrocosros Used for Chemical Fate Studies (continued)
Reference Transport of
Size
(liters)
Equipment Mode of entry Replicates
Duration
(days)
Complexity
Parameters
measured
Experimental
variables
Comments
Ultkamp and
Frank (1968)
Ultkamp and
Frank (1970)
"'Cs
"7CS. K. Hg.
nass
1.7
1
Transparent
ilastlc box
Transparent
ilastlc box
Leaves from
trunk-labelled
trees
Leaves from
trunk-labelled
trees
Factorial. 3
microcosms per
condition
Factorial. 1
microcosm per
condition
149
112
;ombt nation of
leaf litter.
otcroflora.
nillipedes,
mails, plants
soil; simulate!
-ainfall
.eaf litter.
land, milll-
>edes. simu-
lated rainfall
Distribution of
"7Cs in leach-
ate and micro-
cosm components,
rate constants
C02 evolution.
distribution of
elements and
nass in leach-
ate and micro-
cosm components.
rate constants
lombtnattons of
Dtllipedes,
snails, plants
) temperature
levels. 3 rates
»f rainfall. 3
:ensltles of
nlllipedes
-------
49
In another study, temperature, rainfall pattern, and millipede den-
sity were all found to be important in controlling mineral flow in a
four-compartment system in which mass and the transfer of Cs were
being studied (Witkamp and Frank, 1968). This is not surprising to soil
scientists, but most other scientists have conducted their ga^rocosms at
^*TB^r
single, constant temperatures and at single, arbitrary organism densities,
They enlarged the scope of their microcosms to include snails, green
plants, and both mineral and organic soil (Witkamp and Frank, 1970).
The same techniques were used. The effect of a compartment was often
disproportionate to its content: microflora turned over only 10% of
the litter, but contained one-third of the Cs of the community, while
millipedes turned over one-third of the litter but contained less than
137
1% of the Cs. A similar stepwise approach was used to evaluate the
role of various decomposers in the recycling of detritus to primary
producers (Witkamp, 1972). Plant growth was used as a measure of trans-
fer, as well as Cs movement.
The fate of microbially fixed nitrogen was determined in a model
microcosm with N as a tracer (Visser, 1971; Visser, Witkamp, and
Dahlman, 1973). This study is similar in method to the earlier Witkamp
work, except that it was done within a respirometer apparatus. Since
it was sealed much of the time, it could qualify as a closed ecological
system. It could also qualify as a terrestrial-aquatic system, since
the nitrogen fixing bacteria were largely associated with a sediment
which was partially submerged, and an aquatic plant floated in an
aquatic phase.
The potential evaluation of soil microcosms as predictors of actual
environmental events must rest on the ability of the transfer rates de-
rived from the microcosms to predict occurrences on the actual forest
floor. The validity of extrapolating from the microcosm to the forest
should be testable.
A new tool, X-ray energy spectroscopy (or microprobe analysis), is
currently being used on terrestrial microcosms by Dr. Sidney Draggan at
Oak Ridge National Laboratory. This tool maps the presence of heavy
atomic nuclei in electron photomicrographs. To date, it has been shown
that Co is accumulated largely in fungal spores.
-------
50
Semi-closed soil-decomposer-litter-plant-leachate microcosms are
also being used by Draggan and Witkamp to study iodine transfers. These
studies have been limited to the transfer of elements. Radionuclides
were used not only because of their interest to that laboratory, but
also because very small amounts could be measured.
An apparatus for soil percolation studies has been described, which
might be useful in constructing systems involving continuous and repeated
leachate exposure to soil (Goswami and Green, 1971).
14
C-labeled 4-chloroaniline transformation products were compared
when (1) generated in soil or when (2) generated by treatment with soil
fungus enzymes (Bordeleau and Bartha, 1972). Although it would be desir-
able to conduct degradation studies directly in soil, the authors point
out that most of the resultant aniline transformation products are
complex polymers of unknown chemical structure. They are adsorbed to
soil particles to various degrees, which prevents detection or quanti-
fication of results. Several extracts of Geotrichum candidum, and a
horseradish peroxidase preparation gave autoradiograms similar to the
soil incubation of the compound. The authors thereafter used the cell
enzymes to explore the susceptibility of substituted anilines to poly-
merizing transformations. In support of their statement that soil binds
that group of chemicals, only 41% of the total added radioactivity could
be extracted from the soil.
Two Two other microcosm systems are also being investigated. The first,
under grant to Dr. Robert Metcalf (University of Illinois), is a terres-
trial "monoculture" system in a 20-liter glass jug fitted for atmospheric
sampling. Cotton, soybeans or corn are shown in either of two typical
Illinois soils to which a variety of invertebrates have been added. The
C-labeled pesticide (DDT, methoxychlor, dyfonate, aldrin, trifluralin,
or atrazine) is applied to the leaves or soil and, after 30 days, a
prairie vole (Microtus sp.) is added as the top level consumer. Although
still in the developmental stage, preliminary results from this system
suggest that atmospheric losses, in addition to the gross bioaccumulation
and biodegradation ratios can be ascertained in a screening mode.
The second system, yet to be completed and become operational, is
under the direction of James W. Gillett at the National Ecological
-------
51
Research Laboratory at NERC-Corvallis. Eight glass chambers (1m x 0.75m
x 0.64m) with Plexiglas lids have been fitted with controlled air supply
and total air exhaust monitoring package, a controlled "rain" supply,
and a "ground water" to "spring" circulation system for monitoring of
soil leachates, exposure via surface waters, etc. The soil (20cm) is
composed of synthetic potting mixture (peat moss, perlite,.,and nutrients)
amended with variable proportions of sand and clay mineral. Preliminary
tests have employed 12'crop plant species, a variety of soil inverte-
brates, and a number of phytophagous and predaceous insects with varying
success up to 6 weeks. Planned biota will include 10-15 species of crop
and non-crop plants, earthworms, isopods, diplods, aphids (3 species),
lady bug beetles (two species), Diabrotica sp.t a carabid beetle, a small
garden slug, German cockroaches, and either a rodent (one of two Northwest
Microtus sp.) and/or a young Japanese quail. This terrarium system is
being designed, operated, and tested according to a conceptual model of
chemical movement in the environment in an attempt to develop a labora-
tory system capable of assessing effects of toxic chemicals.
B. Terrestrial-Aquatic Microcosms
Farm-Pond •(-" Metcalf Microcosms")
Aquaria containing partly submerged substrates were first used for
chemical transport and degradation studies by Metcalf and his associates
at University of Illinois. Extensive single organism studies involving
14
uptake and degradation of C pesticides had already, been conducted on
most of the components of the "model ecosystem." The microcosm is
described most completely by Metcalf e_t al_. (1971a_). As described
briefly by Kapoor et. ah (1970):
"A model ecosystem for evaluating pesticide biodegradability
has been developed in this laboratory. It consists-of a TO x 12
x 8 in. glass aquarium containing a shelf of 15 kg of washed white
quartz sand which is molded into a sloping soil-air-water interface.
The lower portion is covered by 12 1. of 'standard reference water1
which provides satisfactory mineral nutrition for the growth of
Sorghum halpense on the aerial portion, and the algae Oedogonium
-------
52
cardiacum in the aquatic portion. The latter is seeded with a
compliment of plankton, and contains Daphnia magna and Physa
snails. The aquarium is provided with aeration and is kept in
an environmental plant growth chamber at 80° F (26° C) with 12
hr daylight exposure to 5000 ft candles.
"In operation, the Sorghum seeds were planted and the aquarium
-allowed to equilibrate for 20 days until the Sorghum plants are
about 6 in. high. The leaves were then treated through a micro-
pipette so that only the plant surface was contaminated. Ten large
Estigmene acrea caterpillar larvae were placed in the chamber and .
allowed to feed until the plants were consumed. The radidlabeled
fecal materials thoroughly contaminate the aqueous portion and are
taken up into the several food chains. After 26 days, 300 C illex
quinquefasciatus mosquito larvae were added to the chamber, and
after 30 days three Gambusia affinis fish were added. The experi-
ment was terminated after 33 days, when weighed samples of fish,
snails, mosquito larvae, algae, and water were removed and assayed
to total radioactivity. These samples were homogenized and extracted
with diethyl ether, and both water and ether layers examined by thin
layer chromatography to determine the qualitative and quantitative
nature of the degradative products present, using radioautography
and serial scintillation counting of the areas containing radio-
activity. The results of the total examination of the model system
provide evidence of the relative biodegradability of the pesticide."
A consistent theme is clearly stated in the ten or more Metcalf
publications (Table 4). DDT is a highly effective insecticide, but has
three serious ecological disadvantages: it and its degradation products,
ODD and DDE, (1) bioaccumulate, (2) do not readily degrade, and (3) are
efficient inducers of microsomal oxidases in vertebrate livers. They
bioaccumulate because their solubility is low in water but high in
lipids. Their resistance to biodegradation is due to their lack of
"handles" for attack by enzymes. Since the insecticidal and degradational
properties of modified DDT compounds are not predictable, they must be
measured empirically. Metcalf and his associates have a program for
synthesizing and testing various analogues of DDT, or other insecticides.
-------
Table 4. Terrestrial-Aquatic Microcosms Used for Chemical Fate Studies
Reference
Size
Transport of (liters)
Equipment Mode of entry
Duration
Replicates (days) Complexity
Parameters Experimental
measured variables
Comments
Booth. Vu, and
Hansen (1973]
Brown (1970)
Huckabee and
81 ay lock
(1974)
Kapoor et al.
(1970)
iantazon
(utrlents.
lollutants
'"Hg
"*mcd
"Se
lethoxychlor,
nethlochlor.
)OT
7
500- 100(
60
porio
12
Hquarium with
terrestrial
>ortion
Series of
artificial
lakes and
streams
\quartum with
terrestrial
lortlon
Aquarium with
terrestrial
>ort1on
*pply to
sorghum leaves
Continuous
Input
Simulated
rainfall
kpply In ace-
tone to
sorghum leaves
2
2
1
20
27-129
33
Freshwater.
sand, caterpil-
lars, fish.
snails, mosqui-
tos. Daphnia.
clams, crabs.
Aquatic plants
Freshwater,
soil, algae,
grass, fish.
insects
Freshwater,
stream bank.
stream sedi-
aents, fish.
snails, ter-
restrial and
aquatic plants
Freshwater.
sand, sorghum,
algae, Daphnia
snails, fish.
nosquito larvae
caterpillar
Concentration
in water and
aquatic organ-
isms, concen-
tration factor.
naterials
lalance
Oxygen, biota.
«/ater hardness.
light, pH. flov
rate, tempera-
ture, soil
composition.
pollutants
Concentration
in water, sub-
strate, plants
and animals;
materials
balance
Concentration
of compounds
and their me-
tabolites in
water, fish.
algae, snails.
nosqultos.
Materials
balance
Time
::.-.
:hemical addi-
tives, filter
>eds. 1 1 limi-
tation. pH
rime
rime
Also examines
toxicity of
these compounds
to mice and
Insects
en
OJ
-------
Table 4. Terrestrial-Aquatic Microcosms Used for Chemical Fate Studies (continued)
Size Duration Parameters Experimental
Reference Transport of (liters) Equipment Node of entry Replicates (days) Complexity measured variables Comments
He teal f (1971)
Me teal f et al.
()9?la)
Hetcalf et al.
(1971b)
Metcalf et al.
(1972)
DOT.
nethoxychlor,
ethoxychlor.
nethylchlor.
nethlochlor,
nethoxy-
nethlochlor.
nethyl-eth-
oxychlor,
chlor-methyl-
chlor
DDT.
DOE.
ODD.
nethoxychlor
DDT.
DOE.
nethoxychlor.
nethylchlor.
nethlochlor.
CH,S - CH.O
DDT.
DOE.
nethoxychlor.
nethylchlor.
nethlochlor,
CH.S - CH,0
7
tquarlum with
terrestrial
jortion
Apply 1n
acetone to
sorghum leaves
2
33
:reshwater,
>and, sorghum.
ilgae. Oaphnla.
inall. fish.
:aterpillar.
msqulto larvae
Concentration
>f compounds
ind their me-
tabolites in
vater. snail.
nosquito, fish;
naterial bal-
ance; ecologi-
cal magnifica-
tion; biode-
sradablllty
Concentration
of compounds
and their me'
tabolites in
iater. snails.
nosquito, fish;
naterials
lalance
Btodegrada-
>ility
Concentration
of compounds
and their me-
tabolites in
vater. snails.
fish, mosquito:
naterial
>a lance
Time
See Metcalf et
al. (I97la) for
description of
experimental
procedure
Describes the
organisms in
the microcosm
and the experi-
mental proce-
dure
See Kapoor et
al. (1970)
See Hetcalf et
al. (1971a) for
description of
experimental
procedure
en
-------
Table 4. Terrestrial-Aquatic Microcosms Used for Chemical Fate Studies (continued)
Size Duration Parameters ' Experimental
Reference Transport of (liters) Equipment Mode of entry Replicates (days) Complexity measured variables Comments
Hetcalf (1973)
He tea If et al.
(19?3a)
Met calf et al.
(1973D)
aldrln.
dleldrln.
endrin. ml rex,
lindane.
lexachloro-
>enzene. DOT.
DOE, ODD
organochlorldes
organophos-
|>hate$.
:arbamate.
wnnone.
plmics.
terblcides.
esters, PCS.
rCBD, hexa-
chlorobenzene
91-2-ethyl-
lexylphthalate
•
Concentration
of compounds
and their me-
tabolites in
water, algae,
snails, fish.
mosquito; mate-
rials balance,
ecological mag-
nification!
biodegrada-
billty
Concentration
of compounds
and their me-
tabolites In
the water and
several organ-
isms In the
model ecosystem
materials bal-
ance; ecologi-
cal magnifica-
tion, biode-
gradability
Concentration
of compound and
metabolites In
water, algae,
fish, snail.
clam, Oaphnia.
mosquito; raate-
rials balance
!
£»
M
w
See Hetcalf et
al. (1971a) for
description of
experimental
procedure
See Me teal f et
al. (1971a) for
description of
experimental
procedure
See Hetcalf
(1971a) for de-
scription of
experimental
procedure
tn
en
-------
Table 4. Terrestrial-Aquatic Microcosms Used for Chemical Fate Studies (continued)
Size Duration Parameters Experimental
Reference Transport of (liters) Equipment Node of entry Replicates (days) Complexity measured variables Comnents
Retngold et al.
(197U
lethoxychlor.
IDT
5
Jars
)ne Initial re-
lease
Factorial. 2
iars oer con-
31
Freshwater
)aohn1a. one ot
Concentration
In water and
2 concentrations
if Insecticide
en
tft
-------
57
They published a series of papers which starts with the bioaccumulation
and biodegradation properties in the model ecosystem of methoxychlor,
methioch]or, and DDT (Kapoor et al_., 1970), and continues to compare
other compounds to these. Data for compounds with known beneficial
properties are repeated in subsequent papers; data for new compounds are
published only once. More complex and different biota, and additional -
pesticides are studied in the more recent publications, e.g., Metcalf
et al_. (1973b) and Booth et al_. (1973). The compounds tested are shown
in Table 4. ..
The "Metcalf microcosms" are ingenious and well suited for their
goal of comparing the biomagnification and the biodegradation of related
compounds. The diversity of the target organisms and of the contaminat-
ing microbiota are likely to provide most of the degradation capabilities
available in a natural environment. The physical diversity is advanta-
geous because it provides microhabitats which may be favorable to organ-
isms which degrade a compound only under particular oxygen concentrations
or in the presence of an adequate energy source; The diversity of organ-
isms also provides a diversity of organic compounds in the environment,
so that many kinds of microbes can persist. The diversity of microbes
may increase degradation by the removal of waste products by another
microbe. It would be impossible to examine all these interactions by
studying all possible isolates, all possible combinations, under all
possible 0- and substrate concentrations. Thus the Metcalf system ap-
pears highly effective as a tool to compare the properties of compounds.
However, there are problems in adapting bioaccumulation and biode-
gradation indices to predicting events .in natural environments:
1. The reproducibility of the results is largely unknown.
2. No set of organisms is completely typical of any other set.
If local organisms are used corresponding to each locality,
the results would likely differ, but would be more applicable.
3. Soil has been excluded because it would interfere with the
chemical analysis. Would these results be unchanged by soil?
4. The system is completely insensitive to effects, even those
that would alter movement, e.g., if the caterpillars die, they
are replaced.
-------
58
5. The relative amounts of sand, water, and biota are strictly
arbitrary. Do they influence the parameters measured?
6. Bioaccumulation is the result of complex processes: uptake,
release, and biodegradation. Uptake can occur via physical
processes on the dead organism [Gushing and Rose (1970) showed
higher accumulation on dead phytoplankton], and through food
and water uptake in the live animal. Internally, it may be
transported to different organ systems, each of which has its
own turnover.and degradation rate. Degradation may be affected
by age, health, and nutritional state of the organism. Release
will depend on all of the above, but especially on the concen-
tration in the organism. It is a gross simplification to con-
sider the organism/water relationship akin to a solvent/water
extraction process. Is the bioaccumulation factor constant
even for a given chemical, given variables such as temperature,
competing substrates such as soil, alternate feeding pathways
influenced by selective mortalities and reproductive rates?
7. Since shifts in biota are prevented, their effects on chemical
fate are not measured. The prevention of new, potentially more
successful forms being introduced may give misleading results
if extrapolated to natural environments where diversity is much
greater. This is especially important if pesticide-resistant
forms could become dominant. In the farm-pond microcosm, the
caterpillars eat all of the Sorghum leaves, while much of the
leaf material, caterpillars, and feces drop into the water. If
caterpillars were replaced by beetles that ate only terminal
buds and then flew away from the water, would that influence
the results?
8. Metcalf and his colleagues made no attempt to assess the amount
of pesticide that remained on the "terrestrial" area and the
amount that got into the water. If a storm washed the terres-
trial surface, would the chemical fate have been unchanged?
9. No total budget or accountability is routinely done in a Metcalf
microcosm. This leaves the potential C0? distillation and atmos-
pheric transport unmeasured. Metcalf reports that they have
-------
59
tried to measure this, but little was recovered from the atmos-
phere. Only about 60% of the initial label was recovered. He
attributes the loss to sorption on the aquaria walls and the
sand (personal communication).
Rain-Streambank-Pond
This study (Huckabee and Blaylock, 1974) differed from "Metcalf
microcosms" in mode of entry, complexity, and expression of the data
(Table 4). A simulated "rain" was applied to the terrestrial and aquatic
areas. The complexity included moss, higher plants, litter, soil, water,
sediments, fish, snails, watercress, and the plastic liner of the con-
tainer. The presence of soil and sediments was extremely important be-
cause most of the isotopes were found there. The data were presented
as the distribution of the radioisotopes, i.e., the proportion in each
compartment of the initial label. This means of data display may be
very sensitive to the relative proportions of the various components.
A biomagnification factor, even within the aquatic phase, would have
been very difficult to justify. Since the fish and snails might have
been ingesting portions of the sediment, the sediments might be consid-
ered a more valid donor compartment. The concentration of the isotopes
in the water was extremely low, presumably because the soil provided an
unsaturated sink.
Soil-Stream-Lake
A more ambitious apparatus was constructed and tested by Brown
(1970). It occupied an entire greenhouse. In addition to the soil, it
contained an input lake of 106 liters, a fast-moving stream, a 106-liter
semi-stagnant lake, a marsh, a wide, slow-moving stream, a 65-liter
seepage lake, a narrow, deep stream, a 103-liter semi-stagnant lake, a
winding stream, and a 95-liter catch lake. The great potential of this
system is described, but only a few examples of a nitrate enrichment
study are shown. The report is in the form of a research proposal. The
prototype was built at the C. F. Kettering Research Laboratory, Yellow
Springs, Ohio. The cost of building a new 75 ft fiberglass system was
listed at $1,000.
-------
60
C. Laboratory Stream Microcosms
Only four studies actually used river'or stream microcosms for
transport studies (Table 5), but additional information could be gathered
from biological effect studies and apparatus design reports.
The effects of clays on the transport of Cs and Sr in a model
river was studied by Purushothaman (1971). No biological data are given,
although several biological aspects were mentioned in the study: (1)
macrorooted aquatic plants were planted in the sediments, (2) the ground
water was stored to allow the development of biota, and (3) mention was
made of plants being sampled for analysis. The suspended clays in the
model river resulted in a significant reduction in the downstream trans-
port of Cs. The Cs sorbed on clays which subsequently settled.
The uptake of Cs by attapulgite was higher than that by kaolinite.
85
The downstream transport of Sr was not affected to a considerable
85
degree since-most of the Sr remained in solution. Unfortunately, the
competition for Cs and Sr between clays and biota was not considered.
The accumulation of six radionuclides by the bivalve, Anodonta
piscina!is. was studied under natural temperature conditions from June
to October (Gardner and Skulberg, 1965). The input of the radionuclides
was continuous and was adjusted so that their concentration was constant
during the experiment. Accumulation of the radionuclides occurred in
both the body and the valves (shell). The first phase consisted of a
steady increase lasting several days. A second phase consisted of a
quasi-steady state, when uptake and loss were approximately equal, though
fluctuations did occur presumably due to seasonal and physiological
circumstances. Since the valves have been suggested as model indicators
of past contamination on the assumption that the annual layers are not
subject to subsequent metabolism, the authors noted that their concen-
trations of radionuclides in valves varied as greatly as those in the
body. Therefore, they caution that the metabolic turnover of elements
in the valves may be slow, but must be taken into account.
Usually only one specimen was analyzed at each time. Thus it is
not clear if some of the above-mentioned seasonal variation could have
been due merely to differences between animals. With the exception of
-------
Table 5. River Microcosms Used for Chemical fate Studies
Reference Transport of (liters) Equipment Mode of entry Replicates
Duration
(days)
Complexity
Parameters
measured
Experimental
variables
Comments
Gushing and
Watson (1971
Gardner and
Shulberg
(1965)
Purushothaman
Rose and
Mclntlre
(1970)
"Zn
"P. "Sr.
"''Ce. "'Ru.
'"Cs/'Zr/^Nb
"Sr. '"Cs
dleldrln
85
3600
7700
146
Hatchery trougt
*1th paddle
< heels
Channel
Flume
Trough with
paddle wheel
:ontinuous for
96 days; then
latch for 35
days
Continuous In-
put as liquid
Two Instantan-
eous releases
Continuous
Input from
column of sand
1 (2 channels.
3 Isotopes/
channel)
2
Factorial (2
troughs per
treatment)
180
125
19
60-120
Freshwater
rocks, peri-
?hyton, carp
Freshwater,
pebbles.
natural biota
jlvalve
iacroplants.
sediments.
freshwater
Freshwater
rocks ,
periphyton
Concentration
In carp and
>ertphyton,
specific activ-
ity, accumula-
tion, loss
Concentrations
In water, bi-
valve bodies.
livalve shells
Sorption coef-
ficient, con-
centration In
-------
62
32 89
P and possibly Sr, which continued to accumulate slowly after the
first month, the fluctuations found in the others (137Cs, 144Cs, 95Zr/
N,b, Ru) seemed random, contrary to the authors' interpretation.
The cycling of Zn was studied in a water-periphyton-fish food
web (Gushing and Wat^j, 1971). By continuous additions, Zn concen-
trations were maintained at 1 and 10 pCi/ml in the experimental streams,
as compared to a background level of approximately 0,05 pCi/ml in the
Columbia River water used for a control stream. The accumulation in
the periphyton resembled the pattern of accumulation in the bivalve
Anodonta described above—uptake followed by steady state after about
28 days. The Zn concentration in fish (carp) rose more slowly,
reaching steady state inapproximately 43 days. When the Zn input
was cut off, the concentration in the periphyton gradually decreased,
whereas in the carp, concentration was virtually unchanged from its
prior steady state at the end of the experiment two months later. Con-f
centration data are reported from three samples taken at a given time.
The range of concentrations taken on a given day sometimes varied by a
factor of four.
The accumulation of dieldrin by benthic algal communities was stu-
died in laboratory streams by Rose and Mclntire (1970). Dieldrin con-
centrations of 0.05 to 7.0 ppb were maintained in the water of the
streams by elution from treated sand. Analysis of algal samples showed
concentration factors as high as 30,000. Filamentous algae accumulated
greater amounts of insecticide than unicellular diatom communities.
High current velocity increased accumulation. Dried (dead) algae had
higher accumulation.
D. Aquatic - Batch Microcosms
By far the greatest number.of transport studies have been conducted
in batch or recirculating conditions (Table 6). The studies are highly
diverse in purpose and approach, and range from extremely complex
sediment-water-plant-animal systems to simple two-compartment (donor-
recipient) systems. The simpler studies were often preparatory to, or
part of, more complex systems of interest to investigators.
-------
Table 6. Aquatic-Batch Microcosms Used for Chemical Fate Studies
Size Duration Parameters Experimental
Reference Transport of (liters) Equipment Mode of entry Replicates (days) Complexity measured . variables Comments
Confer (1972)
Crosby and
Tucker (1971)
Cross et al.
(1971)
Gushing and
Rose (1970)
DeKontng and
Mortimer
(1971)
"f. »'P
IDT
"Zn.
stable Zn
"Zn
"C labelled
)OT
200
.1
2000
.2
.07
Aquarium
Beaker
Fiberglass
tank
Water circu-
lating through
glass tube
Flask
Continuous or
spike
Inject In ace-
tone below
iurface of
:ulture
Single
application
Single applica-
tion in recir-
:u1ating and
continuous ap-
>11cat1on In
flow through
experiment
Single applica-
tion in ethano
ir benzene
2-4 aquaria per
condition
1
1
1
Factorial . 3-6
cultures per
condition
180-135
1
270
1.S-3
6
Freshwater.
river perl-
phyton. plank-
snatls, Ostra-
cods
Freshwater
Daphnia
Sewater.
planktonic and
benthlc algae
River water.
periphyton
Freshwater
medium.
Euqlena
Concentration
of P
Concentration
of DOT in'
iaphnja. water.
containers;
biological mag-
nification
Concentration
of "Zn and
total Zn in
benthlc algae.
particulate
material. 3
water fractions:
biological mag-
nification
Continuous mon-
itoring of "Zf
concentration
in water and
periphyton
Cell numbers.
DOT In cells
and in cell
free medium
> Input
Various concen-
:rattons of DOT
rioe
Ight intensity,
live or dead
leriphyton. con-
•etlng cations.
Initial "Zn
concentrations .
time
rime, various
concentrations
if DOT and
ithanol
•
en
00
-------
Table 6. Aquatic-Batch Microcosms Used for Chemical Fate Studies (continued)
Size
Reference Transport of (liters) Equipment Mode of entry Replicates
Duration
(days) Complexity
Parameters Experimental
measured variables
Comments
01 Salvo (1971]
Duke et al.
(1969)
Emst (1970)
Fillp and Lynn
(1972)
Focht and
Alexander
(1970)
Fujita and
Hashizune
(1972)
"S-labelled
bacteria
"Zn
"•C- label led
DOT
Hg
Dlphenylme thane
Radioactive Hg
20
1000
.5
Plastic vessel
containing cir-
culating sea
water
Fiberglass
tank containln;
live traps
Culture flasks
Baffled flasks
Culture flask
single applica-
tion to water
Single applica-
tion with peri-
>dic renewal
)aily oral
application
Jingle applica-
tion of NgCl
Initial consti-
tuent of medium
Single applica-
tion of HgCl
1
actortal
1
1
1
2
IS (con-
trols
ibservec
for 450
lays)
7-14
.08
*
.7
7
liato
lac
Sea water,
living coral,
labelled bac-
teria, reef
invertebrates
Sea water.
sediments,
aysters. clams,
scallops, mud
crabs
Flatfish
Freshwater
green alga, no
>acteria
:reshwater
nedium
freshwater
diatom, no
>acteria
ladtoactlvlty
if water, de-
tritus, coral,
invertebrates.
fate of >«S tn
3 organisms
Concentration
af "Zn In
sediments and
irganisms. bio-
logical magni-
fication
Concentration
of DDT and
netabolites in
various tissues
Concentration
of Hg in cells
as percentage
of concentra-
tion in whole
cul ture
Dxidation of
dtphenylme thane
and related
compounds
Diatom numbers;
radioactivity
of whole cul-
ture, diatoms.
filtrate
Iptake by whole
iwauunlty and
>y a single bi-
valve, time
Salinity, tem-
terature, pH,
!n
(tiled cells.
Ive cells In
lark, live
:ells in light
-'•
•ells grown on
llucose or dl-
iheny line thane,
5 compounds
•elated to dl-
ihenylnethane
'toe, concen-
ratton of Hg,
11 at oo skele-
:ons
In German
en
-------
Table 6. Aquatic-Batch Microcosms Used for Chemical Fate Studies (continued)
Size Duration Parameters Experimental
Reference Transport of (liters) Equipment Mode of entry Replicates (days) Complexity measured variables Comments
Isensee et al.
(1973)
Johannes (1964
Johannes (1965
Johnson et al.
(1971)
Kawatskl and
Schmutbach
0971)
"C-labelled
alky) arsen-
icals
"P (organic)
"P
"C-labelled
aldrtn and DOT
"C- label led
aldrin and
dieldrin
4
.025-. 2!
.05-. 2
1
.5
Aquarium
Flasks
Flasks
Continuous flo*
through glass
chambers
Jar
1 .
itngle applica-
tion
Initial constl-
;uent of roedluo
n bacteria
nltial consti-
:uent of mediun
n bacteria
r
Continuous
input
Single applica-
tion in acetone
actorlal (2
iquaria per
:ondition
1
1
1
1
3-32
2-41
30
3-7
2
:reshwater,
algae, Oaphnia,
snails, fish
Artificial sea
•liter, amphtpod,
lenthic diatom,
nixed bacteria
Seawater. bac-
teria, ct Hates,
flagellates.
plant detritus
Freshwater, one
of ten aquatic
invertebrates
:reshwater.
ostracod
)oncentrat1on
if compound in
io1ogical mag-
ilfication and
iccumulation
'opulation size
>f organisms.
iptake and
•el ease of dis-
solved phos-
>horus (organic
md Inorganic)
Concentration
if "P in
organism and
regeneration oi
inorganic,12?
:oncen trail on
3f compound ant
netabolites in
tater and or-
lanlsms, bio-
logical magni-
Mcation, ,
Concentration
>f compounds
in water and
istracod <• f
rime, three coin-
rounds
Fine, various
:omb1 nations of
irganisms
>
Various -combin
jtions of bac-
teria, ciliates,
flagellates.
dant detritus;
time
\\dr\n or DOT.
me of ten In-
vertebrates .
time
! • '
rine. combina-
tions of -aldrin
irid dieldrin
CTt
tn
-------
Table 6. Aquatic-Batch Microcosms Used for Chemical Fate Studies (continued)
Stze Duration Parameters Experimental
•Reference Transport of (liters) Equipment Mode of Entry Replicates (days) Complexity measured variables Comnents
King (1964)
Kokke (1971)
Kolb et al.
(1973)
Morgan (1972)
Pfaender and
Alexander
(1972)
Parcel la et al
(1970)
"7Cs
Radio-labelled
chlorinated
hydrocarbons ,
lipyrtdylium
herbicides.
heavy metals
Ha stable.
'''Hg
DOT. PC8
"C-labelled
DDT
"P. "P
1
.1
to .6
.5
03-1.0
10
Small aquaria
Flasks
Jeakers ,
some sealed
Jar
Flasks (anaero-
bic conditions]
Cylindrical
container
Single applica-
tion
Initial consti-
tuent of medium
.(quid, nixed
In media, or
alga or paphnia
Single applica-
tion
I
2
1-5
1
1-3
1-16
Var.
168
13
Freshwater
algae, Oaphnia
fish
Hicrobial com-
munities from
soil or waste
matter
Air, freshwater.
algae, Oaphnia
fish
Freshwater,
alga. Oaphnia.
guppies
Freshwater,
sewage or sedi-
ment connunity
of microorgan-
isms, or slnglt
species
Freshwater.
sediment, algae
bacteria
Concentrations
of M7Cs in
rfater and or-
ganisms, bio-
logical magni-
fication
Concentration
of compound in
cells and in
cells free
supernatant
Evaporation.
concentration
factor
Concentration
factor
Concentration
of DOT and its
netabolltes
Concentrations
of several sub-
stances (espe-
cially nutri-
ents in sedi-
nents (blomass)
Various "7Cs
:oncentrations
md algae cell
:oncentrations.
temperature,
:oM>tnations of
Fish and paphnia.
time
farlous glucose
:oncentrations
md pollutants
llxing, food
lathway. Hg con-
:entration
Alternate modes
if entry
rime, various
olcrobial
:onnunities
Hue, four
types of sedi-
nent. aerobic
md anaerobic
cr>
-------
Table 6. Aquatic-Batch Microcosms Used for Chemical Fate Studies (continued)
Size Duration Parameters Experimental
Reference Transport of (liters) Equipment Mode of entry Replicates (days) Complexity measured variables Comments
Relnert (1972)
Sanders and
Chandler
(1972)
Short et al.
(1973)
Stewart et al.
(1971)
Taub and
Pearson
(1973)
lieldrin
"Cl-labelled
>CB
"Ho
rltlun
ig. ca.
:oxaphene, PCB
7.4. .26
1
75
20
.5
Iquarium with
:ont1nuous flov
;ont1nuous flov
through glass
:hamfaers
'olyethylene
•anfc containing
live traps
ilass battery
jar
lars
:ontinuous
application In
ice tone
:ont1nuous
Input * '
iingle applica-
tion
Single applica-
tion
iingle applica-
tion
• •
1
1
1
1
10 pairs
[pair • control
and toxicant)
.5 to 3;
21-35
24
7-56
7
21
35
Freshwater;
combinations oi
ilgae, Oaphnia,
guppies
:reshwater, one
of eight aqua-
tic inverte- '
irates
Freshwater.
sediments.
amphlpods.
algae, trout,
crayfish.
nussel
Freshwater,
algae, tadpoles
or snails
Freshwater,
ilgae. proto-
zoa, rotifers,
laphnla. ostra-
coas. Serratia,
other bacteria
Concentrations
of dleldrin in
ttter, algae,
)aphnia. gup-
)1es; biological
nagnif (cation
Concentration
of compound and
netabolites in
ers. optical
density, ash
free dry weight.
;hlorophyll.
concentration
if toxicants
farious concen-
ratlons of
Iteldrtn in
(dter and food.
:ime
Irae, one of
tight inverte-
irates
'::,
'•• •
toe
•
f
'ine; tadpoles,
wo snails
ime.
oxicant
en
-------
Table 6. Aquatic-Batch Microcosms Used for Chemical Fate Studies (continued)
Size
Reference Transport of (liters) Equipment Mode of entry Replicates
Duration
(days) Complexity
Parameters Experimental
measured variables
Comments
Tioofeeva and
Kullkov
(1967)
WhHUker
(1961)
Utlhm (1970)
Zlofain (1971)
Zlobin and
Perlyuk
(1971)
"Sr
• ip
l"Ru. "'Cs.
"Co
.»,pu
'"Pu/'-CM-
1 to 5
228 and
20000
2
160
160
Aquarium
Aquarium,
outdoor pond
Flasks
Aquarium
Aquarium
Single applica-
tion of "Sr
salt
Single applica-
tion
In detritus and
algae from
settling basin
Single applica-
tion of *"Pu
salt
Single applica-
tion of s"Pu
and "CN salts
1
1
! microcosms
>er sampling
.\ate per ex-
>er1ment
1
1
10-90
46-78
8-28
IS
IS
Freshwater.
lake sand.
aquatic plant
Freshwater
sediment.
algae, fish.
snails.
zooplankton
Freshwater,
algae, detritus
combinations o
jenthtc inver-
tebrates
Sea water.
algae
Sea water.
algae
Concentration
of "Sr In
water, sand.
plants; coeffi-
cient of accum-
ulation
Biomass. trans-
fer rates, bio-
logical magni-
fication, con-
centration of
"P In compon-
ents of micro-
cosm
Concentration
of radio-iso-
topes in detri-
tus, algae,
Invertebrates.
water; biologi-
cal magnifica-
tion
Concentration
of 21'Pu in
water and algae
Concentration
of "'Pu in
water and algae,
distribution of
"£N- in amino
acids
la, Kg. various
"Sr concentra-
tions. 32
>pecles of
>laoU. living
md dead plants
rime. P concen-
tration, tem-
terature, com-
tinattons of
irganisras
lombl nations of
Invertebrates;
letritus or
ilgae; time
J respiratory
inhibitors,
time
iodlum cyanide.
time
en
CD
-------
69
In some studies results were only reported on a single population of
organisms. Only in the case of microorganisms were axenic (pure) cul-
tures used; such studies were generally not referred to as microcosms.
Species-defined (gnotobiotic)-communities have usually been studied for
their biological behavior, but not for chemical transport. Microco||s
studied for biological effects, and which could be adapted to transport
studies, are listed in Appendix A.
The studies most relevant to chemical fate are catalogued in Table
6. Many of these cover aspects which have been discussed in the preced-
ing sections. The text which follows will dwell mainly on those aspects
of microcosm studies which have not been hitherto discussed.
No single aquatic-batch type microcosm serves as the prototype for
all such microcosms, in contradistinction to the farm-pond "Metcalf
microcosms." The Metcalf studies have addressed themselves to the par-
ticular problem of comparing the behavior of related chemicals in a
bench-top mimic of a naturally-occurring ecosystem. On the other hand,
aquatic-batch type microcosm studies have addressed themselves to
answering an array of questions, such as establishing the validity of
certain field methods or identifying and analyzing important transport
Nutrient Cycles
Whittaker (1961) appears to have done the first aquarium study of
32
P cycling in complex microcosms, and considers most of the problems
of methods, measurement, and interpretations of data. His systems in-
cluded water-sediment-plankton and attached algae-snails-zooplankton-fish.
32
Samples were analyzed periodically for P content. The data have been
32
presented in several modes: the proportion of P in each compartment
of the commmity at various times, maximum activity density, time of
maximum activity density, concentration ratio at that time, gross uptake
rate, activity uptake rate, and turnover rate. Additional studies ex-
32
plored the P movement in aquaria with nutrient loads ranging from
oligotrophic to eutrophic, at different temperatures. Nonliving uptake
32
of P by sediment and aquaria surfaces was found to be significant.
32
The uptake and loss of P by Daphnia and guppies were studied in more
detail in simpler systems.
-------
70
In a pond study, this type of analysis was applied to a more com-
plex system. It was not an attempt to validate the predictive value of
32
the aquarium studies. The P in the pond responded similarly to that
32
in the aquaria, i.e., the P left the water rapidly and accumulated in
the sediments and organisms. One of the inadequacies of the investiga-
32
tion of large ponds was the "deficit" of P unaccounted for in the
community compartments sampled. After 24 hours only 55%, after 16 days
25%, and after 44 days 12% of the P introduced could be accounted for.
Much of this was presumed to have accumulated in the sediments below the
bottom rocks. Although compartment models with transfer rates were pre-
sented for the aquaria and pond experiments, they were used essentially
32
to analyze the data rather than to predict P behavior in new or dif-
ferent experiments and conditions.
Confer (1972), in a study of P cycling in flowing aquaria, con-
cluded that phosphorus cycling was inadequate to sustain the community,
and a continual influx of phosphorus was necessary. He presents a case
that the epilimnion contributes phosphorus to the littoral area, and
that an influx of phosphorus is necessary to sustain biological activity
in the epilimnion. This is in contrast to other authors who assume that
the littoral area is in equilibrium with, or contributes phosphorus to,
the epilimnion.
Other nutrient recycling studies proceeded more directly from field
observations to semi-isolated process studies, e.g., the uptake and re-
lease of dissolved organic phosphate by algae, amphipods, and bacteria
(Johannes, 1964), and the transformations of phosphorus compounds by
bacteria alone or with protozoa (Johannes, 1965).
Another approach to sediment-water-biota exchange of phosphorus
was made by Porcella ejt al_. (1970). Sediments of various lakes were
placed in contact with a P-free growth medium which was slowly replaced
so that the medium had a 10-day replacement'time. Periodic analysis of
the water, biota, and sediments provided information on the rate of P
release from the sediment, and its inclusion in the water and biota.
32
Both aerobic and anaerobic conditions were tested. A P tracer was
used in one study. The method provided a comparison between different
sediments and established that the sediment can be a source Of phosphorus
-------
for algal growth in low P waters; the amount of P contributed by the
sediment was related to the amount of phosphorus measured in the sedi-
ments. Qscillatoria mats increased the movement of P into the water.
The authors describe their system as an approach to understanding the
natural environment, but not as a predictive tool. A similar microcosm
technique was useful for studying Hg distribution (Kolb e_t aJL, 1973).
The recycling of S was studied in a microcosm of coral reef com-
ponents (DiSalvo, 1971). Labeled bacteria were used to introduce the
tracer. The radioactivity of the various components of the community
provided information on the principal transfer processes. A flow dia-
gram was used to express the major pathways, but no compartment model
was presented.
99
The distribution of Mo among lake biota and sediments was studied
by Short et a]_. (1973), as part of the Fern Lake Study. The data were
presented as concentrations and accumulation factors gathered over 24
days. The laboratory data aided in understanding the distribution pat-
tern in the lake, especially the rapid uptake by Mi tell a, but no predic-
tions were made or tested.
Continuous Monitoring; Diurnal Cycles
A particularly ingenious technique was developed by Gushing and
Rose (1970) for evaluating Zn recycling by periphyton. The periphyton
was permitted to grow on the outside of a test tube which fit over a
CaF- detection crystal. Zinc-65-labeled water was circulated over the
periphyton community and over a duplicate bare control tube with, detec-
tor. The first detector registered radioactivity in the periphyton and
water, the second registered the radioactivity in the water only; the
periphyton level was calculated as the difference. Either an open or
closed recirculating arrangement was possible; data were represented
from the closed arrangement only. The continuous monitoring of periphyton
and water permitted much more detailed information to be gathered. The
uptake was faster and reached a higher concentration in continuous light
than in continuous dark; both live communities were higher in Zn than
a killed community. Under a 12-hour light, 12-hour dark period, uptake
was faster during each of the lighted periods, but dropped rapidly during
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72
the dark periods. This cyclic behavior was discussed as related to photo-
synthesis or pH shifts. It is important to note that the Zn concen-
tration at the end of each dark period was even lower than that in the
killed community and therefore much lower than that in the communities
held in continuous light or darkness. Diurnal shifts in concentration
have not been discussed by other authors and although they may be aware
of the time of their observations, they report them only as hours elapsed
from the tracer introduction. Rose and Gushing (1970) have also examined
surface adsorption of Zn in greater detail using autoradiography.
Further influences of environmental factors were shown to affect
65
Zn uptake in a community of oysters, clams, crabs, scallops, and their
associated sediment (Duke et al., 1969). High salinity and zinc concen-
65
tration suppressed the concentration of Zn in animals and sediments,
whereas high temperature and pH had the opposite effect. Temporal
changes in accumulation factors were reported in the control animals,
and were discussed, but not explained. The authors cautioned against
extrapolating results obtained at a particular time of the year to other
times, even under the same or similar conditions.
Equilibrium
The problems associated with a tracer reaching equilibrium condi-
tions were discussed by Cross e_t a_L (1971). After nine months, Zn
had not yet come to equilibrium in the water and particulate material.
After discounting (1) possible isotopic effect, and (2) the potential of
a large inorganic, slowly mixing reservoir, and discussing (3) a slow
exchange of Zn between particulate material and water, they found a
fourth explanation most likely: the presence of a nonexchangeable
"pool" of organically complexed Zn that was available to the phytoplank-
ton, but which did not exchange readily with the dissolved inorganic
Zn, including Zn. Their data did not provide validation of this
explanation, and they agreed on the need to study the behavior of stable
elements in addition to their radioisotopes in tracer experiments.
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73
Route of Entry
The multiple entry of chemicals into aquatic animals, i.e., inges-
tion, skin absorption and adsorption, and gill uptake introduce more
complexity into aquatic food chain studies. Isensee e_t al_. (1973) have
attempted to distinguish between bioaccumulation, which they define as
uptake from all processes, and biomagnification, which they define as
concentration "through the consumption of lower by higher food chain
organisms with a net increase in tissue concentration." In practice
these processes are difficult to distinguish in complex systems. :
Duration of Zooplankton-Fish Studies
The voracious appetite of fish and their great search capabilities
relative to the size of most aquaria, have caused most aquatic food
chain studies to be done in two phases: water-algae-zooplankton followed
by zooplankton-fish (e.g., King, 1964; Reinert, 1972; and Morgan, 1972), .
or alternatively, adding the fish at the very end of the experiment
after the zooplankton, etc., have been sampled (Isensee et.aU, 1973).
Stepwise experiments are useful in their own right to isolate processes,
but in the case of zooplankton-fish studies, long-term stable relation-
ships are generally not possible in aquaria.
Single vs Continuous Input
The mode of entry in most aquatic studies is limited to an initial
(acute) dose. In such studies, the concentration in the water is de-
creasing during the duration of the experiment. Continual (chronic)
input has been used to maintain a quasi-constant concentration even when
the organisms were retained (e.g., Reinert, 1972).
Equivalency of Similar Organisms
Although most authors state that they chose "typical" trophic chain
representatives, there has been little effort to test if all algae, all
microcrustacea, all little fish, have the same properties. King (1964)
reported that Daphnia pulex and D. magna differed in Cs uptake.
Daphm'a pu1 ex uptake increased and D. magna uptake was not affected when
available labeled Chlamydomonas was increased. In direct uptake studies
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74
at 22°C, jh pulex concentrated three times the amount of Cs concen-
trated by D. maqna. They also differed in their ability to remove Cs
from their food.
Detritus Feeders
Although many studies assumed that the water-algae-zooplankton was
the major aquatic food pathway, Wilhm (1970) has exploded detritus as
the transfer source of Ru, Cs, and Co into macroinvertebrates
(midges and snails). The transfer via detritus was altered by the size
of the detritus.
Volatile Chemicals
Sealed systems were used for the study of Hg by Kolb e_t a]_. (1973).
A semi-closed system (air pumped in, trapped at exhaust) was used for
tritium (Stewart et al_., 1971). Many, perhaps most, microcosm experi-
ments fail to account for all of the added chemical. Some of this loss
is explained as adsorption on surfaces, but co-distillation, evaporation,
or metabolic degradation to volatile compounds are seldom measured.
Lu and Metcalf (1975) have designed and used an aquatic microcosms
for evaluation of volatile chemicals which are discharged into aquatic
ecosystems. It consists of a 3-liter round-bottomed flask with three
necks to which are fitted: 1) a condenser to reduce losses of volatile
compounds and to retain a constant water level in the flask, 2) a re-
movable filter for constant air flow and for sampling water and 3) traps
for the collection of volatile metabolites or parent compound. This
microcosm has been used to study the environmental fate and biodegrad-
ability of benzene derivatives in aquatic food chains consisting of
algae, snails, mosquito larvae and mosquito fish. The entire microcosm
may be maintained in an environmental growth chamber to provide control
of light and temperature. Such a system could be used to measure reten-
tion and losses of volatile chemicals in water with or without sediment
or biota. Adapting the microcosm for use with chemicals other than
benzene derivatives would involve using suitable absorbents in the off-
gass traps for analysis of volatilization losses.
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75
Accumulation of Organic Pesticides by Components of Aquatic Microcosms
Numerous studies have reported the accumulation of organic pesti-
cides by aquatic organisms. Most of these are toxicity studies anci are
beyond the scope of this report, but a few typical studies are cited
because they relate to the accumulation by a major component of an
aquatic microcosm. Biomagnification of various organic pesticides by
aquatic invertebrates has been well documented and the invertebrates
are usually regarded as a major source of such compounds to fish. The
accumulation of Cl-labeled Aroclor 1254 by eight types of aquatic
invertebrates was studied by Sanders and Chandler (1972). They measured
concentrations in water and organisms in order to calculate 1, 4, 7, 14,
and 21 day magnification (bioaccumulation) factors. The concentration
in water was kept below 3 ppb by continuous flow of the medium. Smaller
invertebrates approached their maximal concentration more rapidly; but
the crayfish was still increasing its concentration between days 14 and
17. They reported a magnification factor for Daphm'a magna of 24,700
for 1 day and 47,000 for 4 days (corresponding to 52+2.0 ppm). For this
same species, Crosby and Tucker (1971) reported a magnification factor
of 16,000 (8 ppb in water) to 23,000 (50 ppb in water) within 24 hours.
DDT concentration was the experimental variable in this latter study and
exposure was tested only at 24-26 hours. Dead Daphm'a or moulted cara-
paces accumulated half as much DDT as live Daphm'a (26 hr exposure).
Crosby and Tucker reported the body burden of DDT to be proportional to
the logarithm of DDT content of the medium. Assuming that natural con-
centrations of 10 ppb DDT are not unusual, 10 mg of Daphm'a exposed:for
a day would accumulate enough DDT to provide an acute oral, dose of about
1.5 mg per kg in a 1-gram fish. This is almost 1/3 of the acute toxic.
dose of cutthroat trout. In larger animals, such as fish, the concen-
tration varies from organ to organ (Ernst, 1970).
Degradation or Chemical Modification
Many invertebrates and fish metabolize organic pesticides to some
degree. Ostracods convert aldrin to the epoxide, dieldrin (Kawatski and
Schmulbach, 1971). Degradation products of DDT were demonstrated in
seven species of aquatic invertebrates (Johnson et al_., 1971). Isomer
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76
changes of PCB residues were found in scut (Gammarus) by Sanders and
Chandler (1972). These studies utilized batch microcosms consisting of
jars, flasks or continuous flow glass chambers. Although the focus of
the experiments was on degradation, uptake and bioaccumulation was de-
termined for the test organisms.
Microorganism Uptake (Including Solvent Effects)
Organic pesticides are usually insoluble in water and are therefore
dispersed with an organic solvent, e.g., alcohol, acetone, or benzene.
While most investigators insure the solvent is not toxic and/or run the
pesticide-and-solvent along with a solvent control, few check on a pos-
sible stimulatory effect of the solvent. The solvents generally used
are suitable organic substrates for microbial growth. In bioassays or
transport studies of higher organisms, microbes are usually present, but
ignored. Their response to the solvent is usually overlooked. Euglena
has both autotrophic and heterotrophic properties, and deKoning and
Mortimer (1971) reported that with 1.0 ml of ethanol in 70 ml of water
there was evidence of a heterotrophic growth response.
The Euglena also responded differently to DDT depending on the
level of ethanol. The rate of DDT uptake was parallel to the increased
number of cells during growth. Cells which had accumulated DDT were
washed and resuspended, and DDT concentration in the cells was followed
for 5 days. The DDT/cell decreased, but the total DDT in the cell mass
remained equal to the inoculum level.
Extensive metabolism of DDT by bacteria has been demonstrated by
Pfaender and Alexander (1972). They used small flasks containing micro-
bial communities maintained under anaerobic conditions in freshwater,
sewage and sediment. Concentration of DDT and its metabolites were de-
termined in the microbial communities over various times. In speculating
why DDT is so resistant in the environment although it shows extensive
biodegradation with cell-free extracts, they proposed that cyclic 02 .
changes may not occur in all environments, but should occur frequently
in soil during a wetting and drying cycle. The alternative explanation,
which they felt was more plausible, is that DDT is only degraded by a
small number of organisms, none of which uses the compound as an energy
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77
or carbon source. Therefore, they have no competitive advantage in the
presence of DDT and do not proliferate at its expense. An increase in
their population caused by other energy sources would accelerate DDT
degradation. This hypothesis is supported by observations that DDT de-
gradation is increased when nutrients are added to the soil. This effect
has been termed co-metabolism (Focht and Alexander, 1970). Phenomena
such as these, succession of.microzones, and co-metabolism, would be more
likely to be shown in a complex microcosm than in a homogeneous pure
culture.
Autoradiography of bacterial colonies has been used by Kokke (1971)
to demonstrate microbial accumulation of radioactivity tagged pesti-
cides. The methods have been applied to members of two microbial eco-
systems, garden soil and aerated waste water. The method detects colonies
that have a more concentrated radioactivity than that in the agar medium.
The method can be used to screen mixed bacteria populations or to study
the effects on, or distribution in, pure strains.
Reproducibility
In the words of Abbott (1966), "Although reproducibility has been
a major postulate justifying the microcosm approach to trophic studies,
only two works dealing with deliberate attempts to replicate generalized
microcosms appear to have been published (Mclntire et_ aJL, 1964; Beyers,
1963)." If we add to this one study on toxic effects (Taub and Pearson,
1973), we have four microcosm studies from which to draw conclusions
concerning replicability.
Beyers, Mclntire e£ a_L, and Abbott all used .naturally-derived com-
munities, and all were interested in community metabolism. Beyers added
the alga Vallisneria to 1 liter of bottom sediments and 3 liters of river
water. After a month the dominant plants were Vallisneria and Oedogonium.
The only macro animals were the lumbricoid oligochaete Sutroa and the
snails Physa and Goniobasis. The microzoa were sparse. Abbott placed
bottom sediments and estuarine water into 19-liter carboys, and after
seven days inoculated the microcosms with an aliquot of phytoplankton
from a nearby bayou. After a month there was visible growth of algae
on the bottle walls, "a few small growth of blue-green algae, scattered
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78
areas of green algae, and brownish patches which were presumed to be
diatoms." Several barnacles (Balanus) had settled and begun active
feeding. Mclntire eit aK flowed river water (2.1iter/min) past bottom
material consisting of rubble and smaller gravel in a lab stream 3 m
long, 25 cm wide, and 20 cm deep. The communities developing on the
substrate were dominated by two diatom species, and at various times of
the year Oedogonium and a blue-green alga, Phormidium.
Replicability is tabulated below where coefficient of variation
(standard deviation/mean) is used as the measure of reproducibility.
Investigator
Beyers (1963)
Mclntire et al .
Number of
replicates
12
6
Coefficient of variation (%)
Production
14.5
13.5
Respiration
14.3
32.7
Biomass
22
_„
(1964F
Abbott (1966) 18 13.9 31.6
Taub and Pearson endeavored to measure the effects of marginally
lethal doses of mercury, cadmium, toxaphene, and PCB on mixed aquatic
communities. The communities were inoculated with known concentrations
of the green alga Chlamydomonas, the protozoa Tetrahymena. Daphnia
magna. ostracods, rotifers, and bacteria. For each of the four toxi-
cants, 30 matched pairs (control and toxicant) of 500 ml communities
were set up, 10 pairs being analyzed after one week, 10 pairs at three
weeks, and the final 10 pairs at five weeks (making 240 communities in
all). Counts of all organisms were taken at these times, along with
optical density, ash-free dry weight, chlorophylls a and b, and phaeo-
pigment. Reference should be made to Taub and Pearson (1973) for com-
plete details of mean values, standard deviations, standard errors, and
the•t-test for treatment-control differences for each of the measured
quanties. Co-efficients of variation for optical density were generally
less than 30%, and often below 20%. Counts for various organisms often
resulted in coefficients greater than 100%.
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79
E. Aquatic - Continuous Cultures
Introduction
"A continuous culture is a flow system in which individual cells
are suspended mk (nearly) constant volume, at or near steady state
of growth established by the continual addition of fresh growth medium,
and the continual renewal of part of the culture." (Kubitschek, 1970).
Continuous cultures have been used for research in biochemistry,
genetics, and cell physiology - whenever some aspect of cell function
has required elucidation. Introduced in 1950 [see the classic papers
by Monod (1950) and Novick and Szilard (1950)], research was often
limited by its expense and difficulty in maintenance, as well as a
reluctance among some researchers to abandon the more traditional batch
culture techniques. Although most studies using continuous culture
techniques do not fall under the heading of transport of chemicals, •
these techniques have been used to study uptake and bioaccumulation by
organisms (Table 7). The techniques also are useful for studying
effects of chemicals.
In general, continuous culture techniques have the following advan-
tages over batch culture studies (Kubitschek, 1970):
1. Growth and division rates are more easily controlled and main-
tained.
2. Cell concentration can be set and maintained, independent of
growth rate.
3. Cells can be grown for Tonger periods in a constant chemical
environment.
4. Low levels of nutrients, mutagens, or toxicants can be main-
tained.
5. Growth rates can be held constant while physical conditions
and nutrient inputs are varied.
6. Cell sizes and biochemical composition can be maintained for a
given strain, since these depend on growth rate.
7. Metabolites are not built up, nor do pH and C02 concentrations
change with time, as in batch cultures.
-------
Table 7. Aquatic Continuous Culture Microcosms Used for Chemical Fate Studies
Size Duration Parameters Experimental
Reference Transport of (liters) Equipment Node of entry Replicates (days) Complexity measured variables Comments
Button et *1.
(1973)
Fisher et al.
(1973)
Fisher et al.
(1974)
Fisher and
Wurster
(1973)
Grlce et al.
(1971
Kunicka-Gold-
flnger and
Kunickl-
Goldflnger
(1972)
Phosphate.
arsenate
PCB
PCB
PCB
Prone tone
C. N. P
.25-. 5
.15
.2
1.0
2. B
.2
0.0125
Continuous
culture
Sealed glass
Dottles
Flasks for con-
tinuous culture
and glass bot-
tles for batch
Sealed glass
jottles
Jltra filtra-
tion cell
Semi -continuous
culture on mem-
irane filters
Continuous
Input
Single applica-
tion
Continuous in-
jection In
nethanol or
iingle appli-
:at1on
Single applica-
tion
Continuous
Input
Continuous 1n-
)ut as consti-
tuent of median
1
1 cultures per
:ondition
2
) cultures per
:ondtt1on
1
i cultures per
:ondhosphate and
irsenatei pH
>CB, geographic
location of
samples, time
Continuous and
>atch cultures.
latural and
gnotobiotic
>hytoplankton
communities,
temperature,
fCB. time
3 temperatures.
3 phytoplankton
species, 3 PCB
concentrations.
time
Eluate volume
Tine, various
concentrations
and sources of
C. N. and P
'rocedure nay
>e applicable
co studying
idsorption onto
ioll and sedl-
nent particles
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Table 7. Aquatic Continuous Culture Microcosms Used for Chemical Fate Studies (continued)
Size Duration Parameters Experimental
Reference Transport of (liters) Equipment Mode of entry Replicates (days) Complexity measured variables Comments
La lost et al.
(1973)
Mosser, Fisher,
Teng. and
Uurster
(1972)
Mosser. Fisher
and Uurster
(1972)
Murray and
Murray (1973]
Newell et al.
(1972)
Riclca et al.
(1967)
Sinclair and
Toplwala
(1970)
Sddergren
(1968)
'opulation
Interactions
>CB. DOT
>CB. DDT
"Co. "Zn.
...Ag
<
Homass
llomass
"C-labelled
M)T
.2
.2
.1
.1.
1.5
1
1
Two-stage
:hemostat
iealed glass
>ottles
iealed glass
lottles
lark brown
lottles
tasks.
:hemostat
wo- stage con-
tinuous cultun
:hemostat
:ontinuous flon
and batch cul-
;ure
:ontinuous
tingle applica-
tion
Single applica-
tion
Single applica-
tion
:onstituent of
nedium
Continuous
nput
:ontinuous
nput
Continuous or
single mixed
1
!-3 cultures
ier condition
J cultures per
:ondltion
> bottles per
:ondit1on
1
1-2
2
3-28
3-10
4
&
13-90
1-6
Protozoan; two
bacteria, glu-
cose limited
medium
Sea water and
freshwater, one
phy top lank ton
Sea water, two
species of
phy topi ank ton
Sediments.
river water
One of eight
species of
marine algae
Single
bacterium
Single
bacterium
Single -algal
species
Concentration
>f glucose; nun
>ers of organ-
Isms
:ell numbers
Cell numbers
Adsorption.
Jesorptlon,
sediment weight
Seven forms of
litrogen in
cells and cell-
Free super-
natant
Dptical density,
dry weight.
RNA and DNA.
enzyme activity
>er cent via-
>111ty; cell
dry weight
loncentration
of cells; con-
centration of
'"Co in cells
and water
lonbinatlons of
irganisiu; time
rime, concen-
tration of PCB
>r DOT. one of
rive phy topi ank-
ton species
rime. PCB or
)OT
rime. pH. sedi-
nent type.
salinity, con-
: en t rat ton of
element, sedi-
nent formation
irowth rate.
light. pH. C02
Mlutlon rate.
two media
lilution rate
rime, living
and dead cells
CO
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Table 7. Aquatic Continuous Culture Microcosms Used for Chemical Fate Studies (continued)
Size
Reference Transport of (liters) Equipment Mode of entry Replicates
Duration
(days) Complexity
Parameters
measured
Experimental
variables
Comments
Taub (1969c)
Taub and Oollai
(1965)
van den Ende
(1973)
Van Gemerden
and Jannasch
(1971)
Vosjan and
van der Hoek
(1972)
Biomass
Biomass
Predator-prey
interactions
Sulfur
Hg. Cu
.5
1
.34
Two-stage con-
tinuous culture
1 to 6 stage
:onttnuous
:ulture
Continuous
:ul ture
Jingle-stage
:ontinuous
culture
:ontinuous
Input
Continuous
Input
:onttnuous
Continuous 1n-
>ut as consti-
;uent of mediun
Continuous
'. units per
:ondition
I
1
7.0
13
45
10
Mgae. proto-
zoan, two
>acteria
Single algae
Protozoan, bac-
teria; sucrose
limited medium
Sulfur bac-
terium, sulfidi
Halted sea
«ater medium
Single bacter1<
Optical density,
cell numbers,
growth rate.
dry weight
) rote In
)ry weight
irotein,
iltrate-nitrltt
In supernatant
lumbers of
organisms
Cell numbers.
protein concen-
tration, growtt
rate
Biomass (as
protein); con-
centration of
netals
.Ight intensity.
uitrlent con-
:entratlon.
lilution rate
)llut1on rate.
light intensity
rime
rime
00
ro
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83
Some disadvantages of continuous culture vis-el-vis batch culture
are:
1.. Greater equipment complexity and cost.
2. There is generally more surface area for wall growth.
3. There is selection pressure favoring fast-growing mutants
(Ghosh and Pohland, 1971).
4. Air bubbling may carry off chemicals under test if they evapo-
rate or co-distill with water (e.g., DDT), necessitating
system elaborations (Sodergren, 1968).
Apparatus
For a fuller treatment of the hardware necessary in a continuous
culture experiment, see Chapter 2 of Kubitschek (1970). The basic
components necessary to continuous culture include the following:
1. A culture vessel—ranging in size from 10 ml to 50 1. Shapes
vary, though the best design is one which minimizes the surface area
available for wall growth.
2. Nutrient supply--a reservoir and a means for supplying nutrient
at a constant rate, such as a peristaltic pump or a Mariotte bottle.
3. Agitation--at low cell concentrations, air bubbling may provide
adequate mixing; for culture volumes greater than 500 ml, a magnetic
stirrer or shake table is necessary.
4. Drainage—an overflow tube of one kind or another. Normally
this is a tube extending down to the desired surface level of the culture,
excess fluid being forced out by a positive pressure maintained within
the culture vessel.
5. Light source and detection in turbidostats—normally in pairs,
one pair passing light through the medium, one pair passing light around
the medium to correct for light source fluctuations. Less than 5% changes
in turbidity can be sensed (Munson and Jeffrey, 1964; Eisler and Webb,
1968).
Principles of Operation
The following summary of continuous culture principles has been
taken primarily from Chapters 1-4 Kubitschek (1970), and the papers by
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84
Herbert, Elsworth, and Telling (1956), and Herbert (1958). These works
comprise an excellent introduction to the theory and operation of con-
tinuous culture.
Perhaps the single most important advantage of chemostat continuous
cultures as a research tool is their ability to control the growth rate.
By contrast, organisms in batch culture undergo an initial lag phase,
then grow at the maximum rate until the limiting substrate is depleted,
then level off again during the stationary phase. The culture is
growing sub-maximally in a very brief time span during the transition
from the exponential phase to the stationary phase. Chemostat operation
permits an expanded look at this transition interval, which probably
corresponds more closely to growth conditions in a natural system.
Multi-Stage
Single Stream. Herbert (1964) has analyzed multi-stage continuous
cultures for both single-stream and multi-stream operation (additional
chemical or nutrient input at each stage), but only for a single organism
and with a view toward industrial optimization of yield. As a laboratory
tool, multi-stage continuous culture has its greatest potential as a
means of controlling growth rate of higher trophic level organisms. For
example, as has been done by Taub and McKenzie (1973) and Taub (1973),
a steady-state algal concentration (Chlamydomonas reinhardtii) achieved
in the first stage of a two-stage culture, was introduced into a second-
stage culture vessel seeded with the protozoan Tetrahymena pyriformis,
which was able to survive and grow on this single food source.
Multi-Stream. Multi-stream systems are potentially valuable as
laboratory tools for the study of fates and effects of chemicals. Growth-
inhibiting or -accelerating chemicals could be continuously introduced
at any stage for the purpose of isolating its effects on and accumulation
in the set of organisms in that stage, as a function of growth rate.
Parameters Measured — Applicability
A vast majority of work in continuous culture has been devoted to
the measurement of nutrient uptake rates and various other cell prop-
erties (such as size, RNA and DNA content) as a function of limiting
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85
substrate type and concentration (Herbert, 1958; Pirt, 1972) largely
falling under the heading of "effects of chemicals," rather than "accu-
mulation and'transport of chemicals." The remainder of this section •
covers those relevant parameters most amenable to measurement by con-
tinuous culture techniques, even though they may not in practice have
been so measured. We relegate to the next section consideration of those
cases in which parameters relevant to accumulation, transformation, and -
transport of chemicals have been actually measured in continuous culture.
It should be recalled that the main advantage that continuous cul-
ture confers is the ability to maintain cell growth rate constant at
values considerably less than maxima and to maintain low levels of nu-
trient or other introduced chemicals for a considerable time. Thus
while maximum chemical uptake rate and accumulation may best be measured
in batch culture, those rates corresponding to low doses, and even more
subtly, those rates involving mutually inhibitory processes between a
limiting nutrient and a toxicant [e.g., phosphorus and arsenic (Button
e_taj[., 1973)], can only be measured in continuous culture. The impor-
tance of such studies is revealed by the Button group's results, which
showed virtually no arsenic uptake in the presence of sufficient phos-
phorus, with significant arsenic uptake when phosphorus was depressed
to low levels.
Those parameters relevant to the accumulation of chemicals [e.g.,
bioconcentration, bi concentration factor (Kenaga, 1972), specific
activity (Nelson and Kaye, 1968)] which are commonly .measured in multi-
trophic level laboratory ecosystems and single organism batch culture,
are amenable to measurement in continuous culture, with the added advan-
tage that the organism can be maintained at steady state, permitting
the study of the influence of an added substance under defined condi-
tions. Of even greater importance in the study of transport across
cell boundaries, continuous culture permits the measurement of transport
rates as a function of chemical concentration in both the culture solu-
tion and the cells of the culture.
Several mechanisms of transport have been reported. For example,
Sodergren (1968) has shown that DDT, with its high solubility in lipids,
is absorbed by both living and dead Chlorella cells at a rate which is
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86
14
equal to the rate of diffusion of C-DDT in water. He has measured the
uptake rate in continuous culture and found that uptake was completed in
less than 15 seconds. ,
Other parameters which may be measured best with continuous culture
techniques are endogenous metabolism or:^rtality (Sinclair and Topiwala,
1970), and excretory products (Newell e^aU, 1972), including those
products resulting from the biotransformation of the chemical under
study.
Though the principal reason for use of continuous culture tech-
niques is to study and accurately measure in isolation the parameters
of transport, accumulation, and the effects of closely controlled amounts
of chemicals, it should be noted that the environmental analog of such
a system is the continuous (as opposed to pulse) dumping of waste efflu-
ent into rivers and streams.
Cases from the Literature
Sodergren (1968) has measured the uptake and accumulation of C-
DDT in living and dead cells of Chlorella sp.» using both batch and con-
tinuous culture techniques. Irreproducibility of the batch culture
results led to the use of continuous flow techniques. This is a repre-
sentative example of the use of continuous culture for measuring and
studying the mechanism of toxicant bioaccumulation. It is also repre-
sentative of the common malpractice of taking only one sample and then
having to make unsupportable statements concerning sampling variation.
Button ert al_. (1973) have demonstrated the use of continuous cul-
ture in measuring the coefficients of competitive inhibition. Low
levels of radioactively labeled phosphate and arsenic were introduced
to a culture of the pink yeast Rhodotorula rubra for a number of phos-
phate limited concentrations. A significant feature was the development
of a means to estimate background phosphate levels (from glassware)
necessary to the interpretation of the very low introduced phosphate
levels. Significant findings included the discovery that maximum
phosphate uptake was approximately three times that measured in batch
culture, indicating that batch cultures may give unrealistically low
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87
predictions of growth in a natural stream ecosystem. Though 120 con-
tinuous culture runs were made, no information on repeatability or
statistical estimation of parameters and their variances was reported,
which is, unfortunately, the common practice.
An excellent example of the use of continuous culture to study the
biotransformation of toxic metals is seen in the work of Vosjan and van
der Hoek (1972) on the anaerobic sulfate reducing organism, Desulfo-
vibrio des u1 furicans. In some fjords a large amount of hydrogen sulfide
is produced which reacts with metals to form insoluble sulfides, sometimes
to the extent that the metals can be commercially exploited. Previous
attempts in batch culture to form sulfides of copper and mercury in con-
tact with Desulfovibrio failed to produce any HgS, and Cu-S was formed
only with difficulty. The presence of Cu impedes growth of Desulfovibrio
altogether.
To illustrate the use of continuous culture for studying toxic ef-
fects of chemicals on organisms in competition, Fisher et. al_. (1974)
measured the effect of 0.1 ppb PCB on the diatom Thalassiosira pseudonana
when grown in pure and mixed culture with the estuarine green alga
Dunaliella tertiolecta. Results from both batch and continuous cultures
were compared. The low dosage of PCB was found to halve the Thalas-
siosira population when grown in continuous culture with Dunaliella,
whereas in pure continuous culture Thalassiosira was unaffected by PCB.
In batch culture there was little effect due to PCB introduction. A
single repetition gave results within 10% of the first measurements.
Additional examples of the use of continuous culture are:
1. Porcella (1969) — luxury uptake.
2. Taub and Dollar (1965) — protein level vs growth rate.
3. Newell e_t.aj_. (1972) — excretory products.
4. Sinclair and Topiwala (1970) — exogenous metabolism at low
dilution rates.
5. Ricica e_t ,al_. (1967) — a two-stage, two-stream study, to
examine culture stability near washout.
6. van den Ende (1973) — two species predator-prey interaction.
7. LaJost e_t al_. (1973) — three species interaction.
8. Taub (1969c) ~ a two-stage, four species interaction.
-------
9. Grice e_t a1_. (1972) — adsorption of pesticide onto a humic
acid preparation.
Reproducibility
Regrettably there is little information on the replicability of
continuous culture systems. Rarely do investigators repeat a given
experiment, though considering the duration of a continuous culture run,
it is understandable. Nonetheless it is practical to check such things
as sampling error, and to assess the variance introduced by laboratory
measuring technique, but this is rarely done either. Organism varia-
bility, and unassessable variations introduced by the continuous culture
apparatus, can be estimated from the single repetitions performed by
Fisher et al_. (1974), Sodergren (1968), and Taub and Pearson (1973),
which gave 10-30% variations in results.
F. Special Types
Species Defined (Gnotobiotic) Microcosms
These differ from other multitrophic level microcosms by the exclu-
sion of unknown microbiota. Such systems eliminate uncontrolled factors
such as growth stimulants or inhibitors deriving from unacknowledged
bacteria and unexpected sources, or losses of nutrient by nitrogen fix-
ation or denitrification, or variable degradation of compounds by hap-
hazard contaminants. The documented microcosms were developed primarily
for effect studies, but could be ideal for transport studies (Table 8).
An alga-herbivorous protozoan-3 bacteria microcosm was reported by Taub
(1969a). A more complex series of microcosms tested the effect of the
addition of rotifers (Taub, 1969b). A marine alga-brine shrimp-bacteria
microcosm was studied by Nixon (1969).
In Situ Bioassay
Some in situ bioassay techniques could be adapted to transport stu-
dies. Even minute samples can now be analyzed for heavy elements by
means of microprobe analysis (X-ray energy spectroscopy). The suspension
of known biota in fine-pored containers and protozoan substrates are
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89
examples of minute samples that could be used to measure bioaccumulatlon
factors and transfer rates. ,
Naturally-Occurring Microcosms
Small, isolated aquatic communities are sustained in the internodes
of plants such as bromeliads, Spartina. and broken stems of bamboo.
Studies of these microcosms may be of interest as descriptions, of proper-
ties of microcosms.
Closed Ecological Systems (Bioregenerative Life Support Systems)
Most space studies involved 02/C02 exchange between algal cultures
and mammals; some included the transfer of nutrients from waste products
to algal production. That literature, recently reviewed by Taub (1974),
has little bearing on chemical transport other than on gas exchange. The
experimental systems might be adaptable to chemical transport, and a
supplemental bibliography is provided (Appendix A).
-------
Table 8. Special Microcosms: Species Defined (Gnotoblotic)
Size
Reference Transport of (liters) Equipment Node of entry Replicates
Duration
(days) Complexity
Parameters
measured
Experimental
variables
Comments
Nixon (1969)
Taub (1969a)
Taub (1969b)
tlxed organic
ind inorganic
tutrients
titrate to cell
lumbers
titrate to cell
lumbers
0.75
0.5
0.5
Flasks
Flask
Flasks
initial
initial, mixed
initial, nixed
1
2
3
200
170
77
Alga, brine
shrimp, 5 bac-
teria
Alga, proto-
zoan, 3 bac-
teria
Alga, protozoa,
rotifers, 6
bacteria
Cell/organism
density
Cell densities
Cell densities
Sea water.
>rlne water
Mga only, algi
>lus protozoan
Mga only,
various combin-
ations
Urtenta died
>etween 150 and
>00 days
Vellotnary
study, lack of
>reda tor-prey
:ycles
to long- tern
reduction in
algal density
due to herbi-
vores
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91
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-------
100
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-------
101
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-------
102
V. APPENDIX
-------
103
Appendix A. Comparable Data on Microcosms Adaptable to Chemical
Transport Studies
1. Laboratory microcosms
2. Closed (Bioregenerative life support)
-------
Table A-). Laboratory Microcosms Adaptable to Transport Studies
Size Duration Parameters Experimental
Reference Transport of (liters) Equipment Mode of entry Replicates (days) Complexity measured variables Comments
Allen and Broct
(1968)
Beyers (1962)
Beyers (1963a)
Beyers (1963b)
Beyers (1965)
Cooke (1967)
"C-labelled
glucose
C02
CO,
C02
C02
:o2
.04
3
3
.006-
5930
.166-
4.02
.3
Test tubes
Aquarium
Aquarium
Test tubes,
flasks, aquaria,
concrete ponds
Test tubes,
flasks, aquarli
leakers
Single applica-
tion
1
12 microcosms
12 microcosms
(3 with herbi-
vorous snails)
1
1
1
30
J
300
Variable
farlabl*
93
latural coomun-
ity of bacteria
ind protozoan
tatural river
jommunity
[algae, worm,
inail. crusta-
:eans)
latural river
community
[algae, worm,
inail. crusta-
:eans)
fariable (bac-
teria, algae,
lydra. coral ,
>yster. crusta-
:eans. proto-
toans)
fariable (bac-
teria, algae,
>rotozoan,
:rustaceans)
tatural coumun-
Ity of algae,
lacterta, crus-
taceans, proto-
coans
tptical density,
•adloactlvity,
>ptiflium temper-
iture
let photosyn-
thesis, night
•espi ration
:0i and 02 di-
irnal rates of
:hange. net
>hotosyn thesis,
lighttlme res-
>i ration
tet photosyn-
thesis, night-
time respira-
tion
:0z diurnal
•ate of change
llghttime res-
>irat1on. net
>hotosynthesis,
>1omass
Tenperature
Temperature,
Line
'ho to period.
>resence of
lerbtvorous
inalls. time
rioe. 9 differ-
ent balanced
tquatlc nicro-
:osns
'hoto period.
> different
>a lanced aqua-
tic communities
Succession In
nicrocosm. time
See Beyers
(1963a)
>ee Beyers
(1963a.b)
-------
Table A-l. Laboratory Microcosms Adaptable to Transport Studies (continued)
Size
Reference Transport of (liters) Equipment Mode of entry 'Replicates
Duration
(days) Complexity
Parameters
measured
Experlnental
variables
Comments
Mitchell
(1971)
Richardson
(1930)
Ruthven and
Cairns
(1973)
Taub (1963)
Uhlmann
(1969)
Uhlmann
(1971)
Phosphate
detergents
Btomass
Cr. Cu. Phenol.
Pb, Mn, In, Co,
UNO,. A,. Sn
Biomass
Blonass
Biomass
6
8-80
.08
3.5-. 7
4-16
Jars
Aquaria
Plextglas
troughs with
circulating
«ater
Aquaria
Series of
aquaria with
semi-continuous
flow
Two to five
stage cultures
xith semi-
continuous floti
latch with per-
odic replace-
tent
lontinuous in-
>ut on single
ippllcatlon
iewage effluent
iynthetlc
iewage
iewage
1
2-8
1
1
98
1-7
93
33
Lake water, mud.
natural algal
and bacterial
community
River water.
aquatic plants.
fish, natural
plankton, sedi-
ments
Natural epi-
phytic commun-
ity or specific
components
Fish, algae,
and brine
shrimp or Oaph-
nla with other
Invertebrates
Bacteria and
phytoplankton.
sediments
Combination of
algae and In-
vertebrates
herbivores
Concentration
>f several
:heo>1cals. al-
)al diversity
Index, cell
lumbers
irowth of fish
>er cent
iurvlval
>H. N concen-
tration, micro-
>rganisn counts
fish growth
liochemlcal anc
:hemical oxygen
lemand
I) gal cell
lumbers, bio-
nass
rime. 3 deter-
lents
:omb1n«ttons of
oxtcants and
>rganisms. time
laphnia orbrlne
ihrtnp. medium
dth and with-
mt sewage <
lombtnatlon of
Illuminated and
lark aquaria
(Ithin series
linking rate.
lilution rate.
lerbivores.
:1me, nutrient
Input-
-
lotes on estab-
Ishnent of
ia lanced aquaria
&
w
IT
n German
o
en
-------
Table A-]. Laboratory Microcosms Adaptable to Transport Studies (continued)
Size Duration Parameters Experimental
Reference Transport of (liters) Equipment Mode of entry Replicates (days) Complexity measured variables Comments
Cooper (1973)
Ferens and
Beyers (1970)
Goodyear et al.
(1972)
Gorden et al.
(1969)
Gorden and
Hill (1971)
McConnell
(1962)
CO,, blomass
COj, blomass
Moroass. CO*
COi. blomass
"•C,H120,.
laH COi
B,
IS
.25
12.8
.25
.25
18
Aquaria
Flasks
Outdoor pools
Flasks
Flasks
Carboys
'eriodlc addi-
tion of fer-
;111zer
lomponent of
tedium
'eriodlc addi-
tion of
mtrlents
2 nlcrocosms
>er condition
1 (5 flasks pei
sampling day
>er condition)
3 nlcrocosms
?er condition
1
1
80
40
70
75
70-125
181-222
iediment, natu-
ral phytoplank-
ton. herbivor-
>us fish
lacterta, algae
'lank ton, her-
>ivorous fish
Bacteria,
ilgae
Lake water con-
taining bac-
teria and algae
latural commun-
ity of bacteria,
jlgae, inverte-
>rates
let photosyn-
thesis, night-
time respira-
tion, stomach
rontents of
fish
let photosyn-
thesis, night-
time respira-
tion, blomass
let photosyn-
thesis, night-
time respira-
tion, fish and
sediment bio-
nass
tet photosyn-
thesis, night-
time respira-
tion, dissolvec
carbon, parti-
:ulate blomass,
>acter1a and
ilgae numbers
Concentration
>f bacteria ant
ilgae, pH. I%C
iptake
iross photosyn-
thesis, resplr-
ition, blomass,
rate of 02 dif-
fusion
Stocking den-
sity of fish.
time
Icute gamma ir-
•adtatfon. time
:lve levels of
Fertilizer
Initial medium.
) types of
>actera
rime, two lakes
fine, nutrient
:oncentration
.
o
o»
-------
Table A-2. Closed (Bloregeneratlve Life Support)
Size Duration Parameters Experimental
Reference Transport of (liters) Equipment Mode of entry Replicates (days) Complexity measured variables Comnents
Bowman and
Thomas (I960;
Eley and Hyers
(1964)
Golueke et al.
(1959)
Golueke and
Oswald (I960!
London and
West (1962)
Oswald et al.
(1962)
It. CO,
Da
iaO. COa. Oa
Ba. CO,.
organic wastes
COa. 0>. Hi.
4H,
Da. COa
irgantc wastes
4
.26 li-
quid.
14.3 gas
2
4
1
Continuous cul-
ture of algae;
nice In desic-
:ator; recircu-
latlng atmos-
>here
Continuous cul-
ture of algae;
recirculating
itmosphere be-
tween algae and
nouse chamber
:ont1nuous
:ulture
:ontinuous cul-
ture apparatus
ilth algal and
>acter1al units
:our connected
rhambers with
•ectrculatlng
itmosphere,
turbidostat
llcroterella,
:losed atroos-
iheric condi-
tions '
:ontinuous 1n-
mt of nutrient:
Continuous 1n-
lut of nutrients
iewage .
Continuous 1n-
iut of liquid
irganic wastes
iewage. fluid
ted turn ,
)ontinuous 1n-
>ut of liquid
irganlc wastes
8
3
6 parallel
mlts
3
7-45
1-15
8-10
2-30
10-43
Mice, algae.
Inorganic
medium
House, algae,
inorganic
medium
Sewage, bac-
teria, algae
Algae, bacteria.
organic wastes
Rabbit or rat.
sewage, fungi,
algae
House, algae.
bacteria
Mgae cell vol-
me. CO, and 0,
volume
/eight of
ilgae, volume
if Oa and COa
Concentration
of algae, bac-
teria, mass,
titrogenous
compounds
field of algae
per day
\lgal concen-
tration, gas
composition
Volume of 0,
and CO,, algae
iroductlon
rine
tedium flow
rate, light in-
tensity, time
.Iqutd flow
rate, tempera-
ture, photo
period, light
Intensity, gas
flow rate
.ight intensity.
nedluBi flow
rate, tempera-
ture
rime, medium
flow rate.
uilmals
-------
Table A-2. Closed (Bloregenerattve Life Support) (continued)
Size Duration Parameters Experimental
Reference Transport of (liters) Equipment Mode of entry Replicates (days) Complexity measured variables Comnents
Zuraw (1962)
i>i. CO,
57
Two Intercon-
nected vessels
reclrculating
atmosphere
Continuous in-
put of nutrients
2
2
Algae, primate
Inorganic
medium
Concentration
of algae. COj.
lutrient con-
centration and
nedium flow
rate, light in-
tensity, combin-
ations of algae
and primate
O
CO
-------
109
"STATE-OF-THE-ART" OF THE USE OF FIELD TESTS
TO DETERMINE ENVIRONMENTAL FATE OF CHEMICALS
I. GENERAL CONSIDERATION OF FIELD TESTS
Laboratory studies of the transport of toxic substances yield gen-
erous amounts of data that must stand the test of field validation to
demonstrate the sensitivity and reliability of the laboratory technique
that generated it. The main shortcoming of laboratory studies is the
inability of the researcher to duplicate, or even recognize, the extreme
complexity of the natural environment. Of necessity, laboratory studies
are conducted under conditions of strict control. Haydu (unpublished
report) has stated that field tests afford the researcher the greatest
leverage in assessing the effects and behaviour of pollutants. This is
obvious, since testing in the natural environment provides the best
measure of transport. Reliable estimates of chemical transport in the
field are difficult because data are gathered under conditions of ex-
treme biological and physico-chemical heterogeneity.
The term field test conjures images of the classical experimental
designs developed from agricultural research (e.g., randomized blocks,
latin squares, graeco-latin squares, etc.). In truth, field testing has
been adopted in numerous disciplines other than agricultural research,
and each has in some manner fostered novel approaches to the experimental
design of these tests. Operationally, field tests may be defined as
extra-laboratory, natural site studies comparing observed parameters of
at least two adjacent areas.
-------
no
II. SURVEY OF FIELD TEST STUDIES
A. Terrestrial Studies
Sites of field tests include agricultural plots (Barrett and Darnell,
1967; Cliath and Spencer, 1971, 1972; Draggan, 1973; Harris and Sans,
1972; Harris et aK, 1971, 1973a, 1973b; Heagle et a].., 1973; Korschgen,
1970; Marcuzzi and Dalla Venezia, 1972; Schulz and Lichtenstein, 1971),
meadow plots (Rogowski and Tamura, 1970; Dahlman e_t al_., 1969), grassland
plots (Barrett, 1968; Bulan and Barrett, 1971; Dahlman, 1972; Dodd and
Van Amburg, 1970), and old field ecosystems (Malone, 1969; Odom and
Kuenzler, 1963; Shure, 1971). Forested ecosystems have also been uti-
lized in field studies (Belousov, 1970; Humphreys and Pritchett, 1971;
Marcuzzi and Dalla Venezia, 1972; Overrein 1971, 1972; Waller and Olson,
1967) with watershed ecosystems providing a novel approach to the gath-
ering of information on element transport within large, relatively closed
systems (Bingham e_t al_., 1971; Gosz, 1972; Mattraw, 1972; Rolfe e_t aJL,
1972; Taylor and Kunishi, 1971; Wilder, 1972). Integrated studies of
watersheds have increased the understanding of land-water coupling and
nutrient cycling in forested ecosystems in New Hampshire (Likens and
Bormann, 1969, 1972; Bormann et. al_., 1967, 1969; Hobbie and Likens, 1973;
Johnson et ajL, 1968; Likens ejt al_., 1967, 1970) and East Tennessee
(Curlin e_t aU, 1967; Henderson et a].., 1973; Nelson, 1970; Reichle
et ai.f 1973).
B. Aquat.ic Studies
Field testing, on a large scale, has been primarily utilized within
the terrestrial environment. However, studies conducted in the aquatic
(Dindal, 1970; Evans and Duseja, 1973; Haydu [unpublished]) and esturine
environments (Albone et al_., 1972; Duke, 1967; Duke e_t aU, 1966) are
extant. Table 1 provides a listing of field studies, in the terrestial
and aquatic environments, that have been used for determing the fate of
chemicals.
-------
Table 1. Field Tests Used for Chemical Fate Studies
Reference Ecosystem type Process observed Transport of Mode of entry Size Duration Replication Cements
Albone et al.
(1972)
Barrett (1968)
Barrett and
Darnell (1967)
Belousov (1970)
Binghan et al.
(1967)
Bonnann et al.
(1967)
Bonnann et al.
(1969)
Bui an and
Barrett (1971)
Estuarlne
Grassland
Clover field
Forest (humid
subtropic)
Watershed
(citrus)
Watersheds
Watershed
Grassland
tegradatton.
transformation
Insect, vegeta-
tion species
:ompos1t1on and
lynamlcs; litter
decomposition;
Insecticide
legradatlon
(airmail an popu-
lation dynamics;
Insecticide
legradatlon
lynamlc behavior
ilement leaching
lutrlent loss as
if fee ted by
:lear-cutt1ng
Jutrlent losses
inder blotlc
regulation
Insect populattoi
•esponses
DDT and degrada-
tion products
Sevln (carbaryl)
Dimethoate
Insoluble phos-
phates
Nitrate
H05, HH*. K*.
M***, SO,"
Organic, tnor-
ian1c,partlcu-
late matter.
elements, tons
Remlnerallzed
nutrients
Subsurface Incor-
poration In
sediment
Foliar spray
Foliar spray
Urea In Irriga-
tion water or In
foliar sprays;
it so sheep manure
tecomposltlon-
nlnerallzatton
Various nutrient
cycles
Various nutrient
cycles
10 x 6 cm
! one-acre plots
) 360 x 320 ft
alots
160 acre
13.2 ha and IS. 6
ta watersheds •
13.2 ha watershei
! one-acre plots
46 days
6 months
28 days
3 years
3 years
3 years
5 months
None
None
None
Not feasible
Not feasible
Not feasible
None
Mcroblal contri-
bution determined
Insecticide de-
nonst rated long-
term side effects
an decomposition.
Insects and
uanuals
Change In Insect
population yielded
Indirect changes
In vegetation and
namnals
Soil moisture ef-
fect dominant
Details hazards
of groundwater
Infiltration
teasurenents of
itoMss, species/
area diversity.
1° consumer diver-
sity and equlta-
>11lty were most
sensitive indexes
of environ, stress
-------
Table 1. Field Tests Used for Chemical Fate Studies (continued)
Reference
Cliath and
Spencer (1971)
Cliath and
Spencer (1972)
Curl In et al.
(1967)
Dahlnan (1972)
Dahloian et al.
(1969)
Olndal (1970)
Dodd and ran
Amburg (1970)
Draggan (1973)
Ecosystem type Process observed Transport of Mode of entry Size Duration Replication Comments
Irrigated agri-
cultural plots
Corn field
Watershed
Grassland
plots
Meadow
Harsh
Grassland
Soybean field
Redistribution
and recovery of
insecticides and
degradation pro-
ducts; volatili-
zation
Loss of pesti-
cides by volatil-
ization of degra-
dation products
Land-water
coupling; nutri-
ent cycles
Simulated fall-
out retention
and behavior
Radiation ef-
fects; inter-
compartmental
mineral cycling
Bioaccuinulatlon;
excretion
Transport;
distribution
Insect, plant.
vegetation,
microbe
populations;
decomposition
Dleldrln.
1 indane
Lindane, DDT
lutrient ele-
nents
*Rb
'3'CS
36C1-ODT
134Cs
Dlazinon
carbaryl
Soil removal;
mechanical
mixing with insec-
ticides; return
of soil to plots
Residual from DDT
foliar spray In
past
Various nutrient
cycles
Mechanical fall-
out simulation
Mechanical fall-
out simulation
Aerial dusting
Dew drip; through-
fall; root exuda-
tion; litter fall
Soil treatment
foliar spray
9 x 3 neter*
39 hectare
and
>8 hectare water-
sheds
2.5 x 5 meters
100 meters2
t acre
>0 x 60 meters
! years
15-24 hours
!0 days
5 months
I years
18 months
I years
Split plot.
randomized
ilock
tone
tot feasible
t
tot feasible
None
2
.
Volatilization
was significant
pathway for loss
and degradation
Volatilization
was significant
pathway for loss
and degradation
Significance of
proper radio-
labeling of
organic compounds
Demonstrates
necessity of
replication to
account for field
variability
ro
-------
Table 1. Field Tests Used for Chemical Fate Studies (continued)
Reference Ecosystem type Process observed Transport of Node of entry Size Duration Replication Comments
Duke (1967)
Duke et al.
(1966)
Evans and
Ouseja (1973)
Fisher et al.
(1968)
Gosz (1972)
Harris and
Sons
Harris et al.
1972, 1973a.b
Heagle et al.
0973)
Estuary
Estuary
Streambank
Watershed
Watershed
Agricul-
tural plots
Rye field
Agricul-
tural plots
Food chain
dynamics; bio-
accumulation
Transport and
bloaccumulatton
Physical trans-
port
Precipitation
Input and
stream dis-
charge of
minerals
Nutrient
transfer
rates
Persistence.
vertical move-
ment btoaccu-
mulatlon
Phytotoxlcity
«Zn
«zn
Dluron,
sunmltol,
atrazlne,
2.4-D. 2.4-T;
Plcloram
Sulfate. ammo-
nium, nitrate.
silica, alu-
minum, bicar-
bonate
P. H. K
Oleldrln
Various organo-
phosphate Insec-
ticides
Photochemical
oxldants
Surface spary
Surface spray
Spray appli-
cation
Precipitation
Acid weathering
of soil and bed-
rock
Mechanical mixing
into soil
Foliar spray
Air pollution
30 x 60 meters
pond
36 square feet
12 to 43 hectare
watersheds
3 x 1.5 meters
1/2000 acre
2.4 meters high
x 3 meters diam.
100 days
100 days
2 years
3 months
21 days
None
None
Not feasible
Not feasible
None
6x6 latin
square
ixchange of In be-
tween sediment and
nater dominated
:yc11ng
loll filtration.
lilution, adsorp-
tion are primary
nodes of herbicide
loss
-------
Table 1. Field Tests Used for Chemical Fate Studies (continued)
Reference Ecosystem type Process observed Transport of Mode of entry Size Duration Replication Comments
Henderson et al.
(1973)
Hobbie and
Likens (1973)
Humphreys and
Pritchett (197)
Johnson et a).
(1968)
Korschgen
(1970)
Likens and
Bormann (1972)
Likens et al.
(1967)
Likens et al.
(1970)
Watershed
Watershed
Slash pine
plantation
Watershed
Corn fields
Watershed
Watershed
Watershed
Blogeochemical
cycles; decom-
position effects
Nutrient output;
effect of defores-
tation; precipi-
tation input
Soil Adsorption
and movement
Weathering of
silicate
minerals
Compartment
interaction
and burdens
after long
tern appli-
cation
Nutrient cycles
Nutrient budgets
Effects of
deforestation
by herbicide
and clear cutting
on nutrient budgets
1, P. K. Ca.
«9
', dissolved
trganlc carbon,
;ine partlcu-
ate organic
:arbon
>
:a, Na. Hg. K
lldrin
'•a, Mg. K,
la
tost major
Ions
Various nutrient
cycles
Various nutrient
cycles
Surface appli-
cation
Various nutrient
cycles
treatment during
previous years
See Curl in et
al. (1967)
See Bonnann et
al. (1967)
57 acre and
68 acre fields
See Bormann et
al. (1967)
See Bomann et.
al. (1967)
2 years
1 year
4 years
3 years
2 years
3 years
Possible in that
there are two
forks in water-
shed
None
Data from 6
watersheds
pooled
None
•
Demonstrated
effective
conservation of
P.
Excellent
rationale for
watershed studies
Details effects
of ecosystem
stress with
mineral transport
-------
Table 1. Field Tests Used for Chemical Fate Studies (continued)
Reference Ecosystem type Process observed Transport of Mode of entry Size Duration Replication Comments
Ma lone (1969)
Marcuzzi and
Dalla Venezia
(1972)
Mattrav.
(1973)
Nelson et at.
(1970)
Odura and
Kunzler
(1963)
Overrein
(1971)
Overrein
(1972)
Reichle et al.
(1973)
Old-field
Poplar stand
and annual
crop field
Watershed
Watershed
aid-field
torthem forest
toll
torthem forest
soil
Watershed
Insect, vegeta- piaztnon
tlon density.
species diver-
sity, herb pro-
duction
Insect density
microfloral
response
Cation exchange
capacity and
exchangeable
metals In soil
Food chain
isolation
and dynamics
teachability
of N
teachability
Ca caused by
sulphur diox-
ide pollution
Photosynthesis,
transpiration.
decomposition.
animal -grazing
nutrient flows
and cycles
'arathion
Fosferno SO
32P
I5H
Ca
C. H20. M.
>. K. Ca
loll surface
ipray
latural input
'rora plants
labeled with
>32
latural
Natural
latural element
:ycles
14 acre
81-100
meter'
field plots
See Curl in ..
(1967)
2 years
1 year
3 months
40 months
40-80 days
None
None
None
No
No
No
Observed Increased
rate of nutrient
cycling
Details
measuring and
sampling methods
Significant because
of natural route
of entry
,
Influence of
acid precipi-
tation of Inor-
ganic ion trans-
port
Development of
mechanistic models
of ecosystem pro-
cesses
-------
Table 1. Field Tests Used for Chemical Fate Studies (continued)
Reference Ecosystem type Process observed Transport of Mode of entry Size Duration Replication Comnents
Rolfe et al.
(1972)
Rogowski and
Tamura (1970)
Schulz and
Uchtenstein
(1971)
Shure (1971)
latershed
:rabgrass-
escue meadow
Agricultural
ilots
lid field
Accumulation of
lead In watershed
ecosystem compo-
nents
Mobility by run-
off and erosion
Persistence and
movement
See Ma lone (1969)
Pb
137Cs
Oyfonate
Diazinon
>b in auto emis-
sions
Soil surface and
Foliar spray
Soil surface
spray
Soil surface
76 mileZ
2.3 meters2
plots
15 x 20 foot
plots
3 acre
585 days
22 months
2 years
No
No
No
Radiotrace
cn
-------
117
C. Substance Categorization and Analysis
Numerous substances have been tested in the field and they can be
grouped in three major categories:
1. elements
2. radioelements ;
3. inorganic and organic compounds.
Testing procedures for determing the transport and behavior of substances
depend heavily on available analytical techniques rather than purely
observable effects. This fact is especially important when consideration
is given to the potential for biological or physico-chemical transfor-
mations that may occur in the natural environment.
Within the scope of this evaluation, identification of field testing
procedures for the determination of organic compound transport and be-
havior is most important. This statement is supported by:
1. The large number of organic compounds presently in use and
presently dissipated to the environment;
2. The expected potential for synthesis and dissipation of many
new organic compounds;
3. The demonstrated potential for biologically and physico-
chemically mediated initiation or enhancement of organic
compound degradation and transformation in the natural
environment.
Field studies of organic compound fate have largely concerned the
behavior and effects of pesticides (Albone e_t al_., 1972; Barrett, 1968;
Barrett and Darnell, 1967; Cliath and Spencer, 1971, 1972; Dindal, 1970;
Draggan, 1973, 1974; Evans e_t al_., 1973;.Harris and Sans, 1972; Harris
e_t aK, 1971, 1973a, 1973b; Korschagen, 1970; Likens e_t aj_., 1970; Malone,
1969; Marcuzzi e_t al_., 1972; Schulz and Liechtenstein, 1971; Shure, 1971;
Wilder, 1972). In light of the rapid growth of nuclear fuel cycle facil-
ities, and the potential for greater releases of radioelements to the
natural environment, some field studies that have followed radioelement
transport are included (Dahlman, 1970; Dahlman e_t al_., 1969; Dindal,
1970; Duke, 1967; Duke e_t ^1_., 1966; Odum and Kuenzler, 1963; Overrein,
1971; Rogowski and Tamura, 1970; Shure, 1971; Waller and Olson, 1967).
-------
118
The techniques described in these studies have provided sound methodol-
ogies for determining radioelement transport under field conditions.
An extremely useful extension of these techniques for determining the
fate of organic compounds has been radioisotope labeling of the compounds
where feasible. The high sensitivity of radioassay techniques has in
many instances, overcome difficulties encountered in other analytical
methods for minute quantities of orqanic compounds. Thorough knowledge,
however, of potential degradation products of organic compounds and
location of the radioisotope label is necessary. For example, in a field
marsh study, Dindal (1970) utilized chlorine-36 labeled DDT so that known
DDT metabolites remained labeled during the study.
D. Size and Its Relation to Field Variability
The size of experimental areas used in field testing cover an ex-
tremely wide range. Harris (1972, 1973a,b) reports the use of micro-
plots, 1/2000 of one acre; while watershed studies have utilized areas
of 960 acres (Bingham, 1971), 76 square miles (Rolfe, 1972), and 162
square miles (Taylor, 1971). As the size of the experimental area in-
creases, so does the environmental heterogeneity the researcher must
recognize and attempt to account for. In areas as limited as a few
acres, varying environmental factors and microclimate may interact
with pollutants to seriously influence the degree of ecosystem modifi-
cation (Draggan, 1973; Malone, 1969; Shure, 1971). Very few field
studies, including those utilizing systems analysis (Waller and Olson,
1967; Rolfe et_al_., 1972) or measurement of ecological parameters such
as community structure and function of species (Barrett, 1968; Barrett
and Darnell, 1967; Bui an and Barrett, 1971; Malone, 1969; Odum and
Kunzler, 1963; Shure, 1971), have attempted replication of treatments.
In field tests one must account for many complex and interacting
variables in delineating the impact of pollutants on ecosystems if re-
producible results are to be achieved. In many of the studies mentioned,
the magnitude of field variability has been grossly underestimated and
conclusions were made which may not be statistically valid. The non-
replication of treatments may be more serious when organisms associated
-------
119
with the degradation and transformation of pollutants (i.e., soil mi-
crobes) are used to measure transport of chemicals.
E. Mode of Entry
-------
120
erosion on natural landscapes may be limited. Wischmeier and Smith
(1965) derived a "universal" soil loss equation which has been used to
predict the magnitude of erosion for various agricultural management
schemes. Their model, based on data collected from 47 different sites,
2
has been favorably compared with measured soil losses from 100 m dense
grassland plots (Dahlman and Auerbach, 1968).
The Wischmeier and Smith model estimates soil loss per unit area,
A, by the equation
A = RKLSCP
where R is a rainfall factor, K is a soil erodibility factor, L is a
slope length factor, 5 is a slope gradient factor, C is a cropping man-
agement factor, and P is an erosion control factor. Numerical values
for these factors are given in their handbook.
Transport by erosion is most important on cultivated land. Losses
by this mechanism would be much smaller from forested or densely vege-
tated land. Therefore, measurement or prediction of transport of chem-
icals by rain erosion may be important only for chemicals associated
with agricultural practices or those that have such widespread usage
that cultivated land will be contaminated.
Atmospheric Transports
The transport of substances into, through and out of the atmosphere
has received much attention. Many of the predictive models that are
used to estimate atmospheric transport were developed for estimating the
impact of gaseous releases from energy production facilities. Validation
of these models is beginning to receive more attention as field measure-
ments are made to monitor the levels of pollutants in the environment.
Basically, these models consider inputs from point, line or area
sources into the atmosphere; dispersion of substances in the atmosphere
by dilution and losses to landscape by wet and dry deposition. The
theories, formulation and use of atmospheric transport models are given
in Slade (1968), Briggs (1969) and Culkowski and Patterson (1976). It
is not within the scope of this study to review atmospheric transport
-------
121
models but the Gaussian plume model (Slade, 1968) Is the most widely
used in environmental assessment work. It is applicable to estimation
of the dispersion of volatile chemicals from 1-ocal ground or water
sources or for dispersion of gaseous and particulate releases from
ground level or elevated sources. Concentration of chemicals in the
atmosphere and on the land surface can be estimated by this method.
A comprehensive version of the Gaussian plume model has been formu-
lated and used by Culkowski and Patterson (1976) to estimate values for
atmospheric concentration and wetfall and dryfall deposition in the
vicinity of three power plants. Correlations between model predictions
for S02 and monitored values were r = 0.80 for ground values and r =
0.91 for atmospheric plume values.
F. Processes Observed
The selection of processes to be observed in field tests is critical
to gathering useful information on chemical transport. Dependent on the
medium comprising the point of entry, the most important processes af-
fecting transport are:
1. persistence of the chemical in the medium
2. mobility of the chemical in the medium
3. physical adsorption and absorption to, and desorption
from the medium
4. biological uptake and accumulation from, and
biological loss to the medium; and, food chain
magnification of the substance from the medium.
Also important to the delineation of chemical fate, especially in
field testing, is the measurement of ecological parameters that may be
correlated with the transport process. Ecological parameters that have
been used include:
I. Biotic
a. respiration
b. decomposition (and in a larger sense biological
degradation and transformation in situ)
c. nutrient and mineral cycling
-------
122
d. population, community, and food chain or food web
structure and dynamics
1. biomass
2. diversity
II. Abiotic
a. temperature
b. moisture
c. pH
d. dead organic matter content
G. Duration
The amount of time expended on field testing for determining toxic
substance transport has been dictated largely by economics. A workable
time span for such testing appears to be no more than six months to one
year. Nevertheless, it has been noted that field testing over such time
spans may be inadequate to indicate long-term transport due to acute or
chronic stresses to the environment (Barrett, 1968, personal communica-
tion).
-------
123
III. LITERATURE CITED
Albone, E. S. 1972. Fate of DDT in Severn esturary sediments.
Environ. Sci. Techno!. 6(10):914-919.
Barrett, G. W. 1968. The effects of an acute insecticide stress on
a semi-enclosed grassland .ecosystem. Ecology 49(6):1019-1035.
Barrett, G. W., and R. M. Darnell. 1967. Effects of dimethoate
on small mammal populations. Am. Midi. Nat. 77(1):164-175.
Belousov, V. S. 1970. Dynamics of phosphorus compounds in brown
forest soils of the humid subtropics on the black sea coast
of the Caucasus. Agrokhimiya 11:31-38.
Bingham, F. T. et al. 1971. Water relations, salt, balance, and
nitrate leaching losses of a 960-acre citrus watershed. Soil
Sci. 112(6) :410-418.
Bormann, F. H. et al. 1967. Nutrient loss acclerated by a clear-
cutting of a forest ecosystem. Science 159:882-884.
Bormann, F. H. et al. 1969. Biotic regulation of particulate and
solution losses from a forest ecosystem. Bioscience 19:600-610.
Briggs, C. A. 1969. Plume rise. AEC-TID-25075.
Bulan, C. A., and 6. W. Barrett. 1971. The effects of two acute
stresses on the arthropod component of an experimental grassland :
ecosystem. Ecology 52(4):597-605.
Cliath, M. M., and W. F. Spencer. 1971. Movement and persistence of
dieldrin and lindane in soil as influenced by placement and
irrigation. Soil Sci. Soc. Am. Proc. 35:791-795.
Cliath, M. M., and W. F. Spencer. 1972. Dissipation of pesticides
from soil by volatilization of degradation products. I. Lindane
and DDT. Environ. Sci. Technol. 6(10):910-914.
Culkowski, W. M., and M. R. Patterson. 1976. A comprehensive
atmospheric transport and diffusion model. ORNL/NSF/EATC-17.
117 p.
Curlin, J. W. et al. 1967. Watershed aquatic habitat interactions
(93-101). ^Auerbach, S. I. (ed.), Health Physics Division
Annual Progress Report for Period Ending July 31, 1967, Oak Ridge
National Laboratory, Oak Ridge, Tennessee.
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124
Dahlman, R. C. 1972. Prediction of radionuclide contamination of grass
from fallout-particle retention and behavior (492-508). USAEC
Symposium Series: Survival of Food Crops and Livestock in the Event
of Nuclear War. CONF-7000909, Jan. 1972.
Dahlman, R. C., and S. I. Auerbach. 1968. Preliminary estimation of
erosion and radiocesium redistribution in a fescue $Mdow.
ORNL/TM-2343. 24 p. ^*
137
Dahlman, R. C. et al. 1969. Behaviour of Cs-tagged particles in a
fescue meadow. Environmental Contamination by Radioactive
Materials. International Atomic Energy Agency, Vienna, 1969.
Dindal, D. L. 1970. Accumulation and excretion of Cl DDT in mallard
and lesser schaup ducks. J. Wild!. Mgmt. 34(l):74-92.
Dodd, J. D., and G. L. Van Amburg. 1970. Transfer to and distribution
of cesium-134 in the soil of two grassland habitats. Can. J. Soil
Sci. 50:1211-129.
Draggan, S. J. 1973. Diazinon and Carbaryl: Effects on the soil biota
of a soybean ecosystem. Ph.D. Thesis. Rutgers University, New
Brunswick, New Jersey.
Draggan, S. J. 1974. Diazinon and Carbaryl; Effects on the vegetation
of a soybean ecosystem. Submitted to: Am. Midi. Nat.
Duke, T. W. 1967. Possible routes of Zinc 65 from an experimental
estuarine environment to man. J. Water Pollut. Contr. Fed.
39:536-542.
Duke, T. W. et al. 1966. Cycling of trace elements in the estuarine
environment. I. Movement and distribution of Zinc 65 in
experimental ponds. Chesapeake Sci. 7(1):1-10.
Evans, J. 0., and D. R. Duseja. 1973. Herbicide contamination of
surface runoff waters. Environmental Protection Agency Report
No. EPA-R2-73-266, June 1973.
Fisher, D. W. et al. 1968. Atmospheric contributions to water
quality of streams in the Hubbard Brook experimental forest,
New Hampshire. Water Res. Res. 4(5):in5-1126.
Gosz, J. R. 1972. Nutrient cycling strategy in a northern hardwood
forest ecosystem. Colorado-Wyoming Acad. Sci. J. 7(2-3):28.
Harris, C. R., and W. W. Sans. 1972. Behavior of dieldrin in soil:
Mi crop!ot field studies on the influence of soil type on
biological activity and adsorption by carrots. J. Econ.
Entomol. 65(2):333-335.
-------
125
Harris, C. R. et al. 1971. lexicological stuides on cutworms. VII.
Microplat field experiments on the effectiveness of four
experimental insecticides applied as rye cover crop and soil
treatments for control of the dark-sided cutworm. J. Econ.
Entomol. 62(2):493-496.
Harris, C. R. et al. 1973a. lexicological studies on cutworms. IX.
Laboratory and microplot field studies on effectiveness and •
persistence of some experimental insecticides used for control of
the dark-sided cutworm. J. Econ. Entomol. 66(1):199-203. ... ..
Harris, C. R. et al. 1973b. Toxicological studies on cutworms.. X.
Laboratory and field microplot studies on effectiveness and
persistence of some experimental insecticides used to control
the black cutworm in organic soil. J. Econ. Entomol. 66(1):
203-208.
Haydu, E. P. The use of experimental streams to determine environmental
factors responsible for the productivity of aquatic communities.
Preprint, 34 pp. Weyerhaeuser Company; Longview, Washington.
Heagle, A. S. et al. 1973. An open-top field chamber to assess the
impact of air pollution on plants. J. Environ. Qua!. 2(3):365-368.
Henderson, G. S. et al. 1973. Walker Branch Watershed: A study of
terrestrial and aquatic system interaction (9-19). ORNL-4848.
Oak Ridge National Laboratory, Oak Ridge, Tennessee.
Hobbie, J. E., and G. E. Likens. 1973. Output of phosphorus, dissolved
organic carbon, and fine particulate carbon from Hubbard Brook
Watersheds. Limnol. Oceanogr. 18(5):734-742.
Humphreys, F. R., and W. L. Pritchett. 1971. Phosphorus adsorption
and movement in some sandy forest soils.. Soil Science Society of.
America Proceedings 35:495-500.
Korschgen, L. J. 1970. Soil-food-chain-pesticide wildlife relationships
in aldrin-treated fields. Journal of Wildlife Management 34(1):
186-199.
Johnson, N. M. et al. 1968. Rate of chemical weathering of silicate
minerals in New Hampshire. Geochimica et Cosmochimica Acta
32:5310545.
Likens, G. E., and F. H. Bormann. 1972. Nutrient cycling in ecosystems
(25-67). ln_ J. A. Wiens, (ed), Ecosystem Structure and Function.
Oregon State University Press, Corvallis.
Likens, G. E. et al. 1967. The calcium, magnesium, potassium, and
sodium budgets for a small forested ecosystem. Ecology 48(5):
772-785.
-------
126
Likens, G. E. et al. 1970. Effects of forest cutting and herbicide
treatment on nutrient budgets in the Hubbard Brook Watershed-
Ecosystem. Ecological Monographs 40(l):23-47.
Malone, C. R. 1969. Effects of diazinon contamination on an old-
field ecosystem. Am. Midi. Nat. 82(l):l-27.
Marcuzzi, G., and L. Dalla Venezia. 1972. First results of the
study of the soil fauna of two Italian artificial ecosystems.
Revue Ecol. Biol. du Sol 9(2):229-233.
Mattraw, H. C., Jr. 1973. Cation exchange capacity and exchangeable
metals in a Florida watershed. Ph.D. thesis. Florida State
University.
Nelson, D. J. 1970. Measurement and sampling of outputs from
watersheds (257-267). Jn. D. E. Reichle (ed.), Analysis of
Temperate Forest Ecosystems. Volume I of Ecological Studies,
Analysis and Synthesis. Springer-Verlag, New York.
Odum, E. P., and E. J. Kuenzler. 1963. Experimental isolation of
food chains in an.old-field ecosystem with the use of phosphorus-
32 (113-120). JJT. V. Schultz, and A. W. Klement (eds.), Radioecology-
First National Symposium on Radioecology. Reinhold Publishing
Corporation, New York.
Overrein, L. N. 1971. Isotope studies on nitrogen in forest soil.
I. Relative losses of nitrogen through leaching during a period
of forty months. Meddr. Norske Skogfors. 29(5):261-280.
t
Overrein, L. N. 1972. Sulfur pollution patterns observed. Leaching
of calcium in forest soil determined. Airtn'o 1(4):145-147.
Reichle, D. E. et al. 1973. International Biological Program:
Oak Ridge Site (20-33). ORNL-4848. Oak Ridge National
Laboratory, Oak Ridge, Tennessee.
Rolfe, £ L. et al. 1972. Modeling lead pollution in a watershed-
ecosystem. J. Environ. Sys. 2(4):339-349.
Rogowski, A. S., and T. Tamura. 1970. Environmental mobility of
Cesium-137. Radiat. Bot. 10:35-45.
Schulz, K. R., and E. P. Lichtenstein. 1971. Field studies on
the persistence and movement of Dyfonate in soil. J. Econ.
Entomol. 64(l):283-287.
Slade, 0. H..(ed.). 1968. Meteorology and atomic energy. U.S.
Energy Research and Development Administration, TID-24190.
-------
127
Taylor, A. W., and H. M. Kunishi. 1971. Phosphate equilibria on
stream sediment and soil in a watershed draining an agricultural
region. J. Agr. and Food Chem. 19(5):827-831.
•
Waller, H. D., and 0. S. Olson. 1969. Prompt transfers of Cesium-137
to the soils of a tagged Liriodendron forest. Ecology 49(l):15-25.
Wilder, H. B* 1972. Investigation of the occurance and transport of-
arsenic in the upper sugar creek watershed, Charlotte, North
Carolina. Geological Survey Research Professional Paper (No.
800-D):205-210.
Wischmeier, W. H., and D. D. Smith. 1965. Predicting rainfall-erosion
losses from cropland east of the Rocky Mountains. Agricultural
Handbook No. 282, ARS/USDA, Washington, D. C. 47 p.
-------
128
RECOMMENDATIONS FOR TESTING ENVIRONMENTAL FATE OF CHEMICALS
I. INTRODUCTION
Review of the "state-of-the-art" of soil, microcosm, and field tests
for determining the transport of chemicals in the environment reveals
that no simple, standardized, test systems exist. Systems which have
been described in the literature do not allow flexibility in many
physicochemical parameters, organism diversity, or competition between
biotic components for toxicant accumulation. Furthermore, it is evi-
dent from our experience with some toxic chemicals in the environment
that no reasonable system of pre-market evaluation will ensure absolute
safety to man or other biota. Any system of screening (test protocols)
can, at best, minimize the hazards associated with the release of toxi-
cants into the environment. Therefore, any testing sequence should be
provisional and flexible enough to permit continuous updating as scien-
tific understanding advances and new procedures become available.
We recognize the fallibility of test systems in terms of degree of
predictability for "real world" situations. No single test procedure
provides an exact measure of the effects that we wish to predict. Since
we can measure only a limited number of natural phenomena, a test may
give both false positive and false negative results. Accordingly, it
would be unwise to base a regulatory decision on anything less than
information provided by several steps in an evaluation scheme.
-------
129
II. TEST PROTOCOLS
A. Value of Short-term Tests
Within the constraints discussed in the previous section, short-
term tests for environmental transport of toxic chemicals are useful
in. cases where probable exposures in the environment are relatively
small. This will be the case for many new industrial chemicals. Even
so, there may be cases where short-term tests made early in a sequence
of evaluation may need to be repeated with greater depth or precision if
subsequent findings give rise to doubt the original test results.
B. Cost Effectiveness of Tests
An important consideration in selection of tests is cost effective-
ness. The value of information obtained in relatively simple, inexpen-
sive tests must always be weighed against the costs (in time and resources)
of further testing. In some cases, the results of several inexpensive,
but relatively imprecise, laboratory tests may be more valuable than the
result of one expensive field test of longer duration. In other cases,
it may be more cost effective to delete laboratory tests of 30 to 60 days
duration and screen a potential toxicant in expensive field tests of 1
year or longer in duration.
Assigning cost values to test procedures is difficult in terms of
estimating both equipment and time requirements. In addition, the level
of expertise required to conduct and/or interpret test results is not
always evident. It is especially difficult to estimate costs of test
procedures from the literature, particularly when considerable time may
have gone into developing the reported test method. For these reasons,
estimates of test costs given in this study are based primarily on the
time required for the test and the salary of persons conducting the test.
This implies a complete in-house capability of equipment and manpower
for performing the tests. However, where custom-made equipment is
required, the cost of such equipment is estimated. The selection of
tests which are recommended in this study was made with an attempt to
minimize equipment and manpower costs.
-------
130
The cost of analytical equipment is not considered because it will
vary with the kinds of chemicals to be analyzed. For manpower cost esti-
mates it is assumed that two levels of expertise are required for con-
ducting tests. Designation of salary for these levels is arbitrary and
is as follows:
1. A technician with a general background in laboratory
techniques—$9,5QO per year; $40 per day.
2. A specialized analyst or professional with a background
in experimental techniques and data interpretation-
Si 7,000 per year; $70 per day.
C. General Testing Hierarchy
Testing for environmental transport of toxic substances should be
preceded by other tests which, to a large degree, dictate the nature
and extent of transport tests. Information on degree of toxicity, per-
sistence, and dispersion, coupled with basic physical and chemical infor-
mation on the toxicant, can be used to select both the type and duration
of transport tests needed to augment the decision making process.
Obviously, many important decisions can be made on the basis of
analogies with other known chemicals. Structure-activity relationships
are reasonably well understood for some groups of chemicals. For ex-
ample, the tendency for organometallics to form complexes with natural
bases and for persistent hydrocarbons to accumulate in organisms as a
function of their partition between fat and water give some basis for
predicting bioaccumulation.
Table 1 is a listing of attributes of toxic substances that could
be used to indicate the necessity and extent of chemical transport testing
at an early stage of decision making. If the product of attribute ranks
(AXBXCXDXE) is equal to or less than 36, tests for mobility, bioaccumu-
lation, and biomagnification are strongly recommended. If the product
is between 36 and 72, decisions on transport testing are dependent upon
C (dispersion). If the product is greater than 72, transport testing
probably is not necessary. The use of 36 and 72 as cutoff points is
arbitrary and is based on examination of various ranking combinations
that give these products.
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131
Table 1. Scheme for Indicating the Necessity
of Environmental Transport Tests
Attribute Rank
A. Toxicity
High 1
Medium 2
Low 3
B. Persistence
High 1
Medium 2
Low 3
C. Dispersion
Widespread, high level 1
Widespread, low level 2
Local, high level 3
Local, low level 4
D. Solubility in lipid
High 1
Medium 2
Low 3
E. Solubility in water
High 1
Medium 2
Low 3
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132
An example of the use of this scheme is the subjective ranking of
the herbicide 2,4-0. It has low persistence in soil, 3; widespread,
low level use, 2; low solubility in lipid, 3; high solubility in water,
1. This gives a product of 18. The chemical has a high toxicity in
plants, 1 which gives a total product of 18. It has a relatively low
toxicity in animals, 3 which would give a product of 54. Based on
toxicity in animals the chemical should still be tested because its
intended use is widespread.
The usefulness of a rating scheme such as this would be greatly
increased if numerical values could be given for the high, medium, and -
low ranks. It is not within the scope of this study to devise appropriate
numerical values here but some suggestions for their selection are of-
fered.
Toxicity data are available on a wide range of chemicals. However,
most data are for .a cute or sub-chronic exposures. Acute toxicity tests
may be sufficient to determine values for the high, medium, and low ranks.
For example, LD5Q values (mg toxicant/kg rat) for oral intake could serve
to establish an acute toxicity hierarchy such as
High <50
Medium 50-500
Low >500
Since chronic toxicity may be unrelated to acute toxicity, the decision
to perform chronic studies may depend on results of tests to estimate
the environmental fate of the chemical.
Data are also available which correlate solubility in lipid (solu-
bility in non-polar organic solvent) with potential bioaccumulation.
A quantitative approach to the structure of organic compounds with re-
spect to their bioaccumulation has been established by Hansch (1969).
He derived a positive linear correlation between solubility and bioaccu
mulation in tissue.
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Some information is available on the relationship of chemical struc-
ture and persistence. It is known that many chemicals degrade in the
environment through chemical, photochemical or microbial processes.
While general, standardized tests for persistence have not been devised,
it may be sufficient here to determine whether a chemical degrades in
days (low persistence), weeks or months in the environmental medium
(soil or water) in which it is expected to be initially released.
For dispersion, it is expected that most chemicals would be locally
dispersed or, at most, dispersed at low levels over widespread areas.
Ranking dispersal characteristics may be quite subjective. A pesticide
such as DDT, for example, may be ranked as a chemical with a widepsread,
.low level dispersion due to its general use. The "widespread" category
should be considered as at least a regional area.
Having made a decision on the necessity of transport testing, the
type of tests can be related to the source of entry of the toxicant into
the environment. Potential pathways of exposure to man may be from
atmospheric, soil or water sources. In addition to potential exposure
to man from these sources, there is potential exposure to any biota (via
food chain and bioaccumulation processes) in ecosystems that may be
subject to one or more of the sources of entry. Critical testing points
for environmental transport are mobility.of the toxicant in soil, solu-
bility in water, and bioaccumulation and biomagnification in food chains.
Aquatic entry sources may, of course, be freshwater or saline water and
pathways may be from water or sediment.
D. Specific Testing Hierarchy
Following determinations of toxicity, persistence and dispersion,
which have indicated the necessity for transport testing, mobility of
the toxicant in soil and bioaccumulation along simple food chains may
be tested in the laboratory. If results from laboratory tests indicate
high bioaccumulation potential or biomagnification along food chains,
field testing should be required.
The tests which are proposed in this study are short-term tests
which can be used to screen toxic chemicals that follow the general
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pathways depicted in Fig. 1. While all of the methodology for these
tests was not developed originally for screening toxicants, it is be-
lieved that it is applicable for this purpose. While varying physical
parameters and other assemblages of organisms may give different results
in terms of absolute quantities, these tests can be used to ascertain
whether toxicants:
1. are similar to known compounds which have caused unintended
damage in the environment,
2. are likely to bioaccumulate in terrestrial and aquatic
food chains,
3. are likely to spread from sources of entry, and
4. should be subjected to long-term testing in the pre-market
evaluation.
Since no one microcosm or test system adequately represents the
complexities of nature or more than one type of ecosystem, tests for
toxicant transport are with relatively simple food chains (two or three
compartment transfers). These food chains are representative of those
in larger, more complex ecosystems and they represent a hierarchy of
trophic levels common to all ecosystems. It is assumed that if toxi-
cants do not bioaccumulate to a great degree or biomagnify in the higher
trophic levels of these simple food chains test systems, they are not
likely to do so in other food chains with other species in a similar
period of time.
To assure comparable results in any of the proposed tests, one or
more reference chemicals should be tested with the toxicant. Reference
chemicals, may be selected on the basis of having similar chemical or
structural properties to the unknown. They also may be selected on the
basis of known behavior in similar test systems. In testing toxicants
in the proposed test systems, application levels of the toxicant should
approximate expected exposure levels from the source of entry into the
environment.
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1. Tests for mobility in soil and sediments
Objectives and general comments
The recommended test for evaluating mobility is the soil thin-layer
chromatography method. The utilization of this approach will enable
reliable evaluation of relative movement and desorption characteristics
of mobile compounds. Standard (reference) compounds are required in lieu
of a standard soil to ensure that individual test results are comparable.
Two reference compounds, 2,4-dichlorophenoxyacetic acid (2,4-0) and ODT,
14
are recommended. Both are available in C labeled form. The two com-
pounds represent fairly mobile (2,4-0) and immobile (DDT) references.
Metabolites or degradation products can be tested along with the parent
substance on the same plate if required. Inorganic substances can be
tested as well. In general, however, the movement of cations will be
very slight and will follow the valence order 1 > 2 > 3 > 4. Some
anions will be mobile and some not. Compounds like nitrate, sulfate,
dichromate, halides, etc., which form fairly water-soluble alkaline
earth (Ca, Mg) salts, are more mobile than phosphates, for example, which
react with Ca, Mg, Fe, etc. An important use of soil TLC can be the
compan'son of the effects of structural alteration on compound mobility.
Helling (1971b) illustrated this by comparing acidic and esterified
forms of four pesticides. Chelating organics (NTA, EDTA, etc.) can also
be tested both for inherent movement and their ability to move toxic
metals through soil or exchange them for native soil cations.
The mode of entry of a chemical into the environment does not in-
fluence the basic test system proposed. The higher the "application"
rate or concentration, the more rapidly the chemical will become dispersed
in soil or sediments* This response in due to concentration gradients
and is not due to changes in sorption of individual molecules. While soil
TLC does not specifically apply to sediments, the same principles apply.
Compounds found to be mobile in the soil test will not be strongly sorted
in aquatic systems.
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At least three soil types should be considered for inclusion in
standardized tests. A single, nationally recognized soil would be ideal
for insuring comparative correlations of diverse chemicals. However,
such a soil has not been designated. The three soils should have a
range of pH values as measured by a 1:1 soil-water extraction. The
best general soil for this test should have a sandy loam texture with
about 10 to 20% clay. The pH should range between 6.0 and 7.0 and the
soil should have an organic matter content of between 0.5 to 1.5%. Two
other soils should be used to enhance the evaluation of mobility. An
alkaline soil (pH 7.0 to 8.0) should be tested to represent semi-arid
regions as well as calcareous sediments. This soil should be a sandy
loam, loam or silt loam (i.e., less than about 20% clay) with between
1 to 3% organic matter. A sandy loam or loam soil with a pH range of
5 to 6 and organic matter content of less than 1% should prove valuable
for understanding movement in acid soils. Usually high organic soils
(greater than 6% organic matter) or high clay soils (greater than 40%
by weight) should not be used since most organic compounds will be rela-
tively immobile in such matrixes. Such soils are mainly important only
in local situations and their use would yield little information on the
likelihood of chemicals to move in soils of more general distribution.
Soil test protocol
«
The basic procedures for soil TLC are identical to conventional
silica gel TLC. The soil is presieved to remove coarse materials. For
sandy loam soils, Helling and Turner (1968) removed particles greater
than 0.50 mm by dry sieving. For finer textured soils (loams, silt loams,
etc.) the fraction greater than 0.25-mm is removed.
A moderately fluid soil-water slurry is then prepared and applied
to 20 x 20 or 20 x 10 cm plates with a variable thickness applicator.
Helling and Turner (1968) and Helling (1971a,b,c) discussed plate prep-
aration which is very similar to conventional TLC. After air drying,
the substances to be tested are applied as spots about 1.5 cm from the
14
bottom. Amounts can range from 0.5 to 2 yg if C labeled. Higher
amounts can be used, but streaking will increase with concentration. If
non-radio!abeled compounds are being tested, a number of spots can be
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applied along the bottom of the plate to increase the amount subsequently
recovered for extraction and analysis. After application, the plate is
placed in a development tank containing about 0.5 cm water. The plate
is allowed to develop until the ascending water front has moved 10 cm
from the origin (application spot). The plate is then removed and air
dried. Autoradiography or scanning techniques are used to locate the
radiolabeled material on the plate. The compound's movement is determined
by measuring the farthest distance moved, even if the substance streaks
on the plate. The size of the leading spot, as well as streaking, should
be noted since streaking indicates that part of the compound, even if
fairly mobile, is not readily desorbing from certain adsorption sites
on the soil. Substances that move as compact spots are not interacting
strongly with the soil, even though they may not move fast due to charge
or polar characteristics. The R,. value is the direct distance moved
since the total distance is 10 cm. A compound which moved 6.5 cm thus
has an R, of 0.65.
Cost and time of soil tests
The TLC procedure is not time consuming or expensive. Excluding
initial soil sieving and plate preparation, the average time for devel-
opment of the soil TLC plate (i.e, the ascending water development step)
is 4 hours.
Following overnight air drying (for nonvolatile compounds), medical
X-ray film is placed in direct contact with the plate for 3 days or
longer as needed. Alternatively, a thin layer chromatogram scanner can
be used. Non-radiolabeled compounds .wi11 need to be extracted from
successive 1-cm vertical zones on the plate and assayed by other .methods.
For volatile compounds, the plates need to be frozen during film exposure.
the special equipment required include a radiochromatogram scanner (in
lieu of autoradiography) and a gas or liquid chromatography system where
extractions are required. Manpower requirements can be met at the tecn-
nical level with the exception of analytical determinations such as gas
or liquid chromatography. Total estimated time needed for the soil test
is 4 days. The estimated cost is $160, excluding equipment.
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Degree of predictability of soil tests
The TLC approach certainly allows a reasonable assessment to be
made of a substance's mobility in soil. The use of sandy loam soils
with pH's between 6.0 and 8.0 allows maximum mobility to be determined.
An examination of the results reported by Helling (1971c) reveals that
when 14 soils were used to test 13 pesticides, compound mobility was not
substantially altered in various textured soils provided the soils were
not strongly acid. Clay loam and silty clay soils usually retard move-
ment more than coarse textured soils, and organic matter content is
important for most compounds (mobility decreasing with increasing organic
carbon content). As a general rule, compounds which move as fast or
faster than 2,4-0 on nonacid soils should be classed as mobile. Com-
pounds which move at R 's around 0.00 - 0.10 (DDT) are immobile, and
those in between can be considered slightly mobile. Acid soils (pH
. 4.5 - 6.0) can be used to screen those compounds which behave like weak
acids in soil. Movement should be less on acid soils than in more neu-
tral or alkaline soils, particularly if the compound is acidic. If pH
is not critical, movement should be similar on nonacid soils (pH 6.0 -
8.0), taking into account any differences in texture. The reference
compound 2,4-D had an average mobility (R,) of 0.72 on the 14 soils
tested by Helling, with the lowest Rf values (0.41 and 0.50) occurring
on two strongly acid soils (pH 4.3 and 4.4). As long as such acid soils
are recognized as retarding movement of weakly acid compounds, reasonable
estimates of relative mobility of even the most mobile group of organics
(the acidic substances) can be obtained.
Required soil and sediment information
The following information is necessary to describe the soils or
sediments tested:
1. pH: 1:1 soil/water slurry
2. Particle size analysis (% sand, silt, clay)
3. Organic carbon content. Organic carbon x 1.724 = organic
matter content
4. Cation exchange capacity (determined at pH 7.0 with ammonium
acetate)
5. Description of site soil sampled, including soil survey
nomenclature if available (e.g., Hagerstown silt loam).
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These standard procedures (1-4) can be found in Jackson (1958) or Black
(1965a,b).
Sediment test protocol
The soil TLC method measures mobility. The mobil$^«concept is not
specifically applicable to sediments, however. Soil TLC results indicate
the affinity of the tested chemical for the soil matrix. Chemicals, which
are immobile or slightly mobile in TLC tests will be strongly sorted to
sediments. Conversely, very mobile chemicals will be weakly sorbed and
will appear in water in significant concentrations (greater than 1%) of
the total in water and sediments.
Batch-type equilibrium tests should be used, however, when sediment
testing is desired. Sediment samples should be air dried or frozen after
collection. One gram samples (oven-dried at 105°C) are placed in screw-
cap centrifuge tubes. The test compound is then added along with 20 mis
of distilled water. The mixture is equilibrated for 2 hours or longer
at 25°C. A preliminary test is run to insure that 2 hours is enough
time to achieve a constant solution phase concentration.
To provide a standard reference K value determined by applying the
Freundlich equation (State-of-the-iArt, soils and sediments). C should
be 1 ug compound per ml of aqueous phase. By conducting a series of
equilibrations at varying contaminant concentrations, a log-log plot of
X/m vs C can be made. From this plot, X/m at C =1 yg/ml is read
from the graph and K is calculated from the Freundlich equation. The
slope, 1/N should approach unity under these conditions (Poinke and
Chesters, 1973). Reference compounds like.DDT and 2,4-D are included
in the test protocol for comparisons.
Using the above test system, a compound which sorbs only to the
extent of 10% has a K value of about 2. A compound which sorbs to the
€>
extent of 99% has a K of about 2000, and for 99.9% adsorption, K is
about 20,000. A general guide which could be used for evaluating gen-
eral transport is: K 3 < 100, the compound is mobile. For K = 100 -
1,000, slightly mobile; K = 1000 - 10,000, relatively immobile, and for
K = >10,000, immobile. This classification is not derived from docu-
mented studies but is suggested as a guide for interpretation of results.
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Cost and time of sediment tests
About one day should be required for equilibration and sampling.
The cost is estimated at about $50 per test.
Degree of predictability of sediment tests
Both soil TLC and Freundlich K values can be used to estimate the
degree of sorption of chemicals to sediments. Rates of movement, however,
are not described by the Freundlich K value and no estimate is available
for desorption rates when fresh (uncontaminated) solution flows over a
contaminated sediment.
Volatility test protocol •
The calculation proposed by Mackay and Wolkoff (1973) appears to
evaluate evaporative losses of low solubility (in water) compounds. If
it is assumed that the rate of water evaporation from 1 m of water is
2740 g/day, then the half-life of a sparingly soluble substance at 25°C
in water (assuming constant is
T = 0.1082 C1s/P1s M.
where
T = half-life in water in units of days.
C. - compound's solubility in water,
P. = compound's vapor pressure,
M. = compound's molecular weight.
The constant 0.1082 incorporates the constants at 25°C of Mackay and
Wolkoff
354.
koff's equation (see "State-of-the Art"). For example, DDT (M^ =
k4; C.s = 1.2 x 10"3 mg/liter; P.$ = 1 x 10"7 mm Hg).
T = 3.7 days
The insecticide dieldrin, which has a vapor pressure of 1 x 10" mm Hg
(same as DDT) but is more soluble in water (0.25 mg/liter), had a calcu-
lated T of 710 days (see Mackay and Wolkoff). The precise time is of less
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Importance than its order of magnitude since assumptions are made about
2 3
the rate of water loss (2740 g/nr/day) and volume (1 m). The values
of T calculated from the equation assume no adsorption occurs to partic-
ulates. Compounds which yield calculated T values of minutes or hours
should be conside^ld extremely volatile. More with T values in days are
of intermediate volatility.
An experimental apparatus which allows the evaluation of volatil-
ization los-ses from soils or sediment-water systems could be set up as
schematically outlined by Spencer and Cliath (1973) or Oilling e_t al_.
(1975). Spencer and Cliaths1 apparatus consists of a relative humidity
control, a water-tension regulation system to control the moisture con-
tent of the soil, a soil column, and an ethylene glycol trap. The tend-
ency of a chemical to be lost from .soils by gas-phase diffusion or
mass-flow induced surface volatilization can be experimentally compared
to the estimated potential based on the water/air partition coefficient.
Chemicals which should be tested by this approach would be those which
have TLC mobilities greater than 0.1 (RJ. Substances with low mobili-
ties on TLC but also low water/air distribution coefficients should also
be tested. The soils used in the volatility tests should be the same
as those suggested for the TLC test.
The apparatus described by Dilling et aJL (1975) offer a simple
method of determining residence times in water. The chemical, in water,
is placed in a closed container and vigorously stirred. The aqueous
phase is periodically monitored to evaluate the time required for one-
half of the chemical to disappear. The half-time is obtained by plotting
concentration of the chemical in solution against time. Sediment may
be added to this test system.
Cost and time of volatility tests
The apparatus diagramed by Spencer and Cliath or Oilling et al.
will cost less than $500. The time required for testing the compounds
would be about one week per soil type and less than one week for water
solutions. Technical help and analytical requirements will be no more
than that required for TLC work.
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Degree of predictability of volatility tests
The Mackay and Wolkoff equation classes substances in terms of min-
utes, hours, days, and months with respect to evaporative half-lives
from water. This evaluation appears to be the best single measure of
how fast a chemical is likely to be lost from water. The presence of
soil or sediment will retard the rate of loss to the extent that the
solution phase concentration is affected by adsorption.
There needs to be a great deal of research devoted to developing
better guidelines for interpreting volatilization losses. However, in
the absence of further guidelines, it would appear safe to predict
that substances with evaporative half-lives (calculated or experimentally
determined) of less than several days should be considered to dissipate
to the atmosphere rapidly.
2. Laboratory tests for terrestrial bioaccumulation
and biomagnification
Objectives and general comments
Toxicants can enter terrestrial ecosystems via deposition from the
atmosphere or via soil contaminated by irrigation water or seepage
(leachate) from landfill. If the source of entry is the atmosphere,
vegetation may accumulate the toxicant through foliar absorption or up-
take from soil via roots. If the source of entry is landfill seepage
or irrigation water, uptake by plant roots will be the dominate mechanism
for accumulation by plants.
If the toxicant is not mobile in soil there will be little leaching
from landfills into either terrestrial or aquatic ecosystems. Further,
erosion or transport due to volatility should not be significant trans-
port mechanisms if the toxicant is buried in a landfill. Therefore,
terrestrial tests for transport are necessary only for atmospheric
sources of entry (deposition from stacks, spraying, dusting) or for
water soluble toxicants in landfills or irrigation water.
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Terrestrial test protocol
The proposed terrestrial test system is air and/or soil -»• producer
food base (Phaseolus vulgaris, common green bean) •*• invertebrate con-
sumer (Acheta domesticus, brown cricket) -»• vertebrate preditor (Colinus
virginianus, quail). This entire producer-to-herbivore-to-omnivore
food chain has not been used experimentally. The bean plant was re-
elected because it is easy to culture and it is one of the classic plants
used for- nutrient uptake studies. The work of Van Hook and Crossley
(1969) demonstrated the utility of the brown cricket in food chain stud-
ies. The quail was selected because it is easy to culture and it is
being used in experimental food chain work by the National Ecological
Research Laboratory at Corvalis, Oregon.
Fifty bean seeds, soaked in water overnight, are planted (2.or 3
per pot) in quartz sand in pots provided with bottom drainage. A nutri-
ent solution is periodically supplied to the tops of pots and allowed
to drain out before fresh nutrient is added. Nutrient may be added one
or several, times daily depending upon pot size and rate of drying.
Simple techniques for sand cultures and preparation of nutrient solutions
are given by Hewitt (1966).
When plants are about 5 inches high (14 days) and have two or more
pairs of leaves, the toxicant may be added with the nutrient solution
to determine uptake via roots or it may be added as a spray or dust to
foliage to simulate atmospheric sources of entry. The sand culture
method will give an overestimate of uptake via roots from soil. If
application is by nutrient solution, the toxicant should be supplied
for several days to compensate for drainage losses. If the toxicant is
a gas (originally or via volatilization), the application procedure may
be modified by enclosing the potted plants in a bell jar, or other suit-
able chamber, during the application period. If the toxicant is avail-
able in radio-labeled form (see soil test protocol), use of this form
may facilitate analysis by increasing sensitivity and ease of detection.
Following applications, several plants are harvested and chemical
assays should be performed on pooled, replicate samples of root and
foliage which has been rinsed in clean water. Preparation of plant
tissue for chemical analysis is given by Jackson (1958).
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Fresh root and foliage material from non-harvested plants are next
fed to 100 adult brown crickets (Acheta domesticus). This cricket is
available commercially as the "bait cricket" used by fishermen. Also
it is easily colonized in the laboratory. Feeding should continue for
about a week.^jten ten crickets should be sacrificed for bioaccumulation
determinations. Van Hook and Crossley (1969) recommended that crickets
be fed in separate cages (pint ice cream cartons with screen bottoms).
However, it should be adequate to house two crickets per cage.
The remaining crickets are fed to three young adult Bob-whites
(quail). Each bird should receive 10 crickets per day for three days,
after which the birds are sacrificed for analysis. It may be necessary
to supplement the diet of crickets with commercially available food
to maintain healthy birds during the test period.
Bioaccumulation factors may be expressed as ppm in the test orga-
nisms and biomagnification factors are calculated by relating the*con-
centration in the organism to that in the organism's food base.
Cost and time
The duration of this test is about 30 days and it will require a
full-time technician who can operate the test and perform the necessary
chemical analyses. The estimated cost for this test, excluding time
spent in culturing the animals, is $1200. An initial expenditure of
about $200 will be required to purchase plant culture materials, cages,
etc. If test organisms are purchased, an additional $50 should cover
this expenditure.
Degree of predictability
This simple test protocol should indicate whether a toxicant is
likely to move rapidly or accumulate along a terrestrial food chain.
If a tested toxicant is very soluble in lipid and was found not to
accumulate in the predator organism, additional testing will be nec-
essary in the recommended food chain or in another food chain. Testing
with higher level predators or mammals should not be necessary. If the
toxicant is soluble in lipid and was found to biomagnify in the test
predator, it should appear in higher trophic levels.
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It is known that biological factors such as age, size, physiological
state, etc. and physical factors such as temperature, periodicity, etc.
effect bioaccumulation. However, the test animals selected have been
shown to give reasonably uniform results over a range of environmental
conditions common to those in a typical laboratory (Van Hook and Crossley,
1969).
It is believed that this test protocol offers a maximum chance for
the toxicant to accumulate over the period of testing. Most parameters
(environmental or biotic) which could be added in a more complex testing
scheme would tend to obscure the test results, thereby reducing the
value of a simple scheme for rapidly estimating the potential hazard
of the toxicant.
It is possible in a short-term, simple test for a toxicant to "slip
through" or behave differently than it will in nature or over longer
time periods. Mercury, for example, is .converted in some natural systems
from a relatively immobile (inorganic) form to a highly mobile (organic)
form. Therefore, if a toxicant to be tested is suspected of having this
conversion potential, it may be wise to test both an inorganic and organic
form.
3. Laboratory tests for aquatic bioaccumulation
and biomagnification
General comments
Freshwater food chains acceptable for use in environmental trans-
port screening procedures have been described by Metcalf e_t a\_. (1971,
1973), Isensee et al_. (1973) and Johnson (1974). Many more could like-
wise be adapted from material available in the literature, but none of
these have been tested extensively enough to be included in protocols
proposed in the present report. The "Metcalf" system has been used to
evaluate the potential environmental effects of a wide spectrum of toxi-
cants, including insecticides, herbicides, fungicides, heavy metals,
etc. (Also see State-gf-the-Art section). An adaptation (and simplifi-
cation) of the aquatic portion of the "Metcalf" system will be described
later for use as the freshwater aquatic test protocol.
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The situation is much less clear for marine and estuarine systems,
in which the problem of reproducibility appears more acute. Investiga-
tors have relied primarily on culture systems which contain naturally
occurring (and highly variable both in space and time) assemblages of
biota and sediments.(see Movement of Chemicals into Biota). One such
system which appears to be suitable as an experimental food chain system
is that derived from culture techniques'for bivalve mollusks (particularly
the oyster Crassostrea virginica.) described by Loosanoff and Davis (1963)
and Loosanoff (1965). Uptake of trace metals by.shellfish has been
studied in a number of bivalves, both in laboratory culture (Pringle
et al_., 1968) and in a microcosm situation (Kerfoot, 1973). In both
cases, the oyster was observed to bioaccumulate heavy metals to a greater
extent than the other organisms tested. An adaptation of the oyster
culture technique and bioaccumulation models will likewise be described
later as the estuarine-marine test protocol. This simple food chain can
be used under either an estuarine or marine salinity-temperature regime
and offers the additional advantage of year-round availability for envi-
ronmental transport testing. In order to help account for, and perhaps
somewhat compensate for, the obviously greater diversity of environmental
conditions and biotic diversity in the real world versus the test systems,
compounds with known properties should be run through all test procedures
as controls in parallel with the unknown material.
Toxicity and bioaccumulation by direct uptake via water or by inges-
tion are irrevocably linked. Simple bioaccumulation factors, i.e.,
water •*• organism and food •*• organism, can be obtained in uptake studies.
It is expected that the classes and species of organisms chosen for
toxicity studies would be essentially identical to those used in food
chain bioaccumulation determinations. Further, it would represent a
considerable waste of effort not to perform these tests simultaneously
at the same facility. Toxicity testing regimes which can be readily
adapted for concurrent measurements of bioaccumulation have been described
by Brungs (1969), Arthur and Leonard (1970) and Biesinger and Christensen
(1972).
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Freshwater test protocol
Because of a lack of information at present, a lotic (flowing water)
protocol cannot Be proposed for use. Johnson (1974) has presented a
lotic, cold-water, food chain model which appears promising but which will
require further study.
Both Isensee £t al_., 1973 and Johnson (1974) have described simplifi-
cations of the "Metcalf" system for aquatic use. A major source of vari-
ability in the "Metcalf" or "Metcalf-derived" systems is to be found in
the "plankton" compartment whose species composition is not controlled.
In order to reduce this source of variation, it is recommended that
food chains be based on more rigidly controlled energy sources.
The following food chains, classified according to food source are
proposed: 1) primary producer base - algae (Chlamydomonas reinhardi) -»•
zooplankton (Daphnia magna) •*• fish (Gambusia affinis) and 2) detritus
base - detritus (trout food mixture) -»• zooplankton (Daphnia magna) •*•
fish (as above). Each chain would be augmented by a scavenger-grazer, .
the snail, Physa sp., whose food source would be dead algae, feces, etc.
The use of the detritus-based food chain should somewhat alleviate the
questions posed by the absence of soils and other organic sources in the
food-chain systems. These food chains offer the advantages of year-round
availability, extensive prior testing and culture experience (see
Movement of Chemicals into Biota), and offer a choice between a detritus-
based or algal-based system made on the basis of lipid-water solubility
and soil mobility tests.
The details of the proposed protocol are as follows: first, a 10 .
gallon aquarium is filled with 30 liters of standard reference water
(Metcalf, 1971). Then, either an aliquot for a Chlamydomonas culture
(Frank, Boll, and Kelly, 1957; Rlchman, 1958; Christensen, 1973) or the
trout food mixture (Biesinger and Christensen, 1972; Robertson et al.,
1974) is added to bring the concentrations to 10"2 cc/fc NO5 cells/ml)
and 1 ml/£, respectively. A sublethal amount of the toxic compound under
study is then introduced and the system is allowed to equilibrate (water-
food concentration). Then, 20 Daphnia/liter of aquarium water are added,
along with 20 snails, and the systems are observed for 4 days. After
4 days, 10 Gambusia are added and, then after 4 additional days, the
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systems are sacrificed. Components should be assayed periodically during
the study-period and all components should be assayed at termination in
order to perform a materials balance on the system. Sampling intervals
and analytical procedures are described by Metcal f e_t al_. (1971) and
Isensee et_ al_. (1973). Bioaccumulation and b^iagnification factors
may then be calculated and the relative hazard of the toxicant can be
estimated.
Cost and time
This test should require about 30 technician days once cultures of
test organisms are set up. Estimated cost is $1200 per test for personel.
About $500 for aquaria and associated equipment will be required to set
up the test.
Estuarine - marine test protocol
The following system is proposed: primary producer base - algae
(Monochrysis 1utheri & Isochrysis galbana) •* oyster (Crassostrea virginica)
This food chain should be extended by feeding the oysters to a marine
predator such as the starfish Asterias forbesi or the green crab Carcinus
maenas. Since work with the proposed food chain has not involved the
addition of a predator (see Movement of Chemicals into Biota), this ex-
tension must be examined experimentally before its inclusion. Until that
time, the food chain may best be terminated by feeding the oyster meats
produced to laboratory animals (mice). In fact, this method of food
chain termination could be retained in addition to the marine terminus.
The details of the protocol are as follows: First, an aliquot of
an algal culture containing mainly Monochrysis 1utheri and Isochrysis
galbana is added to 300 liters of filtered sea water (nutrients added)
in a 200 gallon tank to produce an algal density of 10 cells/ml. Then,
the system is treated with a sublethal concentration of toxicant and
allowed to equilibrate. After equilibration, racks containing 20 medium
oysters are added. This should reduce the algal concentration to neg-
ligible levels within 2 days [see Loosanoff (1965) for filtering rates].
After 4 days, the system components are sacrificed for bioassay and
feeding to laboratory animals. Laboratory animals should be sacrificed
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149
4 days after feeding. Urine and feces should be collected and assayed
during the 4 day period. Care should be taken to separate the G.I.
tract and skin from other tissues prior to analysis.
Bioaccumulation potential is estimated as in the previous case.
Bioaccumulation data may be compared to a reference library of similar
information already obtained for known compounds. Such comparisons,
along with those made at other steps in the hazards-testing protocol,
'i.e., toxicity, mobility, persistence, etc., can then be integrated to
reach an initial decision on the status of the compound in question.
This decision might well be, as noted previously, that the compound
should be tested further before making a final decision as to its suit-
ability for use. Estimated cost and time are the same as for the previ-
ous test.
4. Field tests for transport of toxicants
General comments
It is obvious that no one field testing protocol will be suitable
for determining the fate of toxicants in the environment as a whole.
The following list will aid in categorizing natural ecosystems that can
be considered for transport testing in the field.
1. Terrestrial ecosystems
a. Agricultural
b. Natural
i. Forests
ii. Old fields-Grasslands-Meadows-Tundra
iii. Deserts
2. Terrestrial-Aquatic
a. Watersheds
b. Riverbanks-Streambanks-Lakeshores-Irrigation Ditches
c. Swamps
3. Aquatic
a. Rivers-Streams
b. Lakes-Ponds
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4. Estuarine
a. Estuaries
b. Bayshores
c. Uetlands
5. Marine
a. Oceans
b. Oceanfronts
In addition to specific information (persistence, dispersion, tox- •
icity, physical properties, similarity to other chemicals) already men-
tioned in this report, the following information is useful in designing
field tests.
a. Recommended use-levels
b. Expected or potential dissipation levels
c. Specific entry point into environment (e.g., into a river,
soil treatment, foliar spray, gaseous effluent)
d. Other expected or potential entry points into the environment.
This general information will allow resolution of the point or
points of entry to ecosystems where dissipated chemicals might be con-
centrated and potentially transported to other areas. Of greater impor-
tance, the general information on use and dissipation levels will allow
the use of realistic applications to areas chosen for field testing.
During the production of a chemical, data on identification and
monitoring of residues of products that are disposed of within manu-
facturer-owned areas such as waste holding ponds, land dumps, and
landfills, may be useful in design of subsequent field tests.
Although the purview of this report is not limited to the deter-
mination or organic chemical transport, it is realized that organic
chemicals will comprise the bulk of chemicals requiring environmental
screening for transport. Since minute quantities of toxicants, especially
organics, may become diluted when transported, detection of low-level
quantities poses a very real problem. It is recommended that whenever
possible, especially with regard to organic compounds, candidate toxi-
cants for transport testing be radioisotope-labeled. Care must be taken
in determining degradation product pathways and the radiolabel must in-
sure retention of the label on any degradation product arising from abiotic
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or biotic chemical transformation. Radioisotopes that have been success-
fully utilized in organic compound radiolabeling include:
1. tri ti um
2. carbon-14
3. chlorine-36
4. sulfur-35
5. phosphorus-32
6. iodine-125, -131
7. mercury-203
Field tests for chemical transport are amenable to at least three
modes of measurement:
1. initial sampling
2. time-series sampling
3. terminal and harvest sampling.
Initial sampling is important in that it allows for the determination
of actual levels of chemical input to the test system. Time-series
sampling allows for the direct determination of transfer rates, deg-
radation rates, and transformation rates; indirectly it allows determi-
nation of turnover rates, bioaccumulation factors, and biodegradability
indices. However, if data collected from, time-series studies are. used ....
for rate calculation, sampling periods should be evenly spaced within
time. .Harvest (terminal) sampling allows final mass balance determi-
nations within compartments of the particular field test system utilized.
The large size and the composition of most field test compartments
disallows total harvest, and such sampling is usually done through
selective sub-sampling and iterative procedures to provide total material
balance.
The time of year has profound effects on physico-chemical (e.g.,
precipitation, insolation), geological (erosion and deposition), bio-
logical (temperature, moisture, etc.) and ecological activity (annual
population, community structure changes and succession). If field
studies of chemical transport are to be comparable, data for the tests
must be gathered during the same annual time periods. Due to the impor-
tance of biotic effects on chemical transport, field studies should be
performed during periods when the natural biota of a given field test
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area are at or near their peak activity. For temperate terrestrial and
aquatic ecosystems, this would correspond roughly to either the agricul-
tural or natural growing season. Desert ecosystems generally experience
peak activity during rainy season months, while tundra biota tend to
flourish during the summer months.
The extreme heterogeneity of natural environments makes it neces-
sary to replicate field test treatments to achieve not only valid data
on chemical transport, but to provide an estimate of field (or envi-
ronmental) variability. Environmental variability makes up the largest
component of experimental error in field data; and if test plots are not
replicated, only the largest treatment effects can be ascertained with
a high degree of confidence.
Cochran and Cox (1957) have classified methods for increasing the
accuracy of experiments as follows:
1. increase size of experiment (by provision for more
replicates or by inclusion of additional treatments)
2. refinement of experimental technique
3. handle experimental material so that effects
of variability are reduced (done by careful
selection of material, additional measurements
that provide information about material, or by
skillful grouping of experimental units in such
a way that the units to which one treatment is
applied are closely comparable with those to which
other treatment is applied.
Probably the simplest way of achieving reduction of variability
in field tests, or at least identification and quantification of vari-
ability, is by pretreatment sampling of replicates within a given area.
Judicious observation will help to identify the major sources of envi-
ronmental variability and provide estimates of this variability. From
this knowledge, adequate samples of field test-ecosystem compartments,
and replicates of chemicals treatments may be chosen that are economical
in both time and expense.
Rationales for decreasing experimental error, while optimizing
efficiency have been covered in detail by Cochran (1963), Cochran and
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153
Cox (1957), and Hansen et aiL (1953). Resolution of field test site
variability will require the services of a statistical consultant who
can provide meaningful input into design, execution, and analysis and
interpretation of field data.
Terrestrial field test protocol
Site. Despite the apparent differences in terrestrial ecosystems
that can be included in field testing schemes, the following protocol
can be used in agricultural or natural ecosystems. Site selection will,
of course, be based on point of entry to the environment of the chemical
of interest. Size of the experimental area will depend on the magnitude
of local environmental variability and the necessity to replicate all
treatments.
Replicate, adjacent field areas no more than 100 meters square each
are recommended. Although the number of replicates will in general be
no more than two, identification of extreme environmental variability
and cost considerations may prompt decisions to increase the number of
replicate field areas. The selection of replicate test areas having
similar elevation and drainage is an important factor in the reduction
of variability. Floristic similarity serves, in many cases, to identify
plots that have a minimum degree of variability. Agricultural field
test areas (monocultures) possess the ideal in floristic similarilty.
Inclusion of border areas surrounding both replicate field areas7
and treatment plots within these areas will reduce or eliminate edge....
effects and the possibility of chemical trans location. Borders also
provide convenient walk-ways that reduce the effects of trampling. An
example of the physical layout of a field test having two replicates
and allowing the determination of the transport of one toxicant at
five different concentrations; or, of five different toxicants each
at a single concentration is shown in Figure 2.
Sampling parameters. Previous to treatment, collection of soil
cores from within the treatment plots and their analysis for commonly
determined elements (N, P, C, Ca) and determination of physical prop-
erties (water holding capacity, exchange capacity, pH) will allow an
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154
3.5
10
DIMENSIONS IN METERS
10
Figure 2. Schematic of a terrestrial ecosystem field area with
replicate treatment plots. C denotes control treatments,
T! Ts are chemical treatments or levels of a chemical
treatment.
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155
estimation of environmental variability of the areas selected for test-
ing transport. Sampling of plant species will allow both determinations
of biomass and diversity, and will aid in the identification and quan-
tification of environmental variability.
After treatment, randomly selected soil cores within the treatment
plots should be taken to a depth of one meter. Analysis for the chem-
i",1
ical of interest, its degradation products, or its transformation pro-
ducts within predetermined increments of the core will provide the
following information:
a. validation of the initial input when done immediately
following application of the chemical of interest
b. mobility in soil when taken as time-series
c. persistence in soil when taken as time-series
d. uptake or bioaccumulation of microarthropods
and microinvertebrates when first 5 cm of core
is subjected to Tullgren-type extraction procedure
(Mai one, 1969).
Systematic sampling of vegetation over time and in a terminal sam-
pling, with subsequent analysis of plant material that has been classi-
fied as to species, biomass, and plant part (root, stem, leaves), will
allow determination of uptake rates, and bioaccumulation.
Sampling over time of grazing insects (crickets, grasshoppers,
aphids, other foliage feeders) and predaceous insects (selected spiders,
centipedes, beetles) will allow determination of bioaccumulation and
biomagnification in consumer trophic levels.
Small, native mammals will not usually be present in the relatively
small field test area. I.f they are added, the plots will have to be
made escape proof. In addition, overstocking a small experimental area
can result in unrealistic densities of animals and may result in destruc-
tion of the plots. It is probably better to exclude vertebrates and to
extrapolate bioaccumulation data from toxicity tests in the laboratory.
Volatilization of a chemical may be an important pathway in trans-
port; such volatilization of a chemical, its degradation product, or
its transformation product may be measured with the aid of vapor collec-
tion systems engineered to the characteristics of the chemical being
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156
tested. One such collection system could be placed at random within
each treatment plot to identify and quantify this mode of transport.
Proposed laboratory method for testing volatility (see Volatility test
protocol) could be used in lieu of field testing.
Transport by erosion is most important on cultivated land. Losses
by this mechanism would be much smaller from forested or densely vegetated
land. Therefore, measurement or prediction of transport of chemicals by
rain erosion may be important only for chemicals associated with agricul-
tural practices or those that have such widespread usage that cultivated
land will be contaminated. Wischmeier and Smith's (1965) method of
estimating soil loss due to erosion could be used in such situations.
Cost and time
One professional level field biologist or ecologist with experience
in experimental design (or a statistical consultant) and two technical
level biologists with experience in field work, spectrophoto-chemical,
gas chromatographic and radio- analysis are required. The professional
is expected to devote about one-quarter time and the technician about
half-time for the duration of field testing. Fifteen months should be
allocated to the field test. Three months should be devoted to pretreat-
ment sampling and 12 months to sampling after treatment. Of course, if
the environmental variability of the test area had been previously de-
fined, an adequate number of replicates and samples to be taken may be
determined without a pretreatment sample period. Transport sampling
should begin in late spring and continue to the following spring. This
strategy will provide two sets of vegetation, that have grown under the
influence of the toxicant, for sampling. The estimated cost for a field
test is about $13,500.
No specialized equipment requirement is envisioned for the conducting
of terrestrial field tests. The same costs are envisioned for terrestrial-
aquatic ecosystem and aquatic-estuarine-marine ecosystem field test proto-
cols.
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157
Terrestrial aquatic field test protocol
The same general considerations given to terrestrial field test
systems also apply to terrestrial-aquatic field tests. Identification
of environmental variability and replication of experimental areas are
seen as important aspects in the performance of these tests.
Chemical input should be to the terrestrial portion of the field
test system. Soil cores to one meter and systematic vegetation sampling
will provide information similar to that collected from terrestrial field
plot tests.
Collection and analysis of soil and runoff leaving the terrestrial
portion and entering the aquatic portion of the field test system will
determine the extent of transport of the toxicant from the terrestrial
to the aquatic portion.
Sediment cores to approximately 20 cm should be taken and analysed
in the same manner as soil cores to detail mobility, persistence, and
effect of biota collected from the uppermost 5 cm of sediment cores.
The extracted benthos may then be analysed to determine uptake rate
and bioaccumulation..
Periodic sampling of water, algae, periphyton, higher organisms
(snails, aquatic insects, fish).and plants will demonstrate the magni-
tude of uptake by the biota and aid in calculation of transfer rates,
and turnover rates between compartments (e.g., soil <==> water <==>
sediment <==> biota) and bioaccumulation in the biota of the test system
(e.g., soil —> water —> sediment —> biota).
One professional level aquatic biologist or ecologist with experi-
ence in land-water interaction and statistical design of experiments
(or a statistical consultant) and two technical level biologists or
ecologists with experience in terrestrial-aquatic field work and ana-
lytical procedures are required. The duration of testing is again 15
months.
Aquatic-estuan'ne-marine field test protocol
Rivers-streams-estuaries. The application of toxicants to contin-
uously flowing systems can be either chronic or acute dependent on
expected release rates. It should be noted at this point that field
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158
test methods in aquatic, estuarine, and marine ecosystem have not been
as highly developed as in terrestrial ecosystems.
Monitoring of the toxicant in water should be done continuously,
(with automated water sampling devices) at randomly selected points
within .a predetermined distance of river, stream, or estuary, above and
below the point of toxicant input. The number of monitoring stations
can be dictated by flow rate and identification of environmental vari-
ability within the watercourse. The concurrent monitoring of water
quality will allow possible correlation of changes in chemical transport
with fluctuations in water chemistry. Initial and time-series sampling
will provide validation of input and rate data, respectively; and infor-
mation on how changes in the physico-chemistry of the water under study
affects the availability of the candidate chemical to sediments and biota.
Sediment core sampling over time will demonstrate the effectiveness
of this component as a sink for chemicals undergoing field testing. Ex-
traction of benthic organisms will allow determination of chemical trans-
fers to and from bottom dwelling organisms and the potential for chemical
transport to organisms feeding on benthic organisms.
Knowledge of food chains and measured concentrations of the can-
didate chemical in biotic components of the field test area will allow
determination of uptake rates, bioaccumulation and biomagnification of
introduced toxicants.
Ponds-lakes-bays. Relatively still water ecosystems are more
amenable to compartmental transport study since flow (a major factor
in physical transport) is not present. Basically, the same procedures
for water sampling, sediment sampling, and biotic sampling given for
flowing water ecosystems should be followed. Input, either continuous
(chronic) or acute, should be dictated by expected release rates of
the toxicant.
Experimental ponds. Impoundment of water in aquatic, estuarine,
or marine environments may facilitate studies of chemical transport.
Ponds may be designed to have continuous flow or no flow at all. Such
impoundments, whether used in rivers, estuaries, lakes, or littoral
zones, would provide nearly natural, easily manipulate test systems
for the experimental introduction of toxicants. The main advantage
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159
of an experimental pond is that it affords the researcher captive, but
natural, biotic communities in which food chain transport, bioaccumu-
lation, and biomagnification of chemicals may be determined.
Personnel required
Dependent on the natural ecosystem under consideration: One pro-
fessional level aquatic or marine biologist or ecologist with experi-
ence in experimental design (or a statistical consultant) and two
technical level aquatic or marine biologists with experience in field
sampling and analytical procedures are required.
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160
III. LITERATURE CITED
Arthur, J. W., and E. N. Leonard. 1970. Effects of copper on Gammarus
pseudolimnaeus, Physa Integra, and Campeloma decisum in soft water.
J. Fish. Res. Bd. Canada 27(7):1277-1283.
Biesinger, K. E., and G. M. Christensen. 1972. Effects of various
metals on survival, growth, reproduction, and metabolism of Daphm'a
magna. J. Fish. Res. Bd. Canada 29(12) :1691-1700.
Black, C. A. (ed.). 1965a. Methods of soil analysis, Part I. Agronomy
9. Am. Soc. Agron., Madison, Wisconsin.
. 1965b. Methods of soil analysis, Part 2. Agronomy 9.
Brungs, W. A. 1969. Chronic toxicity of zinc to the fathead minnow,
Pimephales promelas Rafinesque. Trans. Amer. Fish. Soc. 98(2):
272-279.
Christensen, S. W. 1973. Filtration, Ingestion, and Egestion of
Different-Sized Algae by Daphnia magna Straus, Ph.D. Thesis, Yale
University New Haven, Conn.
Cochran, W. G. 1963. Sampling Techniques. John Wiley and Sons, Inc.,
New York. 413 p.
Cochran, W. G., and G. M. Cox. 1957. Experimental Design. John Wiley
and Sons, Inc., New York. 611 p.
Dan1 man, R. C., and S. I. Auerbach. 1968. Preliminary estimation of
erosion and radiocesium redistribution in a fescue meadow. ORNL-
TM-2343. 24 p.
Dilling, W. L., N. B. Tefertiller, and G. J. Kallos. 1975. Evaporation
rates and reactivities of methylene chloride, chloroform, 1,1,1-
trichloroethane, trichloroethylene, tetrachloroethylene and other
chlorinated compounds in dilute aqueous solutions. Environ. Sci.
Technol. 9(9):833-838.
Frank, P. W., C. D. Boll, and R. W. Kelly. 1957. Vital statistics of
laboratory cultures of Daphnia pulex DeGreer as related to density.
Physiol. Zool. 30:287-30Jjt
Hansch, C. 1969. A quantitative approach to biochemical structure-
activity relationships. Accounts of Chem. Res. 2:232-239.
Hansen, M. H., W. N. Hurwitz, and W. G. Madow. 1953. Sample Survey
Methods and Theory. John Wiley and Sons, Inc., New York. 638 p.
Helling, C. S., and B. C. Turner. 1968. Pesticide mobility: Deter-
mination by soil thru layer chromatography. Science 162:562-563.
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Helling, C. S. 1971a. Pesticide mobility in soils. I. Parameters of
soil thin-layer chromatography. Soil Sci. Soc. Amer. Proc. 35(5):
732-737.
. 1971b. II. Applications of soil thin layer-chromatography.
Soil Sci. Soc. Amer. Proc. 35(5):737-743.
. 1971c. III. Influence of soil properties. Soil Sci.
Soc. Amer. Proc. 35(5):743-748.
Hewitt, E. J. 1966. Sand and water culture methods used in the study
of plant nutrition. Commonwealth Agricultural Bureau, Farnham
Royal, England.
Isensee, A. R., P. C. Kearney, E. A. Woolson, G. E. Jones, and V. P.
Williams. 1973. Distribution of alkyl-arsenicals in a model eco-
system. Environ. Sci. Technol. 7(9):841-845.
Jackson, M. L. 1958. Soil chemical analysis. Prentice-Hall, Inc.,
Englewood Cliffs, N.J. 498 p.
Johnson, B. T. 1974. Aquatic food chain models for estimating bio-
accumulation and biodegradation of xenobiotics, paper presented
at the International Conference on Transport of Persistent Chemi-
cals in Aquatic Ecosystems, Ottawa, Canada, May 1-3.
Kerfoot, W. B. 1973. Cadmium accrual in a flowing marine microcosm.
pp. 1-9 in Ryther, J. H., WHOI-73-2, The Use of Flowing Biological
Systems in Aquaculture, Sewage Treatment, Pollution Assay, and
Food-Chain Studies, Woods Hole Oceanog.raph.ic Institution. Technical
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in Advances in Marine Biology, Vol. 1, Academic Press, New York.
Mackay, D., and A. W. Wolkoff. 1973. Rate of evaporation of low-
solubility contaminants from water bodies to atmosphere. Environ,
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Malone, C. R. 1969. Effects of diazinon contamination on an old field
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Metcalf, R. L., G. K. Sangha, and I. P. Kapoor. 1971. Model ecosystem
for the evaluation of pesticide biodegradability and ecological
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Metcalf, R. L., I. P. Kapoor, P. Lu, C. K. Schuth, and P. Sherman.
1973. Model ecosystem studies of the environmental fate of six
organochlon'ne pesticides. Environ. Health Perspectives, 35-44,
June.
Pionke, H. B., and G. Chesters. 1973. Pesticide-sediment-water inter-
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Pringle, B. H., D. E. Hissong, E. L. Katz, and S. T. Mulawka. 1968.
Trace metal accumulation by estuarine mollusks. J. Sanit. Eng.
Div., Amer. Soc. Civ. Engrs. 94:455-475.
Raymont, J. E. G., and R. S. Miller. 1962. Production of marine
zooplankton with fertilization in an enclosed body of sea water.
Int. Revue ges. Hydrobiol. 47(2):169-209.
Richman, S. 1958. The transformation of energy by Daphnia pulex.
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Robertson, A., C. W. Gehrs, B. D. Hardin, and G. W. Hunt. 1974.
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Spencer, W. F., and M. M. Cliath. 1973. Pesticide volatilization as
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Wischmeier, W. H., and D. D. Smith. 1965. Predicting rainfall-erosion
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Acheta domesticus. Health Phys. 16(4):463-467.
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TECHNICAL REPORT DATA
(Please read laaruciions on the reverse before completing)
1. REPORT NO.
EPA-560/5-76-001
3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE
S. REPORT OAT!
State-of-the Art and Proposed Testing for Environmental
Transport of Toxic Substances
ORT DATE
June, 1976
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
8. PERFORMING ORGANIZATION REPORT NO.
J.P. Witherspoon, E. A. Bondietti, S. Draggan,
F. B. Taub, N. Pearson and J. R. Trabalka
9. PERFORMING ORGANIZATION NAMg AND ADDRESS
Environmental Sciences Division
Oak Ridge National Laboratory
Oak Ridge, Tennessee 37830
10. PROGRAM ELEMENT NO.
-2LA32
11. CONTRACT/GRANT NO
EPA 40-404-73
12. SPONSORING AGENCY NAME AND ADDRESS
Environmental Protection Agency
Office of Toxic Substances
4th and M Streets, S.W.
Washington, O.C. 20460
13. TYPE OF REPORT AND PERIOD COVERED
Final Report
14. SPONSORING AGENCY CODE
IS. SUPPLEMENTARY NOTES
18. ABSTRACT
This study is a review and evaluation of the use of soil, laboratory microcosm,
and field tests to determine transport of chemicals. The soil thin-layer chroma-
tography test appears to be the best method for evaluating mobility of chemicals
in soil. Laboratory results with this method have been verified in a number of field
studies. Review of studies on sediment-water interactions indicated that batch
equilibrium techniques may be used to test mobility of chemicals in sediments. Review
of laboratory microcosm studies revealed that microcosms have served as useful tools
in the -tudy of movement of nutrients, toxic substances, and energy. Although experi-
mental microcosms have employed a'wide variety of organisms and-levels of complexity,
all of them omit large-scale natural processes. Microcosms nf reduced complexity are
suitable for measuring rate processes over a short time range, but more complex
microcosms present difficulties in measurement of some rate processes due to the.
importance of mutualistic and competing processes. It is proposed that, until re-
search on. microcosms progresses, simple food chain tests should be used to-determine
whether toxicants are likely to bioaccumulate or biomagnify. Field test systems con-
tain a large (frequently unknown) number of species; and the number of toxic effects,
chemical transfers, and species interactions that can be studied is limited only by
the time and resources available to the experimenter.
17.
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79. Laboratory Records, ORNL-RC
80. ORNL Patent Office
81. ORNL-Y-12 Technical Library
EXTERNAL DISTRIBUTION
82. Research and Technical Support, ERDA-ORO.
83-183. M. J. Prival, Office of Toxic Substances, U. S. Environmental
Protection Agency, 4th and M Streets, S. W., Washington, DC
20460.
184-210. Technical Information Division, Oak Ridge, TN 37830.
» US. GOVERNMENT PRINTING OFFICE: 1976-748-189/435
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