Document text
AD-A262 226
AD
TECHNICAL REPORT 9205
*
EVALUATION OF THE MILITARY EFFECTIVENESS OF CHLOR-FLOC
WATER PURIFICATION TABLETS FOR TREATMENT OF
WATERBORNE MICRO-ORGANISMS
Stephen A, Schaub
Helen T. Hargett
Kurt I. Kamrud
and
Charles R. Sterling *
Marilyn M. Marshall *
OCT 1992
U S ARMY BIOMEDICAL RESEARCH & DEVELOPMENT LABORATORY
Fort Dotiick
Frodofick, MD 21702-5010
♦DEPARTMENT OF VETERINARY SCIENCE
UNIVERSITY OF ARIZONA
TUCSON, AZ 85721
Approved for public release;
distribution unlimited.
dg > 6 056
93-06400
U 8 ARMY MEDICAL RESEARCH A OEVELOPMEN T COMMAND
Fort Dotrick
FYodorICk, MD 21702-5012
ECurity classification of this page
REPORT DOCUMENTATION PAGE
Form Approved
OMBNO 0704-0188
1» REPORT SECURITY CLASSIFICATION
iiMrt AccTrTfn
2* SECURITY CLASSIFICATION AUTHORITY
2b declassification /DOWNGRADING SCHEDULE
4 PERFORMING organization REPORT NUMBER($)
lb RESTRICTIVE MARKINGS
3 OlSTfliBUTlON/AVAILABiLITY OF REPORT
APPROVED FOR PUBLIC RELEASE;
UNLIMITED DISTRIBUTION
5 MONITORING ORGANIZATION REPORT NUMBER<S)
. 9205 _
6a. NAME OF PERFORMING ORGANIZATION
U.S. ARMY BIOMEDICAL RESEARCH
AND DEVELOPMENT ^LABORATORY _
6c. ADDRESS {City, Start, end ZIP Code)
FORT DETRICK
FREDERICK, MD 21702-5010
8a. NAME OF FUNDiNC/SPONSORiNG 8b. OFFICE SVM6
ORGANIZATION ,j _ 5 ^ MEDICAL
RESEARCH & DEVELOPMENT COMMAND _
8c ADDRESS (Oty. State, end ZIP Code)
6b OFFICE SYMBOL 7a NAME OF MONITORING ORGANIZATION
{rt eppikeble)
SGRD-UBG-0 _ _
7b ADDRESS (Oty. Start, end ZIP Code)
8b. OFFICE SYMBOL I 9 PROCUREMENT INSTRUMENT IDENTIFICATION NUMBER
10 SOURCE OF FUNDING NUMBERS
PROJEa
NO A878
3M162787
WORK UNIT
ACCESSION NO
FORT DETRICK
1C. MD 21702-5012 _ _
1 1 . title (include Security CletsJficetion)
(U) EVALUATION OF THE MILITARY EFFECTIVENESS OF CHLOR-FLOC WATER PURIFICATION TABLETS
FOR TREATMENT OF WATERBORNE MICRO-ORGANISMS _ .
12. PERSONAL AUTHOR(S)
haub, Helen T. Hargett. Kurt I. Kamrud, Charles R. Sterling & Marilyn M. Marsha
I3b TIME COVERED 114 DATE OF REPORT {Year, Montfi, Oey) |15 PAGE COUNT
FROM ifiRQ TO OQqi I 1992 OCT I 58 _
16. supplementary notation
18- SU8J6CT TERMS {Continue on re^crw if ntcfsssry snd khntif/ by btock nufnbor)
Water, purification, CHLOR-FLOC tablets, micro-organisms,
Cryptosporidium, Klebsiella, echovirus, latex beads,
protozoan cysts, bacteria, disinfection, coagulation
17. COSATi CODES
GROUP Sue-GROUP
09
04
«at«r purification tablets for
treating microbiological contaminants In drinking water. The test waters represented
various physical/che:nical challenge conditions. Microbial challenges consisted of enteric
bactc’ia (Klebsiella terrigena) , enterovirus (Echovirus 1), protozoan oocysts (Crypto-
sporldium parvum) , and cyst simulant (latex beads). Studies used the U.S. Environmental
Protection Agency's interim "Guide Standard and Protocol for Testing Microbiological Water
Purifiers" for guidance. Results indicated that the CHLOR-FLOC system effectively reduced
both bacterial and viral challenge components of the study to the required levels under all
conditions, but failed to physically remove the oocysts and cyst simulant to the required
•levels under all conditions. Viability studies also indicated that the chemical disinfectior
component of the CHLOR-FLOC tablets had minimal capabilities to kill the encysted organisms
over the 20— minute contact time. The overall study results revealed that the CHLOR— ILOC
^system vas not adequate to physically remove, or to provide adequate chemical disinfection
of, Cryptosporidium oocysts to the required level of 99.9 percent reduction.
ZO OISTR.aUTlON 'AVAILABILITY OF ABSTRACT jZ1 ABSTRACT SECURITY CLASSIFICATION
CSuNClASSlFIEOrtJNLIMlTEO □ SAME AS RPT □ DTlC USERS ! _ _ _
lie. NAME OF responsible INDIVIDUAL fZZb TELEPHONE (Include Arte Code) 22c. OFFICE SYMBOL
STEPHEN A. SCHAUB I (301) 619-2624 SGRD-UBG-0
)0 Form 147?, JUR< 86
Prevlout editions ere obsolete
The findings in this report are not to be construed as an
official Department of the Army position unless so designated by
other authorized documents. Citations of commercial
organizations or trade names in this report do not constitute an
official Department of the Army endorsement or approval of the
products or services of these organizations. Research was
conducted in compliance with the Animal Welfare Act, and other
Federal statues and regulations relating to animals and
experiments involving animals and adheres to principles stated in
the Guide Ific Care and Sil Laboratory NIH
publication 86-23, 1985 edition.
Disposition
Destroy this report when it is no longer needed. Do nor.
return it to the originator.
LTIC Q
"■•rrHD t
• AcCfcsIon for
NTIS CHA&I
one TAB □
Unannounced Q
jiisiitication _ _ _
By _ _ _
QiStribuilon/
- -
Availability Code*
Avail and (or
OKI Special
TABLE OF CONTENTS
LIST OF TABLES . ii
LIST OF FIGURES . iii
PREFACE . iv
SUMMARY . V
INTRODUCTION . 1
USABRDL IN-HOUSE STUDIES MATERIALS AND METHODS . 3
UA COLLABORATIVE STUDIES MATERIALS AND METHODS . 12
RESULTS . 19
DISCUSSION . 42
CONCLUSIONS . 45
APPENDICES . 46
REFERENCES . 48
DISTRIBUTION . 50
1
LIST OF TABLES
1. Microbiological Challenges and Removal Endpoints . 3
2. Water Quality Challenge Conditions . 5
3. Klebsiella terriaena Disinfection with Two
CHLOR-FLOC Tablets at 5 ‘C . 21
4. Klebsiella terriaena Disinfection with One
CHLOR-FLOC Tablet at 10 ‘C . 22
5. Echovirus Disinfection with Two CHLOR-FLOC
Tablets at S *C . 24
6. Echovirus Disinfection with One CHLOR-FLOC
Tablet at 10 *C.... . 25
7. Disinfection of Klebsiella terriaena and Echovirus
with One CHLOR-FLOC Tablet at 10 *C with pH
Adjusted to Maintain 9.0 . 27
8. Cryptosporidium parvum Oocyst Removal with Two
CHLOR-FLOC Tablets at 5 ‘C . 28
9. Cryptosporidium parvum Oocyst Removal with One
CHLOR-FLOC Tablet at 10 ‘C . 29
10. Protozoan Simulant Removal with Two CHLOR-FLOC
Tablets at 5 *C . 31
11. Protozoan Simulant Removal with One CHLOR-FLOC
Tablet at 10 *c . 32
12. Reduction of Cryptosporidium Oocysts and Protozoan
Simulant with One CHLOR-FLOC Tablet at 10 ’C
with pH Adjusted to Maintain 9.0 . 34
13. Inf activity of Positive Controls . 3 5
14. Infection of Mice Receiving CHLOR-FLOC or
Globa line Treated Oocysts . 37
15. Positive Control Data . 39
16. CHLOR-FLOC Inf activity Data for Phase II . 40
17. Recovery and Viability of CHLOR-FLOC Treated Oocysts... 41
ii
LIST OF FIGURES
1. CHLOR-FLOC Water Purification System Components . 8
2. Flocculation/Coagulation with Two CHLOR-FLOC Tablets
in Tapwater and wc Water . 8
3. CHLOR-FLOC Treated Waters at 5 ‘C
a . Tapwater . 9
b. Worst Case Water . . . 9
4. New CHLOR-FLOC System with Worst Case 5 *C Water
a. Five Minutes After Treatment . 14
b. Settled Flocculated Material 20 Minutes
After Treatment . 15
c. Filtration/Collection of Treated Water After
Settled Materials Were Discarded . 16
5. Klebsiella terriaena Disinfection with CHLOR-FLOC . 2 3
6. Echovirus Disinfection with CHLOR-FLOC . 26
7. Cryptosporidium parvuro Oocyst Physical Removals
with CHLOR-FLOC . . . 30
8. Protozoan simulant (Latex Beads) Removals with
CHLOR-FLOC . 3 3
»
iii
PREFACE
The U.s, Army is Interested in alternative drinking water
disinfectants for treatments of individual soldier water supplies
in the field as a replacement for the current iodine (Global ine)
tablets. The use of iodine tablets is adversely impacted by
reduced disinfection efficiency at low temperature and pH,
especially for the enteroviruses, and at high pH for certain
protozoan cysts such as Giardia and Cryptosporidium. Also,
because of limitations in disinfection effectiveness, newer
military doctrine requires two iodine tablets per liter of
drinking water with a contact time of 35 minutes before human
consumption. The use of two tablets contributes significant
adverse organoleptic (taste and odor) properties to the treated
water. These problems and the potential avoidance of water can
be a serious concern where personnel may be required to consume
up to 15 liters a day to prevent dehydration (especially in hot,
arid climates) . A new commercially available disinfectant-
flocculating agent, CHLOR-FLOC, may provide improved water
purification over a broad range of pH and temperature, and may
not suffer from interferences by organic chemical demand in field
water supplies.
iv
SUMMARY
The U.S. Army is interested in innovative methods for the
microbiological purification of drinking water from highly
contaminated sources. This study evaluates the efficacy of
CHLOR-FLOC tablets for treating microbiological contaminants
(bacteria, enteroviruses, and protozoan cysts) utilizing the U.S.
Environmental Protection Agency's (USEPA) interim "Guide Standard
and Protocol for Testing Microbiological Water Purifiers" as
guidance in testing.
The tests, to determine the efficacy of CHLOR-FLOC for water
purification, examined various physical/chemical challenge
conditions, incorporating temperatures of 5 and 10 *C, pH of 4.5,
7.0, and 9.0, and general water qualities imparted by
distilled-deionized halogen demand free, tap, and complex
synthetic waters. The microbial challe-^ges consisted of
Klebsiella teiriaena bacteria, Echovirus 1, Cryptosporidium
parvum oocysts, and latex beads (a protozoan cyst simulant) .
CHLOR-FLOC purification of challenge waters was provided for
a period of 20 minutes, using two tablets for the 5 *C studies
and one tablet for the 10 'C experiments. At the end of the
disinfection-flocculation period, the samples were filtered
through flannelette bags, treated with sodium thiosulfate to
neutralize the chlorine, and then assayed for surviving
micro-organisms. Suggested minimal microbial removal
requirements to be considered effective for military use are: 6
logs for enteric bacteria; 4 logs for enteric viruses; and 3 logs
for protozoan cysts (or simulant) over the prescribed contact
period.
The study demonstrated that the USEPA guide standard was
appropriate for use in determining the effectiveness of the
CHLOR-FLOC disinfectant for the removal of typical waterborne
microbial indicators and pathogens at low temperatures. The
results indicated that both bacterial and viral challenge
components of the study were effectively reduced to the required
levels under all conditions. This study also showed that
physical removals (coagulation and filtration) of the protozoan
oocysts and latex beads (cysts simulant) were essentially
equivalent; hov/ever, their physical removal alone was not
adequate to meet the military removal requirements. Experiments
examining the effectiveness of CHLOR-FLOC 's disinfection
component for viability of Cryptosporidium oocysts showed that
the disinfectant had very minimal capabilities to kill the
encystfjd organisms over the 20-minute contact time.
INTRODUCTION
The U.S. Army Biomedical Research and Development Laboratory
(USABRDL) conducted low temperature efficacy studies on the
disinfectant tablet CHLOR-FLOC, for the disinfection and physical
removal of typical waterborne enteric microorganisms (bacteria,
virus, and protozoan cysts), to determine if CHLOR-FLOC meets
► military requirements for individual soldier water purification.
CHLOR-FLOC contains a disinfectant, sodium dichloro-s-
triazinetrione dihydrate, at a concentration of 2.5 percent; and
it also contains a complex coagulant mixture to clarify the water
of turbidity, larger micro-organisms, and some organic chemicals
such as humic and fulvic acids. The components of this complex
coagulating mixture are: aluminum sulfate, sodium carbonate,
bentonite, sodium carboxy-methylcellulose, Syloid-244, and
Superfloe (a polyacrylamide) . After coagulation, an important
step in the CHLOR-FLOC treatment process is final filtration
through a tightly-woven flannelette filter bag, after the
disinfection process is complete, to remove flocculated materials
which may contain some residual microbiological population.
While CHLOR-FLOC has been approved by the U.S. Environmental
Protection Agency (USEPA)^ especially for recreational and
emergency uses at higher water temperatures (e.g., 20 *C) , few
studies have been conducted at low temperatures. No studies have
been conducted under the USEPA's new interim "Guide Standard and
Protocol for Testing Microbiological Water Purifiers"^ (hereafter
referred to as the guide standard and protocol) , which, in the
future, may be mandated for the marketing of point of use
microbiological water purifiers in the United States. Because
the military requires microbiological purification of waters of
wide diversity and quality at all temperatures, the following
study was performed to assess CHLOR-FLOC in typical-use
scenarios. The study conditions imposed may not fully account
for the very worst quality waters which the individual may be
required to treat and consume on a worldwide basis, but should
provide a representative challenge for most of the
microbiological contaminant levels expected in the more typical
water resources.
Collaborative research efforts between the USABRDL and the
University of Arizona (UA) , Department of Veterinary Science,
Tucson, AZ, were also conducted to determine the relative
effectiveness of CHLOR-FLOC and Army issue Globaline (iodine)
tablets for disinfection of Cryptosporidium parvum oocysts,
utilizing animal infectivity as the measure of effectiveness.
These studies were necessary when it became apparent from USABRDL
in-house efforts that the oocysts were not effectively removed
physically by the CHLOR-FLOC filtration component of the
' treatment process. Studies were conducted in two phases: phase I
- evaluation of the viability of oocysts solely by disinfection
1
using CHLOR-FLOC (without filtration) with comparative tests
using Globaline tablets for disinfection; phase II - evaluation
of the residual infectivity of oocysts after disinfection and
subsequent filtration through flannelette material using
CHLOR-FLOC. (The flannelette bag material was provided by the
U.S. Army Natic)c Research, Development and Engineering Center (a
prototype material developed by their laboratory).] All
CHLOR-FLOC operational and testing procedures were identical to
those used during the USABRDL in-house tests except that the
strain of Cryptosporidium oocysts used was that provided by the
UA.
2
USABRDL IN-HOUSE STUDIES MATERIALS AND METHODS
1. MICROBIOLOGICAL CHALLENGE
The CHLOR-FLOC disinfection studies utilized the USEPA's
guide standard and protocol for testing guidance. However, the
selection of the microbiological challenges was somewhat
different than those indicated in that document. Table 1 shows
the waterborne microbiological challenges and their removal
endpoint requirements. It should be noted that the challenge for
viruses is limited to a single virus, Echovirus l, and excluded
rotavirus (which was necessary because of limitations in the time
and availability of the appropriate cell cultures) . Also, the
Giardia sp. cyst challenge was replaced in the test protocol by
Cryptospor idium oarvum oocysts because it was felt that the
Cryptosporidium represented a worse challenge to the CHLOR-FLOC
by their smaller size and supposed resistance to common water
disinfectants. Also, military exposures to Cryptosporidium are
thought to be of worldwide significance.
TABLE 1. MICROBIOLOGICAL CHALLENGES AND REMOVAL ENDPOINTS
Test Orqanisms
Challenge
Levels/Liter
Minimum Removal
in Loq (%)
Bacteria - Klebsiella terrigena
(overnight culture)
10®
CFU
6
(99.9999)
Enteric virus - Echovirus 1
10*^
PFU
4
(99.99)
Protozoan cvsts - Crvotosooridium
PAJLYMII!
10®
cysts
3
(99.9)
Latex bead cyst simulant - 3.7 ^m
AccuBsads"*
lo"^
beads
3
(99.9)
a. Klebsiella terrioena bacterium preparation
Klebsiella terriaena (#33257) was obtained from the American
Type Culture Collection (Rockville, MD) , grown in nutrient broth,
and frozen at -70 'C in 1.0 ml volumes for the testing seed
stock. To prepare seed inoculum for each test day, Klebsiella
was grown overnight in nutrient broth and centrifuged at 8U00 rpm
for 10 minutes using a Sorvall GSA rotor. The pelleted bacteria
were resuspended and washed three times in demand-free phosphate
buffered saline (DFPBS) , and then filtered through a Whatman # 2
filter pad to remove bacterial clumps. The filtered Klebsiella
cells were diluted in DFPBS and adjusted to a scale reading of 35
us'ng a Klett-Summerson colorimeter. A 1 ml volume of this
fru. oension was added to each liter of test water to provide a
cha lenge of approximately 1.0 X 10^ CFU/L.
3
b. Echovirus l test preparation, purification and
g.eparatjgn
Echovirus 1, V239 strain, (obtained from Dr. Mark Sobsey,
University of North Carolina) was used to prepare the challenge
virus. A 150 cm^ monolayer of confluent BGMK cells (Whittaker
Bioproducts, Walkersville, MD) was inoculated with 0.5 ml of
stock seed virus at a multiplicity of infection (MOI) of 10
plaque forming units per cell (PFU/cell) . After incubation for 1
hour at 36 *C, 50 ml of Earles Minimum Essential Medium (MEM)
containing 2 percent fetal calf serum (FCS) was added to the
infected cells. When total cytopathic effect (CPE) with minimal
cell detachment was observed, the liquid was collected and
centrifuged at 10,000 X g for 30 minutes. The supernatant fluid
was discarded, and the pelleted cells were saved. Twenty ml of
DFPBS, pH 7.2, was added to the flask; and the attached cells
were removed from the surface with a cell scraper and combined
with the pelleted cells from above. The BGMK cell suspension was
"freeze-thawed" three times in an ethanol-dry ice bath to release
the virus and then extracted three times in a Waring blender for
1 minute with 1, 1, 2-trichlorotrif luoroethane at a ratio of 4 ml
per 6 ml of virus suspension. After each extraction, layers of
the mixture were separated by centrifugation at 800 X g for 10
minutes in a refrigerated Sorvall centrifuge, and the aqueous
phases were collected and pooled. The aqueous volume containing
the virus was concentrated to a final volume of 10 ml with an
Amicon Centriprep" concentrator (30,000 MW cutoff) by
centrifugation at 1500 X g in an lEC refrigerated centrifuge.
The virus particles were separated by rate-zonal centrifugation,^
using 10 to 30 percent sucrose gradients prepared in phosphate
buffered demand-free water (0.05 M PO^ buffer, pH 7.2), at 90,000
g in a Beckman SW28 rotor for 2 hours and 15 minutes. The
gradient was collected in 2 ml fractions and assayed on BGMK
cells by the plaque assay titration method. Fractions F-9
through F-14 and F-20 were combined to give a proportional number
of small, medium and large aggregates. This combination yielded
3.15 X 10^ PFU/ml when assayed on BGMK cells.
c. Cryptosporidium parvum oocvst preparation
Calf feces (50 percent in 2.5 percent potassium dichromate)
containing Cryptosporidium parvum oocysts was obtained from the
University of Idaho, Dept, of Veterinary Science, Caldwell, ID,
and purified using the method of E. Waldman si The calf
feces suspension was dispensed into 50 ml polypropylene conical
centrifuge tubes in 10 ml volumes, and an equal volume of pH 7.0
PBS containing 0.1 percent Tween 20 was added to each tube. The
contents in the tubes were thoroughly mixed and centrifuged at
750 X g for 15 minutes. The liquid portions were discarded, and
the pellets were resuspended in 15 ml PBS with Tween 20.
Anhydrous ether (5.0 ml) was added and mixed with each of the
suspensions for 1 minute. The tubes were then centrifuged at 500
4
X g for 10 minutes. The top three layers (ether, debris plug and
PBS with Tween 20) were removed and discarded. The pelleted
cysts were resuspended in 10 ml of PBS and combined; then they
were Cwsntrifuged again. A small volume of the liquid was
retained in the tube to resuspend the pelleted cysts. Cysts were
further purified by the Percoll** (Sigma # P-1644) discontinuous
density gradient method. The Percoll* was diluted in 0.15 M NaCl
to densities of 1.04 and 1.08. The gradients were prepared in 10
ml centrifuge tubes by layering 3 ml of the 1.04 density on top
of 3 ml of the 1.08 density Percoll.** A volume of 0.5 ml of the
oocysts was layered on the surface of each gradient in the
centrifuge tube, and the tubes were centrifuged at 250 X g for 10
minutes at oom temperature. The upper bands, lower bands, and
pellets were collected separately from the tubes, diluted in 10
volumes of PBS, and then centrifuged at 500 X g for 10 minutes.
The PDS-Percoll* fluids were discarded; then the pellets were
resuspended in a small volume of PBS with 0.01 percent Tween 20
and examined microscopically for oocysts. The oocysts, which
were concentrated in the resuspended pellet from the lower bands,
were diluted in PBS with 0.01 percent Tween 20 to contain
approximately 1.66 X 10^ oocysts/ml. Oocysts were added to each
test beaker to furnish 1. 5-2.0 X 10°/ 1.
d. Latex _bead (protozoan simulant) preparc.tion
A suspension of AccuBead”* particles (latex beads) with a
mean diameter si/e of 3.7 fjr. (geometric standard deviation =
1.03), was obtained from FASTEK (A Kodak Company, Liverpool, NY).
The latex beads were prepared in sterile deionized-distilled
water (dd H2O) containing 50 f*g/ml of sodium dodecyl sulfate
(added to reduce bead clumping caused by electrostatic
attraction) , The beads were added to the l~liter test beakers of
water to provide a final concentration of 1. 5-2.0 X 10^ beads/ 1.
2. PHYSICAL,/ CHEMICAL WATER CHARACTERISTICS AND TEST PROCEDURES
The tests to determine the effectiveness of CHLOR-FLOC
exan ined a range of physical/chemical challenge conditions in
various disinfectant free waters (Table 2) .
TABLE 2. WATER QUALITY CHALLENGE CONDITIONS
Water
Temp( ’C)
pH
Turbidity
NTU
TDS
mg/1
TOC
mq/1
DF^
5 & 10
5.0,
7.0,
9.0
<5.0
50-500
<5.0
Tap2
5 & 10
5.0,
7.0,
9.0
<5.0
50-500
<5.0
WC^
5 4 10
5.0,
7.0,
9.0
>30.0
>1500
>10.0
tapwater
worst case water
5
Halogen demand^f ree water (HDFW) was prepared by addition of
10 mg/l chlorine to dd H2O. After storage in the dark at room
temperature for 24 hours » the water was dechlorinated by exposure
to sunlight. Buffer solutions used to adjust the pH of the test
waters were prepared in HDFW. All glassware items for these
tests were washed, cleaned in a solution of sulfuric acid
containing No-Chromix,* washed again in detergent, rinsed with
distilled water, soaked overnight In a 20 mg/l chlorine solution,
and rinsed in HDFW. The glassware was sterilized in a dry>heat
oven at 150 ‘C for 90 minutes.
b. Test waters
Water characteristics, pH and temperature were adjusted for
each run according to each set of conditions being tested. For
each run, four beakers were filled with 1 liter of challenge
water. All challenge organisms were added to three of the
beakers (two for replicate CHLOR-FLOC disinfectant challenge and
one control to determine viability of all challenge organisms
over the test period) ; and the fourth beaker of water served as a
control to me«tsure CHLOR-FLOC disinfectant levels over the test
period. Challenge organisms were added to provide the following
approximated concentrations per liter of water: 1.0 X 10®
Klefcgi?U9 tgrriqeng ceils; 2.0 :: 10® Crvptosporidium parvum
oocysts; 2.0 X 10 latex beads; and 1.0 X 10° PFU Echovirus 1.
(1) Tg.pwa.t:e|r
Tapwater was dechlorinated by continuous stirring at room
temperature for 24-48 hours. Four beakers, each containing
1 liter of. the dechlorinated water, were placed in a precooled
circulator waterbath. After the water in each beaker reached the
appropriate temperature, the pH was adjusted accordingly with l.O
N NaOH or 1.0 N H2SO4.
(2) Worst case (WC) water
Tapwater was dechlorinated by continuous stirring at room
temperature for 24-48 hours. After measuring 1 liter of the
dechlorinated water into each of four beakers, the following
components were added to each beaker: 1500 mg sea salt; 10 mg
humic acid; 150 mg AC Test Dust to give a turbidity of 30 NTU.
When the water reached the appropriate temperature, the pH was
adjusted accordingly with NaOH or H2SO4 as above.
(3) pemand-frfts (PF) yatgr
Four 1-liter beakers of HDFW were adjusted to the
appropriate temperature and pH for each test with NaOH or H2SO4
as above.
6
c. Test procedures
Components of the CHLOR-FLC<c individual water purification
system used in these tests are shown in Figure 1. CHLOR-FLOC
tablets were added to the test beakers according to the
manufacturer's recommendations for treating natural resource
waters (as represented in this study by DF, Tap, and WC waters) .
Two tablets were added to 5 *C water, and one tablet was added to
10 *C water. In all tests the CHLOR-PLOC tablets were added to 1
liter of the prescribed water and dissolved by stirring the water
for 1 minute. The water components were allowed to settle for 4
minutes, then stirred vigorously for a few seconds, followed by a
IS-minute interval for coagulation and flocculation. After a
total contact time of 20 minutes, the entire sample was filtered
through a flannelette bag provided by the manufacturer into a
separate container. Figure 2 shows the flocculation and
coagulation in tap and worst case 5 *C waters 5 minutes after
treatment with two CHLOR-FLOC disinfectant tablets. Figure 3a
shows the appearance of the final product tapwater compared with
the 5-minute treated tapwater, while figure 3b compares the worst
case final product water with the S-minute treated wor<«t case
water. Microbiological samples were taken from the reaction
mixture just before CHLOR-FLOC addition, at 5 minutes upon
redispersing the floe after the initial mixing/settling period,
and after filtration through the flannelette material (which
provided a total reaction time of 22 minutes) . Samples were
taken frem the viability control beaker, which contained the
prescribed water and organisms without the CHLOR-FLOC, at 0 time
and after filtration through the flannelette bags at 22 minutes;
and similarly from the disinfectant control beaker, which
contained only the CHLOR-FLOC in the water to measure residual
disinfectant. In all cases the test samples were immediately
neutralized with sodium thiosulfate except for the CHLOR-FLOC
measurement control. After each use, the flannelette bags were
backf lushed with tapwater to remove trapped organisms and beads,
immersed briefly in a mild detergent solution, and thoroughly
rinsed in deionized distilled water. The bags were randomly used
throughout the test runs and were replaced when they became thin
or torn.
7
Figure 1. CHLOR-FLOC water purification system components
Figure 2. Flocculation/coagulation with two CHLOR-FLOC tablets
in tapwater (left) and worst case water (right)
8
Figure 3. CHLOR-FLOC treated waters at 5 *C
a. Tapwater (before and after bag filtration)
Figure 3. CHLOR-FLOC treated waters at 5 ’C
b. Worst case water (after and before bag filtration)
9
A modification to the above procedure was used to conduct
additional experiments to determine if the maintenance of high
test water pH would interfere with the coagulation, filtration,
or disinfection of the test micro-organisms. This was considered
necessary because the CHLOR-FLOC tablets are buffered to lower
the pH of the water to less than pH 7.0. However, under typical
conditions it is not unrealistic to expect that field drinking
waters could be naturally buffered at higher pH which would
overcome the low pH buffering capacity of the tablets. For this
evaluation, test water pH values were adjusted and maintained at
pH 9.0, using an inorganic base (KOH) , after CHLOR-FLOC addition.
The test organisms were added 1 minute after CHLOR-FLOC addition,
when the waters had stabilized at pH 9.0, to insure that the
initial high pH of the water (approaching pH 11.0) before
CHLOR-FLOC addition was not deleterious to the test virus and
bacteria .
3 . SAMPLE ANALYSES
a. Chlorine analysis
Free available chlorine (FAC) was determined by the
LaMotte-Palin DPD chlorine test procedure. Samples from each
test water were collected for FAC level determinations 1 minute
after addition of CHLOR-FLOC tablets to the challenge waters and
immediately following filtration through the flannelette material
after the 20 minute treatment period.
b. Bacteriological analysis
Sample analyses were performed using the m-Endo medium for
the membrane filtration procedure as described in the USEPA guide
standard and protocol. Ten ml samples of each test water were
collected before CHLOR-FLOC was added; and after the disinfectant
was added, samples were collected at 5 minutes and after
flannelette bag filtration at 20 minutes. Samples were
immediately placed in tubes which contained sodium thiosulfate to
neutralize the chlorine. Serial 10-fold dilutions were made in
PBS, and triplicate l ml volumes of each dilution were filtered
through the 0.45 pm porosity Millipore bacteriological filters.
Colonies were counted after 24 hours incubation at 35 *C.
c. Virus analysis
Test samples were taken from each beaker before CHLOR-FLOC
was added; and after CHLOR-FLOC was added, samples were collected
at 5 minutes and after the 20-minute treatment period following
bag filtration. The 5 ml samples were immediately added to 5 ml
of 2X MEM containing 2 percent newborn calf serum and 1 percent
sodium thiosulfate. Subsequent dilutions were made in IX MEM
containing 2 percent newborn calf serum. A volume of 0.25 ml of
each virus dilution was inoculated onto triplicate 72-hour BGMK
10
cell monolayers grown in 60 mm tissue culture dishes; the cells
were then incubated in 5 percent CO2 at 37 *c, rocked at
15-minute intervals for 1 hour, and overlaid with 5 ml of Medium
199 containing antibiotics (5 units/ml Nystatin, 0.05 mg/ml
Gentamycin, and 5 units/ml Penicillin-Streptomycin) , 2 percent
newborn calf serum, 1.4 percent Difco purified agar, 0.125
percent sodium bicarbonate and O.Ol M Hepes buffer. Following
incubation in 5 percent CO, for 48-56 hours at 37 *C, each plate
was overlaid with 4 ml of Hanks BSS containing 5 percent neutral
red stain. The stain was allowed to absorb for 1 hour and then
poured off. Plaques were counted 12 hours after staining.
d. Protozoan cvst and cvst simulant analyses
Before adding the CHLOR-FLOC tablets, 100 ml samples were
collected from each beaker of the untreated water which contained
micro-organisms and latex beads. At the end of the CHLOR-FLOC
treatment period the seeded waters were filtered through
flannelette bags, and 500 ml samples were collected
(approximately 22 minutes after disinfectant tablets were added) .
All samples were immediately dechlorinated with sodium
thiosulfate. After addition of 0.1 percent Tween 20, each sample
was separately filtered through a 47 mm Nuclepore 1.0 ^m
polycarbonate membrane filter to collect the oocysts and beads.
The filter membrane was then cut into four sections and placed
into a polypropylene centrifuge tube. Oocysts and beads were
washed from the membrane filter surface by adding 10 ml of PBS
(containing 0.01 percent Tween 20) to the tube and vigorously
mixing the tube contents on vortex mixer. The membrane filter
was washed five times. After each wash, the liquid portion,
which contained the suspended oocysts and beads, was removed from
the tube and combined in a new tube. The oocysts and beads,
collected in the wash solutions, were concentrated by
centrifugation at 1200 X g for 20 minutes. The liquid portion
was removed and discarded except for approximately 1 ml which was
used to resuspend the pellet of cysts and beads. The resuspended
cysts and beads were stained with 0.5 percent Malachite Green for
20 minutes at room temperature and then decolorized with 0.25
percent sulfuric acid prior to quantitation with a hemacytometer
using Nomarski differential interference contrast with a 63X
objective. Background debris retained the green color, whereas
the oocysts and beads appeared colorless.
11
UA COLLABORATIVE STUDIES MATERIALS AND METHODS
For the collaborative studies, the USABRDL provided all
necessary personnel, supplies, equipment, and the CHLOR-FLOC
water purification tablets; and USABRDL personnel performed the
actual treatment procedures on the waters, including oocysts
dosing, as described for in-house efforts. The studies were
physically conducted at the UA Veterinary Department of Science
Laboratories. The UA collaborators provided the Cryptosporidium
oocysts, neonatal test animals, animal per diem, test animal
holding facilities, and reagent waters used in the study. The UA
veterinary staff also determined the appropriate cyst dosages for
the infectivity studies, prepared the various disinfectant-
treated test water samples for animal infectivity studies,
prepared and infected the animals with control and treated cysts,
and performed histological examinations of the gastrointestinal
tract of the animals for evidence of Cryptosporidium infection.
1. MICROBIOLOGICAL CHALLENGE
Cryptosporidium parvum oocvst production and PunillcatLOU
Cryptosporidium oocysts were recovered from the feces of
experimentally infected 2- to 5-day-old Holstein bull calves by a
previously developed method.^ Briefly, Cryptosporidium parvum
infection was produced in calves by feeding 1. 0-2.0 X 10®
infective oocysts suspended in 1 liter of reconstituted
commercial milk replacer. The calves were isolated in vealer
pens; and the feces excreted during the peak oocyst shedding
period were collected, mixed with ar. equal volume of 5 percent
potassium dichromate (K2Cr207), ana stored at A ’C. The
collected feces were sieved sequentially through stainless steel
screens of decreasing aperture ending with 63 pm pore size (230
mesh). Sequential discontinuous sucrose gradient (1.064 to 1.103
g/ml) centrifugation, followed by isopycnic Percoll"’ gradient
(1.091 g/ml) centrifugation, completed oocyst purification. The
purified oocysts were stored in 2.5 percent potassium dichromate
at 4 *C. Oocysts were withdrawn from storage as needed for
experimentation and washed with PBS (0.025 M, pH 7.4) by
filtration through polycarbonate filters (1-3 pm pore size) or by
centrifugation to remove the dichromate storage solution.
2. PHYSICAL/CHEMICAL WATER CHARACTERISTICS AND TEST PROCEDURES
The designated 1-liter test waters were prepared as
described in the USABRDL in-house studies above.
12
USABRDL Staff dosed the waters with the oocysts in
accordance with the standard test procedure described earlier,
except that the CHLOR-FLOC product was not filtered through
flannelette material. CHLOR-FLOC or Globaline tablets were added
at the prescribed levels — two Globaline tablets with a
disinfection contact time of 35 minutes; and one CHLOR-FLOC
tablet at 10 ’C, or two CHLOR-FLOC tablets at 5 ‘C with a
disinfection contact time of 20 minutes. The iodine ar d chlorine
disinfectant levels were monitored in each sample to ensure that
proper disinfection levels were attained and maintained
throughout the appropriate disinfection contact time. The
chlorine /iodine were rapidly neutralized with sodium
thiosulfate. The samples were then handed over to the UA staff
where the oocysts were concentrated from the treated water,
physically enumerated, diluted for infectivity studies, and
provided to the neonatal mice by gavage.
During phase I expjriments, .1-1 iter volumes were collected
from each of the Cryptosporidium seeded test waters (untreated,
CHLOR-FLOC treated, and iodine treated) . After the chlorine and
iodine were neutralized, the samples were filtered through 1 /im
pore size Nuclepore polycarbonate membrane filters. The filters
were then carefully removed, placed in a 50 ml tube (tube A) with
10 ml of washing solution (1 liter of nanopure water containing
10 pi of Tween 20) , and t^ixed with a vortex mixer for 15 seconds.
The filter was removed and placed in another 50 ml tube (tube B)
with zn rrl-^iricnal 10 ml of washing solution and mixed. The
contents tube B was added to tube A, and the filter in tube B
was was!, r *g&in. The filter in tube B was removed while the
second vash from that tube was also combined with tube A. Tube B
was washed twice with 7.5 ml of nanopure water and combined with
tube A. The entire contents of tuba A were centrifuged at 3000
rpm in a Sorv^ll T-6000B centrifuge with a HIOOOB rotor for lO
minutes. Th'i liquid port. ion was aspirated down to 1 ml, and the
pellet was thoroughly mixed with the ml of residual wash water.
Appropriate dilutions of this concentrated sample were used to
measure resiOual cyst concentrations and diluted for oral gavage
of test anima.’.s.
c . Phase ri test procedures
Testing procedures fo.llov.Md the same basic protocols as
described above with the r^^'wne division of responsibility. During
these trials iodine disinfection was not evaluated. This
research evaluated the capabilities of new prototype plastic
reaction bags for floe formation and settling by the coagulating
agents contained in the CHLOR-FLOC tablets. These bags have a
spout on the bottom through which settled flocculated material
can be wasted before the main bulk of water is filtered through
flannelette. Figures 4a, b, and c depict the disinfection
process for a worst case water at 5 ’C treated with two
CHLOR-FLOC tablets as follows; (a) flocculation 5 minutes after
treatment, (b) settle? coagulated-f locculated material 20 minutes
after treatment, and (■;) filtration and collection of the product
water after settled materials were discarded. The bags and also
the flannelette material used in this effort were prototypes
provided by the U.S. Army Natick Research, Development, and
Engineering Center.
Figure 4. New CHLOR-FLOC system with worst case 5 ’C water
a. Five minutes after treatment
14
Figure 4b. Settled flocculated naterial 20 minutes
after treatment
For phase II efforts a modified protocol for oocyst recovery
from worst case water samples was utilized to circumvent problems
obcerved with the recovery of cysts during phase I. The
procedure was as follows: After neutralization of residual
chlorine with sodium thiosulfate, the 1 liter samples were
transferred equally into two 750 ml centrifuge bottles; the
original bottles were washed with 10-20 ml of washing solution,
and this material was added to the centrifuge bottles. The
samples were centrifuged in a Sorvall T-6000B centrifuge with a
TIOOOB rotor at 3500 rpm for 15 minutes. The supernatant fluid
was aspirated to a few ml in the bottom of the bottles, and the
pellet was resuspended into the residual fluid. The resuspended
material was transferred into 50 ml tubes and centrifuged again
at 3000 rpm for 10 minutes. The fluid was aspirated and the
pellets were combined. Finally, the residual pellet was
centrifuged as before and aspirated to 1.0 ml. This provided the
material used for neonatal mouse dosing.
15
Figure 4c. Filtration/collection of treated water
after settled materials were discarded
3. SAMPLE ANALYSES
Late-term pregnant female BALB/c mice were purchased from
Harlan Sprague Dawley (Indianapolis, IN) . Within 24 hours of
birth, mouse pups were randomized and placed h>ack with the
mothers (6 to 7 pups/litter) to minimize maternal effects on
experimental outcomes. Animals were maintained in mlcro-isolate
cages throughout the experiments. Jtoom temperature was
maintained at 18~26 *C with a 12-hour light/dark cycle and a
relative humidity of 40-70 percent. Mice were fed Tekland
Mouse/Rat Chow and sterile water libitum.
Previous experience with this mouse infectivity model
indicated that neonatal mice can be routinely infected orally
with 10^ to 10^ Cryptosporidium oocysts at 5 days of age.
Gastrointestinal (GI) colonization and oocyst shedding develops
2*‘5 days later and resolves in about 5-7 days. BALB/c mice have
been used for numerous experiments with Cryptosporidium , .
Sterling's laboratory has just completed a susceptibility
dynamics infection in relation to the BALB/c neonatal mouse.
They were able to infect 100 percent of the neonatal BALB/c mice
through 9 days of age with 10^ oocysts by oral intubation.
a. Phage I
Median infectious dose range determinations for the phase I
study were conducted by infecting neonatal mice by gavage in
which a range of Cryptosporidium oocysts were administered to
groups of neonatal mice. The dose was determined by
extrapolation from the dose range studies in which infection of
intestinal villi was determined.
b. Phase II
During phase II studies the baseline ID^q was determined by
an ill vitro excystation method® (Appendix 1) . The method
involved triplicate determinations of excystation in which the
relative numbers of intact oocysts, exeysted shells, and
sporozoites were counted; and the percent of theoretical
sporozoite yield was produced. Then estimation of the IDcq from
previously determined linear regression analysis of neonatal
mouse infectivity yg. percent theoretical sporozoite yield was
performed .
The protocol for experimental dosing utilized centrifuged
sample preparations from 50 ml centrifuge tubes. The volume was
increased to 5 ml (2 ml for centrifuged worst case water) with
nanopure water, and the sample was mixed on a vortex mixer to
uniformly disperse the oocysts. The dilutions needed to obtain
the "high dose," "median dose," and "low dose" in 100 ><1 of
inoculum, based on the previously determined ID^q and the
recovery efficiencies were as follows:
0 of oocyst seeded per liter X (recovery efficiency)
divided by (IDcq X 10^) = # of ml of di3v>tion needed for
10,000 X IDgQ in 100 pi. This was the ' iigh dose."
Dilute the above suspension 1:10 to obtain 1,000 X ID^q in
100 Ml. This was the "medium dose."
Dilute the "medium dose" suspension 1:10 to obtain 100 X
IDgQ in 100 This was the "low dose."
For experimental studies, CHLOR-FLOC treated oocysts (ID50
dose and sequential 10-fold higher levels) were also administered
by oral gavage to 5- to 7-day-old neonatal BALB/c mice. This was
accomplished with a blunted slightly bent, 1/2 inch, 25-26 gauge
hypodermic needle fitted with a short piece of polyethylene
17
tubing mounted on a 1 cc tuberculin syringe. The animals were
sacrificed 7 days post inoculation; and approximately 3 cm of the
terminal ileum was removed, fixed in 10 percent formalin,
embedded in paraffin, and sectioned. Hematoxylin and eosin
stained paraffin sections were examined microscopically for
evidence of Cryptosporidium infection in the microvillous region
of villous enterocytes. Specimens with parasitic stages present
were scored as positive; those without were scored as negative.
Positive specimens always showed numerous parasitic stages (at
least 50-60 per lOOX microscope field), while no parasites could
be found on any sections taken from negative tissue samples.
Infection was scored by the relative concentration of
Cr vptospor id ium in the ileum ranging from a 0 to 4+ level of
infection. Any level of infection was scored as a positive
result.
During phase II, companion tests using fluorescent
monoclonal antibody (OW 64 MAb) directed at the suture line of
Cryptosporidium oocysts were utilized as a measure of infectivity
for comparison against results from neonatal infectivity
procedures. The suture line in oocysts appears only when the
oocyst has experienced some degradation, and the antibody was
prepared against this suture material. In this case the numbers
of oocysts and their infectivity were determined microscopically
using both a fluorescent monoclonal antibody for the cyst wall
(total cysts) and one for the suture line. Because the
antibodies were tagged with different color fluorescing dyes, the
full cysts and cysts with suture lines could be discriminated and
each could be counted.
18
RESULTS
1. USABRDL IN-HOUSE STUDIES
a. Free available chlorine
FAC levels produced by CHLOR-FLOC did not differ
significantly between control and challenge waters or between
halogen demand free, tap, and worst case waters. Nor was FAC
affected significantly by pH, temperature or turbidity. The
1-minute readings from test waters ranged from 6-10 mg/1 for a
single CHLOR-FLOC tablet, and lO-ninute sample readings ranged
from 5-9 mg/1. It is assumed that two tablets would give
proportionately the sane results. Th>> resuspended floes
interfered somewhat in visually determining color depth from the
DPD tests and may have been the cause of the variable range in
FAC levels for each tablet. However, all 20-minute samples
including controls consistently showed a 1-2 mg/1 decrease in FAC
level when compared against their l-minute level. The decreased
levels of FAC could have been due to chlorine dissipation while
the containers were uncovered during sampling, combination with
water constituents, or impaired visual acuity from increased
flocculation. If present, combined chlorine was not detected by
increased color intensity using the DPD chlorine comparator.
b. Bacteria and virus removals
Disinfection/physical removal of Klebsiel? a terriuena in all
waters at pH 7.0 exceeded the six-logwj removai. requirements at
both 5*c and lO’C (Tables 3 6 4). TaMes 566 reveal that
Echovirus 1 inactivation also exceeded the 4-logj^Q removal
requirements for all waters and conditions at pH 7.0 for both
temperatures. Both the Klebsiella tglfAgenfl and Echovirus 1
removals were attained within 5 minutes of contact with
CHLOR-FLO and did nov require physical filtration of the
flocculated mixture through the flannelette bag to achieve these
removals. Similarly, the bacteria and virus removals from the
variouc waters at pH of 4.5 and 9.0 were equivalent to the pH 7.0
results (e.g., no detectable organisms present at 5 minutes).
These overall bacteria and virus removals (averaged from the
initial pH 4.5, 7.0, and 9.C waters) are shovn in Figures 5 and
6. Not unexpectedly, the flannelette bag-filtered test samples
taken at 22 minutes were also negative for the bacteria and
viruses in all cases. The micro-organism control samples without
CHLOR-FLOC, which received only filtration through the
flannelette material after 20 minutes, experienced low, but
variable, removals which indicate that the flannelette material
had little effect in filtering out or adsorbing the bacteria and
virus in the absence of CHLOR-FLOC. The reason for the
variability seen in the controls in both the bacterial and viral
removals was not determined. However, there are several
19
possibilities for the variability, such as initial aggregation of
organisms in the unbuffered challenge waters which were dispersed
during the sample dilutions in PBS, handling techniques by
different operators, manufacturing differences in the flannelette
material, or the number of times the filter bags were used.
As shown in duplicate tests, artificial maintenance of the
pH at 9.0 in the various waters upon CHLOR-FLOC treatment did not
interfere with the removal characteristics of either the
Klebsiella terriaena bacteria or Echovirus 1 (Table 7) ; both were
removed to the required levels within 5 minutes of contact, again
without filtration. These averaged removals are also shown in
Figures 5 and 6.
c. Cvst and cvst simulant removals
The removal of Cryptosporidium cysts (Tables 8 & 9) and the
3.7 fixa AccuBeads"' (Tables 10 & 11) was determined only for
physical removal as provided by coagulation and subsequent
filtration through the flannelette material. In these
experiments only pre-disinfection and 20-ir.inute reaction mixtures
(after filtration) were analyzed. For all three test waters at
pH 7.0, two CHLOR-FLOC tablets at 5 *C achieved cyst removals
ranging from 80.40 to 96.20 percent and latex bead removals
ranging from 78.20 to 90.40 percent. Experiments conducted under
identical conditions but at 10 *C with a single CHLOR-FLOC tablet
produced similar results. Here, cyst removals ranged from 92.58
to 99.80 percent, while the cyst simulant removals ranged from
96.97 to 99.42 percent; only minor differences from this were
noted in waters tested at pH 4.5 and 9.0. As seen in Figures 7 &
8, there was a general trend toward improved removals of both
type particles as water quality decreased, i.e., worst case > tap
> demand free distilled waters. Also, there was a tendency
toward better removals of both cysts and beads at the higher
temperature, possibly because of increased coagulation.
When pH 9.0 was maintained after CHLOR-FLOC addition, the
cyst and simulant removals were slightly better (Table 12,
Figures 7 & 8) compared against the ambient test pH results.
Under these high pH conditions cyst removals ranged from 97.80 to
>99.90 percent, and bead removals ranged from 93.49 to 99.71
percent. The comparison between the Cryptosporidium cyst and the
3.7 pm AccuBead** removals indicates that the removals are very
similar and that the beads are a credible simulant for evaluating
the physical removal of these cysts. Beads could be used in
future studies on large-scale batch water treatment with
CHLOR-FLOC to determine Cryptosporidium cyst removals.
20
TMIE 3. KlEtSttLLA TtWlCTtA OiSIRFECTKM WITH TUO CHLM-FLOC TMLETS AT S
21
cni/L: colony fonolng units/liter
TMIF 4. nCBStEUA TEMtGEM OISIMFECriON WITH ONE CHLOR-FIOC TABtET AT 10
CFU/L*: colony fomlng unlts/llter
** Average of duplies
TAKE S. ECNOVIMS DISItEECTICM Wtm TWO CMW-ELOC TMLCTS AT 5
Ik
PFU/L: plaqut fenilnt wt<tt/(<lt*r
Tme 6. ecmviws oisiNrtcTiM with ok cKioK-rioc tmlct at io
ATU/L*: pl«qM feniing w(ts/ttt*r
Figure 6. ECHOVIRU8 DiaiNFBCTION WITH CHLOR-FLOC
T LEGEND
WATER CHARACTERISTICS
Averages of pH A. 5, 7.0, and 9.0 waters
Averages of duplicate tests
tA»LC r. OmufCCTIOT Of WMlCtHA AMD tCMOVIAU* WITH
OME CHLM-rLOC TAtlET AT 10 WITH pH ADJUSTED TO NAIHTAIN 9.0
Motor
TVPO
toapto
_ f _ lin _
KlOfrlttUO HfftHTW
_ CfSCA _ I Koduetlon
Ichovlrut 1
9eu»«/l _ % Urtittim
•e’
1
0
1.20 K 10*
1.04 X 10*
S
0
>99.9999
0
>99.99
DE
z
0
1.20 K 10*
1.04 X 10*
$
0
>99.9999
0
>99.99
OE
Control
0
1.05 X 10*
9.75 X 10^
22
8.50 X 10^
19.42
7.72 X 10^
20.44
top
1
0
1.97 X 10*
5.00 X 10*
5
0
ClUOO
>TnF*innnr
0
>99.99
top
2
0
1.97 X 10*
5.00 X 10*
)
0
91PF»yYYT
0
>99.99
top
Control
0
1.27 X 10*
2.52 X 10*
22
5.00 X 10^
74.58
2,51 X 10*
•8.19
mc2
1
0
1.15 X 10*
1.40 X 10*
s
0
>99.9999
5.55 X 10*
>99.99
22
-
0
>99.99
VC
2
0
1.15 X 10*
1.40 X 10*
s
0
>99.9999
1.07 X 10*
>99.99
22
-
0
>99.99
wc
Control
0
9.70 X 10^
1.05 X 10*
22
8.70 X 10^
10.51
4.90 X 10^
53.55
* CEUi colony ferainp wtUe
** 9ni: ploojo fenilns
DE t dMMnd froo Motor
MC^t Morot cooo Mocor
27
rf90°i»
OOCTSt KMOVAl WITH TWO Cm.ai*rt.OC TAtlCTS AT S
42.35
T«M.€ 9. OryTOSWUtOlUW 9A9WW OOCTST KWVAl WITH OMC CNLOt-FLOC THtltT AT 10
29
1.54 X
Figure 7.
CRYPTOSPOmPIUM PARVMM OOCYST PHYSICAL REMOVALS
WITH CHLOR-PLOC
» M I -I I > - > > ■ -M - > - ♦
o O Y f) n ^
l/SlVA0W9y ISAOOO
30
* Averages of pH A. 5, 7.0, and 9.0 waters
** Averages of duplicate tests
TAILE 10. MIOTOZOM StNUtMT KCMOVAL WITH TWO CHIM-FIOC TABLETS AT 5
i
8
8
S
R
S
:S
&
I
» ml
IS
I
st
i.**
I
II
I
t -S
-
IS
i k
‘I
8
VN
i8
8
e
I
s
u
31
8
K <o
o o
8^ «0
O O
o o
••
o o
«A
8 8
»*«►
o o
K lA
O O
•* »•
(V «n
o e
K K
X X
X X
X X
X X
X X
X X
X X
X X
^ fW
K.
^ m
a s
S 8
8 :$
8 8
lit 8
z s
oi rj
nJ ^
^4 lA
•- ^
«» ^
AJ
8
8
8
8
8
8
8
8
IS
R
8
8
«Q
«>
8
8
O
o o
fw Pw
O O
o o
O O
K.
o o
e o
Is. P^
O O
M X
X X
X X
X K
X K
K X
X X
X K
X X
•-
fe 8
fc s
8 a
8 ::
^ K
« ^
8 2
a 8
S 8
•-
•“ IV
•- K
8
8
a
8
o
O
8
O
5
8
R
>
kJ
8
»
'"o'«o
O O
'"o'*o
•• #•
%,"o
*^"*0
M X
X X
X X
X X
X X
X X
X X
X X
X X
n 8
a 8
S 8
e js
2_R
i: 5
8 1!>
$ s
iD H#
*#
Al ^
fsi •-
Al •-
^ *»
IV
O «Vf
#v
e
fM
O 4^i
rw
e «NA
fM
O IV
fV
o rv
IV
e fv
«v
o IV
rv
e IV
ry
?
«k
m
s
i
t
c
8
p-
■
8
**
c
9
9
S
U
TABLE PROTOZOAN StMUANt REMOVAL WITH ONE CHLOR-FLOT lABlE* AT 10
o
fsj
o
«0
o
fw
BA
t
IX
S
s
s
i
k!
«0
K)
8i
r
IX
R
R
z
K T©
O O
K
O O
K.
O
*- ^
^ ••
• lA
o o
K K
O O
K ^
O O
^ •*
K s#
e o
o o
•• ••
M K
X X
X X
X X
X X
X X
X X
X X
X X
s s
^ IX
S !S
IX IX
S| 'S
V ^
o IX
lx O*
^ IX
s.
IX A*
lA
S#
•- S#
»- BA
§
c-
o
«
•o
S«
IX
IX
IX
IX
o
BA
i
vX
IX
fw
K
<^
IX
rx
i
IX
o
o
fO
K «A
o o
BT
Q
Is.
o o
O O
lA
O O
•“ ^
N. K
O O
O O
I'* BA
o o
^ ©
e o
X X
X X
X X
X X
X X
X X
X X
X X
X X
s ;s
c «
C R
IX ••
O' IX
g s
CO O'
♦“ 'O
-■» 1".
e •-
O 'X
IX »*
IX sO
IX ^
IX ^
n A©
© <o
s
o
o
o
lA
s
s
O
o
BA
o
«
i
o
o»
sO
<o
>
IX
O'
•A
IX
IX
o
s
o
♦A
fw ^
o o
♦» ^
fw
o e
o o
o o
*•
fs. K
O O
K nO
O O
A. lA
o o
K K
O O
•» ^
X X
X X
X X
X X
X X
X X
X X
X X
X X
*
^ K»
x> ^
^ **y
IX IX
S 5
z z
O BA
o •#
IX •-
R g
«M» ^
••
^ IX
R »
IX o
#2 o
IX ••
0
22
O IX
IX
O IX
IX
e IX
IX
o IX
IX
O IX
IX
O IX
lx
O IX
IX
O IX
IX
e
«»
IP
KJ
P>
U
z
3^
WATER CHAR ACT
TABLE 12. REDUCTION OF CRYPT0SF>08IDIUM OOCYSTS AND PR0T02OAN SIMULANT WITH ONE
CHLOR-FLOC TABLET AT TO **C WITH pH ADJUSTED TO MAINTAIN 9.0
Water
Tvpe
Sample
_ ! _ Use
Cytt Sleulant
_ Accufeeacte/L _ X RetAxtion
of’
1
0
1.69 X 10*
2.52 X
10^
22
1.20 X 10*
99.29
1.51 X
10*
93.49
DF
2
0
1.77 X 10*
2.54 X
10^
22
9.50 X 10’
99.47
1.59 X
10*
95.74
DF
Control
0
1.77 X 10*
2.41 X
10^
22
1.41 X 10*
20.54
1.90 X
10^
21.16
Tap
1
0
1.73 K 10*
1.45 X
io"
22
1.10 X 10*
99.36
7.12 X
10*
95.09
Tap
2
0
1.73 X 10*
1.45 X
10^
22
3.77 X 10*
97.60
9.17 X
10*
93.70
Tap
Control
0
1.73 X 10*
1.45 X
10^
22
i.eo X 10*
•4.0S
9.89 X
10*
31.79
WC^
1
0
1 .63 X 10*
1.51 X
10^
22
4.00 X 10^
99.75
4.40 X
10*
99.71
WC
2
0
1.63 X 10*
1.51 X
10^
22
<1.00 X 10^
>99.90
6.8'* X
10*
99.55
wc
Control
0
1.63 X 10*
1.51 X
10^
22
8.00 X 10^
50.90
M*
61.00
DE : dcmnd fr«« wattr
WC‘: worst CM* KRter
34
2. UA STUDY-PHASE I
a. Median infective dose validation
Initial studies were perfomed to validat ; the median
infective dose (ID^q) for Cryptosporidium oocy^' ^ in the neonatal
mouse model. Two groups of 32 neonatal mice, 5 to 7 days old,
were challenged with doses of oocysts which had been exposed to
DF water at 5 and 10 *C for 20 minutes. These data showed that
the ID5Q was 67 oocysts in S *C water and 80 oocysts for
10 *C water. The combined IDcq was 72 oocysts. This combined
value was used to determine the experimental and positive control
challenge doses because the resulting larger group sizes would
provide a more reliable measure of the ID5Q for these oocyst
preparations.
b. PggitiYfr ggntrplg
Two groups of neonatal mice were challenged with oocysts
exposed to each of the test water conditions without
disinfection. One group was given the ID5Q of 72 oocysts (low
dose) while the other received 720 oocysts (high dose) . Complete
data are shown in Table 13. All mice challenged with the high
dose showed a high percentage and level (intensity) of infection.
Mice challenged with low doses of oocysts exposed to DF water
showed the lowest infectivity, while those challenged with
oocysts in worst case water showed high infectivity even at the
low dose.
TABLE 13. INFECTIVITY OF POSITIVE CONTROLS
Water Cyst Percent Neonatal
Treatment Dose Mice Infected
5
•c
DF
r
720
100
(6
of
6)
5
•c
DF
72
43
(3
of
7)
5
•c
WC
720
100
(6
of
6)
5
•c
WC
72
86
(6
of
7)
5
•c
WC
pH
9.0
720
100
(5
of
5)
5
*c
WC
pH
9.0
72
100
(7
of
7)
5
•c
WC
pH
5.0
720
100
(7
of
7)
5
*c
WC
pH
5.0
72
80
(4
of
5)
* Unless designated otherwise the pH was approximately 7.0.
35
c. Experimental
The goal of the test was to determine if there was a
significant Cryptosporidium oocyst disinfection capability by
CHLOR-FLOC when used at recommended concentrations. Desired
levels of disinfection were three orders of magnitude (3 log^^Q) .
For comparison some testing was also conducted on Army issue
Global ine (iodine tablets) to determine the relative differences
in effectiveness of the two chemicals. Test waters were seeded
with 1.0 X 10° oocysts per liter before either CHLOR-FLOC or
Global ine tablets were added. After the disinfectant contact
period was reached, the chlorine and iodine in the test waters
were neutralized; and the oocysts were concentrated as described
earlier (recoveries greater than 99 percent by the method
described) . Microscopic counts of the final sample concentrate
were performed using phase microscopy or monoclonal fluorescent
tagged antibody (antibody for the cyst wall) counting procedures
to quantify cysts for animal dosing. Two groups of neonatal mice
were challenged with oocysts exposed to each of the test water
disinfectants. One group was given a "low dose" of 7,200 oocysts
(2 logs above the IDcq of 72 oocysts) while the other received
the "high dose" of 72,000 (3 logs above the ID^q) . According to
this dosing protocol if any of the mice challenged with the low
dose became infected, then a 3-log reduction in cyst infect ivity
would not have been achieved. This indeed appears to be the case
(Table 14). Except for two CHLOR-FLOC treated groups, involving
5 and 10 *c worst case water, all mice showed unmistakable signs
of Cryptosporidium infection. This appears in contradiction to
controls when worst case was highly infective.
36
TABLE 14. INFECTION OF MICE RECEIVING CHLOR-FLOC
OR GLOBALINE TREATED OOCYSTS
Water Cyst Neonatal Mice
Treatment _ Dose _ _ Percent Infected
5 'C DF CHLOR-FLOC
72,000
100
(14
of
14)
5 ’C DF CHLOR-FLOC
7,200
100
(10
of
10)
10 ’C DF CHLOR-FLOC
72,000
100
(9 of 9)
10 *C DF CHLOR-FLOC
7,200
100
(11
of
11)
5 ’C WC CHLOR-FLOC
72,000
100
(10
of
10)
5 ‘C WC CHLOR-FLOC
7,200
91
(10
of
11)
5 ’C WC CHLOR-FLOC pH 9
72,000
100
(12
of
12)
6 'C WC CHLOR-FLOC pH 9
7,200
100
(12
of
12)
10 ‘C WC CHLOR-FLOC
72,000
91
(10
of
11)
10 ‘C WC CHLOR-FLOC
7,200
100
(13
of
13)
5 ’C DF I,
72,000
100
(12
of
12)
5 ‘C DF I2
7,200
100
(13
of
13)
5 ’C WC I,
72,000
100
(13
of
13)
5 ‘C WC Ij
7,200
100
(14
of
14)
* Unless specified the pH during the test was approximately 4.S
to 5.0 due to the buffering capacity ox the CHLOR-FLOC and
Globaline tablets.
37
3. UA STUDY-PHASE II
a. Mean infective dose
The median Infective dose was determined for the oocyst
preparation used in this study by the excystation method
discussed in the materials and methods. Excystation was
accomplished in triplicate according to the protocol. The
percent theoretical sporozoite yield was determined according to
the following formula:
Percent Theoretical
Sporozoite Yield - _ Sporozoites counted X 100
4 (Intact cysts -*■ Shells)
ID5Q Excystation Data:
Trial Intact
1 56
2 46
3 49
Mean 50.3
% Theoretical Sporozoite
Shells
Sporozoites
193
298
162
284
158
324
171
302
ield * _
302 X 100
4(50.3 + 171)
The log^Q of this value was used to determine the expected ID^q
from the chart. The regression line on the chart is based on the
log change of sporozoite yield yft. the log change in ID5Q.
Therefore, the regression line equation was used to calculate the
ID50 (Appendix 2).®
b. Positive controls for oocvst recovery efficiency and
intectivitv
The four test waters (DF, 5 and 10 'C; WC, 5 and 10 ’C) were
seeded with 1.19 X 10® oocysts per liter. After 20 minutes, the
preparations were stirred, and l ml was withdrawn for the
positive infectivity control challenge doses. The remaining test
waters were filtered through 1 ^m polycarbonate filters (for DF
distilled water) or centrifuged (for WC water) to recover the
oocysts. Centrifugation was required for the WC waters due to
filter clogging.
DF mean recovery » 7 1.1 percent
WC mean recovery ■ 45.0 percent
These values were used to determine the volume of concentrate
needed to prepare the challenge doses for each experiment.
Two groups of neonatal BALB/c mice were challenged with
oocysts exposed to each of the test waters. One group was given
the ID50 of 115 oocysts (low test dose) while the other received
1150 oocysts (high test dose) . Complete data are shown below
38
(Table 15) . All DF nice challenged vith ^he high dose showed a
high level of infection. Mice challenged with WC 5 *C water
showed the 50 and 100 percent infection expected for the low and
high dose respectively. Mice challenged with the WC
10 *c water did not show this pattern, however. In this case 75
percent of nice challenged with low dose were infected, while
only 50 percent of nice challenged with high dose becane
Infected. Since oocysts tend to adhere to and clunp with the
particles present in worst case water, it is possible that one
■ouse in each of these groups received less than the expected
dose. The outcone of the positive control experinent (Table 14)
supported the expected ID^q and established the infectivity of
the oocysts used in the tests.
TABLE 15. POSITIVE CONTROL DATA
Dose
- i Qt gQgYOtg _ Treatnent _ % Infected
DF
5 ’C
115
Control
50
(2/4)
DF
5 ‘C
1150
Control
80
(4/5)
DF
10 *C
115
Control
60
(3/5)
DF
10 ‘C
1150
Control
100
(5/5)
WC
5 ’C
115
Control pH
5
50
(2/4)
WC
5 ’C
1150
Control pH
5
100
(2/2)
WC
10 ‘C
115
Control pH
5
75
(3/4)
WC
10 ‘C
1150
Control pH
5
50
(1/2)
c. CHLQR»FLQC infectivitv test
The results of disinfection/ filtration evaluations with
CHLOR-FLOC and the associated reaction bag/flannelette filtration
procedures to clarify the water of the flocculated naterials
(including organisns and debris) are shown in Table 16. As in
the phase I studies, one tablet was used at tenperatures of 10 *c
and two tablets were provided at 5 *C. After the CHLOR-FLOC
treatment, sample processing, concentration, and oocyst
quantification were completed, neonatal mice were infected with
the control ID.q oocyst dose as well as 100, 1000, and 10,000
ID5Q dose levels. According to this infectivity protocol, no
mice becoming infected would confirm at least a 4 -log decrease of
infectivity. The data shown below indicate that the experimental
treatments caused no measurable reduction in oocyst infectivity.
A 99.9 percent (3 logs) reduction in viable oocyst concentration
was not achieved. This is supported by the approximately 50
percent infection in mice challenged with the ID5Q and the almost
universal 100 percent Infection of mice challengeo with doses of
oocysts that exceed the ID^q.
TABLE 16. CHLOR-FLOC INFECTIVITY DATA FOB PHASE II
Dose
_ t gl gggYgtfi _ Tr^atn^nt _ I .^nrfistcA
DF 5 *0
2
Tablets
60
(6/10)
DF 5 'C
100 ID50
2
Tablets
100
(10/10)
DF 5 ‘C
1000 ID50
2
Tablets
100
(10/10)
DF 5 *C
10000 ID50
2
Tablets
100
(10/10)
DF 10 ’C
1
Tablet
60
(6/10)
DF 10 ’C
100 IDgn
1
Tablet
80
(8/10)
DF 10 ’C
1000 ID50
1
Tablet
100
(9/9)
DF 10 ‘C
10000 ID5Q
1
Tablet
100
(10/10)
HC 5 ‘C
1^50
2
Tablets
pH 5
100
(10/10)
HC 5 ’C
100 ID50
2
Tablets
pH 5
100
(10/10)
HC 5 •€
1000 ID50
2
Tablets
pH 5
100
(10/10)
HC 5 'C
10000 IDgQ
2
Tablets
pH 5
100
(10/10)
HC 10 ‘C
1
Tablet |
pH 5
100
(10/10)
HC 10 ‘C
100 ID50
1
Tablet pH 5
100
(10/10)
HC 10 *C
1000 ID50
1
Tablet |
pH 5
100
(10/10)
HC 10 *C
10000 ID50
1
Tablet ;
pH 5
100
(10/10)
Tap 5 ’C
100 ID50
1
Tablet
60
(3/5)
Tap 5 ‘C
1000 ID50
1
Tablet
100
(10/10)
d. Alternative viability fst _Qf CMLQR«FLQC trsafd Qocyg.ts
A snail portion of the final concentrated sanple after
CHLOR'FLOC treatment was evaluated for viability of the residual
oocysts using nonoclonal antibody. The physical oocyst renoval
by the CHLOR'-FLOC system included the settling and removal of the
floe that was formed by the coagulant component contained in
CHLOR-FLOC with the particulates contained in the challenge water
using the flannelette filters. The viability of the oocysts was
determined using a nonoclonal antibody, OH 64 HAb, that was
developed by Dr. Sterling's laboratory at the UA Department of
Veterinary Science to determine the degradation or partial
opening of cyst suture lines. Previous studies by that
laboratory^ have provided a linear regression analysis
astablishing a correlation between binding of the OH 64 MAb
monoclonal antibody to the cyst suture and loss of cyst
infectivity. Table 17 shows only a small portion of OH 64
positive oocysts found after CHLOR-FLOC treatment, thus
indicating that the disinfectant treatmant was not effactive in
reducing the viability of the oocysts. This finding supports the
results of the neonatal mouse infectivity studies. The results
also show that only a very small component of the cyst challenge
40
was physically ranoved by the coagulation, sedinantation, and
filtration processes of the CKLOR-FLOC treatment, it was
observed in the trials that the CHLOR-FLOC sediment had a
tendency to hang up on the sides of the plastic bag and was not
readily settled, thus impacting on the amount of material that
could be wasted before filtration through the flannelette bag.
The bags obviously provided little clarification of the residual
floe as can be seen in Figure 4c.
TABLE 17. RECOVERY AND VIABILITY OF CHLOR-FLOC TREATED OOCYSTS
Treitment
Oocyst
Challenqe/L
Oocysts
Recovered/L
% Oocysts
Recovered
% Oocysts
0W64 Positive
DF 5 'C
1.0 X 10®
3.72 X loj
37.2
0.42
DF 10 ‘C
1.0 X 10®
3.32 X lo;
33.2
0.0
HC 5 ‘C
1.0 X 10®
1.90 X ic;
19.2
0.25
HC 10 ‘C
1.0 X 10®
3.81 X 10'
3fl.l
0.14
41
DISCUSSION
1. USABROL IN-HOUSE STUDIES
The results of in-house studies indicated that the
CHLOR-FLOC tablets were very effective at temperatures of 5 *C,
using 2 tablets, and 10 *C, using 1 tablet, for the destruction
of both the Klebsiella terriaena and Echovirus 1; achieving over
a 10^ reduction in the bacterial challenge and over 10^ reduction
in the virus challenge. These removals were achieved within the
first 5 minutes of contact even before the materials were
filtered through the flannelette materials at 20 minutes. The pH
of the initial waters before CHLOR-FLOC addition did not appear
to have a significant impact on the effectiveness of the
disinfectant; however, in all cases the initial pHs, even as high
as pH 9.0, were quickly reduced to less than pH 5.0 due to the
low pH buffering capacity of the tablets. The artificial
maintenance of pH 9.0 did not hamper the ability of either one or
two tablets to provide the required removals of the bacteria and
viruses still within the 5-minute contact period. This was
somewhat surprising because higher pH typically reduce the
effectiveness of chlorine based-disinfectants. The residual
chlorine levels even using one tablet remained in the range of
6-10 mg/liter at the 5-minute sampling time and and 5-9 mg/liter
after 20 minutes, regardless of the water quality challenge
(including worst case water). Control tests showed that the
flannelette material had no significant removal potential for the
organisms; but because of the activity of the disinfectant
component, the effectiveness of the bags in the presence of the
coagulant component alone of the CHT,OR-FLOC system was not
evaluated.
The above results are somewhat different than observed for
the bacteria used in studies by Powers.^® He did not detect any
residual bacteria in natural waters dosed with £. coli or
Pseudomonas when disinfected with either CHLOR-FLOC or iodine
tablets over a 10-minute contact time. However, in further
studies^^ he indicated that even after 20 minutes there were a
few residual £. coli survivors in the flocculated material.
These organisms were not found in the water, whereas we had total
destruction of Klebsiella within 5 minutes in the water and floe.
It cannot be ascertained if the £. coli were more resistant or
somehow became entrained in the flocculated material, thus
contributing to their survival. Additionally, studies by Dugway
Proving Ground^^ on Poliovirus 1 (Chat strain) and simian
rotavirus (SA-11 Strain) Indicated that virus disinfection was
not entirely adequate. In their study, using the EPA guide
standard and protocol, they attained over the required 4-log
removal of rotavirus, but only achieved several logs removal of
Poliovirus 1. Poliovirus removals ranged from 2.5 to 2.7 logs at
5 *C using two CHLOR-FLOC tablets within 40 minutes. Companion
studies with two iodine tablets showed only 1.0 to 2.2 log
42
removals in a similar time period. It cannot be determined why
there was such a discrepancy in virus disinfection between our
efforts and those of the Dugway Proving Ground. It is possible
that the strain of poliovirus they were using was more resistant
to the CHLOR-FLOC than the echovirus strain of our Laboratory.
Also, it is not knovm what procedure they used for preparing
their stock virus; possibly, their virus preparation had a high
ratio of large virus clumps, which is known to change the
disinfection kinetics of similar enteroviruses due to the
protective effects of the outer virions for the inner ones of the
aggregates .
The physical removal of the Crvptospor id ium oarvum oocysts
by the CHLOR-FLCC system indicated less than required removals of
10^ cysts. This was also noted for the latex beads (simulant of
cyst particles) . It was apparent that the coagulant/filtration
component did not work effectively in providing either adequate
size floes or small enough filter matrix to trap the cyst and
simulant particles. As would be expected, however, the worse the
challenge water quality, the better the floe formation and the
better the cyst-size particle removal. Similarly, the increased
temperature experiments showed slightly improved removals
probably because of improved flocculation. Maintenance of pH 9.0
did allow the removals to approach the 10^ target for both cysts
and simulant. The relative removal of the simulant latex beads
was very comparable to that for oocysts under all of the
challenge conditions and appeared to be a good model for cyst
removal.
2. UA PHASE I STUDY
Cr VDtospor id ium infection was noted in all groups of
neonatal mice given oral doses of oocysts treated by either
CHLOR-FLOC (not filtered through flannelette material) or
Globaline water disinfectants. The results demonstrated that the
activity of these chemicals used to treat the CrvDtosporidium-
seeded test waters did not achieve 3-log reductions in oocyst
viabilities. The high level of infection observed in the groups
challenged with the low dose also may allow the ruling out of a
2-log reduction. There may have been a reduction of less than 2
logs of infective oocysts with CHLOR-FLOC, but the challenge dose
levels used in the tests tend to mask any reduction of lower
magnitude. During microscopic examination, a few experimental
histology specimens were observed to have lower levels of
infection than others. This phenomenon was not seen in any of
the control specimens which all showed heavy Infection. Reduced
levels of cysts in the animal specimens may have indicated some
level of degradation in the cyst integrity by the CHLOR-FLOC,
thus leading to reduced levels of infection in the animal host.
Infectivity studies with the Globaline disinfectant containing
iodine indicated no susceptibility of the oocysts to the
disinfectant at either low or high doses. Also, there was no
43
indication of reduced infectivity level in histological samples
for those mice fed Globaline-treated oocysts. The results would
indicate that neither CHLOR-FLOC nor Globaline were effective
disinfectants for Cryptosporidium oocysts, and that neither could
approach a 3-log reduction in infectivity as required in the
USEPA's "Guide Standard and Protocol for Testing Microbiological
Water Purifiers."
3. UA PHASE II STUDY
The results of the phase II tests revalidated the very
minimal capabilities of CHLOR-FLOC to disinfect Cryptosporidium
oocysts in either distilled water, tapwater, or worst case water
at 5 and 10 ’C. Also the parallel tests of infectivity using the
OW64 HAb monoclonal antibody against the oocyst suture (which
show up after loss of viability of the cysts) indicated a very
low percentage of oocysts positive for this suture directed
antibody. The results also indicated that physical removal of
oocysts by the coagulation-flocculation process and filtration
through flannelette material had little capability to physically
remove oocysts. The experimental CHLOR-FLOC reaction bags tended
to not allow proper settling of the floe that formed by
coagulation; thus, the bulk of this material containing oocysts
could not properly be voided prior to the filtration step.
The overall study resulrs revealed that the CHLOR-FLOC
system, as presently configured, was not adequate to physically
remove, or provide adequate chemical disinfection of,
Cryptosporidium oocysts to the required levels of 99.9 percent
reduction recommended by the USEPA's "Guide Standard and Protocol
for Testing Microbiological Water Purifiers." Improved removal
of floe and filtration would significantly enhance the ability of
the CHLOR-FLOC to physically remove protozoan cysts and thus help
meet the criteria. The disinfection of oocysts by io<^ine was
negligible, thus indicating that some sort of filtration or other
procedure would be necessary to ensure that the oocysts were
physically removed before consumption.
44
CONCLUSIONS
1. The results support the efficacy of major components of the
USEPA guide standard and protocol for the evaluation of the
disinfection/removal effectiveness of CHLOR-FLOC.
2. CHLOR-FLOC will provide adequate removals of typical
waterborne test indicator bacteria and the enteroviruses at low
temperatures, even when water is maintained at high pH.
3. The bacterial and virus removal results support the USEPA 's
acceptance of this chemical disinfectant/coagulant mixture for
recreational and emergency disinfection of water.
4. While no direct comparison of CHLOR-FLOC against calcium
hypochlorite or iodine tablet disinfection was made, results from
previous contract studies^^ for iodine disinfection indicate
CHLOR-FLOC provides at least equal or better results for viruses.
5. The physical removal of Cryptosporidium parvum cysts and
3.7 iim AccuBead"* cyst simulant did not continuously meet the
reductions required by the USEPA guide standard.
6. If it were necessary for the Army to use CHLOR-FLOC at this
time, improvements in the effectiveness of water filtration after
CHLOR-FLOC treatment (such as filtration through 3.0 ^m absolute
rated pore size filters) would provide the increased cyst removal
capabilities needed to meet field use considerations.
45
Appendix 1
EXCYSTATION OF CRYPTOSPORIDIUM OOCYSTS
Materials: Freshly washed oocysts (Pelleted in a 15 ml conical tube)
tissue Culture PBS (TPBS)
2x Excystation Medium (Frozen medium must be allowed to reach room
temperature before use.)
Procedure: (Note; This procedure may be adapted for use with very small volumes. In
this case, use 1 .5 ml Eppendorf tubes, wash with 1 mi TPBS, centrifuge at setting #7,
aspirate down to 1 00 ^1. and add 1 00 m* of 2x excystation medium.
1 . Carefully add 5 ml of TPBS to the freshly washed oocysts without disturbing the
pellet. Centrifuge at 3000 rpm for 3*5 minutes. Repeat this step once. (Note:
centrifuge at 3000 rpm for 10 minutes if the pellet was disturbed.)
2. Aspirate down to 0.5 ml, trichurate with a pasteur pipette to break up the pellet,
and bring the volume to 1 .0 ml with 2x excystation medium. Place in 37* C water
bath for 60 minutes. Remove from the water bath and let the sample sit at room
temperature for 30 minutes before counting. Place on ice if counting cannot
proceed immediately.
3. Count and record at least 200 intact cysts + shells and the sporozoites in the
same area of the counting chamber. Repeat this once using another sample from
the excystation mixture.
4. To calculate the percent excystation, divide the number of shells by the sum of
intact cysts and shells and multiply by 100. Calculate the number of sporozoites
produced per shell by dividing the number of sporozoites by the number of shells.
Percent Excystation - - Shelte - ^ ^qq
Intact Cysts + Shells
Sporozoites per Shell = Sporozoites
Shells
4. Calculate the percent theoretical sporozoite yield by dividing the number of
sporozoites observed by 4x the sum of the number of shells and the number of
intact cysts.
Percent Theoretical
Sporozoite Yield
^ Sporozoites ^ ^00
4(lntact Cysts + Shells)
Ref: Woodmansee. 1987. J. Protozool. 34(4):398*402.
Appendix 2
Infectivity v Sporozoite Yield
Log Change Sporozoite Yield
Regression line; y ■ 1.39(x) - 0.066
r • 0.995. P«0.004n
REFERENCES
1. ERA Registration No. 57425“!, CHLOR-FLOC. U.S. Environmental
Protection Agency, Office of Pesticide Programs, Registration
Division (TS-767) , Notice of pesticide registration. 1988. U.S.
Environmental Protection Agency, Washington, DC.
2. Report of Task Force. “987. Guide standard and protocol for
testing microbiological water purifiers. U.S. Environmental
Protection Agency, Office of Drinking Water and Office of
Pesticide Programs, Washington, DC.
3. Sharp, D.G. and J.D. Johnson. February 1977. inactivation
of viruses in water by bromine and its compounds: Influence of
virion aggregation. Final Technical Report ADA048622. Contract
No. DAMD-17-74-C-4013 , University of North Carolina, Chapel Hill,
NC: Contractor.
4. Waldman, E. , S. Tzipori, and J.H.R. Forsyth. 1986.
Separation of Cryptosporidium species oocysts from feces by using
a Percoll discontinuous density gradient. J. Clin. Micro. 23(1):
199-200.
5. Arrowood, fl.J., and C.R. Sterling. 1987. Isolation of
Cryptosporidium oocysts and sporozoites during discontinuous
sucrose and isopycnic Percoll gradients. J. Parasitol. 73;
314-319.
6. Novak, S.M. and C.A. Sterling. 1991. Susceptibility
dynamics in neonatal BALB/c mice infected with Cryptosporidium
EfiJUaUD. J. Protozool. 38 (6) : 102s-104s.
7. Korich, D.G., J.R. Mead, M.S. Madore, N.A. Sinclair, and C.R.
Sterling. 1990. Effects of ozone, chlorine dioxide, chlorine,
and roonochloramine on Cryptosporidium parvum oocyst viability.
AppI. Environ. Microbiol. 56(5) : 1423-1428.
8. Woodmansee, D.B. 1987. studies of in vitro exeystation of
Cryptosporidium parvum from calves. J. Protozool. 34(4):
398-402.
9. C.R. Sterling. Personal communication with Dr. S. Schaub.
Subject: Use of innovative methods for the determination of
Cryptosporidium oocyst viability. 1990.
10. Powers, E.M. Letter, Subject: Bacteriological efficacy of
iodine tablets, (Globaline) compared to CHLOR-FLOC tablets.
1990.
48
11. Powers, E.M. Personal conununicatlon with Dr. S. Schaub,
Subject: Residual viability of coliforn indicator organisms in
CHLOR-FLOC floe. 1991.
12. Harper, B.G. , A.K. Schwedler, D.R. winters, and I.G.
Resnick. March 1991. Final test record report for virucidal
efficacy of developmental water purification tablets. TECOM
Project No. 8-EG-225-WPT-001. Life Sciences Division, Materiel
Test Directorate, U.S. Army Dugway Proving Ground, Dugway, UT.
13. Sobsey, M.D. June 1990. Annual and Final Report,
Inactivation of Hepatitis A virus (HAV) by chlorine and iodine in
water. Annual and final report. Contract No. DAMD17-86-C-6053 .
University of North Carolina at Chapel Hill, Chapel Hill, NC:
Contractor.
49
DISTRIBUTION
No. of
4 Commander
U.S. Army Medical Research and Development Command
ATTN: SGRD-RMI-S
Fort Detrlck
Frederick, MD 21702-5012
2 Defense Technical Information Center
ATTN: DTIC-FDAC
Cameron Station
Alexandria, VA 22304-6145
3 Commander
U.S. Army Medical Department Center and School
ATTN: HSMC-FC
Fort Sam Houston, TX 782,34-6100
2 Commander
U.S. Army Biomedical Re.m4<^ch and Development
Laboratory .■
ATTN: SGRD-UBZ-IL '
Fort Detrick
Frederick, MD 21702-5010
1 Commander
U.S. Army Biomedical Research and Development
Laboratory
ATTN : SCRD-UBZ-P
Fort Detrlck
Frederick, MD 21702-5010
1 HQDA (SGPS-PSP)
5109 Leesburg Pike
Falls Church, VA 22041-3258
1 HQDA (DALO-TSE-W)
The Pentagon, Room 1D600
Washington, DC 20310-0561
1 Commander
U.S. Amy Environmental Hygiene Agency
ATTN : HSHB-ME-HR
Aberdeen Proving Ground, MD 21010-5422
CoBunander
U.s. Army Natick Rasearch, Development and
Engineering Center
ATTN: STRNC-WEB
Natick, MA 01760-5018
Commander
U.S. Army Belvoir Research, Development and
Engineering Center
Fuel and Water Supply Division
ATTN: STRBE-FSE
Fort Belvoir, VA 22060-5606
Commander
U.S. Army Special Warfare Center and School
ATTN: ATSU-CD-ML-M
Fort Bragg, NC 28307-5000
Commander
U.S. Army Quartermaster School
ATTN : ATSM-CDM
Fort Lee, VA 23801-5000
Commander
U.S. Naval Civil Engineering Laboratory
Code L-66
Port Hueneme, CA 93046
Commander
U.S. Air Force Engineering and Services Center
ATTN: DEOP
Tyndall Air Force Base, FL 32403-6001