United States
Environmental Protection
Agency
Municipal Environmental Research EPA i
Laboratory
Cincinnati OH 45268
Research and Development
&EPA
Determination of
Giardia Cyst
Viability
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series These nine broad cate-
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This report has been assigned to the ENVIRONMENTAL PROTECTION TECH-
NOLOGY series. This series describes research performed to develop and dem-
onstrate instrumentation, equipment, and methodology to repair or prevent en-
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This document is available to the public through the National Technical Informa-
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EPA-600/2-79-063
July 1979
DETERMINATION OF GIARDIA CYST VIABILITY
by
Ernest A. Meyer
Department of Microbiology and Immunology
University of Oregon Health Sciences Center
Portland, Oregon 97201
Grant No. R 804898
Project Officer
John C. Hoff
Drinking Water Research Division
Municipal Environmental Research Laboratory
Cincinnati, Ohio A5268
MUNICIPAL ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
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DISCLAIMER
This report has been reviewed by the Municipal Environmental Research
Laboratory of the U.S. Environmental Protection Agency, and approved for
publication. Approval does not signify that the contents necessarily re-
flect 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 for use.
ii
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FOREWORD
The Environmental Protection Agency was created because of increasing
public and government concern about the dangers of pollution to the health
and welfare of the American people. The complexity of that environment and
the interplay between its components require a concentrated and integrated
attack on the problem.
Research and development is that necessary first step in problem solu-
tion and it involves defining the problem, measuring its impact, and search-
ing for solutions. The Municipal Environmental Research Laboratory develops
new and improved technology and systems for the prevention, treatment, and
management of wastewater and solid and hazardous waste pollutant discharges
from municipal and community sources, for the preservation and treatment of
public drinking water supplies, and to minimize the adverse economic, social,
health, and aesthetic effects of pollution. This publication is one of the
products of that research; a most vital communications link between the re-
searchers and the user community.
This research provides a method for determining Giardia cyst viability
which is apparently more sensitive than the previously-used dye exclusion
methods. The development of this method makes possible its application in
a variety of future studies, including:
1) the efficacy of presently-available water treatment methods
in inactivating Giardia cysts,
2) the study of protozoan excystation, and
3) the establishment in culture of strains of Giardia tropho-
zoites from cysts, rather than from sacrificed animals.
Francis T. Mayo, Director
Municipal Environmental
Research Laboratory
iii
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ABSTRACT
The principal objective of this research was the development of a
sensitive, standardized method of determining whether or not Giardia cysts
are viable. The availability of such a method is necessary to determine
the effect of chemical and physical agents on the viability of these organ-
isms in water.
An asymptomatic human carrier of Giardia was found and provided the
principal source of cysts for these experiments. A standard method of con-
centrating and purifying Giardia cysts from feces was developed.
A major contribution of this research has been developing a method of
inducing, and determining the factors involved in, Giardia excystation.
This method involves exposing cysts to pH 2.0 HC1 for one hour at 37°C, and
transferring the cysts to Giardia growth medium HSP-3 for one hour at 37°C.
Variation of these conditions has revealed that the time of acid exposure,
the incubation temperature and the composition of the post-acid incubation
medium influence excystation levels.
Another contribution of this research has been the application of the
excystation procedure as a method for assessing the viability of cysts.
The effect of various storage temperatures on cyst survival in water has
been examined. This study showed that survival time decreases with in-
creasing temperatures above 0°C; near-freezing temperatures permitted
longest survival. Freezing and thawing cysts resulted in an almost com-
plete loss of viability.
This report was submitted in fulfillment of grant no. R804898 by
Ernest A. Meyer under the sponsorship of the U.S. Environmental Protection
Agency. The report covers a period from October 20, 1976 to October 19,
1978.
iv
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CONTENTS
Foreword ill
Abstract iv
Figures vi
Tables vii
Acknowledgements viii
1. Introduction 1
2. Conclusions and Recommendations 2
3. Materials and Methods 3
Cyst sources 3
Cyst purification and storage 3
Growth medium 3
Excystation solutions 3
Excystation procedure 4
Quantitation of excystation and statistical analysis ... 4
Photomicrography 5
Effects of temperature on excystation 5
4. Results and Discussion 6
Induction and description of excystation 6
Identification of inducing factor 8
Effect of variation of physical environment 12
Hydrogen and other ions 12
Time of acid exposure 12
^emperature 14
Post-acid incubation medium 20
Cyst variation 21
Maturation, daily variation and viability 21
Variation in sensitivity to physical environment . . .21
Culturing 23
Other reports based on this research 23
References .25
Bibliography 26
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FIGURES
Number Page
1 Pattern of the eight fields examined to determine percentage
of excystation 4
2 Representative steps in Giardia excystation. (A) Typical cyst;
(B through F) sequential emergence of trophozoite(s) [arrows
(E) indicate ventral adhesive discs of daughter trophozoites];
(G) excystation completed, division of daughter trophozoites
continuing; (H) empty cyst. Scale bar = 10 um 7
3 Excystation of Giardia exposed to pH-varied synthetic gastric
juice. Vertical bars represent standard error of the mean . . 10
4 Excystation of Giardia exposed to pH-varied HC1. Vertical
bars represent standard error of the mean 11
5 Effect of storage at 8°C on Giardia cyst viability as deter-
mined by excystation. Vertical bars represent standard
error of the mean 15
6 Effect of storage at 21°C on Giardia cyst viability as deter-
mined by excystation. Vertical bars represent standard
error of the mean 16
7 Effect of storage at 37°C on Giardia cyst viability as deter-
mined by excystation. Vertical bars represent standard
error of the mean 17
8 Effect of storage at -13°C on Giardia cyst viability as
determined by excystation. Vertical bars represent stan-
dard error of the mean 18
9 Effect of storage at 37°C on Giardia cyst viability as deter-
mined by excystation over a 24-hour period 19
10 Giardia excystation pattern for cysts stored at 8°C for an
11-week period 22
vi
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TABLES
Number Page
1 Excystation of Giardia exposed to human upper alimentary
tract fluids 8
2 Excystation of Giardia exposed to pH-adjusted human upper
alimentary tract fluids 9
3 Excystation of Giardia exposed to complete and component-
varied synthetic gastric juice 11
A Excystation of Giardia exposed to inorganic acids at
PH 2.0 12
5 Excystation of Giardia by varying pH and exposure time 13
6 Excystation of Giardia by varying the temperature of
HC1 (pH 2.0) and HSP-3 14
7 Excystation of Giardia exposed to HC1 (pH 2.0) and transferred
into solutions of varying complexity and pH 20
8 Excystation of Giardia exposed to HC1 (pH 2.0) and transferred
into component-varied HSP-3 at pH 7.0 21
9 Optimum time of acid exposure for Giardia excystation with
aging of cysts 23
vii
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ACKNOWLEDGMENTS
The cooperation of two colleagues who helped identify Giardia-infected
individuals is acknowledged: Dr. Abdul Rashad, Head of the Microbiology
Division in the Department of Clinical Pathology at the University of Oregon
Health Sciences Center, Portland, Oregon, and Dr. Arthur Hall, veterinarian
at the Oregon Regional Primate Research Center, Portland, Oregon. We thank
Dr. Lynette Feeney-Burns, Associate Professor of Ophthalmology at the Uni-
versity of Oregon Health Sciences Center, Portland, Oregon, for making
available microscope lenses used in the photomicrography studies presented
here. We also thank the asymptomatic patient who served.as the donor of the
cysts used in these studies.
viii
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SECTION 1
INTRODUCTION
The problem addressed in the proposed study relates to the control of
human infections caused by parasitic protozoa in the genus Giardia. Organ-
isms in this genus are common intestinal inhabitants of man and a great
variety of other animals. In various surveys in the last 30 years, the in-
testinal incidence of Giardia infection in humans has ranged from 1 to 10
percent. .-.
Humans acquire Giardia infection by ingesting the cyst form of the or-
ganism; excystation takes place in the small intestine and the resultant
trophozoites divide by binary fission and colonize the host. Trophozoites
swept into the fecal stream lose their motility, round up, and are excreted
as dormant, thick walled cysts which, upon being ingested by a suitable host,
are capable of excysting and establishing another intestinal infection.
Giardia infections may be silent or symptomatic. Symptoms, when they
occur, usually include a fatty diarrhea (steatorrhea) with epigastric pain
and gas. Giardiasis may persist for years. The organisms apparently
rarely invade the host's tissues. Children, dysgammaglobulinemic indivi-
duals and those with other immune deficits are particularly susceptible to
symptomatic Giardia infections.
The epidemiology of giardiasis is similar to a number of other intes-
tinal pathogens of man (viral, bacterial, and protozoal) in that the ulti-
mate source of the organisms is (cyst-containing) fecal material and the
portal of entry is the mouth. Drinking water containing Giardia cysts is
believed to have been the vehicle for some of the giardiasis outbreaks in
recent years, including outbreaks in Aspen, Colorado; Leningrad, Russia;
and Rome, New York. The vehicle of a giardiasis outbreak in 1953-1954 in
Portland, Oregon, which involved an estimated 50,000 cases, or one in
Sydney, Australia, in 1975 involving hundreds of cases, has never been de-
termined .
Inasmuch as public water supplies represent a potential vehicle for
the spread of the cyst forms of these organisms, it is important that water
be treated in a way that assures that no viable Giardia cysts are present.
Because disinfection is often the means by which microorganisms are inac-
tivated in water supplies, it would be of value to know whether Giardia
cysts are in fact inactivated by usual methods of water treatment. Such a
treatment method has not yet been obtained, because a reliable method of
differentiating living and dead Giardia cysts is not available. The deve-
lopment of a method for determining cyst viability is an object of this
research.
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SECTION 2
CONCLUSIONS AND RECOMMENDATIONS
This study has demonstrated that Giardia excystation, which normally
occurs in the host gastrointestinal tract as a part of the infection pro-
cess, can be regularly induced in vitro. Factors which affect excystation
have been studied, and standard methods for concentrating and purifying
cysts and inducing excystation have been developed. Preliminary comparison
of eosin-exclusion and excystation as criteria of cyst viability suggests
(1) that eosin-exclusion consistently indicates viability of cysts which
are incapable of excystation, (2) that Giardia cysts are less hardy than
previously indicated, and (3) that earlier criticisms of eosin-exclusion
were well-founded. Excystation would seem to be the method of choice for
determining Giardia cyst viability.
Excystation studies indicate that Giardia cysts survive well in near-
freezing water; viability is reduced at higher temperatures, Freezing and
thawing is detrimental to cyst survival.
Now that a sensitive, standardized excystation method is available for
assessing Giardia cyst viability, it seems appropriate that it be used to
obtain information that applies to the provision of safe drinking water,
including:
(1) determining the effect of current chemical water treatment
methods on Giardia cysts;
(2) determining the effect of other water treatments, including
temperature, on Giardia cysts;
(3) identifying water treatment methods that will kill these
organisms if present water treatment methods fail to inac-
tivate Giardia cysts; and
(4) determining the relationship between excystation, eosin-
exclusion and animal infectivity as indicators of cyst
viability.
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SECTION 3
MATERIALS AND METHODS
Cyst sources. Initial studies on excystation were carried out on cysts
obtained from dogs (University of Oregon Health Sciences Center [UOHSC] Ani-
mal Care Facility, Portland, Oregon), from monkeys (Oregon Regional Primate
Research Center, Portland, Oregon), and from hospital inpatients (UOHSC Depart-
ment of Clinical Pathology). Some additional cyst-bearing animal feces were
provided by other individuals and institutions. Later studies, and all those
presented in this thesis, were performed using cysts obtained from one asymp-
tomatic human male with giardiasis.
Cyst purification and storage. Cysts were purified by a modification
of the procedures of Roberts-Thomson et^ al. (1) and Sheffield and Bjorvatn (2)
as follows: Feces were suspended to a thin consistency in tap water and
filtered successively through 1000, 710, 500, 250 and 177 ym-aperture nylon
meshes. Three to 5 ml of filtrate was placed on 3 ml of chilled 0.85 M
sucrose in a 15 ml conical centrifuge tube and the tube was centrifuged at
600 g for 5 minutes at room temperature in a swinging bucket rotor. The
water-sucrose interface was removed, diluted 1:10 with water, and re-centri-
fuged for 5 minutes. The pellet was resuspended in 3 ml of water and the
sucrose gradient centrifugation repeated as many times as needed to achieve
the desired purity. Following this the water-sucrose interface was again
removed, diluted 1:10 with water, filtered under vacuum through a 20 ym-aper-
ture nylon mesh, and centrifuged for 5 minutes. This pellet was resuspended
and diluted in tap water to a concentration of approximately 50,000 cysts/ml.
In all experiments except those in which the effect of storage temperature
was studied, cyst suspensions were stored at 8°C. The purification procedure
could be accomplished in an hour yielding approximately 30 percent cyst re-
covery, with purity acceptable for light microscopic examination and enu-
meration, and apparently undiminished cyst viability.
Growth medium. Giardia growth medium HSP-3 derived from Meyer's HSP-1
and HSP-2 media (3) was used in the excystation and cultivation of tropho-
zoites. This medium has the following formulation: 85 ml Hanks-phytone
broth, 20 ml Seitz filter-sterilized heat-inactivated human serum, 7.5 ml
NCTC-135 (Gibco), and 1.5 ml 1.0 M NaHC03; final pH 7.0. For culturing
trophozoites, the following antibiotics were added to HSP-3: 250,000 units
potassium penicillin G, 0.02 g streptomycin sulfate, and 0.01 g,gentamicin
sulfate.
Excystation solutions. Human saliva, gastric juice and duodenal-jejunal
fluid were obtained from normal individuals (saliva) and from hospital in-
patients (UOHSC Division of Gastroenterology Diagnostic Unit). The pH of
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these solutions was measured on a Leeds and Northrup model 7401 pH meter
and adjusted to desired pH values by the addition of dilute HC1 or 1.0 M
NaHC03.
Synthetic gastric juice, which was a composite of those described by
Hirschowitz (4) and Konturek (5), contained major components of normal hu-
man gastric juice in aqueous solution as follows: NaHC03 (25 mN), KC1
(12 mN), NaCl (40 mN), CaCl2 (12 mN), HC1 (120 mN), and pepsin (1500 units/
ml; Sigma); final pH 1.6.
Aqueous HC1 solutions were prepared by adding concentrated HC1 to dis-
tilled water to the desired pH.
Excystation procedure. Unless stated otherwise the following excysta-
tion procedure was used in all excystation experiments: One volume of
purified cyst preparation (usually 0.1 ml) was added to at least 10 volumes
of excystation solution (saliva, gastric juice, HC1, etc.), and the mixture
was incubated at 37 C for one hour. Following incubation, the suspension
was centrifuged at 600 g for 5 minutes at room temperature, and the pellet
resuspended in water and re-centrifuged. The pellet was then resuspended
in HSP-3 (usually 0.5 ml) at 37 C. A depression slide chamber was filled
with the suspension, sealed with a cover glass and paraffin-Vaseline, and
incubated inverted at 37 C for one hour. The slide was then examined and
the percentage of excystation determined. Timed experiments showed that a
one hour incubation in HSP-3 was sufficient to allow complete escape of
those trophozoites capable of excysting.
Quantitation of excystation and statistical analysis. Excystation was
quantitated by systematic examination of slide chambers at 300x on a Uni-
tron model PH-BMIC inverted microscope. Eight fields were examined on each
cover glass in the pattern shown in Figure 1.
Depression slide
Fields of
examination
Cover slip'
\
Paraffin-Vaseline
seal
Figure 1. Pattern of the eight fields examined to determine
percentage of excystation.
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The fields were chosen such that they were mid-way between the outer edge
and the center of the circular chamber, thus avoiding the sparsely populated
outer edge and the densely populated center. This pattern was considered to
be representative of the entire cover glass.
The percentage of excystation was determined by counting the number of
intact cysts (1C), partially excysted trophozoites (PET), and totally ex-
cysted trophozoites (TET) , and applying the following formula:
TET/2 + PET
Percent
Excystation
In this formula the number of totally excysted trophozoites was divided by
2 because every cyst in which complete excystation occurred promptly yielded
a pair of trophozoites. Totally excysting trophozoites rather than empty
cysts were counted because: 1) empty cysts were sometimes difficult to de-
tect due to their lack of retractility, 2) excysted trophozoites were easy
to detect because of their active flagella, and yet seldom traveled appre-
ciably, attaching to the glass almost immediately, and 3) the number of
empty cysts present in a preparation due to causes other than excystation
(e.g. death and trophozoite disintegration) were sometimes appreciable,
particularly as the preparation aged. Trophozoite multiplication did not
introduce additional counting errors since the generation time is approxi-
mately 18 hours [in M-5, a Giardia growth medium similar to HSP-3;(6)]
Statistical significance was determined by Student's t-test.
Photomicrography. Photographs were taken under phase contrast at 630x
on a Zeiss Invertoscope D with Kodak SO-115 film.
Effects of temperature on excystation. The effect of cyst storage
temperature on excystation was examined by the following procedure: Puri-
fied cysts were suspended in water and stored at either -13°, 8°, 21° or 37°C;
the effect of the latter three temperatures was studied in a single experi-
ment using cysts purified from one fecal specimen. Cysts stored at -13°C
were frozen in 0.5 ml aliquots by cooling to 8°C followed by transfer to
-13°C; aliquots were thawed at 37°C as needed. At other temperatures, cysts
were stored in 100 ml volumes from which aliquots were removed. Cyst via-
bility was assessed periodically by excystation in HC1 at pH 2.0 by the
procedure described previously.
The effects of the acid and post-acid medium incubation temperatures
were examined together. Cysts were exposed to pH 2.0 HC1 at either 8°, 21°
or 37°C by the usual procedure. The cysts exposed at one temperature were
then divided into three equal portions (a total of nine for the three
temperatures) , and one portion from each temperature suspended and incubated
in HSP-3 at either 8°, 21° or 37°C. These suspensions were observed after
four and 18 hours incubation.
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SECTION 4
RESULTS AND DISCUSSION
I. Induction and description of excystation.
Using the procedure described previously, excystation was induced in
Giardia cysts and followed the course described below.
Excystation was seen minutes after the transfer to cysts into HSP-3,
(Figure 2). The trophozoite first separated from cyst wall and began
flexing movements, then began to emerge from the cyst. The emergence re-
sembled the extrusion of a fluid-filled balloon through a small hole which
was usually located in one end of the oval cyst. Active movement of fla-
gella external to the cyst accompanied and possibly aided the escape of the
trophozoite. Cell division always followed the emergence of a trophozoite
from the cyst. Often the process of differentiation and division was
nearly complete even before the trophozoite was free from the cyst wall.
The entire process of excystation required from five to 30 minutes follow-
ing transfer of cysts into HSP-3.
Abortive excystation was noted in three forms. In the first, the
flexing movement inside the cyst was followed by a cessation of trophozoite
activity and a "re-expansion" of the trophozoite to fill the cyst. In ob-
servations of cysts of this type over several hours, no subsequent activity
was seen, indicating loss of viability. In the second form of abortive
excystation the trophozoites appeared to have died while attempting to es-
cape from the cyst, resulting in a final stage which appeared like one of
those in Figure 2B-D; in this stage cytoplasm of theintra- and extra-cystic
portions of these partially excysted trophozoites appeared structurally
normal. In the third form, cysts appeared similar to the one in Figure 2B,
except that the protruding cytoplasm was devoid of any discernible internal
structure or organelles. In addition, the number and size of the protru-
sions varied; when multiple protrusions were present they were always loca-
lized in one area of the cyst surface. Protrusions apparently "budded-off"
completely from some cysts and could be seen as refractile spheres free in
the medium. The frequency of cysts exhibiting abortive excystation seemed
to increase with increasing cyst age.
Later experiments revealed that aberrant, as well as abortive, excy-
station could occur. Occasionally trophozoites attempted excystation
through the side of the cyst (lateral excystation) rather than through one
end (terminal excystation). In these, excystation was almost always abor-
tive. Unusual extra-cystic structures were rarely observed. These
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Figure 2. Representative steps in Giardia excystation. (A) Typical cyst; (B through F)
sequential emergence of trophozoite(s) [arrows (E) indicate ventral adhesive discs of
daughter trophozoites]; (G) excystation completed, division of daughter trophozoites
continuing; (H) empty cyst. Scale bar = 10 pm.
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included motile trophozoite-like organisms approximately \ to 4 normal tro-
phozoite size, and rod-like or club-shaped structures resembling axostyles
surrounded by cytoplasmic remnants "wiggling" independently in the medium.
The incidence of aberrant excystation and of unusual extra-cystic structures
was greater in some fecal specimens than in others — even from the same
host. In one human case followed from onset of symptoms through eight
months of asymptomatic infection, most fecal specimens contained typical
cysts yielding up to 60 percent excystation and normal, active trophozoites.
Other fecal specimens contained cysts which appeared typical, but in which
the ability to excyst was either poor or absent. Still other specimens
yielded irregularly shaped cysts containing either normal or abnormal-
appearing trophozoites. These atypical cysts were likely to undergo aber-
rant and/or abortive excystation, and were found with increasing frequency
in fecal specimens as the patient approached a three-month period of non-
excretion.
II. Identification of inducing factor.
In an attempt to duplicate the conditions to which cysts are exposed
in vivo, purified Giardia cysts were exposed to human upper alimentary
tract fluids according to the excystation procedure described previously.
Due to a shortage of fluids, only one trial was performed, and approximately
100 cysts were counted at each pH in each fluid. It was found that only
those fluids with a pH of 5.0 or below induced excystation and that the
percentage of excystation appeared to increase with decreasing pH (Table 1).
TABLE 1. EXCYSTATION OF GIARDIA EXPOSED TO HUMAN UPPER
ALIMENTARY TRACT FLUIDS
Fluid
£H_
Percent
Excystation
Water (control)
HSP-3 (control)
Saliva
*
Gastric juice
Gastric juice
Gastric juice
Duodenal- jejunal fluid
Duodenal- jejunal fluid
6.8
6.8
7.6
7.6
5.0
2.4
5.7
4.3
0
0
0
0
10.9
40.8
0
10.6
From gastric resection patient (patient 1).
From patient 1, adjusted to pH 2.4.
8
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The influence of pH on excystation was examined by adjusting specimens
of human saliva, gastric juice, and duodenal-jejunal fluid to various pH
values and inducing excystation in these fluids. Again, one trial was per-
formed and approximately 100 cysts were counted at each pH level, in each
fluid. The results of this experiment are shown in Table 2.
TABLE 2. EXCYSTATION OF GIARDIA EXPOSED TO pH-ADJUSTED HUMAN
UPPER ALIMENTARY TRACT FLUIDS
Fluid
Water (control)
HSP-3 (control)
Saliva
Gastric juice
Duodenal-jejunal fluid
PH
6.8
6.8
7.6
2.3
7.6
6.8
5.9
4.9
4.0
2.9
2.1
7.2
2.3
Percent
Excystation
0
0
0
43.8
0
0
0
18.0
42.5
30.3
37.2
3.1
48.6
All specimens with pH values between 2.1 and 4.9 induced high levels of
excystation; little or no excystation was induced at higher pH values. In
gastric juice the highest levels of excystation were induced between pH 2.1
and 4.0, with reduced excystation at pH 4.9 and none at higher pH values.
A synthetic gastric juice was prepared and used in an attempt to iden-
tify the factor(s) inducing excystation in normal gastric juice. Aliquots
of the synthetic fluid were adjusted to different pH values and excystation
performed in these solutions. Eight trials were performed in which an
average total of 1825 cysts were counted at each pH. The results (Figure 3)
were similar to those found in experiments using human gastric juice. Peak
percentages of excystation were observed between pH 2.0 and 3.2 with pro-
gressively diminishing levels above or below these pH values. Peak per-
centages of excystation were significantly greater than those at other pH
values (p<0.05).
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40
30
20
10
Figure 3. Excystation of Glardia exposed to pH-varied synthetic gastric
juice. Vertical bars represent standard error of the mean.
To identify factors other than pH influencing excystation, complete
and component-varied synthetic gastric juices were prepared and compared
for ability to induce excystation. Eight separate trials were performed in
which an average total of 1459 cysts were counted in each solution. With
the exception of water and HSP-3 controls, all of the solutions induced al-
most identical levels of excystation (p>0.05; Table 3); excystation in the
control tubes was significantly lower than in the other solutions (p<0.05).
The only common factor in the excystation-inducing solutions was HC1 in a
concentration resulting in a pH of 1.6. The presence of salts and pepsin
did not significantly alter the degree of excystation.
10
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TABLE 3. EXCYSTATION OF GIARDIA EXPOSED TO COMPLETE AND COMPONENT-VARIED
SYNTHETIC GASTRIC JUICE
Solution
Compete (HC1 + salts +
HC1 4- salts
HC1 only
Water (control)
HSP-3 (control)
PH
pepsin) 1.6
1.6
1.6
6.8
6.8
Mean * percent
excystation
± S.E.M.
25.8 ± 7.3
23.0 ± 7.0
23.2 ± 7.0
0.1 ± 0.1
0.0 ± 0.0
Means are derived from an average total of 1459 cysts counted in each
solution.
To examine the influence of HC1 on excystation, aqueous HC1 solutions
at various pH values were prepared and eight excystation trials were per-
formed in each solution; an average total of 1564 cysts were counted at
each pH. The results (Figure 4) revealed a pattern similar to those ob-
served when pH-varied gastric juice and synthetic gastric juice were used.
30 r
PH
Figure 4. Excystation of Giardia exposed to pH-varied HC1. Vertical
bars represent standard error of the mean.
11
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The percentages of excystation at pH 1.3 to 2.7, although not significantly
different from each other (p>0.05) were significantly greater than those at
other pH values (p<0.05).
To determine whether the factor inducing excystation was hydrogen ion,
chloride ion, or a combination of the two, several mono-, di-, and trivalent
inorganic acids were diluted in water to a final pH of 2.0 and excystation
attempted. Eleven trials were performed, an average total of 2242 cysts
counted in each acid. Each acid induced almost identical levels of excysta-
tion (p>0.05; Table 4).
TABLE 4. EXCYSTATION OF GIARDIA EXPOSED TO INORGANIC ACIDS AT pH 2.0
Mean * percent
excystation
Acid ± S.E.M.
Water (control) 0.2 ± 0.2
HC1 17.4 - 2.2
HN03 12.5 ± 3.0
HC104 15.5 ± 2.0
H2SC>4 15.5 ± 2.6
H3P04 19.4 ± 2.8
*
Means are derived from an average of 2242 cysts counted in each solu-
tion.
III. Effect of variation of physical environment.
A. Hydrogen and other ions. As shown previously, the induction and
level of excystation is dependent upon pH (Tables 1 and 2, Figures 3 and 4).
Neutral or near-neutral-pH solutions failed to induce excystation regardless
of their complexity (Tables 1 and 2, Figures 3 and 4), with exceptions to be
discussed later. Other than hydrogen, the ions tested did not appear to in-
fluence the degree of excystation (Tables 3 and 4).
B. Time of acid exposure. To examine the influence of acid exposure
time on excystation, cysts were exposed to HC1 at various pH values for
intervals from five minutes to four hours. Five trials were performed
counting an average total of 1449 cysts at each pH-time combination. The
results, shown in Table 5, indicated that: 1) the exposure time required
for excystation is reduced as pH decreases; 2) an optimum range of exposure
times exists at pH values from 0.5 to 6.2, and at any one time an optimum
pH interval can be determined; and 3) the mean optimum exposure time within
this range increases as the pH increases. Additionally, the mean optimum
percentage of excystation at pH 0.5 and 2.0 were not significantly different
from each other, yet were significantly greater than those at 4.0 and 6.2
(p<0.05).
12
-------
TABLE 5. EXCYSTATION OF GIAEDIA BY VARYING pH AND EXPOSURE TIME
Exposure time
PH
0.5
2.0
4.0
6.2
5
*
243
28
0
10 15
256 231
248
1
20 30
__** 127
222 203
5 _28
0
(minutes)
60
38
100
11
3.
90
53
2
8.
Mean optimum1'
120 180 240 exposure time i
20
92 —
i A °
10
20
45
120
Mean optimum „
percent excystation
243
224
26
5
Values represent percentage of excystation with reference to control tube (pH 2.0, 60 minutes)
which was designated arbitrarily as 100 percent. Underlined values represent optimum percentages
of excystation at a given pH which are not significantly different from each other (p>0.05).
fc&
Excystation was not determined at these combinations.
Average of exposure times at which optimum percentages of excystation were not significantly
different from each other at a given pH (p>0.05).
Average of optimum percentages of excystation which are not significantly different from each
other at a given pH (p>0.05).
-------
C. Temperature. The influence of temperature on cysts and excystation
was examined in three aspects: .1) the effect of storage temperature, 2) the
effect of the temperature of the acid incubation solution, and 3) the effect
of the temperature of the post-acid incubation medium (HSP-3).
The effect of storage at -13°, 8°, 21° and 37°C on cyst viability was
assessed periodically as described previously. An overall average of 440
cysts were counted at each temperature on each day tested. Representative
results of these experiments are summarized in Figures 5-8. Cysts stored
at 8 C (Figure 5) had higher levels of excystation for a longer duration
than those stored at the other temperatures. The maximum percentages of
excystation observed at each storage temperature were in the order 8°>21°>
37 >-13°C. Viability at 8°C was observed through day 77 at which time the
cyst suspension was depleted. At 21°C (Figure 6), cyst survival ranged from
5 to 24 days. Cysts stored at 37°C (Figure 7) never survived longer than
four days. Usually the percentage of excystation was greatly reduced, even
after 24 hours, as indicated by an experiment in which excystation was moni-
tored hourly, counting an average of 117 cysts at each temperature for each
hour. In this experiment (Figure 9) the percentage of excystation for cysts
stored ranged from 40 to 60 percent over the 24-hour period; at 37°C, follow-
ing initial peaks of excystation, the percentage of excystation declined to
less than 10 percent after 7 hours, and diminished gradually over the next 18
hours. At -13 C storage (Figure 8) the cysts showed almost a total loss of
viability within 24 hours. Only 0.2% of the cysts survived to day 11 at
this temperature.
An interesting feature of storage at 8° or 21°C was that in all cyst
suspensions obtained from fresh fecal specimens, a period of low excysta-
tion, varying from two to seven days and followed by an increase in excy-
station, was observed.
The effects of the HC1 and HSP-3 incubation temperatures were examined
together as described previously. Five trials were performed, in which an
average total of 379 cysts were counted at each temperature combination.
The results of this experiment (Table 6) showed that excystation is depen-
dent upon both the acid and the HSP-3 incubation temperatures. The highest
TABLE 6. EXCYSTATION OF GIARDIA
Acid temperature
8
21
37
OF HCL (pH 2.0)
8
0
0
0
BY VARYING THE TEMPERATURE
AND HSP-3
HSP-3 temperature*
21
0.5
3.0
15.6
-------
70
60
50
.§ 40
30
20
10
QL /^rtn^-S
V L**! I I I I I I
I \
\0 14 18 22 26 30 34
Storage Time (days)
-A-//
38 42 46 50
77
Figure 5. Effect of storage at 8°C on Giardia cyst viability as determined by excystation. Vertical
bars represent standard error of the mean.
-------
50
40
30
20
10
0
8
10 12 14 16 18 20 22
Storage Time (days)
24 26 28 30 32
Figure 6. Effect of storage at 21 C on Giardia cyst viability as determined by excystation.
Vertical bars represent standard error of the mean.
-------
0
-A_A_A_;
8 10 12 14 16
Storage Time (days)
18
20 22 24
Figure 7. Effect of storage at 37 C on Giardia cyst viability as determined by excystation.
Vertical bars represent standard error of the mean.
-------
Ot>
50 r
o
<3-
o
ro
o
OJ
o
—
OL
A
A - A - A
• III
• 8°C
A -i3°c
J I
-A.
I
46 8 10
Storage Time (days)
12
14
8. Effect of storage at -13 C on Giardia cyst viability as determined by excystation.
Vertical bars represent standard error of the mean.
-------
•—• 8'C
A 37'c
N/V
\
I—I I I I 1 I
J L
0
8 10 12 14 16 18
Storage Time (hours)
20 22 24
Figure 9. Effect of storage at 37 C on Giardia cyst viability as determined by excystation
over a 24-hour period.
-------
levels of excystation occurred when the temperature of .both solutions was
37°C. Lower levels of excystation occurred with other temperature combina-
tions. The percentage of excystation increased as the temperature of the
acid, the HSP-3, or both was increased; the exception was that no excysta-
tion was induced when the temperature of the HSP-3 was held at 8 C, regard-
less of the temperature of the HC1. No change in the excystation percentages
was noted after 18 hours incubation in HSP-3.
D. Post-acid incubation medium. The effect of the composition of the
post-acid incubation medium on excystation was investigated by exposing
cysts to HC1 at pH 2.0 by the usual procedure,followed by their suspension
in solutions of varying complexity and pH. Three excystation trials were
performed counting an average total of 834 cysts in each solution. Signi-
ficant levels of excystation were induced only in HSP-3 at pH 6.2 and 6.8
(Table 7). Further, excystation was significantly greater in medium at
pH 6.8 than at pH 6.2 (p<0.05).
TABLE 7. EXCYSTATION OF GIARDIA EXPOSED TO HC1 (pH 2.0) AND TRANSFERRED
INTO SOLUTIONS OF VARYING COMPLEXITY AND pH
Post-acid
Solution
Water
HC1
Saline (0.85 M)
PBS
HSP-3
PH
7.0
2.0
7.0
7.0
0.5
2.0
4.0
6.2
6.8
Percent
Excystation
0.3
0.8
0
1.1
0
0
0
63.3
100.0
Percentages of excystation with reference to the control tube (pH 6.8
HSP-3) which is arbitrarily 100 percent.
The experiment was repeated as before, except that cysts were trans-
ferred into either 1) Hanks-phytone, 2) Hanks-phytone with serum, or
3) HSP-3 (Hanks-phytone with serum and NCTC-135), all at pH 7.0. Three
trials were performed in which an average total of 1205 cysts were counted
in each medium. It was found (Table 8) that the levels of excystation in
HSP-3 and Hanks-phytone with serum, while not significantly different from
each other (p>0.05), were significantly greater than excystation in Hanks-
phytone alone (p<0.05).
20
-------
TABLE 8. EXCYSTATION OF GIARDIA EXPOSED TO HC1 (pH 2.0) AND TRANSFERRED
INTO COMPONENT-VARIED HSP-3 AT pH 7.0
Mean * percent
excystation
Solution ± S.E.M.
Hanks-phytone 13.1 ± 1.4
Hanks-phytone + serum 42.7 ± 3.1
HSP-3 50.9 ± 4.0
*
Means are derived from an average total of 1205 cysts counted in each
solution.
IV. Cyst variation^
A. Maturation, daily variation and viability. Throughout this study,
inconsistent results were sometimes found when a single purified cyst pre-
paration was used over a period of days or weeks. These results appeared to
be the result of cyst variability due to aging. To study this phenomenon,
purified cyst suspensions were prepared from different fecal specimens from
the same host, stored at 8 C, and excystation in pH 2.0 HC1 followed in each
suspension over a period of weeks. Figure 10 is the excystation pattern
for a single suspension of purified cysts over 11 weeks, and is representa-
tive of patterns obtained with other samples. Three excystation trials
were performed daily; an average total of 451 cysts were counted each day.
This pattern has three important features. First, as noted previously,
cyst storage began with a four day "lag" period of low excystation, after
which an increase in excystation percentages was seen. Secondly, following
the lag period, high levels of excystation were seen until about day 30
during which period extreme fluctuations in excystation occurred almost
daily. Thirdly, following the period of elevated excystation, these levels
decreased rapidly for approximately a week, and then gradually diminished
during the next six weeks, indicating a loss of viability. Although the
specific pattern varied greatly, the following characteristics were observed
in most suspensions with low-temperature storage. A lag period of one to
seven days, followed by an increase in excystation over three days, preceded
a one to four week period of elevated but fluctuating excystation; this was
followed by a drop in excystation and an extended period (usually several
weeks) of gradually diminishing low levels of excystation.
B. Variation in sensitivity to physical environment. Cysts were
examined to determine if sensitivity to the physical environment changes
during aging. Purified cysts stored for 47 days at 8 C were excysted
periodically using pH 2.0 HC1 at various exposure times up to 120 minutes.
Three trials were performed counting an average total of 822 cysts at each
temperature for each day tested. The results are summarized in Table 9.
As the cysts aged, the mean optimum acid exposure time increased from 20 to
70 minutes. From days 34 to 47 the mean optimum time remained at 70 minutes,
21
-------
to
10 14
I t
18 22 26 30 34 38 42 46
Storage Time (days)
Figure 10. Giardia excystation pattern for cysts stored at 8°C for an 11-week period.
-------
TABLE 9. OPTIMUM TIME OF ACID EXPOSURE FOR GIARDIA EXCYSTATION
WITH AGING OF CYSTS
Day
1
5
9
13
16
19
23
34
47
Mean optimum
exposure time
(min)
20
20
35
20
20
35
40
70
70
Range
(min)
20
20
10 -
10 -
10 -
10 -
20 -
20 -
20 -
60
30
30
60
60
120
120
Mean optimum
percent excystation
18.1 ±
20.0 ±
8.5 ±
11.4 ±
8.4 ±
7.4 ±
6.5 ±
3.0 ±
1.9 ±
0.0
0.0
0.6
0.4
0.7
0.4
0.6
0.4
0.2
*
Average of exposure times at which optimum percentages of excystation
were not significantly different from each other on a particular day
(p>0.05).
Average of optimum percentages of excystation which are not signifi-
cantly different from each other on a particular day (p>0.05).
although it may have proved longer if an acid exposure time of more than
120 minutes had been tried. The minimal optimum exposure time (see Range,
Table 9) did not change much during the storage period but remained between
10 and 20 minutes, whereas the maximal optimum exposure time increased from
20 to 120 minutes, resulting in the increasing mean optimum exposure time.
Additionally, the mean optimum percentage of excystation showed a similar
pattern as observed in previous storage experiments.
V. Culturing.
Using the procedure described previously with pH 2.0 HC1, excystation
was induced in Giardia obtained from infected humans, monkeys, dogs, beavers,
rats and mice, and cultures were established from excysted trophozoites of
cysts from humans and monkeys. These cultures were maintained for up to
seven months in HSP-3, and most were cultivated axenically from the time of
excystation.
VI. Other reports based on this research.
Additional reports, based on the research conducted under this grant,
include the following:
23
-------
Bingham, A.K. and E.A. Meyer. The in vitro excystation of Giardia.
Fourth International Congress of Parasitology (abstract), pp. 95-96,
1978.
Jarroll, E.L., Jr., A.K. Bingham, E.A. Meyer and S. Radulescu. Effect
of temperature on Giardia cyst survival as determined by eosin
staining and excystation. Fourth International Congress of Parasit-
ology (abstract), p. 96, 1978.
Bingham, A.K., E.L. Jarroll, Jr., S. Radulescu and E.A. Meyer.
Induction of Giardia excystation and the effect of temperature on
cyst viability as compared by eosin-exclusion and in vitro excyst-
ation. Symposium: Waterborne transmission of giardiasis (in press),
(U.S. Environmental Protection Agency, 1979).
Bingham, A.K. and E.A. Meyer. Giardia excystation can be induced
in vitro in acidic solutions. Nature, 277:301-302, 1979.
Bingham, A.K., E.L. Jarroll, Jr., E.A. Meyer and S. Radulescu.
Giardia sp.: Physical factors of excystation in vitro and excyst-
ation vs. eosin-exclusion as determinants of viability.
Exp. Parasitol. (in press), 1979.
24
-------
REFERENCES
1. Roberts-Thomson, I.C., D.P. Stevens, A.A.F. Mahmoud, and K.S. Warren.
Giardiasis in the mouse: an animal model. Gastroenterology 71(1):
57-61, 1976.
2. Sheffield, H.G., and B. Bjorvatn. Ultrastructure of the cyst of
Giardia lamblia. Am. J. Trop. Med. Hyg. 26(1):23-30, 1977.
3. Meyer, E.A. Giardia lamblia: Isolation and axenic cultivation. Exp.
Parasitol. 39:101-105, 1976.
4. Hirschowitz, B.I. Secretion of pepsinogen. In Handbook of Physiology:
Alimentary Canal (Heidel, W. and C.F. Code, eds.) Section 6, Vol. II,
897, (American Physiological Society, Washington, 1967).
5. Konturek, S.J. Gastric secretion. In MTP International Review of
Science: Gastrointestinal Physiology (Guyton, A.C., E.D. Jacobsen, and
L.L. Shanbour, eds.) Physiology Series One, Vol. 4, 227-243, (University
Park Press, Baltimore, 1974).
6. Danciger, M., and E.A. Meyer. The in vitro growth response of Giardia
trophozoites from the rabbit. J. Protozool. 18(2):311-313, 1971.
25
-------
BIBLIOGRAPHY
Ament, M.E., and C.E. Rubin. Relation of giardiasis to abnormal intestinal
structure and function in gastrointestinal immunodeficiency syndromes.
Gastroenterology 62(2):216-226, 1972.
Armaghan, V. Biological studies on the Giardia of rats. Amer. J. Hyg.
26:236-257, 1937.
Barbour, A.G., C.R. Nichols, and T. Fukushima. An outbreak of giardiasis
in a group of campers. Amer. J. Trop. Med. Hyg. 25:384-389, 1976.
Brodsky, R.E., H.C. Spencer, and M.G. Schultz. Giardiasis in American
travelers to the Soviet Union. J. Infect. Dis. 130(3):319-323, 1974.
Center for Disease Control: Morbidity and Mortality Weekly Report.
Giardiasis - in residents of Rome, New York, and in U.S. travelers to
the Soviet Union. 24:366-371, 1975.
Center for Disease Control: Morbidity and Mortality Weekly Report. Water-
borne giardiasis outbreaks - Washington, New Hampshire. 26(21):169-175,
1977.
Cowen, A.E., and C.B. Campbell. Giardiasis - a cause of vitamin B}2 malab-
sorption. Digest. Dis. 18 (5):384-390, 1973.
Craun, G.F. Waterborne outbreaks. J. Water Poll. Control Fed. 99(6):
1268-1279, 1977.
Deschiens, R. Chimisme gastrique et infections parasitaires du tube
digestif. Ann. Inst. Pasteur, Paris. 43:1353-1369, 1929.
Dobell, C. The discovery of the intestinal protozoa of man. Proc, R, Soc,
Med. 13(3):1-15, 1920.
Hegner, R. Excystation and infection in the rat with Giardia lamblia
from man. Amer. J. Hyg. 7:433-447, 1927.
Hegner, R. Excystation jLn vitro of human intestinal protozoa. Science
65:577-578, 1927.
Hegner, R. The viability of cysts of Giardia lamblia from man in the
stomach of the rat. Amer. J. Hyg. 7:782-785, 1927.
26
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Hoskins, L.C., S.J. Winawer, S.A. Broltman, L.S. Gottlieb, and N. Zamcheck.
Clinical giardiasis and intestinal malabsorption. Gastroenterology
53(2):265-279, 1967.
Lackie, A.M. The activation of infective stages of endoparasites of verte-
brates. Biol. Rev. 50:285-323, 1975.
Lucian, 0. Lambliaza. pp. 1-248. (Editura Academiei Republicii Socialiste,
Bucharest, Romania, 1971).
Moore, G.T., W.M. Cross, D. McGuire, C.S. Mollahan, N.N. Gleason, G.R.
Healy, and L.H. Newton. Epidemic giardiasis at a ski resort. N. Engl.
J. Med. 281:402-407, 1969.
Petersen, H. Giardiasis (Lambliasis). Scand. J. Gastroenterology 7
(Supp. 14):l-44, 1972.
Shaw, P.K., R.E. Brodsky, D.O. Lyman, B.T. Wood, C.P. Hibler, G.R. Healy,
K.I.E. McLeod, W. Stahl, and M.G. Schultz. A communitywide outbreak
of giardiasis with evidence of transmission by a municipal water supply.
Ann. Int. Med. 87(4):426-432, 1977.
Veazie, L. Epidemic giardiasis. N. Engl. J. Med. 281:853, 1969.
Wright, R.A., and T.M. Vernon. Epidemic giardiasis at a resort lodge.
Rocky Mtn. Med. J. 73:208-211, 1976.
27
-------
TECHNICAL REPORT DATA
(I'lcasc read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/2-79-063
3. RECIPIENT'S ACCESSION-NO.
4. TITLE AND SUBTITLE
DETERMINATION OF GIARDIA CYST VIABILITY
5. REPORT DATE
July 1979 (Issuing Date)
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
8. PERFORMING ORGANIZATION REPORT NO.
Ernest A. Meyer
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Department of Microbiology and Immunology
University of Oregon Health Sciences Center
3181 S.W. Sam Jackson Park Road
Portland, Oregon 97201
10. PROGRAM ELEMENT NO.
1CC824, SOS 2, Task 12
11. CONTRACT/GRANT NO.
R-804898
12. SPONSORING AGENCY NAME AND ADDRESS
Municipal Environmental Research Laboratory—Gin. ,OH
Office of Research & Development
U. S. Environmental Protection Agency
Cincinnati, OH 45268
13. TYPE OF REPORT AND PERIOD COVERED
Final 10/76-1/79
14. SPONSORING AGENCY CODE
EPA/600/14
15. SUPPLEMENTARY NOTES
Project Officer: J. C. Hoff (513) 684-7331. See P. 24 of the report for
other published material based on this research.
16. ABSTRACT
The principal objective of this research was the development of a
sensitive, standardized method of determining whether or not Giardia
cysts are viable. The availability of such a method is necessary to
determine the effect of chemical and physical agents on the viability of
these organisms in water. Using cysts collected from an asymptomatic
human carrier, a method for inducing excystation was developed and
optimized with regard to pH, time of acid exposure, incubation temperature
and excystation medium. The method was applied in determining the effects
of cyst storage in water at various temperatures on cyst survival as
determined by the cultural excystation method and eosin dye exclusion.
The results indicate that cyst survival time decreased with increasing
temperatures above 0 C. Freezing and thawing resulted in almost complete
loss of viability.
7.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS
COSATI Held/Group
Giardia, Protozoa, Viability, Cysts,
Resistance, Potable Water, Water Supply
Giardia lamblia
6 C
6 F
57 N
18. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (ThisReport)
UNCLASSIFIED
21. NO. OF PAGES
36
20. SECURITY CLASS (Thispage)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (9-73)
28
> US. GOViRNMENI PRINTING OFFICE 1979-657-060/5427
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