Environmental Monitoring Series
DEVELOPMENT OF A BIOLOGICAL
MONITORING NETWORK
-A TEST CASE
NATIONAL ENVIRONMENTAL RESEARCH CENTER
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
LAS VEGAS, NEVADA 89114
-------
RESEARCH REPORTING SERIES
Research reports of the Office of Research and Develop-
ment, U. S. Environmental Protection Agency, have been
grouped into five series. These five broad categories
were established to facilitate further development and
application of environmental technology. Elimination of
traditional grouping was consciously planned to foster
technology transfer and a maximum interface in related
fields. The five series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
This report has been assigned to the ENVIRONMENTAL MONI-
TORING series. This series describes research conducted
to develop new or improved methods and instrumentation
for the identification and quantification of environ-
mental pollutants at the lowest conceivable significant
concentrations. It also includes studies to determine
the ambient concentrations of pollutants in the environ-
ment and/or the variance of pollutants as a function of
time or meteorological factors.
EPA REVIEW NOTICE
This report has been reviewed by the National Environ-
mental Research Center-Las Vegas, EPA, and approved for
publication. Approval does not signify that the contents
necessarily reflect the views and policies of the U.S.
Environmental Protection Agency, nor does mention of
trade names or commercial products constitute endorse-
ment or recommendation of use.
Document is available to the public for sale through
the National Technical Information Service, Springfield,
Virginia 22161.
-------
EPA-680/4-75-003
June 1975
DEVELOPMENT OF A BIOLOGICAL MONITORING NETWORK
-A TEST CASE-
Suitability of Livestock and Wildlife
As Biological Monitors for Organophosphorus Contaminants
By
W. W. Sutton
Monitoring Systems Research and Development Laboratory
National Environmental Research Center
P. 0. Box 15027
Las Vegas, NV 89114
L. L. Salomon
Test Operations Directorate
Dugway Proving Ground
Dugway, UT 84022
Contract No. B10019
ROAP 22 ACS
Program Element No. 1HA325
Prepared for
OFFICE OF RESEARCH AND MONITORING
U.S. ENVIRONMENTAL PROTECTION AGENCY
:;ASHINGTON, D.C. 20460
-------
ABSTRACT
Upon request by the National Environmental Research Center-
Las Vegas, a review was conducted of a Dugway Proving Ground (DPG)
monitoring network which is designed to establish baseline erythro-
cyte acetylcholinesterase (AChE) levels in the fauna of West Central
Utah, and to evaluate the suitability of using livestock and wildlife
as biological monitors for organophosphorus contaminants.
Wildlife species sampled during these DPG efforts included the
antelope ground squirrel (.Ammospevmoph-ilus leucurus), the Ord kangaroo
rat (Dipodomys OTdii), the deer mouse (Peromysaus manioulatus), and
the black-tailed jackrabbit (Lepus oalifornicus). Individual blood
samples from these wildlife species as well as samples from cattle and
sheep were collected and analyzed for red cell AChE activity. The
analytical method employed was based on the Warburg manometric techni-
que.
Results indicate that the range of red cell AChE activity values
for both livestock and wildlife species is sufficiently compact to
allow observation of the depression of enzymic activity that would
result from organophosphorus exposures. Controlled studies have shown
that, following exposure to organophosphorus chemicals, the red cell
activity recovers in an essentially linear fashion. Additive effects
resulting from the simultaneous exposure to military agent VX and
either toxic plants or commercial pesticides are discussed.
ill
-------
CONTENTS
Page
Abstract ii
List of Figures v
List of Tables vi
Acknowledgments vii
Conclusions viii
Recommendations ix
Background 1
The Pesticide Problem 1
Application of Biological Monitors 1
Overview of Some Supporting DPG Programs 1
Ecological Surveys 2
Toxicology Studies 3
Transport Processes 3
Soil, Vegetation and Water 3
Meteorological Transport 3
Instrument Evaluation 4
Modeling Capability 5
EPA Interest in the DPG Organophosphorus Monitoring Programs 6
Monitoring Methods and Materials 7
Field Techniques 7
Analytical Techniques 10
Results and Discussion 14
Field Survey 14
-------
CONTENTS cont'd
Page
Control Studies in Support of Field Program 19
Acetylcholinesterase Determinations Using Hereford 19
Steers
Toxicity of VX in Hereford Steers and Sheep 23
Cattle as Indicator Animals 25
Acetylcholinesterase Determinations Using Range Sheep 25
Results from Studies Using Laboratory- Animals 25
Studies on Wildlife Species (Small Mammals) 26
Studies on Wildlife Species (Fish) 26
Complementary Toxicological Effects of Plant Poisons and 27
Chemical Agents on Mammals
Evaluation of Complementary Effects Between Military 30
Chemical Agents and Organophosphorus Pesticides
References Cited 32
Appendix 33
vi
-------
LIST OF FIGURES
FIGURE PAGE
1. Schematic representation of a typical trap line 8
for wildlife collections
2. Diagrammatic erythrocyte acetylcholinesterase activity 12
recovery pattern following various degrees of inhibition
3. Mean dose response curve showing the relationship 20
between oral VX exposures of 0.1 and 0.3 yg/kg/day
for 56 days and the level of erythrocyte acetylcholines-
terase activity. Four Hereford steers were used in each
treatment group.
4. Mean dose response curve, depression and recovery phases 21
in Hereford steers showing the relationship between oral
VX exposures of 0.1, 0.2, and 0.3 yg/kg/day for 56 days
plus an exposure of 0.7 yg/kg/day for 75 days and the
level of erythrocyte acetylcholinesterase activity
5. Mean dose response curve, depression and recovery phases, 22
in three Hereford steers showing the relationship
between oral VX doses of 1.0 yg/kg/day for 75 days and
the level of erythrocyte acetylcholinesterase activity
6. Mean dose response curve, depression and recovery phases, 22
in three Hereford steers showing the relationship
between oral VX doses of 2.2 yg/kg/day for 75 days and
the level of erythrocyte acetylcholinesterase activity
viii
-------
LIST OF TABLES
TABLE PAGE
1. Abbreviated Habitat Classification System Used In 9
Categorizing Various Wildlife Collections
2. Erythrocyte Acetylcholinesterase Activity in Four 15
Species of Small Mammals Indigenous to the Bonneville
Basin of West Central Utah - Spring Collections 197.3
3. Erythrocyte Acetylcholinesterase Activity in Four 16
Species of Small Mammals Indigenous to the Bonneville
Basin of West Central Utah - Fall Collections 1973
4. Erythrocyte Acetylcholinesterase Activity Mean Values 17
in Sheep and Cattle in Bonneville Basin of West Central
Utah - Fall 1970
5. Erythrocyte Acetylcholinesterase Activity Mean Values 17
in Sheep and Cattle in Bonneville Basin of West Central
Utah Spring 1971
6. Erythrocyte Acetylcholinesterase Activity Mean Values 18
in Sheep and Cattle in Bonneville Basin of West
Central Utah - Fall 1971
7. Erythrocyte Acetylcholinesterase Activity Mean Values 18
in Sheep and Cattle in Bonneville Basin of West
Central Utah - Spring 1972
8. Toxicity of VX in Sheep 24
ix
-------
ACKNOWLEDGMENTS
This report was prepared through EPA Contract No. B10019 to
Dugway Proving Ground. Selected experimental data from field and
laboratory studies, previously collected by DPG scientists, were
reviewed and discussed in terms of the EPA stated interest in
biological monitoring. Portions of the document were taken from
DPG reports and additional material was provided specifically for
EPA. The document was compiled and written by W. W. Sutton and
L. L. Salomon.
x
-------
CONCLUSIONS
1. The dispersion of baseline erythrocyte AChE activity levels in
cattle, sheep, black-tailed jackrabbits, deer mice, Ord kangaroo
rats and antelope ground squirrels is sufficiently small, relative
to mean values, to allow detection of enzymic depressions resulting
from organophosphorus exposure.
2. The method employed to analyze for red cell AChE, a modified
Warburg manometric technique, has provided reproducible, accurate,
and timely information. Furthermore, this method does not require
highly skilled personnel or expensive equipment. Other relatively
simple methods of AChE analysis are available which may prove
equally valid or more economical for use at other laboratories.
3. Following organophosphorus exposure, red cell activity recovers
in an essentially linear manner that is dependent on erythropoiesis
and the life of the individual red cells. If, for example, red cell
AChE depression is noted in a biannual survey of range cattle, two
or three subsequent collections taken at twenty-day intervals would
establish the slope of the recovery curve. By extrapolation, the
original degree of enzymic depression and the last date of pesticide
exposure could be estimated.
4. Analysis of red cell AChE activity is most applicable as a
screening technique since it does not permit identification of the
individual organophosphorus substance responsible for enzymic depres-
sion. AChE assays do provide a measure of actual effect on the biolo-
gical material as they serve to detect the highly specific biochemical
reaction common to all organophosphorus exposures. Once enzymic
depression is established and the ranchers, wildlife managers, agri-
cultural officials, etc., questioned, a series of more involved and
expensive procedures can be initiated to determine the specific
cholinesterase inhibitor, provided interrogation does not resolve
the problem.
-------
RECOMMENDATIONS
Analogous studies to calibrate the red cell AChE response
in terms of commercial pesticide units should be conducted in
various indicator species, and field programs should be initiated
in areas where intensive agricultural use is made of organo-
phosphorus pesticides. Results from such a combined field and
laboratory effort could be compared with the DPG survey results,
as the DPG perimeter survey provides data from a control zone
known to be essentially free of organophosphorus pollutants.
xii
-------
BACKGROUND
THE PESTICIDE PROBLEM
The continually expanding human population requires an abundant
food supply. This requirement is being achieved partially through the
introduction of high-yield crop varieties, improved irrigation, new crop
management techniques such as multiple cropping, as well as more effec-
tive farm machinery, fertilizers and, of course, pesticides. Pesticides
present a somewhat unique situation as far as environmental protection
is concerned since these particular toxic substances are intentionally
released into the environment with a desired lethal effect, albeit for
a restricted group of organisms. The current concern over DDT and other
persistent halogenated hydrocarbons has resulted in an expanding use of
the generally less persistent organophosphorus and carbamate insecticides
and, therefore, a growing need for monitoring to avert or detect adverse
environmental effects from such compounds.
APPLICATION OF BIOLOGICAL MONITORS
Historically, most monitoring efforts have assessed a single pathway
system only, even when total pollutant exposure was the ultimate goal.
To establish the total potential exposure from air, water, soil, and
food material, monitoring programs are needed that provide an inte-
grating function. Biological monitors appear to be uniquely suited since,
as in the case of many terrestrial mammals, atypical biotic character-
istics can often be detected following various modes of pollutant expo-
sure, i.e., oral (food or water), inhalation and percutaneous. Through
inhibition or alteration of metabolic processes, as well as retention of
many pollutant substances by the tissues, biological organisms can be
employed in many monitoring situations.
OVERVIEW OF SOME SUPPORTING DPG PROGRAMS
Dugway Proving Ground which was activated in 1951 as a permanent
military installation for the testing of chemical and biological weapons
systems developed monitoring programs as a logical operational require-
ment. An integrated monitoring effort was Initiated to detect any adverse
ecological effects that might result from the testing program. This moni-
toring effort included a broad spectrum of detailed ecological, chemical,
meteorological, epidemiological and toxicological studies to provide
sound environmental baseline data for West Central Utah, an area approxi-
mately 15,000 square miles in size.
It was recognized that tb.ere is no universally applicable formula
for an integrated monitoring program. The ecological characteristics
of the specific area or region, the type(s) and source(s) of pollutants,
the nature of the specific objectives to be met, and considerations of
resources and time, all enter into the selection of activitives to
achieve practically and scientifically acceptable goals.
-------
Ecological Surveys
For its purposes, DPG considered establishment of ecological
baseline data to be a key factor in an integrated monitoring program
to determine whether military activities impact adversely upon the
local environment, and to define ecological receptors and transport
pathways that may be critical in the event of pollutant release. The
preliminary hypothesis was that no significant adverse effects on
flora or fauna resulted from the activities, e.g., the employment of
organophosphorus compounds, and this hypothesis was tested by evalua-
tion of comprehensive empirical data.
Information collected on wildlife species includes population
density and diversity, seasonal and migration cycles, predator-prey
relationships, intra or interspecific competition for food, grazing
competition from domestic livestock, depredation by parasites and
disease, plus any additional related effects resulting from variations
in rainfall, temperature, soil type and soil fertility. It was also
important, in certain cases, to determine those factors which influence
the host-parasite relationship within the ecosystem of interest and
which allow for the maintenance of infection and foster or inhibit
epizootics. Studies or literature reviews are also undertaken at DPG
to analyze wildlife feeding habits to assess the potential effects of
agricultural or military pollutants upon the animal feeding patterns,
upon the foods themselves and upon adverse reactions occurring under
natural conditions. Furthermore, an analysis and classification of
various soils according to type, particle size, water permeability,
water content, acidity-alkalinity and the amounts and types of mineral
salts are also a part of the baseline study. Seasonal analyses are
needed to determine the locations and amounts of surface water and to
define the conditions that effect the subsurface water and its movements
and/or drainage. Plant surveys provide data for evaluating the effects
of plants upon wildlife and/or domestic animals (livestock) and for
assessing the effects of poisonous plants in combination with toxic
chemical agents. This program of ecological monitoring and surveil-
lance is being achieved, within the framework of available resources
and time, by careful selection of control zones where all major factors
of ecological consequence are equally and simultaneously operative
except that there is no possibility of influences attributable to
military operations. In short, the need for minutely detailed investi-
gations is being obviated by establishing appropriate control zones
distant to the test zones (sites selected because of their sensitive
location relative to military testing). Significant ecological
divergences between control and test zones, particularly differences in
trends, may then be used to reevaluate the selection of control sites
and/or the occurrence of unfavorable events traceable to military
operations.
-------
Toxicology Studies
A number of toxicology studies were undertaken as an adjunct to
the ecological monitoring network. Toxicity levels have been esta-
blished for various agents and decomposition products using laboratory
and indigenous small mammals. In addition, systematic acute and chronic
toxicity feeding studies have been conducted using large domestic animals
of economic importance. These studies were primarily conducted to deter-
mine the AChE depression effected by administration of lethal agents.
Information gained from these projects was applied to studies in which
animals were used as indicators of residual toxic materials present in
vegetation. Possible synergistic effects between naturally occurring
plant materials and agents were also investigated.
Transport Processes
As was the case with the other studies on organophosphorus military
agents, these DPG investigations on transport pathways were designed to
be comparable with techniques and methods commonly employed to measure
pesticides. In several instances, pesticides were actually used to
verify analytical procedures and in persistence studies a rather exten-
sive data bank related to pesticides, herbicides, etc., has been devel-
oped. It was found that while much information was available on halo-
genated pesticides, only fragmentary information as to methodology and
environmental fate was available for many of the organophosphorus pesti-
cides .
Soil, vegetation and water
The fate of organophosphorus chemical agents has been examined in
soil and vegetation under carefully controlled conditions simulating
various aspects of the Dugway environment. As a result, the hydrolysis
routes were defined and the hydrolysis products of the neuro-toxic
agents were identified. The major mechanisms by which these chemicals
degrade have been established. Investigations into the uptake, trans-
location and metabolic fate of these chemicals by plants have also been
completed.
Detection and measurement of traces of lethal agents and organo-
phosphorus pesticides in water have also been conducted; as little as
0.4 parts per billion can be detected, a level far below that which is
toxic to living matter. Furthermore, even though these materials
decompose in water, the decomposition products were quantitatively
accounted for up to one year after deposition.
Meteorological transport
Pesticides are generally introduced into the ecosystem through
some type of spraying (aerial or surface) operation, therefore, the
air route is especially important. Both the efficiency of the pesticide
-------
application and the pesticide dispersal to non-target areas are influenced
by local meteorological conditions. Meteorogological transport character-
istics are also extremely important for the analysis of military chemicals.
To be effective, a military chemical agent much like an agricultural
poison, must be properly disseminated over a suitable target area. Dis-
semination of a military chemical comprises projection, or delivering the
chemical to the target, and dispersion or spreading of the agent over the
target in an effective manner. Environmental studies involving organo-
phosphorus chemicals at DPG were, therefore, intimately concerned with
the transport and diffusion of airborne vapors and droplets. Knowledge
of the droplet diameter, fall velocity and evaporation rate as well as
the volume fraction of the aerosol which has a high probability of being
subjected to drift have improved the prediction capability of long range
contamination. The size of the droplets directly relates to their ability
to impact on the target and if the droplet size is too small the tendency
of the aerosol to drift is increased. Meteorology programs for the study
of transport of agent clouds were established to (1) evaluate and describe
the influence of complex environmental features such as terrain and
vegetation on the processes of aerosol transport and diffusion, (2) to
select optimum monitoring techniques and (3) to assess the efficiency of
various chemicals and release mechanisms. During the past decade, almost
five hundred major field diffusion experiments were conducted through, a
combination of inhouse (DPG) efforts and contractor support. Emphasis
was placed on mesoscale studies (1 to 100 kilometers) and included topo-
graphic variations consisting of a single or a multiple valley complex,
a single open airshed or a single geographic feature. Open air releases
of organophosphorus aerosols, gases and simulant materials have provided
valuable data for field assessment of various air monitoring networks.
These studies were based on (1) the theoretical development of mathe-
matical models for atmospheric transport and C2) validation of the
theoretical models with data derived from field experimentation. Early
in the process of generating mathematical models and practical opera-
tional information, it was surmised that vast amounts of the necessary
information could be acquired by using a harmless tracer, e.g., fluores-
cent particles, whose behavior upon dispersal in the atmosphere is
identical to that of clouds of agents. The size and number of spray
drops impinging on the target can be determined since the number of
fluorescent particles in each droplet is a direct measure of droplet
size.
Additional field experiments are being conducted to determine the
quantity of toxic material that is resuspended by wind action. Prior
to experiments of this sort, extraction methods and recovery techni-
ques were developed. The decomposition and decay of agents has received
particular attention so that determinations could quantify the amount
of agent available for resuspension.
Instrument Evaluation
Since monitoring networks require high sensitivity samplers, a
continuous program of instrument evaluation is in existence, with
-------
particular emphasis on the detection of organophosphorus aerosols and
gases. In recent studies a flame photometric detector (FPD) was evalu-
ated for use as a field monitor of agent concentration. Analytical
units, such as FPD, are employed in the field during selected tests or
in specifically designed chamber tests. Data from field trials have
been used to assess instrument desirability and to establish engineering
and design changes for the next generation prototype.
In support of air tracer studies for aerosol or particulate trans-
port several surface point sources and aircraft disseminator systems
are being employed together with rotorod (impactor) samplers, millipore
filters, and aircraft sampling devices. Assessment of tracer samples
is routinely accomplished by microscopic techniques with portable
assessment systems; an automated system is now being constructed.
Turbulence statistics measurement programs required to apply diffusion
theories to practical studies of aerosol or air pollutant movement can
be aided by two instrument packages (for aircraft) consisting of the
following components:
(a) Doppler radar (measurements of Vertical Wind Profile)
(b) Temperature probe (ambient air)
(c) Relative humidity probe
(d) Infrared ground surface temperature probe
(e) Air turbulence indicator
(f) Air speed and altitude indicator
(g) Multi-channel strip recorder
This system was used extensively in the past on safari operations in
areas where other methods of data acquisition were either impossible
or impractical.
Modeling Capability
A complete modeling capability has been established at DPG with
particular emphasis on mathematical descriptions for the diffusion
and transport of airborne substances. These models, adapted for
computer solution, have been validated utilizing data from the weapons
test program and the above mentioned fluorescent particle tracer
technique.
Modeling developments, which have been incorporated into capabi-
lities for the field Army, have also been utilized in hazard evalua-
tion for specific Department of Defense problems. Hazard prediction
techniques were developed in case of an accidental release during
the transportation of organophosphorus chemicals by truck, rail and/or
ship. Area source models have also been applied to the evaluation
of downwind concentration levels for planned Herbicide Orange disposal
operations.
-------
EPA INTEREST IN THE DPG ORGANOPHOSPHORUS MONITORING PROGRAMS
Generally, organophosphorus pesticides are similar to organo-
phosphorus military agents in chemical structure, biological activity,
or both. As a result, it is felt that these detailed environmental
investigations may serve as prototypes for corresponding efforts on
the organophosphorus pesticides.
The relationship between these pesticides and the military agents
is seen in the general formula for cholinesterase inhibitors of the
organophosphorus group.
0(S)
"R! and R2 are capable of almost infinite variation. They may
represent alcohols, phenols, mercaptans, amides, or alkyl or aryl
groups attached directly to the phosphorus, etc. Common X radicals
are from fluorine (e.g., in diisopropylfluorophosphate) paranitro-
phenol (e.g., in Paraoxon), and phosphates (in a pyrophosphate,
tetraethylpyrophosphate), but in other inhibitors X may be cyanide,
thiocyanate, carboxylate, chloride or almost any phenoxy or thio-
phenoxy group" (Holmstead, 1963). Generally, organophosphorus com-
pounds containing a double-bonded sulfur atom are low in pesticide
activity, toxicity, and AChE inhibitory activity. Upon metabolic
conversion or rearrangement to the corresponding oxygen derivative,
the toxicity tends to rise dramatically along with their effectiveness
as an inhibitor of AChE.
Military nerve agents are selected from this general group to
meet necessary biological, physical, and chemical properties including
stability and toxicity by a given route of entry into the body. Simi-
larly, organophosphorus pesticide selection is based on a number of
criteria related to their specific use. Some organophosphorus pesti-
cides have LD5Q values close to those of nerve agents, but they are
usually less volatile. An amount of nerve agent giving a certain
degree of biological effect is not intrinsically more hazardous or
toxic than an amount of organophosphorus pesticide giving the same
degree of biological effect.
-------
MONITORING METHODS AND MATERIALS
FIELD TECHNIQUES
Originally the monitoring program utilized such indigenous small
mammals as the black-tailed jackrabbit, Townsend ground squirrel,
antelope ground squirrel, least chipmunk, Great Basin pocket mouse,
Ord kangaroo rat, chisel-toothed kangaroo rat, bushy-tailed wood rat,
grasshopper mouse, long-tailed pocket mouse, desert cottontail,
western harvest mouse, deer mouse, pinyon mouse and desert wood rat.
However, the wildlife monitoring effort for the detection of anti-
cholinesterase substances was condensed to include four sentinel or
indicator species. These were the antelope ground squirrel
(Atmospermoph-Llus leuourus), the Ord kangaroo rat (Dipodomys ovd-ii)s
the deer mouse (Peromyseus manicu'latus) and the black-tailed jack-
rabbit (Lepus californiaus). Selection of the species was based on
their wide distribution, availability in terms of numbers and season,
and ease of sampling. The antelope ground squirrel is active through-
out the year and inhabits the vegetated and semi-vegetated dunes of
the valleys and the sandier parts of the foothills. Pepomysous
manioulatus3 the ubiquitous deer mouse, is the most cosmopolitan and
numerous rodent in the area. It is especially abundant in the vegetated
dunes along the valley floors and in the sandy areas of the foothills.
The Ord kangaroo rat is most prevalent in the vegetated dunes and among
mixed brush in the lower foothills. Finally, the black-tailed jack-
rabbit occurs in essentially all plant communities of the valleys,
foothills and mountains, but the populations fluctuate from year to
year.
Seven collection sites were chosen to include locations of vary-
ing distance from the Proving Ground, so that all directions surround-
ing the test grids would be represented and a variety of microhabitats
would be included. Particular consideration was given to the prevailing
wind conditions for the area. Since seasonal population movements often
occur, especially among jackrabbits, each survey site represented a
fairly large area to insure collection of an adequate number of mammals
throughout the year. Can traps, consisting of one-quart cans with
museum special snap traps, were used for rodent collections. Traps
were baited with a mixture of seeds and were checked daily in the early
morning. During the winter months, cotton batting material was placed
in the traps to avoid exposure fatalities. Lines of 40 can traps, with
an interval of 6 to 8 paces between traps, were set in an elliptical
pattern (Figure 1) in areas which visual reconnaissance indicated would
be productive. The most fruitful areas were found to be vegetated sand
dunes on which adequate cover and numerous burrows were observed. An
abbreviated classification system (Table 1) for the various habitat
types proved useful in categorizing the collection. Jackrabbits were
collected from each sentinel area using firearms. Since night collect-
ing was usually more productive in areas of reduced animal density,
-------
FIGURE 1. Schematic representation of a typical trap line
for wildlife collections
hunting was conducted from the back of a truck equipped with spotlights.
If an area had a large rabbit population the hunting was done on foot
during daylight hours. All blood samples were taken in the field by
cardiac puncture using 10-ml heparinized Vacutainers and 1.5-inch,
20-gauge needles. After the blood was collected, the Vacutainers were
immediately placed in a portable ice chest and transported back to the
laboratory for processing. Rodents, which had been live-trapped, were
placed in small cloth bags and returned to the laboratory for bleeding.
-------
Table 1. ABBREVIATED HABITAT CLASSIFICATION SYSTEM USED IN
CATEGORIZING VARIOUS WILDLIFE COLLECTIONS
Code Recorded on Individual
Biotic Community Mammal Collection
Pond 1
Marsh, 2
Stream 3
Irrigated Area 4
Cultivated Area 5
Greasewood 6
Juniper Brush 7
Juniper Mountain 8
Pinyon - Juniper 9
Mixed Brush 10
Shadscale-Graymolly 11
Shadscale-Graymolly-Greasewood 12
Vegetated Dunes 13
Rabbitbrush 14
Big Sage 15
Big Sage - Rabbitbrush 16
Spring 17
Natural Cave 18
Mine 19
River 20
Lake 21
Grass-Annuals 22
Greasewood - Sagebrush 23
Rabbitbrush - Greasewood 24
Rocky Hillsides Sparse Vegetation 25
Aspen - Fir 26
Shadscale - Sagebrush 27
-------
If the distance to the laboratory was such that this was impractical,
field collections were taken by cardiac puncture using 0.5 ml heparinized
tuberculin syringes with 3/8-inch, 26-gauge needles. The blood was then
transferred to 3-ml heparinized tubes and, as in the case of rabbit
samples, refrigerated until it could be processed. Collections of both
rodents and lagomorphs were catalogued by species, sex, location, habitat,
and date collected.
Wildlife controls were maintained at the DPG Faunal Colony. The
following species were sampled in conjunction with the perimeter survey.
Jackrabbits (Lepus oalifornicus')
Wood rats (Neotoma lep-lda]
Kangaroo rats (Dipodomys sp.~)
Grasshopper mice (Onydhomys leucogastex1')
Harvest mice (Reithrodontomys megalotis')
Canyon mice (Peromyseus ooinitus~)
Pinyon mice (Peromyscus tvuei,)
Deer mice (Pevomysous manieu1atus~)
As was the case with wildlife collections, livestock samples were
teken from several locations of varying distance from the Proving Ground
with particular attention being given to prevailing wind conditions.
In addition to these range animals sampled on the DPG perimeter, blood
collections were taken from sheep and cattle at the DPG Animal Colony.
Lambs, pregnant and nonpregnant ewes, wethers and mature Hereford
steers were kept under controlled conditons to quantitate AChE activity
variations. Lambs were bled weekly for six months to determine if any
change in red cell activity occurred prior to maturity. The DPG Animal
Colony sheep and cattle were, of course, not exposed to military or
agricultural cholinesterase inhibiting agents. Activities recorded
in the spring and fall were of special importance since the field
collections were made at this time.
Perimeter and DPG control livestock blood samples were taken from
the jugular vein using 10-ml 100 X 16 mm heparinized vacutainers with
a 20-gauge X 1-1/2" needle. Samples were cooled and transported to the
laboratory for analysis.
ANALYTICAL TECHNIQUES
Exposure to low levels of organophosphorus pesticides does not
characteristically result in detectable tissue accumulation. However,
since these compounds are degraded metabolically and are excreted in
the urine, determination of the urinary metabolites, e.g., phenols,
phenoxy acids and alkyl phosphates, can be used as a means of assessing
exposure and of establishing with more or less specificity the identity
of the pollutant. A property common to all organophosphorus pesticides
is their inhibitory effect on AChE. This, too, can serve as the basis
of detecting exposure, but not of identification. It remained, therefore,
10
-------
to select that analytical method which best met the requirements of
the monitoring effort.
The physiological effects resulting from the systemic absorption
of organophosphates and carbamates are caused by inhibition of
cholinesterase enzymes of the nervous system, muscles and secretory
glands. Phylogenetically, the earliest function of acetylcholine (ACh)
and related enzymes was probably to modify the passage of various sub-
stances across cell membranes. With the evolution of structural and
biochemical complexity this function was retained by some membranes
and totally lost by others. The greatest specialization has been
achieved in the nervous tissue where the ACh-AChE system, by virtue
of its effect on the ionic permeability of membranes, functions as a
transjunctional mediator. In the case of erythrocyte AChE, the
enzyme appears to be vestigial but it may too affect ionic permeability
in some nonessential way. Cholinesterase enzymes of plasma and red
blood cells are also inhibited by organophosphorus and carbamate chemi-
cals but this has no apparent effect on the animal's health. Acetyl-
cholinesterase may be inhibited in a reversible or an irreversible
manner. While these are not absolute distinctions, reversible inhibi-
tion generally indicates that the enzyme-inhibitor complex dissociates
freely upon removal of the inhibitor and irreversible inhibition implies
that the enzyme activity does not readily return on mere dialysis.
Organophosphorus compounds phosphorylate cholinesterase to form a
dialkylphosphorylated enzyme which is an irreversible complex unlike
the easily dissociated carbamate-enzyme complex. Recovery of activity
from an irreversibly inhibited enzyme will depend on the rate of regen-
eration of new enzyme protein. In the case of red cells, new cells must
be formed and recovery of total erythrocyte activity is dependent on
the rate of erythropoiesis and the normal red cell life span.
Analytical methods for the detection and quantitation of excretion
products derived from organophosphorus pesticides are generally demand-
ing in time, skills, and equipment. By their very nature, these methods
impose a limitation on the information obtainable from the monitoring
effort unless multiple analyses are performed at correspondingly increased
cost. Furthermore, usable concentrations of metabolic residues are found
in the blood and urine for a limited time only after pesticide exposure
and false positives may be recorded if the animals ingest relatively
harmless organophosphorus degradation products. Urine is also difficult
to sample from unconfined animals and effectively useless as a source of
routine monitoring specimens. Measurements of urinary residues are also
difficult to correlate with the actual hazard caused by the organophos-
phorus chemicals, which are more accurately reflected by the degree of
red cell AChE inhibition.
Determination of erythrocyte AChE levels indicate the degree of
actual damage to the biological material as they serve to detect the
highly specific biochemical reaction common to all organophosphorus
pesticide exposures. Admittedly, red cell AChE analysis represents a
11
-------
screening method only since it does not permit identification of the
individual organophosphorus pesticide(s) responsible for the enzymic
depression, but the results are not complicated by prior ingestion of
harmless pesticide degradation products. As the rate of enzymic recovery
is predictable, it is possible to estimate the date of an acute exposure
or the date of the last day of a chronic exposure. If recovery is not
linear and/or of the proper slope, it suggests that the animals are still
being exposed to the toxic materials. In biological monitoring projects
designed for environmental surveillance, a series of field collections
would indicate the slope of the recovery curve if the animals had pre-
viously been exposed to an anticholinesterase substance. If, for example,
red cell AChE depression is noted in a biannual survey of range cattle,
two or three subsequent collections taken at twenty-day intervals would
establish the slope of the recovery curve. By extrapolation, the original
degree of enzymic depression and the last date of pesticide exposure
could be estimated. Figure 2 presents a diagrammatic view of erythrocyte
AChE recovery which could be used to classify the slope of the recovery
curve. The figure could also be viewed as establishing three arbitrarily
selected zones. Activity increases corresponding to Zone I would indi-
cate a need for increased surveillance of a given area, but conclusions
concerning the degree of contamination should be reached cautiously since
>•
>
:3s
LU
u
50
RECOVERY (DAY)
75
100
FIGURE 2.
Diagrammatic erythrocyte acetylcholinesterase activity
recovery pattern following various degrees of inhibition
12
-------
normal AChE activity levels fall within a fairly broad range. An apparent
AChE recovery falling within Zone II would indicate probable exposure to
a cholinesterase inhibitor while a recovery sequence with Zone III bound-
aries would suggest the presence of a significant degree of contamination.
Theoretically, it would be possible to mathematically model the red cell
enzymic recovery rate following various types of organophosphorus expo-
sures. However, for practical field monitoring purposes, the recovery
pattern that would essentially fall within one of these three arbitrarily
selected zones can provide an extrapolated approximation of the last
exposure date.
For these reasons, and since organophosphorus chemicals cause no
other major effect than that attributable to cholinesterase inhibition,
measurement of AChE activity was judged to be the only realistic and
practical approach to monitor for the effectiveness of the ecological
protection program of DPG. An ancillary benefit is also realized from
the wildlife collection part of the screening program because population
density, age distribution, and species composition can reflect many
nonrelated detrimental effects of human operations. Capabilities for
more detailed chemical and pathological investigations were also esta-
blished, but with no plans for routine employment of that support unless
required by findings made during the screening effort.
Witter (1963) and Augustinsson (1954) have reviewed several of the
acetylcholinesterase assay techniques. These reviews included discussions
of methods based on acid production, choline production, chemical deter-
mination of unreacted acetylcholine, and use of non-choline esters.
Additional papers have discussed radioisotopic techniques suitable for
AChE assays (Reed et al.3 1966 and Gaballah, 1968).
A gasometric method of analysis for red cell AChE, employing the
Warburg manometric technique* was selected for the DPG monitoring program
because it, in effect, is the ultimate reference standard. The procedure
is described in Appendix I.
*For a more elaborate and fundamental discussion of the principles and
classic manometric techniques utilizing the Warburg respirometer, the
reader is referred to Umbreit, W. W., R. H. Burris, and J. F. Stauffer,
1949. Manometric Techniques and Tissue Metabolism. Burgess Publishing
Co., Minneapolis, MM. 227 pp.
13
-------
RESULTS AND DISCUSSION
A survey and surveillance program has been maintained to establish
baseline levels of erythrocyte acetylcholinesterase (AChE) activity in
representative wildlife and livestock species. This program has provided
quantitative verification of the concept, solutions to logistical and
analytical problems, development of an appropriate quality assurance pro-
gram, information on fiscal and personnel requirements, and most impor-
tantly yielded sufficient data to establish the baseline information for
the geographic area under investigation. The fact that these collection
efforts were designed to establish baseline information must be stressed,
not only because baseline surveys are an essential part of integrated
monitoring but also because DPG did not test toxic organophosphorus
materials in the open air for many months prior to, nor at any time after
inception of this study. Furthermore, results of residue analysis of
soil, water, and vegetation, concurrently covering the same area as the
livestock and wildlife surveillance effort, did not show the presence of
any organophosphorus compounds.
FIELD SURVEY
Tables 2 through 7 present representative AChE activity data from
livestock and wildlife species collected in areas adjacent to DPG. The
observed variations in erythrocyte activity levels in no way interfere
with the reliability and consistency with which depressed values can
be interpreted. Anderson et al. (1969) presented red cell activity
values for large numbers of sheep and cattle and concluded that the
range of values in these animals was sufficiently small so as to present
no handicap in recognition of reductions in enzymic activity caused by
organophosphorus poisoning. The standard deviation calculated for
these (Anderson's) data revealed a dispersion of AChE activities about
the mean startlingly similar to that noted in the DPG survey. Actually,
the data obtained by DPG for AChE in erythrocytes of sheep are more
closely clustered about the mean, Anderson's data having a number of
outliers (which were disregarded in calculating the standard deviation).
To be useful as sentinels, wildlife species will need to be ade-
quately distributed geographically, and available in terms of numbers,
season and ease of sampling. Furthermore, as with the cattle and sheep,
the baseline activity of AChE in erythrocytes should be sufficiently
compact to allow accurate interpretation of changes owing to encounters
with organophosphorus compounds. The wildlife data obtained by DPG
showed, in most cases, a greater central tendency than did AChE values
from domestic animals, although they were based on a smaller number of
specimens. Lepus adliforn-Lous and Peromysaus man-iculatus are probably
the most successful sentinel candidates both in regard to availability
and dispersion of enzymic activity. Levels of activity in red cells
of jackrabbits are normally low which might produce some difficulty
in determining significant changes were it not for the fact that they
14
-------
Table 2. ERYTHROCYTE ACETYLCHOLINESTERASE ACTIVITY* IN FOUR SPECIES**
SMALL MAMMALS INDIGENOUS TO THE BONNEVILLE BASIN OF
WEST CENTRAL UTAH _ SPRING COLLECTIONS 1973
OF
Area
Callao
Cedar Mtns.
South
Condie
Fish Springs
Gold Hill
Government
Creek
Granite Mtn.
losepa
Wendover
Species
D.o.
F.m.
L.c.
D.o.
P.m.
L.c.
A.I.
D.o.
F.m.
L.c.
A.I.
D.o.
P.m.
L_.£.
P.m.
L_.c_.
D.o.
P_.in.
D.o.
L_.£.
P.m.
L_.£.
A.I
D.o.
P.m.
L.c.
Sample
Size
9
28
10
18
17
9
1
11
19
9
7
12
13
10
33
10
23
15
26
10
27
2
2
5
6
5
Activity
Mean
45.9
47.1
20.9
76.7
49.0
20.4
27.7
65.5
53.6
19.2
68.1
65.7
53.7
22.7
49.7
20.7
61.0
43.7
59.1
21.5
52.1
21.4
82.4
59.7
43.4
20.7
Standard
Deviation
5.7
6.9
2.8
10.9
11.8
2.1
9.9
7.4
3.9
11.0
11.0
10.6
3.3
10.6
2.3
6.8
9.7
18.4
4.3
9.2
0.5
3.8
7.7
9.1
3.4
95% Confidence
Interval
41.5-50.3
44.4-49.8
18.9-23.0
71.3-82.2
42.9-55.1
18.8-22.0
58.9-72.2
50.1-57.2
16.2-22.2
57.9-78.2
58.7-72.7
47.3-60.1
20.3-25.1
46.0-53.4
19.0-22.3
58.1-63.9
38.3-49.0
51.7-66.5
18.4-24.6
48.4-55.7
17.1-25.7
48.0-116.7
50.1-69.3
33.9-52.9
16.4-24.9
*Activities for Lepus ealiformous are reported as yM C02 evolved per
100 yl RBC's per 30 minutes. Activities for all other species are
reported as yM C02 evolved per 50 yl RBC's per 30 minutes.
"Species: A.I. - Ammospevmophilus leuezams, jD_.o_. - Dipodomys ordii
P_.in. - Peromyscus maniaulatus, L_.£. - Lepus californiaus
15
-------
Table 3. ERYTHRQCYTE ACETYLCHOLINESTERASE ACTIVITY* IN FOUR SPECIES**
OF SMALL MAMMALS INDIGENOUS TO THE BONNEVILLE BASIN OF
WEST CENTRAL UTAH - FALL COLLECTIONS 1973
Area Species
Callao
Cedar Mtns.
South
Condie
Fish Springs
Gold Hill
Government
Creek
Granite Mtn.
losepa
Wendover
P_.m.
L.£.
A.I.
D.o.
P_.m.
L.c.
A.I.
D.o.
P_.m.
L_.£.
A.I.
P.m.
L.c.
D.o.
P.m.
L.c.
A.I
D".o.
P".m.
L.£.
A.I.
D.o.
P.m.
F.c.
P.m.
F. c.
A.I.
F.o.
L.c_.
Sample
Size
30
5
2
20
8
5
3
16
11
5
2
28
7
8
14
6
3
25
4
6
3
2
18
6
6
5
4
11
7
Activity
Mean
43.2
20.9
50.6
, 73.4
44.7
21.1
63.0
66.2
47.3
20.0
63.5
48.6
21.1
67.5
31.7
20.4
67.1
60.6
48.3
20.4
77.0
70.2
48.5
19.0
48.6
19.7
69.9
59.9
21.3
Standard
Deviation
11.8
3.5
10.5
16.0
21.4
3.1
8.6
7.7
12.7
3.9
0.0
9.5
3.0
18.6
8.4
2.5
28.1
23.7
8.6
3.7
4.1
3.8
10.2
4.1
7.2
3.1
32.7
20.5
5.4
95% Confidence
Interval
38.8-47.7
16.5-25.1
43.7-145.0
66.0-80.9
26.8-62.6
17.4-25.1
41.7-84.3
62.1-70.3
38.8-55.8
15.2-24.7
63.5-63.5
44.9-52.3
18.3-23.9
52.0-83.1
26.9-36.6
17.8-23.0
-2.8-136.9
50.9-70.4
34.6-61.9
16.5-24.3
66.9-87.0
35.9-104.5
43.4-53.5
14.7-23.4
41.1-56.2
15.9-23.5
17.9-121.8
46.1-73.7
16.3-26.2
"Activities for Lepus oa^formcus are reported as yM C02 evolved per
100 yl RBC's per 30 minutes. Activities for all other species are
reported as yM C02 evolved per 50 yl RBC's per 30 minutes.
**Species: A.U - Ammospermophilus leueurus, D_.£. - Dipodomys ordii
P_.m. - Peromysaus manioulatus, L_.£. - Lepus californious
16
-------
Table 4. ERYTHROCYTE ACETYLCHOLINESTERASE ACTIVITY MEAN VALUES*
IN SHEEP AND CATTLE IN BONNEVILLE BASIN OF WEST CENTRAL UTAH
FALL 1970
Herd
Number
1
2
4
6
28
29
30
Species
Sheep
Sheep
Sheep
Sheep
Cattle
Cattle
Cattle
Summer
Range
Bear Lake
Bear Lake
Bear Lake
Strawberry
Callao
Grouse Creek
Vernon
Winter
Range
E. Dugway Mtns.
Big Davis Mtn.
White Rock
E. Topaz Mtn.
Callao
Grouse Creek
Vernon
Mean
100.5
98.4
97.6
77.2
218.4
253.5
256.7
Sample
Size
28
30
30
24
50
44
30
Standard
Deviation
13.0
14.0
14.9
12.5
41.9
55.2
44.1
95% Confidence
Interval
95.4.105.5
93.2-103.6
92.0-103.2
71.9-82.5
206.4-230.3
236.7-270.3
240.3-273.2
*Activity mean values for sheep are reported as yM C02 evolved per 100 yl RBC's per
15 minutes. Activity mean values for cattle are reported as yM C02 evolved per
50 yl RBC's per 15 minutes.
Table 5. ERYTHROCYTE ACETYLCHOLINESTERASE ACTIVITY MEAN VALUES*
IN SHEEP AND CATTLE IN BONNEVILLE BASIN OF WEST CENTRAL UTAH
SPRING 1971
Herd
Number Species
1
2
4
6
28
29
30
Sheep
Sheep
Sheep
Sheep
Cattle
Cattle
Cattle
Summer
Range
Bear Lake
Bear Lake
Bear Lake
Strawberry
Callao
Grouse Creek
Vernon
Winter
Range
E. Dugway Mtns.
Big Davis Mtn.
White Rock
E. Topaz Mtn.
Callao
Grouse Creek
Vernon
Mean
115.
105.
118.
100.
272.
286.
276.
6
2
9
9
3
9
3
Sample
Size
30
30
30
30
29
30
23
Standard
Deviation
14
18
19
13
37
53
45
.3
.3
.3
.0
.9
.9
.8
95% Confidence
Interval
110.
98.
111.
96.
257.
266.
256.
2-120
4-112
8-126
0-105
9-286
7-306
6-296
.9
.0
.1
.8
.8
.9
.1
*Activity mean values for sheep are reported as yM CC>2 evolved per 100 yl RBC's per
15 minutes. Activity mean values for cattle are reported as uM C02 evolved per
50 yl RBC's per 15 minutes.
17
-------
Table 6. ERYTHROCYTE ACETYLCHOLINESTERASE ACTIVITY MEAN VALUES*
IN SHEEP AND CATTLE IN BONNEVILLE BASIN OF WEST CENTRAL UTAH
FALL 1971
Herd
Number
1
2
4
6
23
33
29
30
Species
Sheep
Sheep
Sheep
Sheep
Sheep
Sheep
Cattle
Cattle
Summer
Range
Bear Lake
Bear Lake
Bear Lake
Strawberry
Lost Creek
Coalville
Grouse Creek
Vernon
Winter
Range
E. Dugway Mtns.
Big Davis Mtn.
White Rock
E. Topaz Mtn.
Gold Hill
W. Dugway Mtns.
Grouse Creek
Vernon
Mean
94.6
99.9
97.6
89.4
87.1
88.1
285.4
285.0
Sample
Size
30
30
30
30
30
30
30
23
Standard
Deviation
12.2
13.9
13.9
15.2
13.4
14.0
42.2
41.7
95% Confidence
Interval
89.9-99.1
94.7-105.1
92.4-102.7
83.7-95.1
82.0-92.1
85.9-96.4
269.7-301.2
266.9-303.0
*Activity mean values for sheep are reported as yM C0"2 evolved per 100 yl RBC's per
15 minutes. Activity mean values for cattle are reported as yM CC>2 evolved per
50 yl RBC's per 15 minutes.
Table 7. ERYTHROCYTE ACETYLCHOLINESTERASE ACTIVITY MEAN VALUES*
IN SHEEP AND CATTLE IN BONNEVILLE BASIN OF WEST CENTRAL UTAH
SPRING 1972
Herd
Number
1
2
4
6
23
33
28
29
30
Species
Sheep
Sheep
Sheep
Sheep
Sheep
Sheep
Cattle
Cattle
Cattle
Summer
Range
Bear Lake
Bear Lake
Bear Lake
Strawberry
Lost Creek
Coalville
Callao
Grouse Creek
Vernon
/.' ,
Winter
Range
E. Dugway Mnts.
Big Davis Mtn.
White Rock
E. Topaz Mtn.
Gold Hill
W. Dugway Mtns.
Callao
Grouse Creek
Vernon
_ , . v
Mean
96.2
97.1
94.5
103.1
87.6
94.8
247.1
227.9
233.7
Sample
Size
30
30
30
30
30
29
30
30
30
Standard
Deviation
12.3
13.8
15.4
14.7
12.8
14.2
37.7
36.5
36.2
95% Confidence
Interval
91.6-100.8
91.8-102.3
88.8-100.3
97.6-108.5
82.8-92.3
89.4-100.2
233.0-261.2
214.3-241.5
220.2-247.2
15 minutes. Activity mean values for cattle are reported as yM CC>2 evolved per
50 yl RBC's per 15 minutes.
18
-------
fall within a narrow range. Somewhat more variable results were obtain-
ed from Dipodomys ordii while Anrnosphermophilus leucurus was often not
collected in large enough numbers to be useful for statistical analysis.
CONTROL STUDIES IN SUPPORT OF FIELD PROGRAM
Results obtained from range sheep and Hereford steers following
acute and chronic oral doses of the military agent VX confirm that the
red cell activity recovery rate can be predicted and suggest that, by
collecting a series of blood samples over a period of time under field
conditions, a post-exposure diagnosis of both the degree of contamina-
tion and the time of exposure could be ascertained.
Acetylcholinesterase Determinations Using Hereford Steers
Hereford steers can tolerate the ingestion of substantial quanti-
ties of nerve agent for an extended period of time. No significant signs
of illness were observed in animals receiving daily VX doses of 0.7 micro-
grams per kilogram body weight for 75 consecutive days, although the
erythrocyte AChE activity was markedly depressed. Following 1.0 and
2.2 micrograms per kilogram per day, the only gross clinical sign was
some salivation, dose-related in its degree, which resulted in frequent
licking and resultant rash on the noses of the animals. Following
daily treatment with 4.4 micrograms per kilogram, signs of impaired
health were noted after the fifth day. At all except the lowest dosage
level used (0.1 micrograms per kilogram per day), the degree of AChE
inhibition was related to the total dose rather than the daily dose,
i.e., a given total dose had the same effect whether administered at
0.2 or 2.2 micrograms per kilogram. However, at the low dosage, 0.1
microgram per kilogram, depression of enzymic activity was only about
half as effective for a given total dose. Packed cell volumes and reti-
culocyte counts remained unaffected relative to control values. The
blood cell observations indicate that neither the amount of circulating
erythrocytes nor their lifespan or turnover time was affected by the
treatment .
Data plotted in Figure 3 illustrate that erythrocyte
cholinesterase responds rapidly and sensitively to low intakes of VX.
As seen in Figure 4, erythrocyte acetylcholinesterase activity was
depressed to 62 percent when 0.1 ug/kg/day was administered for 56 days
(Curve A), and to approximately 19 percent when 0.2 (Curve B) and
0.3 yg/kg/day (Curve C) were fed. At the lower dosage, there is clear
evidence that the activity had reached a plateau within about 40 days,
indicating the existence of a steady state in which the influx of
fully active cells balanced the inhibitory action of the nerve agent.
In Figures 4 (Curve D) , 5, and 6, presenting enzymic activity as
affected by the feeding of 0.7, 1.0 and 2.2 yg/kg/day, respectively,
the AChE levels seem to have attained steady states seen most clearly
in Figure 3, however, this may be more apparent than real. The
activity could have been depressed to as low a value as possible, but
a residuum was consistently measurable. Following a dose of 4.4
kg/day, the acetylcholinesterase was rapidly depressed to nil and
19
-------
V)
CO
20
0-3 lig/kg/DAY
10 20 30 40 50 60
DAYS
FIGURE 3. Mean, dose response curve showing the relationship between
oral VX. exposures of 0.1 and 0.3 yg/kg/day for 56 days and
the level of erythrocyte acetylcholinesterase activity.
Four Hereford steers were used in each treatment group.
20
-------
on several days between treatment day 45 and 75 no activity was detect-
ed. This was the only treatment (4.4 yg/kg/day for 75 consecutive days)
to cause death in one of the test animals. The lack of any detectible
activity was not a consistent finding during this heavy chronic exposure
and this failure of enzymic activity to remain below the detection limit
may be attributed to the continual turnover of erythrocytes in which new
active cells replace those whose acetylcholinesterase activity was
inhibited.
CO
<
OQ
DAYS
20 40
60 80 100 120 140
FIGURE 4. Mean dose response curve, depression and recovery phases in
Hereford steers showing the relationship between oral VX
exposures of Q.I, Q.2, and 0.3 yg/kg/day for 56 days plus an
exposure of 0.7 yg/kg/day for 75 days and the level of
erythrocyte acetylcholinesterase activity.
21
-------
100
90
80
70
* 60
LU
= 50
UJ
g 40
30
20
10
0
0 10 20 30 40 50 60 70
DAYS TREATMENT »
15 25 35 45 55 65 75 85 95 105 125
DAYS RECOVERY
FIGURE 5. Mean dose response curve, depression, and recovery phases, in
three Hereford steers showing the relationship between oral
VX doses of 1.0 yg/kg/day for 75 days and the level of erythro-
cyte acetylcholinesterase activity.
100
90
80
70-
*
uj 60
2
5 50
in
» 40
30
20
10-
* *
-1-
0 10 20 30 40 50 60 70
DAYS TREATMENT >\
15 25 35 45 55 65 75 85 95 105 115 125
DAYS RECOVERY »[
FIGURE 6. Mean dose response curve, depression and recovery phases, in
three Eereford steers showing the relationship between oral
VX doses of 2.2 yg/kg/day for 75 days and the level of erythro-
cyte acetylcholinesterase activity.
22
-------
The repeated daily ingestion of VX resulted in progressive depres-
sion of AChE and, since there was insufficient time for the restoration
of activity between exposures, the effect on the enzyme was virtually
cumulative. Following completion of the organophosphorus treatment,
the erythrocyte AChE activity returned to the original levels in an
essentially linear manner, a process which covered approximately 115 days.
Since erythrocyte AChE remained depressed for prolonged periods after the
disappearance of symptoms, cholinesterase activity in the tissues was
probably restored prior to red cell recovery. Once the restoration of
red cell activity had begun the rates were independent of the amount
originally ingested and the recovery of AChE activity reflected the rate
of erythrocyte replacement.
Toxicity of VX in Hereford Steers and Sheep
It was concluded from a brief series of experiments involving the
feeding of VX that 1.2 mg of VX per 46 kg of body weight constituted
an approximate acute LDso in younger steers (average weight = 251 kg).
The small number of larger steers (approximate weight = 636 kg) avail-
able for this investigation did not permit estimation of an LDso.
There was indication that the LDso value of VX may be somewhat lower
for smaller steers.
The oral LDso of VX in sheep was found to be virtually identical
to that in cattle (Table 8), but it did not depend on body weight.
VX applied directly to the skin was also quite toxic, the acute LDso
being approximately equal to the acute oral LDso, but the time to effect
(or death) was much prolonged. Interestingly, sheep can tolerate large
doses of VX on wool without noticeable effects. It was clear that the
wool served as an excellent protective covering. The toxicity of VX
was greatest by the intravenous route (to simulate inhalation exposure)
in terms of time to effect (TE) , time to death (TD) , and the dose
required to produce death in 50% of the sheep
The lower half of Table 8 summarizes data based on the feeding of
VX until death. The indicated doses were given in three divided, equal
amounts at 08&0, 1200, and 1600 hours daily. Although this was an arti-
ficial condition, the animals being incapable of ingesting VX following
collapse on the range, it was noted that TEso and TDso were not grossly
different at any dosage level and tended to converge as the dosage
increased. TE50 was chosen conservatively, the time to effect being
that when animals first showed signs of weakness (stumbling and loss of
coordination) rather than permanent collapse. The blood cholinesterase
activities of all animals declined drastically. Cholinesterase activity
in animals receiving 150 ug of agent per day were below the detection
limit for several days before any evidence of weakness was noted.
Additional groups of range sheep were fed VX impregnated pellets
in single oral doses, multiple oral doses until collapse, and multiple
oral doses until death. During these studies the sheep were maintained
23
-------
Table 8. TOXICITY OF VX IN SHEEP
ACUTE
Route
I.V.
Skin (nose §
feet)
Wool
Oral
TEa TDb
1-20 min 5-90 min
10-60 min 23-120 min
15-300 min 35 min-72 hrs
LD50
(yg/50 kg)
190
1650
22,500
1,500
CHRONIC*
Oral
(yg/day)
150d
3006
900£
ED50
In50 (yg/50 kg)
288 hrs 1,800
69 hrs 856
26 hrs 986
TDb
1U50 (yg/50 kg)
426 hrs 2,663
88 hrs 1,100
31 hrs 1,155
capsules with feed).
a. TE = Time to effect (loss of coordination, ability
to run or stand)
b. TD = Time to death
c. ED = Effective dose
d. Mean weight - 60 kg
e. Mean weight - 38 kg
f. Mean weight - 54 kg
24
-------
at different locations on, and several miles away from, the proving
ground to ascertain potential complicating environmental effects.
These environmental differences primarily concerned variations in
the type of pasture available to the animals. However, the different
pasture conditions produced no noticeable change in the suscepti-
bility of sheep to the VX pellets.
Cattle as Indicator Animals
For purposes of biological monitoring, cattle would seem to be
excellent indicator animals since chronic daily doses of about
25 micrograms depressed enzymic activity significantly. Based on
an average intake of 20 kg of feed per day*, cattle surveys will
readily detect the persistent presence of 1 part of VX (or toxic
substances equivalent to VX) in 1 X 109 parts of feed (1 part per
billion). At a daily intake of 225 micrograms, less than 1 micro-
gram per kg of body weight or 1 part per 108 (100 million) of feed,
the effect on red cell AChE is rapid and dramatic. Only short-term
exposure is needed for a telling effect. Cattle also tend to consume
preferentially the upper parts of browse which are more likely to be
contaminated with organophosphorus pesticides from spray operations
than parts near the ground.
Acetylcholinesterase Determinations Using Range Sheep
Rambouillet-Columbia range sheep were also used as test animals
to monitor the depression and subsequent recovery of erythrocyte
AChe activity following acute ingestion of VX. As was the case with
Hereford steers, an essentially linear return to baseline AChE acti-
vity was observed following cessation of organophosphorus exposure.
Synthesis of new erythrocytes and the concomitant increase in acti-
vity was responsible for the recovery to baseline levels. Therefore,
the recovery rate is largely independent of the original degree of
depression caused by the cholinesterase inhibitor and the length of
time required for the recovery will be approximately the same whether
the original degree of depression was 20 per cent or 90 per cent.
Results from Studies Using Laboratory Animals
It has been shown that guinea pigs are suitable hioassay- tools.
They reflect repeated oral intakes of about 1 microgram of VX per kg
per day by significant red cell AChE depressions. While this is far
*The Merck Veterinary Manual (1961) estimates the daily consumption of
feed by 600-pound beef steers to be 16 pounds (7.7 kg) of 90% dry weight
material. The present calculation is based on the assumption that dry
matter constitutes 33% of the weight of forage plants.
25.
-------
lower than the sensitivity of chemical assay, VX, or the equivalent
in toxic substances derived therefrom, may be distributed throughout
the relatively bulky amount of food consumed by the animals. As with
domestic animals and wildlife species, the specific identity of the
organophosphorus substances need not be known for this purpose, so
that guinea pigs may serve as a screening mechanism. It is expected
that a drawback in this method will be the dietary fastidiousness of
the animals.
The domestic rabbit appears to be a relatively poor candidate for
bioassay of VX and its toxic products at low concentrations. Quantities
of VX that had a decided effect on AChE in guinea pigs had little effect
on the enzymic activity of rabbit erythrocytes. The conclusion that
the domestic rabbit is a poor bioassay tool is reinforced by the fact
that AChE in rabbit erythrocytes is intrinsically low.
Studies on Wildlife Species (Small Mammals)
Controlled laboratory studies were also conducted using deer mice and
jackrabbits and included a very limited effort with the wood rat and
Ord kangaroo rat. Data resulting from these studies to date have not
revealed the clear sequence of AChE depression and subsequent recovery
following oral VX challenges. The red cell AChE activity levels were
significantly depressed following VX administration but individual
activity values were very erratic. It should be stressed that the
techniques required for the controlled studies on wildlife species are
somewhat more difficult than the relatively simple feeding and sample
collecting steps performed on domestic and laboratory animals. Fre-
quent collection of blood from a small mammal such as the deer mouse
can stimulate erythropoiesis and complicate the red cell AChE picture.
Furthermore, wild mammals such as the jackrabbit can be difficult to
handle during the organophosphorus feeding trials. The only conclusions
that can be reached at this time are that red cell activity is sensitive
to organophosphorus exposure and these wild animals appear to be suitable
field indicators for the presence of such anticholinesterase compounds.
Dose-response curves still need clarification, however.
Studies on Wildlife Species (Fish)
The manometric technique has also been employed to study the base-
line levels of brain acetylcholinesterase activity in rainbow trout
from five hatcheries operated by the Utah Department of Natural
Resources, Division of Fish and Game. Ninety-five samples of trout
ranging in length from 61 to 368 mm and in weight from 3 to 482 g
have been studied as a function of wet brain weight as well as total
brain protein.
Rainbow trout brain acetylcholinesterase activities range from
0.5 to 1.4 micromoles of acetylcholine hydrolyzed per mg wet brain
tissue per hour, and from 6.4 to 16.0 micromoles of acetylcholine
hydrolyzed per mg total brain protein per hour, depending upon the
26
-------
size of the trout. The brain acetylcholinesterase activity has been
found to decrease as the wet brain weight, total fish length and
fish weight increase.
Curves for the limits of rainbow trout brain acetylcholinesterase
activity have been determined by calculating the arithmetic means
(confidence coefficient = 0.95) for activities and wet brain weight,
total length, and weight classes of trout.
With these background data, enzyme inhibition due to unusual
occurrences of organophosphorus compounds in the aquatic environment
can be detected.
Complementary Toxicological Effects of Plant Poisons and Chemical
Agents on Mammals
Poisonous plants are common to the grazing ranges surrounding DPG
and are known to be consumed by livestock, occasionally in lethal quan-
tities. It is therefore essential to establish whether the toxicity of
these poisonous plants and that of VX are additive or even synergistic.
In this way it may be established whether organophosphorus pesticides
or nerve agents represent an especially acute hazard to animals whose
feed contains toxic plant substances.
During a series of studies designed to elucidate this problem (1970-
1972), combinations of organophosphorus agent and plant substance(s)
were administered, consecutively or simultaneously, to determine inter-
actions of the poisonous substances in terms of gross physiological
effects. Data were collected to permit estimation of seasonal risk
factors by location at the Dugway perimeter.
1. Oxalate-containing plants are common in West Central Utah.
Halogeton (Halogeton glomevatus') is a frequent problem to
ranchers and greasewood (SaTOobatus vermiculatus) to a more
limited extent. When appreciable quantities of these plants
are consumed, the soluble oxalate may either unite with the
calcium in blood and upset the mineral balance in the inter-
stitial fluid producing hypocalcemia, be degraded by rumen
microorganisms, or combine with calcium in the rumen to form
insoluble calcium oxalate. The reduction in serum calcium
is mainly caused by the deposition of calcium oxalate in the
soft tissues. Death usually results from a combination of
factors including tissue damage, hypocalcemia, interference
with energy metabolism, asphyxiation, and heart failure because
of changes in membrane potential. In summary, the literature
yielded little information that would suggest any direct
pharmacologic interaction between oxalate and VX. However,
changes in free (ionic) calcium are known to affect neuro-
muscular irritability and the release of acetylcholine from
prasynaptic vesicles, in this way establishing a tenuous
link to the primary action of VX. Furthermore, there remained
27
-------
the strong possibility that a debilitated animal is rendered more
susceptible to low quantities of VX (and other anticholinesterase
compounds) in an essentially nonspecific way.
Over 100 Rambouillet-Columbia sheep were studied at DPG during
1970 and 1971 to investigate potential synergistic responses follow-
ing oral doses of oxalate and VX. The study was supported by labora-
tory 'analyses to determine (1) red cell acetylcholinesterase (AChE)
levels during and after treatment and (2) serum calcium, phosphate
and creatinine values. Numerous necropsies were performed and a
rather extensive series of histological examinations were made on
selective tissues.
The most consistent pathological finding following oxalate
ingestion (with or without a VX combination) was hemorrhage and
edema of the rumen and reticulum with destruction of the rumenal
arteries by oxalate crystals. Hemorrhagic alterations in the
reticulum were neither pronounced nor consistently present. The
crystals were distributed throughout the duct system and cell
degeneration was occasionally noted adjacent to the areas of
crystalization. Blood-tinged froth was commonly present in the
nasal passageways and the lungs were usually congested. Hypocal-
cemia, hyperphosphatemia and slightly elevated creatinine values,
indicating some retention of nitrogenous substances, were also
noted in sheep that had ingested oxalate. Red cell AChE levels
were, as expected, not effected by the oxalate treatments.
Animals receiving various VX challenges showed the classic
responses to organophosphorus intoxication. Red cell AChE recovery
following VX doses was not effected by a simultaneous administra-
tion of oxalate during the treatment period. Total AChE recovery
took approximately 120 days. Post-mortem examinations conducted on
sheep treated with VX alone revealed no signs of tissue damage. In
one case lung congestion was pronounced but the animal in question
was recumbent for 12 hours prior to death.
When oxalate and VX were administered simultaneously there
was some evidence of synergism in the mortality ratio. However,
the apparent potentiating effect was not catastrophic. From the
point of view of chemical safety, the presence of oxalate containing
plants in grazing areas does not create unforeseen chemical safety
hazards to range sheep since only large intakes of oxalate, VX or
combinations of these are likely to eventuate in death. There is
however, a greater risk of misdiagnosis of symptoms. Quantities of
VX far below the fatal level, with or without oxalate, can supress
erythrocyte AChE activities to nil. Therefore, enzymatic activity
is an inadequate criterion in establishing the cause of death, and
major reliance must be placed on other signs and symptoms for a
definitive diagnosis.
28
-------
2. Western false hellebore (Veratrum oalifornioum), a large
coarse, erect herbaceous plant with broad leaves, is found
in mountain meadows and valleys near DPG where it often
invades and dominates moist slopes. In cases of serious
depletion it is frequently one of the last perennial species
in the meadow association to disappear. The toxic substance
is composed of a rather large group of chemically related
alkaloids, and the resulting toxicity following the ingestion
of plant material is influenced by several variables, e.g.,
stage of growth. Following veratrum ingestion the animal
demonstrates symptoms that are similar to those resulting
from VX intoxication and include general weakness, exces-
sive salivation with frothing, and irregular gait, coma and
extensor rigidity. The veratrum alkaloids did not depress
red cell AChE activity. Veratridine, an ester alkaloid,
germine, an alkamine, and veratrine, a mixture of alkaloids
and possibly the most representative test material, were
injected at various dose levels (without and with a VX
combination) into Swiss-Webster mice. The experiments on
mice were pilot studies only and complete dose-response
curves were not established for each veratrum alkaloid,
but possible synergistic responses were noted in the trials
when VX was administered concurrently.
A synergistic response, as revealed by total deaths,
was obtained in sheep that received both VX and veratrine.
However, when the number of severely affected animals was
included for comparison, the results indicated an addition of
of individual toxicities rather than a potentiation. Thus,
while an increased hazard apparently exists for sheep that
are exposed to both VX and veratrum plants, the study is not
entirely realistic because relatively large doses of agent
VX were used. Corresponding levels of contamination would
be considered dangerous in themselves and, therefore, this
study did not suggest a need for revised safety standards.
A major problem in the field would be an incorrect diagnosis
since neither veratrine nor VX cause pathological lesions and
both toxins produce nearly identical gross symptoms. An erythro-
cyte AChE analysis will determine if organophosphorus agents
are involved but will not provide information on complications
due to veratrum plants.
3. Death camas CZigadenus spp.), a poisonous Utah range plant,
is often found near Dugway in areas where, judging by the tracks
and accumulation of feces, sheep have obviously been grazing.
Morphological characteristics of the plant include long narrow
leaves, an unbranched single stem, a terminal raceme or panicle
of greenish white, yellow or pink flowers, and a scaly onion-
like underground bulb (but without onion odor). The ester alka-
29
-------
loids of death camas possess pharmacological activity resembling
the veratrum alkaloid veratridine while the alkamine genuine is
found in both false hellebore and death camas. Since Zigadenus
and Vevatmm species are included in the same subfamily
(Melanth-iaoeae} of liliaceous plants, it is not surprising that
their respective alkaloids are of a similar chemical composition.
The majority of sheep losses attributed to death camas occur in
early spring because the plant furnishes green, succulent feed
in advance of many range species, and because the young plant
stages are the most toxic to livestock.
Initially, a series of field trips was conducted to esta-
blish the relative frequency of death camas in the Dugway area.
Since extraction procedures do not provide a high yield of
toxin, plant material was homogenized in a Waring blender and
fed to sheep with stomach tubes. Feeding poisonous plants with
a stomach tube was apparently successful and the study revealed
some indication of potentiation when oral doses of death camas
and agent VX were given concurrently. However, the probable
result was that the death camas challenge so debilitated the
animal that the additional VX insult caused death without being
the result of classical synergism.
Evaluation of Complementary Effects Between Military Chemical Agents
and Organophosphorus Pesticides
Consideration has been given to the possibility that certain com-
binations of military chemical agents and Organophosphorus agricul-
tural poisons might have additive toxic effects since they often have a
similar mechanism of action. It should be noted that the brief experi-
ments reported here were not conducted in great detail since supporting
information only was required for the DPG monitoring program„ However,
in considering the application of agents attention was directed toward,
among other things, the time of occurrence of maximal depression of red
cell activity for each compound and the individual rates of absorption
and detoxification.
Cattle and sheep were treated with agricultural pesticides follow-
ing the method and dosage recommended by the manufacturer. The response
to the pesticide and military agent given alone was compared to the
response when treatment consists of a combination (Military agent and
pesticide) dose. Modifications of this procedure included the response
to the combination challenge when excessively high doses of pesticide
were given (a condition that might occur, if the ranchers do not follow
the manufacturer's advice).
An indication of potentiation was observed when Bayer 21/199
pesticide and agent VX were simultaneously administered to mature sheep.
Future experiments might apply the pesticide approximately three days
30
-------
prior to the VX challenge since the oral VX dose takes effect more
rapidly than the percutaneous pesticide treatment. While by definition
this experiment revealed a suggestion of synergism, this conclusion
should be viewed with caution because of the small number of sheep per
group and the absence of a complete Bayer dose-response curve.
Selected levels of VX and/or malathion were also administered to
Rambouillet-Columbia sheep for the purpose of monitoring the red cell
acetylcholinesterase response and to investigate the possibility of
synergism resulting from the two organophosphorus substances. These
two compounds did not produce a synergistic action. However, they
did produce an additive effect. Sheep that survived the combination
malathion-VX treatment had typical erythrocyte AChE recovery curves.
31
-------
REFERENCES CITED
1. Anderson, P. H., A. F. Machin, and C. N. Herbert, "Blood
Cholinesterase Activity as an Index on Acute Toxicity of Organo-
ph.osph.orus Pesticides in Sheep and Cattle," Res. Vet. Sci.,
Vol. 1£, pp. 29-33, 1969.
2. Augustinsson, K., "Assay Methods for Cholinesterase," Methods
of Biochemical Analysis, Vol. 5_, pp. 1-63, 1954.
3. Gaballah, S., "A Direct Radioisotopic Microassay for Cholinesterase,"
Proc. Soc. Exp. Biol. Med., Vol. 129, pp. 376-380, 1968.
4. Holmstedt, B., Structure-Activity Relationships of the Organo-
phosphorus Anticholinesterase Agents, p. 428-485, In Cholinesterases
and Anticholinesterase Agents, G. B. Koelle, Ed., Springer-Verlag,
Berline-Gottingen-Heidelberg, 1220 pp., 1963.
5. Merck Veterinary Manual, Second Edition, Merck and Co., Inc.,
Rahway, NJ, 1630 pp., Beef Cattle Nutrition, p. 694-706, 1961.
6. Reed, D. J., K. Goto, and C. H. Wang, "A Direct Radioisotopic Assay
for Acetylcholinesterase," Analytical Biochemistry, Vol. 16,
pp. 59-64, 1966.
7- Umbreit, W. W., R. H. Burris, and J. F. Stauffer, Manometric
Techniques and Tissue Metabolism, Burgess Publishing Co.,
Minneapolis, MN, 227 pp., 1949.
8. Witter, R. F., "Measurement of Blood Cholinesterase," Archives of
Environmental Health, Vol. 6, pp. 537-562, 1963.
32
-------
APPENDIX I
DESCRIPTIVE OUTLINE OF THE WARBURG MANOMETRIC TECHNIQUE USED
FOR THE ASSAY OF RED BLOOD CELL ACETYLCHOLINESTERASE ACTIVITY
1. Preparation and Storage of Erythrocytes Prior to Assay for
Acetylcholinesterese Activity
Blood from large mammals is drawn in 10-ml heparinized
Vacutainer tubes and mixed by inverting each tube several times
to prevent clotting. Small mammal blood is collected in 3-ml
heparinized Vacutainer tubes. The blood is stored at 4° C until
processing. When processed, the tubes of whole blood are
centrifuged in a refrigerated centrifuge (4° C) at 3,000 rpm for
for 12 minutes. The plasma, the white blood cells and any clots
are removed from the surface of the packed red blood cells and
discarded.
Two volumes of 0.9% sodium chloride solution are added to
one volume of packed red blood cells in tubes. The cells and
saline are gently mixed by inverting the tubes several times and
centrifuged at 3,000 rpm for 12 minutes. Supernatant fluid
should be drawn off slowly and carefully in order to remove all
of the saline solution but as little of the red blood cells as
possible. This washing procedure removes serum cholinesterase
activity and other potentially interfering substances.
Red blood cells from cattle and sheep should be washed to
remove all clotting substances even though the plasma has little
or no detectable cholinesterase activity. Whole erythrocytes of
small mammals should be stored at 4° C until time of assay.
For preparation of sheep erythrocytes 2.1 ml of 0.1% saponin
solution is pipetted into clean plastic vials. One ml of the
erythrocyte suspension is added to the 2.1 ml of saponin solution.
Saponin solution lyses the red blood cells and liberates cell
bound enzyme. Rinse the pipette several times by repeatedly
aspirating and expelling the saponin solution-erythrocyte mixture.
Allow the lysed cells to stand 10 to 15 minutes at room tempera-
ture and freeze the tubes at -27° G until time of assay. The
remainder of the non-lysed cells may be stored along with the
lysed cells for future reference.
Sample preparation of cattle erythrocytes consists of
pipetting 2.6 ml of 0.05% saponin solution into clean plastic
vials. Draw up 0.5 ml of the erythrocyte suspension and add
this to the saponin solution. Rinsing the pipette and storage
procedured are the same as for the sheep erythrocytes.
33
-------
2. Preparation and Storage of Enzyme-Inhibited Erythrocytic
Controls
Add 15 ml of distilled water to a clean 50-ml Erlenmeyer
flask. Add one 15-mg tablet of prostigmin (a cholinergic drug
used in the form of the bromide, Roche Laboratories, Nutley, NY)
to the flask. Allow five minutes for the tablet to dissolve,
mix by gently swirling the flask, and filter the solution.
Measure 1.05 ml of 0.1% saponin solution into each plastic vial.
Add 1.05 ml of the filtered prostigmin solution into each vial.
Draw up 1.0 ml of the appropriate erythrocyte suspension (fresh
whole washed rabbit, cattle or sheep erythrocytes) and add this
to the solution in each of the vials. Excess blood must be wiped
from the tip and outer surfaces of the pipette, otherwise, the
sample size will vary considerably. As before, rinse the pipette
and store the vials of enzyme-inhibited erythrocytes at -27 C
until time of assay.
These preparations provide 100 microliters (yl) of enzyme-
inhibited lysate per 0.3 ml, the amount required as a control in
the assay of sheep and rabbit erythrocytic specimens. For speci-
mens from cattle and rodents only 0.15 ml of the lysate is
required to equal 50 yl quantity specified. Because of the
small quantities of blood obtained from rodents, lysates pre-
pared from rabbit erythrocytes are employed as controls for
rodent specimens.
3. Preparation of Reagents for Manometric Assay for
Erythrocyte Acetylcholinesterase Activity
The reagents required for manometric assay of erythrocyte
acetylcholinesterase activity are (1) modified Krebs-Ringer
(KR) solution prepared fresh daily and (2) acetylcholine iodide
prepared fresh daily in KR solution. The Krebs-Ringer solution
is saturated for at least 15 minutes with a mixture of 95% N2 +
5% C02- (Attach a fritted glass stick with plastic tubing to
the gas regulator on the gas cylinder, and release a small but
steady stream of the gas mixture.) The concentration of acetyl-
choline iodide depends on the particular species from which
specimens are taken. For rodent specimens a 0.011 M solution is
prepared. Add 150.2 mg acetylcholine iodide to a 50 ml volumetric
flask, and fill to the mark with gassed Krebs-Ringer solution.
For cattle, sheep and jackrabbit specimens a 0.01 M solution
is prepared. Add 136.6 mg acetylcholine iodide to a 50-ml
volumetric flask and fill to the mark with, gassed Krebs solution.
34
-------
4. Preparation of Samples for Assay
a. Lysate Samples from Cattle and Sheep Erythrocytes
Allow the erythrocytic lysates to thaw at room temperature
for at least one hour. Once thawed, mix the individual samples
gently. With a 1-ml serologic pipette add 0.3 ml of each sample
to be assayed into the sidearm of each individual Warburg flask;
accurate measurement is essential. Fill the pipette and wipe
excess blood from the sides and tip before dispensing. Do not
dispense the last 0.1 ml in the pipette. Add 2.2 ml of 0.01 M
acetylcholine iodide solution to the main compartment of each
Warburg flask. Prepare a negative control in one flask to serve
as a thermobarometer control as follows: for sheep erythrocytic
lysates, pipette 0.3 ml enzyme-inhibited lysate into the sidearm.
Into the main compartment pipette 2.2 ml of 0.01 M acetylcholine
iodide solution. For cattle erythrocytic lysates, pipette 0.15 ml
enzyme-inhibited lysate plus 0.15 Krebs-Ringer solution into the
sidearm. Pipette 2.2 ml of 0.01 M acetylcholine iodide solution
into the main compartment.
b. Erythrocytic Samples from Jackrabbits or Domestic Rabbits
Using a 1.0 ml serologic pipette dispense 0.2 ml of a 0.1%
saponin solution into the sidearm of a Warburg flask. With a
100-yl micripipette draw up 100 ul of whole erythrocytes for each
sample and add this to the 0.2 ml saponin solution in the sidearm
of the appropriate flask. Measure the amount accurately and wipe
excess sample from the sides and tip of the pipette. Rinse the
micropipette carefully by repeatedly aspirating and expelling the
saponin solution-erythrocyte mixture. Avoid creating bubbles.
Let the micropipette remain standing in the sidearm for five
minutes to allow all of the solution to drain. Remove the micro-
pipette and expel any remaining solution into the sidearm. Shake
the flask carefully to mix the solution in the sidearm, but avoid
contaminating the main compartment of the flask with the blood
mixture. If any erythrocytic mixture does contaminate the main
compartment, discard the flask and begin again. Add 2.2 ml of
0.01 M acetylcholine iodide solution to the main compartment of
each flask. Prepare a blank or negative control in one of the
flasks to serve as a thermobarometer control. Pipette 0.3 ml
enzyme-inhibited erythrocytic lysate from rabbits into the side-
arm. Pipette 2.2 ml of 0.01 M acetylcholine iodide solution into
the main compartment.
c. Erythrocytic Samples from Rodents
Using a 1.0-ml serologic pipette dispense 0.2 ml of a 0.1%
saponin solution into the sidearm of a Warburg flask. With a
50_yl micropipette draw up 50 yl of whole erythrocytes and add
35
-------
this to the 0.2 ml saponin solution in the sidearm of the flask.
Measure the amount accurately and wipe excess sample from sides
and tip of the pipette. Rinse the micropipette carefully by
repeatedly aspirating and expelling the saponin solution-
erythrocyte mixture; avoid creating bubbles. Let the micro-
pipette remain standing in the sidearm for five minutes to allow
all of the solution to drain. Remove the micropipette, and expel
any remaining solution into the sidearm. With a 1.0 ml serologic
pipette add 0.25 ml of the saturated Krebs-Ringer solution to the
sidearm of the flask and shake each flask carefully. Pipette
exactly 2 ml of 0.011 M acetylcholine iodide solution to the main
compartment of the flask. Prepare a blank manometric control
by placing 0.15 ml of enzyme-inhibited erythrocytic lysate from
rabbits plus 0.35 ml of gas saturated Krebs-Ringer solution into
the sidearm. Place 2.0 ml of 0.011 M acetylcholine iodide solu-
tion in the main compartment.
d. Assay of Samples for Acetylcholinesterase Activity
A Warburg apparatus is used to measure the evolution of C02.
Check the level of Brodie's solution in each manometer. Add more
solution if necessary to bring the level of fluid to within no more
than 1/3 inch below the black index line. Check to see that all
stopcocks on the manometers are open. Set each micrometer to 450.
Apply lanolin to the ground glass joints of the sidearm stoppers,
as well as to the ground glass joints on the manometers. Assemble
the Warburg flasks to the manometers and secure each flask with a
spring. Insert the venting plugs into the sidearms and check to
see that the gas vents in the sidearms are open. Secure each
plug with a spring. Insert the frames on the Warburg apparatus
as desired. Check the level of water in the bath to insure that
it reaches but does not cover the neck of the Warburg flasks.
Monitor the level of fluid in each manometer as a double check
that the sidearm vents are open and have remained open. Esta-
blish that the temperature of the water bath is 37° C. Connect
a gassing tube to each sidearm plug and flush the vessels with
the 95% N2 + 5% C02 mixture. Adjust the flow of gas carefully.
Use enough pressure to flood the flasks with gas, but avoid exces-
sive pressure, which might expel or mix the separated components.
Continue to flush with the gas mixture for 10 minutes. After-
wards close the gas vent in each sidearm of the vessels by rotat-
ing the plug one quarter C90°) turn, and disconnect the gassing
tube. Remove each frame individually from the Warburg apparatus
and tip the frames three times to mix thoroughly the substrate
and blood preparations. Complete the mixing to assure that the
entire contents are in the main chamber of the flasks. Replace
the frames on the Warburg apparatus and start the shaker. Allow
10 minutes for temperature equilibration and the reaction to
commence. Close the stopcocks on the manometer. Adjust the
micrometers to bring the fluid level to the black index line on
36
-------
each manometer. Read and record the micrometer settings five min-
utes after closing the stopcocks. Continue to adjust the micro-
meters as often as necessary during incubation to maintain the fluid
level at the index line. Adjust the fluid level and record readings
at 5-minute intervals for the duration of the assay. The duration
of assay varies with the species of erythrocytes under test. Speci-
mens from cattle and sheep are followed for 30 minutes; those from
rabbits and rodents, 45 minutes. At the conclusion of the assays
stop the shaker and open the stopcocks on the manometers. Reset
micrometers to their initial reading (450). Remove the frames from
the Warburg apparatus, remove venting plugs from the sidearms and
remove the flasks from the manometers. Wipe the remaining lanolin
from the ground glass joints of the manometers with tissue paper,
and clean the Warburg flasks and sidearm stoppers.
5. Reporting Results
Results for cattle and sheep are recorded as yl C02 evolved
respectively per 50 and 100 pi RBC per 15 minutes. Results for
rodents and jackrabbits are recorded as \il C02 evolved respectively
per 50 and 100 yl RBC per 30 minutes. Prepare a data test chart
as shown in Appendix Table 1. Five-minute time intervals ranging
from 0 to 30 minutes for livestock and 0 to 45 minutes for wildlife
are placed across the top of the chart. Manometer readings are
recorded in the top half of the chart. Values in the lower half
are obtained by subtracting the readings of the later time inter-
val from the earlier reading for each sample.
The system is allowed to stabilize during the first ten minutes
after mixing; no readings are taken during this time period. In
the case of livestock, total the readings taken for the 15, 20,
and 25-minute periods. Then, in a separate step, sum those values
for the 20, 25, and 30-minute periods. Compute a mean for these
two subtotals and subtract the mean for the enzyme-inhibited control
(1C) sample. The difference represents the net AChE activity. For
specimens from wildlife species sum the values for the 15, 20, 25,
30, 35, and 40-minute intervals. Then total the values for the 20,
25, 30, 35, 40, and 45-minute intervals. As before, compute a mean
for these two subtotals and subtract the mean for the enzyme-inhibited
control sample. A standard control (SC) sample is prepared from
sheep (or other available species) erythrocytes and is assayed as a
standard with each assay of unknown. To compute the net standard
control for each assay, first total the readings taken for the 15,
20, and 25-minute intervals; then sum the values for the 20, 25,
and 30-minute periods. Compute a mean for these two subtotals and
subtract the mean of the enzyme-inhibited control. The difference
represents the net standard control for the assay. Values which
were recorded as microliters (yl) are reported as micromoles (yM).
The conversion factor for jackrabbits is 0.68, for rodents and
sheep 1.34, and for cattle 2.68.
37
-------
APPENDIX TABLE 1. ACETYLCHOLLNESTEREASE (AChE) DATA TEST CHART
Wildlife
Readings
from
manometers
Time intervals
Specimen number 1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
S.C.
I.C.
1
2
3
4
5
6
Values 7
from 8
readings 9
10
11
12
13
14
15
S.C.
I.C.
| Livestock
00 05 10 15~ 20 25 "TO 35 40 45
Omit
Avg.
Net
S.C. — ~
I.C. ~ —
Net S.C. is
38
-------
TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-680/4-75-003
2.
4. TITLE AND SUBTITLE
DEVELOPMENT OF A BIOLOGICAL MONITORING NE
Test Case - Suitability of Livestock and W
Biological Monitors for Organophosphorus C
7. AUTHOR(S)
W.W. Sutton and L.L. Salomon
9, PERFORMING ORG -\NIZATI ON NAME AND ADDRESS
Monitoring Systems Research and Developmen
National Environmental Research Center
P.O. Box 15027
Las Vegas, NV 89114
12. SPONSORING AGENCY NAME AND ADDRESS
Office of Research and Development
U.S. Environmental Protection Agency
Washington, D.C. 20460
3. RECIPIENT'S ACCESSION- NO.
5. REPORT DATE
TWORK - A Julie 1975
ildlife as 6. PERFORMING ORGANIZATION CODE
ontaminants
8. PERFORMING ORGANIZATION REPORT NO.
10. PROGRAM ELEMENT NO.
t Laboratory F11082
11. CONTRACT/GRANT NO.
ROAP 22 ACS
13. TYPE OF REPORT AND PERIOD COVERED
FINAL
14. SPONSORING AGENCY CODE
15. SUPPLEMENTARY MOTES
16. ABSTRACT
Upon request by the NERC-LV, a review was conducted of a DPG monitoring network which
is designed to establish baseline erythrocyte acetylcholinesterase (AChE) levels in
tie fauna of West Central Utah, and to evaluate the suitability of using livestock and
wildlife as biological monitors for organophosphorus contaminants. Wildlife species
sampled during these DPG efforts included the antelope ground squirrel, the ORD
Kangaroo rat, the deer mouse, and the black-tailed jackrabbit. Individual blood
samples from these wildlife species as well as samples from cattle and sheep were
collected and analyzed for red cell AChE activity. The analytical method employed
was based on the Warburg manometric technique. Results indicate that the range of
red cell AChE activity values for both livestock and wildlife species is sufficiently
compact to allow observation of the depression of enzymic activity that would result
from organophosphorus exposures. Controlled studies have shown that, following
exposure to organophosphorus chemicals, the red cell activity recovers in an
essentially linear fashion. Additive effects resulting from the simultaneous
exposure to military agent VX and either toxic plants or commercial pesticides are
discussed.
17.
KEY WORDS AND DOCUMENT ANALYSIS
a. DESCRIPTORS
Monitoring
Biological Monitors, Wildlife, Livestock
Organophosphorus
I3. DISTRIBUTION STATEMENT
b. IDENTIFIERS/OPEN ENDED TERMS
Biological Monitoring
Monitoring Network
Animals as Monitors
19. SECURITY CLASS (This Report)
UNCLASSIFIED
20. SECURITY CLASS (This page)
UNCLASSIFIED
c. COSATI Field/Group
0601 0603
0606 0616
0703 1400
21. NO. OF PAGES
52
22. PRICE
EPA Form 2220-1 (9-73)
GPO 693-743/^8
------- |