United States
                   Environmental Protection
                   Agency	
Risk Reduction
Engineering Laboratory
Cincinnati OH 45268
                   Research and Development
 EPA/600/S2-89/019  Mar. 1990
&EPA         Project Summary

                   Protozoan  Sources of
                   Spontaneous  Coliform
                   Occurrence  in  Chlorinated
                   Drinking Water
                   Emmett B. Shotts, Jr. and Richard E. Wooley
                     The spontaneous occurrence  of
                   conforms  in chlorinated drinking
                   waters has resulted in concern over
                   their potential source  and mecha-
                   nism(s) of Introduction into water de-
                   livery systems.
                     Previous observations  related  to
                   protozoa!  resistance  to  chlorine
                   coupled  with  the  ingestion  of
                   bacteria by this group of organisms
                   prompted  an  in-depth study  of
                   bacterial-protozoan interaction in the
                   presence of chlorlnatlon.
                     Bacterial organisms  used  were
                   either from previous "spontaneous
                   coliform outbreaks"  or potential
                   human pathogens. The protozoans
                   utilized were laboratory adapted and
                   wild  strains of  protozoans found
                   naturally In water reservoirs.  These
                   organisms were established In axenic
                   cocultures and the effect of various
                   concentrations of  chlorination and
                   exposure were determined.
                     It was  found  that,  alone, the
                   bacteria were killed by from 0.25-1
                   mg/L of  chlorine; however, when
                   ingested  by protozoans, these  same
                   bacteria survived in significant con-
                   centrations from levels of chlorina-
                   tion 4 to 10 times the above dose.
                     These findings provide a possible
                   and plausible  explanation  of how
                   bacteria may survive chlorination and
                   appear in water delivery systems.  It
                   further suggests a need for increased
                   awareness  in developing improved
                   methods  of processing  drinking
                   water that  include  the  removal of
both viable and dead protozoans from
entering the distribution system.
  This Project Summary was  devel-
oped by EPA's Risk Reduction Engi-
neering Laboratory, Cincinnati,  OH, to
announce key findings of the research
project that Is fully documented in a
separate report of the same title (see
Project Report ordering Information at
back).

Introduction
  The spontaneous occurrence of coli-
forms in  chlorine-treated drinking water
has generated a great deal of concern,
particularly in areas where reservoir water
is  sand filtered  and chlorinated. Prior
work on  this problem addressed  the
production of chlorine resistant clones of
bacteria and also the inability to  readily
isolate chlorine-injured bacteria.  It was
the purpose of this investigation  to
related the sparing  and  enhancement
qualities noted in Legionella-Tetrahymena
interaction into this emerging problem in
water delivery systems. This was ac-
complished by evaluating the bacterial
(coliforms isolated from outbreaks)-proto-
zoan (both laboratory and  wild strains)
relationship  to  determine if ciliated
protozoans that naturally occur in water
impoundments could provide protection
against chlorination for engulfed bacteria,
thus allowing the bacteria to be released
spontaneously by the protozoan at  a later
time in the water distribution system. To
achieve this, techniques were used that
involved enumeration of bacteria  follow-
ing protozoal engulfment with subsequent

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chlorination. Chlorination was done  on
aliquots both before sonication and after
sonication,  respectively,  thus  providing
appropriate controls. From this research,
it  was  determined that  protozoa did
provide protection against chlorination to
engulfed coliform  bacteria,  in  some
cases, for  at  least 24 hr  from initial
exposure  of  2-4  mg/L of  chlorine
although protection to exposures of  20
mg/L  of chlorine was noted  in  some
interactions.

Materials and Methods
  This  investigation examined  the
effects of free and combined  chlorine
upon selected protozoan-bacterial cocul-
tures  that  might  be  found  in  drinking
water  resources.  Comparative  inactiva-
tion studies were done on bacteria alone
and in cocultures with protozoans under
chlorine  demand-free conditions that
simulated those of routine  water treat-
ment.  Selected  coliforms  previously
recovered  from  "spontaneous occur-
rences"  in  water distribution  systems
were obtained from the Risk Reduction
Engineering Laboratory, USEPA, Cin-
cinnati,  OH.  These include Klebsiella
pneumonia, K.  oxytoca,  Enterobacter
cloacae, E. agglormerans,  Citrobacter
freundii  and  Escherichia  coli.  Other
organisms  subsequently  used  included
Campylobacter jejuni, Legionella gor-
manii,  Salmonella typhimurium, Yersinia
enterocolitica, and Shigella sonneii.
  These bacteria were   studied  in
coculture with  Tetrahymena pyriformis
(primary organism used  because of its
ease  in handling) (ATCC 30327), Acan-
thamobea castellanii (ATCC 30234), and
Bodo  edax  (ATCC   30903).  Axenic
cultures  of the latter  resulted  in limited
success; subsequent engulfment studies
were not fruitful and it  was removed from
the study.  Near the end  of  the study, a
wild strain  of Cylidium was studied  in
coculture with £ coli to establish that the
relationship extended  beyond laboratory
strains of protozoans.
   All  studies were  carried out using
chlorine  demand free  buffer (CDFB) and
were  considered as  chlorine  demand
free  (CDF)  when  the  chlorine con-
centration was less than 0.01 mg/L free
available chlorine as  measured by DPD
colorimetric method  read  in a B & L
Spectronic 2000 spectrophotometer.* All
chlorine estimations  were  standardized
to  chlorine quality control samples from
 "Mention of trade names and commercial products
  does not constitute  endorsement  or  recom-
  mendation for use
USEPA,  Environmental Monitoring Sys-
tems Laboratory, Cincinnati.
  Subsequent study of the experimental
components  (i.e.,  protozoa and bacteria
alone) were done prior  to coculture
studies where concentrations of 1 x 104
of the respective bacteria and protozoan
were allowed to interact.
  Following  exposure  to  various con-
centrations of chlorine  at time intervals,
bacterial  viability  and numbers  were
determined  by plate counts  using mT7
agar where possible to permit maximum
recovery of possible  chlorine-damaged
cells.
  Prior to plating, the chlorine containing
suspension of coculture was  neutralized
with sodium thiosulfate, centrifuged, and
resuspended in  sterile culture  medium
for  sonication to release the  bacteria
from the protozoa.
  Samples were obtained  and observed
for  engulfment, vacuole formation, and
multiplication  by electron microscopy
(EM).

Results
  As noted in Table 1,  a great variability
existed  among bacteria isolated  as  to
their survival within respective protozoan
hosts. It was readily established that  all
bacteria  unprotected  by  protozoan
engulfment  were  dead at  1 mg/L  of
chlorine  or  less.  The  protozoa, on the
other hand, were viable after 2 to 5 times
this chlorine dose. These  data indie
that although Tetrahymena was  killec
2 mg/L of  chlorine, the bacteria con
ued to  replicate  within the  protozc
Similarly, the same thing  happened
the Acanthamoeba, and, although re
tance to chlorine was not demonstra
in the wild  type Cylidium, viable bact<
were  present after 24 hr.  It should
noted that  in no case were more than
10  organisms  noted  ingested  in
protozoan,  yet  large  percentages
bacterial survival were obtained  p
viding  evidence of bacterial replicat
within both the viable and  dead prc
zoans.

Discussion and Conclusions
  These studies have  demonstrated
alternate explanation  to  chlorine res
tance  as  a mechanism  of bactei
introduction into water delivery  syster
This  study  further  demonstrates tl
although protozoans may be killed duri
chlorination, viable bacteria  may still
introduced  into water delivery systems
  These occurrences have  been denru
strated  not only  with  isolates frc
previous spontaneous  coliform  occi
rences  but also  with  several  prims
human  pathogens of  gastrointestir
disease and  also  with  Leg/one
gormanii. In the  latter  case, the inti
duction  of viable  organisms  into  t
delivery system with subsequent temp*
 Table  1.   Relative  Survival of Selected Bacteria Engulfed by  Protozoans and Exposec
           Varying Amounts of Chlorination for 24 Hours.*
                                              Protozoans*
Bacteria&
K. pneumonia
K. oxytoca
Ent. cloacae
Ent. agglormerans
Cit. freundii
Esc. coli
Camp, jejuni
Leg. gormanii
Sal. typhimurium
Yer. enterocolitica
Shig. sonneii
Tetrahymena
pyriformis
2/20*
1120
0.51100
2/70
1/30
4/60
0.25/10
4/50
2/65
4/100
41100
Acanthamoeba
castellanii
10/80
4/20
2/80
2/100
0/0
1/100
-@
—
Cylidium
spp.
-
:
—
-
—
 'Percentage survival of engulfed bacteria at highest chlorine concentrations following 24
  exposure.
 Tetrahymena resisted 1 mg/L chlorine for 24 hr; Acanthamoeba resisted 10 mg/L chlorine
  24 hr.
 &AII bacteria (alone) were killed  at 1 mg/L chlorine except for C. jejunt and Ent. cloacae (0
  mg/L chlorine).
 * First number denotes highest concentration of chlorine in mg/L showing surviving bacterii
  24 hr; second number represents the percentage of the original bacterial inoculum (1 x I
  surviving.
 @Not done

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ature selection in hot water tanks could
provide  an  explanation related  to the
spread of this disease  in bath waters. In
other studies, L pneumophila has  been
shown to act similarly.
  From the standpoint  of gastrointestinal
disease, it is  suggested  that, without
further processing, drinking water that is
sand filtered  and chlorinated for  human
use  presents a  potential  source  of
infection. This risk could be reduced by
further processing of the water prior to
distribution.
  In  summary, a mechanism present in
nature has been  demonstrated whereby
bacterial-protozoan interaction negates
usual methods of water treatment for
many  supply systems  and, despite
chlorination, presents the possibility  of
the introduction of measurable amounts
of potentially  pathogenic bacteria into
drinking water delivery systems.
  The full  report was submitted  in
fulfillment of  CR812833-01 by the Uni-
versity of Georgia Research  Foundation
under the sponsorship of the U.S.  Envi-
ronmental Protection Agency.

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  Emmett B. Shotts, Jr. and Richard E. Wooley are with the University of Georgia,
   Athens, GA 30602.
  Eugene W. Rice and Harry 0. Nash are the EPA Project Officers (see below).
  The complete report, entitled " Protozoan Sources of Spontaneous Coliform Oc-
   currence in  Chlorinated Drinking Water,"(0rder No.  PB 89-190 292IAS; Cost:
   $13.95, sub/ect to change) will be available only from:
         National Technical Information Service
         5285 Port Royal Road
         Springfield, VA 22161
         Telephone: 703-487-4650
  The EPA Project Officers can be contacted at:
         Risk Reduction Engineering Laboratory
         U.S. Environmental Protection Agency
         Cincinnati, OH 45268
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
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