LT2ESWTR
Toolbox
Guidance
Manual
Proposal
Draft
June
2003
A­
1
Appendix
A
Site
Specific
Determination
of
Contact
Time
for
Chlorine
Dioxide
and
Ozone
A
water
system
may
perform
a
site
specific
study
to
generate
a
set
of
chlorine
dioxide
or
ozone
CT
values
for
that
site
if
it
believes
those
developed
by
EPA
do
not
reflect
the
true
inactivation
achieved.
Such
a
study
would
involve
measuring
actual
Cryptosporidium
inactivation
under
site
conditions,
with
a
full
range
of
temperature
and
contact
times.
If
accepted
by
the
State,
the
CT
values
may
be
used
instead
of
those
developed
by
EPA.

The
LT2ESWTR
does
not
specify
any
requirements
for
the
chlorine
dioxide
or
ozone
sitespecific
study,
only
that
it
be
approved
by
the
State
(
40
CFR
141.729(
b)(
3)
and
(
c)(
3)).
This
appendix
describes
the
different
elements
of
a
study
and
discusses
some
of
the
issues
involved
in
the
statistical
analysis
of
the
results.

A.
1
Experimental
Design
Experiments
should
be
conducted
with
water
that
is
representative
of
the
water
to
be
treated
with
respect
to
all
conditions
that
can
affect
Cryptosporidium
inactivation.
Inactivation
experiments
should
be
performed
with
water
exerting
the
highest
oxidant
demand
(
i.
e.
spring
run­
off
or
summer
conditions)
at
high
temperature
to
obtain
the
worst­
case
scenario
in
terms
of
chlorine
dioxide
or
ozone
demand/
decay
rate.
In
addition,
experiments
should
also
be
conducted
with
water
obtained
during
the
winter
months
at
the
lowest
temperatures
observed
at
the
treatment
plant.
These
experiments
would
allow
for
the
determination
of
the
highest
CTs
that
would
be
necessary
to
achieve
the
required
level
of
inactivation.
Additional
experiments
may
be
necessary
to
characterize
the
effects
of
other
water
quality
parameters.

In
order
to
obtain
the
most
challenging
water
to
assess
the
chlorine
dioxide
or
ozone
process,
a
predetermined
testing
schedule
should
be
established
based
on
source
water
TOC
and
UV
254
levels.
Testing
can
occur
when
source
water
values
for
these
parameters
fall
within
defined
worst­
case
ranges.
Experiments
should
then
be
performed
in
the
laboratory
at
worst­
case
temperatures
for
a
given
month.

In
order
to
obtain
a
complete
data
set,
testing
should
occur
at
least
every
other
month
over
the
course
of
an
entire
year.
Each
sample
date
should
be
determined
by
the
first
time
the
TOC
or
UV
254
levels
are
within
75
percent
of
the
maximum
historical
value
for
that
month.
At
the
time
of
sampling,
sufficient
water
should
be
acquired
to
allow
for
three
sets
of
experiments
to
be
conducted,
with
each
experiment
having
six
data
points
(
CT
values)
and
a
control.
Two
independent
sets
of
experiments
should
be
conducted
with
the
water.
Should
significant
discrepancies
develop
between
the
data
sets,
a
third
set
of
experiments
would
need
to
be
conducted.
An
example
experimental
matrix
is
provided
in
Table
A.
1.
Appendix
A
­
Site
Specific
Determination
of
Contact
Time
for
Chlorine
Dioxide
and
Ozone
LT2ESWTR
Toolbox
Guidance
Manual
Proposal
Draft
June
2003
A­
2
Table
A.
1
Example
Experimental
Test
Matrix
Date
Temperature
to
be
Tested
(
Historical
Record)
Water
Quality
Criteria
Schedule
of
Experiments
Test
1
Test
2
Test
3
February
Lowest
Annual
TOC
or
UV
254
>
75%
of
max
historical
value
X
X
If
Required
April
Highest
in
April
Same
X
X
If
Required
June
Highest
in
June
Same
X
X
If
Required
August
Highest
Annual
Same
X
X
If
Required
October
Highest
in
October
Same
X
X
If
Required
December
Lowest
in
December
Same
X
X
If
Required
A.
2
Experimental
Procedure
A.
2.1
Preparation
of
oocysts
High
oocyst
quality
is
imperative
to
the
success
of
the
study
because
sub­
standard
oocysts
could
dramatically
affect
the
data
in
a
way
that
would
underestimate
the
CT
required
to
achieve
a
desired
level
of
inactivation.
Traditionally,
Cryptosporidium
parvum
oocysts
are
derived
from
two
host
sources,
bovine
and
rodent.
The
most
common
strain
of
Cryptosporidium
parvum
used
to
date
is
the
Iowa
strain,
developed
by
Dr.
Harley
Moon.
It
is
recommended
that
the
utility
perform
all
experiments
using
fresh
(<
1
month
old)
Iowa­
strain
oocysts
obtained
from
a
reputable
supplier.
The
utility
should
ensure
that
after
purification
the
supplier
stores
the
oocysts
at
4

C
in
a
solution
of
dichromate
or
0.01
M
phosphate
buffer
saline
solution
(
pH
7.4)
containing
two
antibiotics
(
1,000
U/
mL
penicillin,
and
1,000
mg/
mL
streptomycin),
and
an
antimycotic
(
2.5
mg/
mL
amphotericin
B).
The
oocysts
should
be
shipped
in
a
cooler
on
ice
to
the
utility
via
nextday
service.
Upon
arrival,
the
oocysts
should
be
placed
in
a
refrigerator
and
stored
at
4

C
until
needed.

When
ready
for
use,
the
oocysts
should
be
suspended
in
0.01
M
pH
7
buffer
and
centrifuged
at
a
relative
centrifugal
force
of
approximately
1,100
for
at
least
10
minutes.
Appendix
A
­
Site
Specific
Determination
of
Contact
Time
for
Chlorine
Dioxide
and
Ozone
LT2ESWTR
Toolbox
Guidance
Manual
Proposal
Draft
June
2003
A­
3
Following
centrifugation,
the
oocysts
should
be
aspirated
and
re­
suspended
in
the
buffer,
then
centrifuged
again
at
the
same
conditions.
This
step
should
be
repeated
once
more
to
remove
as
much
of
the
antibiotic
or
dichromate
solution
as
possible.
Following
the
last
aspiration,
the
oocysts
should
be
re­
suspended
in
approximately
10
mL
of
the
pH
7
buffer.
The
oocysts
should
then
be
stored
at
4

C
until
the
experiment
is
initiated.
The
oocysts
should
be
vortexed
thoroughly
prior
to
initiation
of
the
experiment.
Additional
details
regarding
this
procedure
can
be
found
in
Rennecker
et
al.
1999.

A.
2.2
Source
Water
Preservation
Testing
should
be
conducted
as
close
as
possible
to
the
date
that
the
experimental
water
is
collected.
If
testing
is
to
be
performed
at
a
location
other
than
the
utility
where
the
water
was
collected,
the
water
should
be
sent
to
the
laboratory
via
an
overnight
delivery
service
and
stored
at
4
degrees
Celsius
until
the
start
of
testing.

A.
2.3
Experimental
Apparatus
A.
2.3.1
Chlorine
Dioxide
It
is
recommended
that
chlorine
dioxide
be
generated
using
the
equipment
and
procedures
outlined
in
Standard
Methods
for
the
Examination
of
Water
and
Wastewater,
APHA
1998.
With
this
as
a
basis,
all
inactivation
experiments
using
chlorine
dioxide
should
be
performed
using
a
batch­
reactor
configuration.
An
example
of
such
a
system
is
provided
by
Ruffell
et
al.
2000.
This
system
uses
an
enclosed
recirculating
water
bath
to
maintain
the
desired
temperature
inside
the
reactor
vessels,
which
consist
of
2­
liter
amber
glass
bottles.
During
the
experiment,
care
should
be
taken
to
minimize
the
exposure
of
the
reactors
to
light.
Mixing
of
the
reactor
contents
is
provided
with
a
magnetic
stir
bar
and
stir
plate.

A.
2.3.2
Ozone
Inactivation
experiments
can
be
performed
with
either
a
semi­
batch
or
batch
reactor
configuration.
When
performing
experiments
with
a
semi­
batch
system,
it
is
recommended
that
analytical
components
similar
to
those
described
by
Hunt
and
Mariñas
(
1997)
be
used.
Using
this
system,
the
reactor
vessel
containing
the
experimental
water
is
maintained
at
the
experimental
temperature
by
immersion
in
a
water
bath.
Ozone
can
be
generated
from
either
compressed
air
or
oxygen
and
passed
through
a
continuously­
stirred
glass
bottle,
which
serves
to
dampen
the
effect
of
fluctuating
ozone
concentration.
The
ozonated
gas
leaving
the
dampening
bottle
is
then
introduced
to
the
experimental
water
via
a
fine­
bubble
diffuser.
The
ozonated
water
is
stirred
continuously
using
a
magnetic
stirring
plate
and
a
stir
bar.
Appendix
A
­
Site
Specific
Determination
of
Contact
Time
for
Chlorine
Dioxide
and
Ozone
LT2ESWTR
Toolbox
Guidance
Manual
Proposal
Draft
June
2003
A­
4
It
is
recommended
that
inactivation
experiments
performed
using
a
batch
reactor
configuration
use
analytical
components
similar
to
those
described
by
Kim
(
2002).
This
reactor
used
a
100­
mL
gas­
tight
syringe
to
prevent
ozone
in
solution
from
volatilizing
into
the
atmosphere.
The
temperature
inside
the
reactor
is
held
constant
by
immersion
in
a
recirculating
water
bath,
and
mixing
is
provided
by
a
stir
bar
in
the
syringe
controlled
by
a
magnetic
stir
plate.
Ozone
can
be
produced
from
either
compressed
air
or
oxygen.
A
concentrated
ozone
stock
solution
should
be
prepared
using
distilled
de­
ionized
or
reverse
osmosis­
filtered
water.

Other,
less
complex,
batch
reactor
systems
are
also
available
which
simply
use
an
open
vessel
such
as
an
Erlenmeyer
flask
or
beaker
(
Finch
et
al.
1993a).
With
these
systems,
the
reactor
containing
the
experimental
water
is
typically
maintained
at
the
desired
temperature
using
a
water
bath.
An
ozonated
solution,
prepared
with
distilled
de­
ionized
or
reverse
osmosis
water,
is
added
to
the
experimental
water,
and
the
ozone
dose
is
measured
from
the
diluted
experimental
water.
When
using
this
type
of
batch­
reactor
configuration
that
is
open
to
the
atmosphere,
the
user
should
take
into
account
that
ozone
is
lost
to
volatilization.
This
loss
of
ozone
should
be
considered
and
minimized
when
performing
any
inactivation
or
demand/
decay
experiment.

A.
2.4
Inactivation
experiments
The
CT
values
obtained
from
each
of
the
site­
specific
inactivation
experiments
are
expected
to
be
similar
to
those
provided
in
the
standard
LT2ESWTR
tables.
Therefore,
utilities
wishing
to
determine
site­
specific
inactivation
data
are
advised
to
use
the
standard
tables
as
a
baseline.
Each
experiment
should
be
designed
such
that
six
data
points
span
the
range
of
the
"
standard"
inactivation
curve
for
a
given
temperature.
One
"
control"
point
with
no
disinfectant
should
also
be
taken.

A.
2.4.1
Chlorine
Dioxide
An
experimental
protocol
developed
from
Ruffell
et
al.
2000
is
provided
here
as
an
example.
The
reactor
bottle
should
be
filled
with
experimental
water
to
a
total
volume
corresponding
to
the
desired
sample
volume
times
the
number
of
samples
expected
per
bottle
(
6
is
recommended).
The
bottle
is
then
placed
in
the
water
bath
and
allowed
to
equilibrate
to
the
target
experimental
temperature.
At
this
point,
chlorine
dioxide
stock
solution
is
added
to
the
reactor
bottle
at
the
target
dose.
The
reactor
bottle
is
then
capped
to
minimize
chlorine
dioxide
volatilization.
The
chlorine
dioxide
concentration
is
measured
approximately
10
min
after
dosing.
An
experiment
was
started
by
adding
approximately
a
pre­
determined
number
of
oocysts
to
the
reactor
that
will
be
sufficient
for
at
least
six
data
points.
Note
the
volume
of
the
oocyst
aliquot
should
be
less
than
1
mL.
Samples
are
then
taken
periodically
at
the
contact
times
that
correspond
to
the
desired
CT.
The
samples
are
immediately
filtered
through
a1
µ
m
filter.
The
filter
is
then
placed
in
a
clean
50
mL
beaker
and
rinsed
with
approximately
15
mL
of
the
dilute
surfactant.
The
resulting
oocyst
suspension
is
transferred
into
a
sterile
15
mL
centrifuge
tube.
Appendix
A
­
Site
Specific
Determination
of
Contact
Time
for
Chlorine
Dioxide
and
Ozone
LT2ESWTR
Toolbox
Guidance
Manual
Proposal
Draft
June
2003
A­
5
These
steps
are
repeated
at
various
contact
times
corresponding
to
target
CT
parameters.
After
the
last
sample
is
taken,
the
chlorine
dioxide
dose
is
measured
again.
"
Control"
samples
are
also
taken
for
each
experiment
by
placing
a
sample
of
oocysts
inside
a
similar
reactor
containing
the
experimental
water
minus
the
disinfectant
at
the
target
temperature.
The
oocysts
are
typically
exposed
to
this
condition
for
the
duration
of
the
experiment
and
subsequently
processed
for
viability
assessment
with
methods
similar
to
those
for
the
disinfected
samples.

A.
2.4.2
Ozone
If
a
semi­
batch
reactor
configuration
is
used,
the
protocol
described
by
Rennecker
et
al.
(
1999)
is
recommended.
The
protocol
is
described
briefly
as
follows.
Ozonated
gas
is
applied
to
the
temperature­
acclimated
experimental
water
via
a
fine
bubble
diffuser.
The
ozone
gas
concentration
is
adjusted
to
achieve
steady­
state
at
dissolved
ozone
concentrations
representative
of
what
would
be
observed
at
the
facility.
The
actual
dissolved
ozone
concentration
achieved
for
each
experiment
is
measured.
Mixing
of
the
ozonated
water
is
performed
with
a
magnetic
stir
bar
and
stirring
plate.
An
inactivation
experiment
is
initiated
by
injecting
a
suspension
containing
a
sufficient
number
of
oocysts
into
the
reactor,
and
ends
by
simultaneously
removing
the
bubble
diffuser
and
injecting
a
quenching
agent.
It
should
be
noted
that
the
number
of
oocysts
necessary
for
each
data
point
is
dependent
on
the
viability
assessment
method
selected.
Oocysts
are
then
removed
from
the
quenched
solution
by
filtration
through
a
1
µ
m
filter.
The
reactor
is
then
rinsed
with
approximately
50
mL
of
a
dilute
surfactant,
and
then
again
with
approximately
100
mL
of
the
experimental
water
to
remove
any
residual
surfactant.
Both
eluents
are
passed
through
the
filter
that
is
then
placed
in
a
clean
50
mL
beaker
and
rinsed
with
approximately
15
mL
of
the
dilute
surfactant.
The
resulting
oocyst
suspension
is
transferred
into
a
sterile
15
mL
centrifuge
tube.
These
steps
are
repeated
at
various
contact
times
corresponding
to
target
CT
parameters
(
i.
e.,
the
product
of
dissolved
ozone
concentration
and
contact
time).

Control
samples
are
prepared
with
each
daily
experimental
set
by
shutting
off
the
ozone
generator,
but
allowing
the
oxygen
gas
to
flow
through
the
system.
Oxygen
gas
is
allowed
to
bypass
the
semi­
batch
reactor
after
shutting
off
the
generator
to
purge
residual
ozone
gas
from
the
system.
All
other
conditions
used
for
the
control
are
consistent
with
the
experimental
conditions
previously
described.
The
"
contact"
time
for
control
samples
is
1
minute.
After
completion
of
the
experiment,
the
samples
are
generally
centrifuged
at
1,100g
for
10
minutes
and
stored
in
a
phosphate
buffer
solution
for
a
period
of
time
not
to
exceed
48
hours
prior
to
viability
assessment
procedures.

Experiments
performed
with
a
head­
space
free
reactor
can
follow
the
following
protocol
(
described
previously
in
Kim
2002).
The
experimental
temperature
is
maintained
by
immersing
the
100­
mL
syringe,
which
serves
as
the
reactor
in
a
water
bath.
Mixing
inside
the
reactor
is
provided
using
a
stir
bar
and
magnetic
stir
plate.
The
syringe
is
filled
with
the
experimental
water
containing
enough
oocysts
for
all
six
data
points.
At
this
point,
an
aliquot
of
temperature­
Appendix
A
­
Site
Specific
Determination
of
Contact
Time
for
Chlorine
Dioxide
and
Ozone
LT2ESWTR
Toolbox
Guidance
Manual
Proposal
Draft
June
2003
A­
6
adjusted
ozone
stock
solution
of
known
concentration
is
added.
Samples
are
then
taken
at
time
intervals
corresponding
to
the
pre­
determined
estimated
CT
using
a
syringe
containing
a
quenching
reagent.
The
samples
are
then
processed
using
filtration
and
centrifugation,
similar
to
those
described
above.
A
"
control"
should
be
performed
for
each
experiment
by
placing
the
sample
number
of
oocysts
in
the
experimental
water
at
the
desired
temperature.
The
oocysts
should
remain
there
for
a
period
of
time
equal
to
the
duration
of
the
inactivation
experiment.
After
this
time,
the
oocysts
should
be
processed
in
a
manner
consistent
with
the
disinfected
samples.

Experiments
performed
with
batch
reactor
components
that
are
not
head­
space
free
typically
follow
a
similar,
although
less
complex
protocol.
An
example
of
such
a
system
and
the
associated
experimental
protocol
can
be
obtained
from
Finch
et
al.
1993a.

It
should
be
noted
that
for
all
batch­
reactor
systems,
a
careful
characterization
of
the
ozone
demand
and
decay
kinetics
of
the
experimental
water
should
be
performed
prior
to
any
disinfection
testing.
In
addition,
it
is
also
recommended
that
ozone
concentration
samples
be
procured
alternately
between
inactivation
samples
to
verify
ozone
concentrations
observed
during
the
disinfection
study.

A.
2.5
Sample
Processing
After
procuring
each
sample
point,
the
samples
should
be
stored
at
4

C
until
the
end
of
the
experiment.
At
the
end
of
each
experiment,
the
samples
should
be
centrifuged
at
a
relative
centrifugal
force
of
1,100
for
at
least
10
minutes
to
remove
quenching
agents
or
surfactants.
Following
centrifugation,
the
samples
should
be
carefully
aspirated
and
re­
suspended
in
0.01
M
pH
7
buffer
solution.
The
samples
should
be
stored
at
4
degrees
until
the
time
of
viability
assessment.

A.
2.6
Viability
Assessment
Determining
the
viability
of
oocysts
for
varying
levels
of
disinfection
is
one
of
the
most
critical
components
of
the
inactivation
experiments.
At
present,
there
are
three
methods
available
to
assess
Cryptosporidium
parvum
viability,
each
presenting
unique
advantages
and
disadvantages.
These
methods
include
the
following
techniques:

°
Animal
infectivity
°
Cell
culture
(
in
vitro
infectivity)

°
In
vitro
excystation
Appendix
A
­
Site
Specific
Determination
of
Contact
Time
for
Chlorine
Dioxide
and
Ozone
LT2ESWTR
Toolbox
Guidance
Manual
Proposal
Draft
June
2003
A­
7
The
most
established
of
these
methods
is
animal
infectivity.
This
viability
assessment
method
typically
involves
inoculating
immuno­
suppressed
neonatal
mice
with
varying
numbers
of
oocysts
exposed
to
a
particular
CT.
After
a
certain
"
incubation"
period,
the
mice
are
then
sacrificed
and
their
intestinal
tracts
are
examined
for
signs
of
Cryptosporidium­
induced
infection
(
cryptosporidiosis).
The
primary
benefit
of
this
method
is
that
it
demonstrates
that
the
treated
oocysts
are
capable
of
reproduction
inside
a
mammalian
host
and
therefore
are
able
to
induce
an
infection.
One
criticism
of
this
method
is
that
although
an
infection
is
capable
of
being
observed,
mouse
infectivity
has
not
been
correlated
to
human
infectivity.
In
addition,
the
protocol
associated
with
this
method
is
difficult
and
expensive.
It
requires
specialized
laboratory
training,
facilities,
and
equipment.
An
example
of
this
protocol
can
be
found
in
Finch
et
al.
1993b.

A
second
method
used
to
assess
the
viability
of
Cryptosporidium
parvum
is
known
as
in
vitro
infectivity
or
cell
culture.
At
present,
cell
culture
methodologies
used
for
this
purpose
are
based
on
either
microscopic
evaluation
(
Slifko
et
al.
1997)
or
polymerase
chain
reaction
(
PCR)
(
Rochelle
et
al.
1997).
The
first
step
in
using
cell
culture
to
assess
oocyst
viability
involves
applying
the
treated
oocysts
to
a
lawn
of
cells
(
typically
derived
from
human
or
canine
cell
lines).
After
an
incubation
period,
using
microscopic
evaluation­
based
culture
methods,
the
cells
are
stained
with
fluorescent
chemicals
and
then
examined
microscopically
for
various
cryptosporidium
life
stages.
The
presence
of
these
life
stages
suggests
that
the
oocysts
were
capable
of
reproduction
and
thus
were
viable
and
likely
able
to
cause
an
infection
in
humans.

When
using
a
PCR­
based
technique,
after
incubation
the
cells
are
processed
and
the
Cryptosporidium
parvum
RNA
is
extracted.
Infectivity
is
then
determined
by
targeting
specific
genetic
sequences
in
the
RNA.
The
primary
advantage
of
using
cell
culture
to
assess
Cryptosporidium
parvum
infectivity
is
that
it
can
measure
very
low
concentrations
of
oocysts.
Therefore,
cell
culture
is
capable
of
demonstrating
high
levels
of
inactivation.
In
contrast,
the
disadvantages
associated
with
using
cell
culture
include
a
lack
of
agreement
over
the
preferred
cell
lines
and
viability
assessment
technique.
In
addition,
there
has
been
no
extrapolation
between
cell
culture
techniques
and
human
infectivity.
Lastly,
cell
culture
techniques
are
complex
and
typically
require
specialized
equipment
and
rigorous
training,
which
makes
this
procedure
somewhat
expensive.

A
third
method
known
as
in
vitro
excystation
has
also
been
developed
to
assess
the
viability
of
Cryptosporidium
parvum
(
Rennecker
et
al.
1999).
This
method
involves
exposing
oocysts
to
a
simulation
of
a
mammalian
digestive
tract.
Following
the
simulation,
the
oocysts
are
then
examined
microscopically
for
oocyst
life
stages
that
are
indicative
of
viability.
The
advantages
of
this
method
are
that
it
is
cost­
effective,
offers
the
ability
to
rapidly
develop
data,
and
requires
minimal
training.
The
main
disadvantage
of
the
method
is
that
of
the
three
methods
described,
in
vitro
excystation
has
the
least
similarity
to
an
actual
infection.
However,
it
should
be
noted
that
in
spite
of
this
fact,
two
published
studies
have
shown
that
inactivation
data
Appendix
A
­
Site
Specific
Determination
of
Contact
Time
for
Chlorine
Dioxide
and
Ozone
LT2ESWTR
Toolbox
Guidance
Manual
Proposal
Draft
June
2003
A­
8
obtained
with
in
vitro
excystation
closely
matches
animal
infectivity
and/
or
cell
culture
data
(
Rennecker
et
al.
2000,
Owens
et
al.
1999).

A.
3
Statistical
Analysis
A
general
approach
for
calculating
a
set
of
CT
values
involves
the
following
steps:

1)
Fitting
an
inactivation
model(
s)
to
the
experimental
inactivation
data
(
for
the
entire
year).

2)
Calculating
the
predicted
average
CT
requirements
from
the
best
fit
model.

3)
Calculating
and
applying
a
factor
of
safety
for
the
average
predicted
CT
requirement.

One
approach
by
Clark
et
al.
(
2002)
used
a
one­
parameter
Chick­
Watson
model
to
fit
experimental
data
sets
and
develop
standard
CT
curves,
relative
to
inactivation
level
and
temperature.
As
described
in
the
LT2ESWTR
Preamble,
EPA
used
the
Clark
et
al.
approach
for
developing
CT
values
but
adjusted
the
analysis
to
account
for
different
types
of
uncertainties
and
variability
inherent
in
the
data.
EPA
wanted
to
account
for
variability
among
different
water
matrices
and
oocyst
strains,
but
not
variability
within
the
same
group
(
i.
e.,
same
oocyst
lot
and
water),
and
uncertainty
in
the
regression.
While
such
a
complex
approach
may
not
be
necessary
for
a
site­
specific
study,
the
water
system
should
be
aware
of
the
uncertainties
and
variability
of
the
experimental
data
and
use
a
statistical
method
that
builds
in
a
reasonable
safety
factor
to
ensure
public
health
is
protected.

Two
types
of
confidence
bounds
that
are
commonly
used
when
assessing
relationships
between
variables,
such
as
disinfectant
dose
(
CT)
and
log
inactivation,
are
confidence
in
the
regression
and
confidence
in
the
prediction.
Confidence
in
the
regression
accounts
for
uncertainty
in
the
regression
line
(
e.
g.,
a
linear
relationship
between
temperature
and
the
log
of
the
ratio
of
CT
to
log
inactivation).
Confidence
in
the
prediction
accounts
for
both
uncertainty
in
the
regression
line
and
variability
in
experimental
observations
 
it
describes
the
likelihood
of
a
single
future
data
point
falling
within
a
range.
Bounds
for
confidence
in
prediction
are
wider
(
i.
e.,
more
conservative)
than
those
for
confidence
in
the
regression.
Depending
on
the
degree
of
confidence
applied,
most
points
in
a
data
set
typically
will
fall
within
the
bounds
for
confidence
in
the
prediction,
while
a
significant
fraction
will
fall
outside
the
bounds
for
confidence
in
the
regression.
Appendix
A
­
Site
Specific
Determination
of
Contact
Time
for
Chlorine
Dioxide
and
Ozone
LT2ESWTR
Toolbox
Guidance
Manual
Proposal
Draft
June
2003
A­
9
References
American
Public
Health
Association,
American
Water
Works
Association,
and
Water
Environment
Federation.
1998.
Standard
Methods
for
the
Examination
of
Water
and
Wastewater.
Washington
D.
C.

Clark,
R.
M.;
Sivagenesan,
M.,
Rice;
E.
W.;
and
Chen,
J.
(
2002).
Development
of
a
Ct
equation
for
the
inactivation
of
Cryptosporidium
oocysts
with
ozone.
Wat.
Res.
36,
3141­
3149.

Finch,
G.
R.;
Black
E.
K.;
Gyurek,
L.;
and
Belosevic,
M.
(
1993a).
Ozone
inactivation
of
C.
parvum
in
demand­
free
phosphate
buffer
determined
by
in
vitro
excystation
and
animal
infectivity.
J.
Appl.
Environ.
Microbiol.
59(
12),
4203­
4210.

Finch,
G.
R.;
Daniels,
C.
W.;
Black,
E.
K.;
Schaefer
III,
F.
W.;
and
Belosevic,
M.
.(
1993b).
Dose
response
of
C.
parvum
in
outbred
neonatal
CD­
1
mice.
J.
Appl.
Environ.
Microbiol.
59(
11),
3661­
3665.

Hunt,
N.
K.;
and
Mariñas,
B.
J.
(
1997)
Kinetics
of
Escherichia
coli
inactivation
with
ozone.
Wat.
Res.
31(
6),
1355­
1362.

Kim,
J.
H.;
Tomiak,
R.
B.;
Rennecker,
J.
L.;
Mariñas,
B.
J.;
Miltner,
R.
J.;
and
Owens,
J.
H.
(
2002).
"
Inactivation
of
Cryptosporidium
in
a
Pilot­
Scale
Ozone
Bubble­
Diffuser
Contactor.
Part
I
I:
Model
Verification
and
Application."
ASCE
Journal
of
Environmental
Engineering,
128(
6),
522­
532.

Li,
H.;
Finch,
G.
R.;
Smith,
D.
W.;
and
Belosevic,
M.
(
2000).
Chemical
inactivation
of
Cryptosporidium
in
water
treatment.
AWWA
Research
Foundation,
Denver,
CO.

Owens,
J.
H.;
Miltner,
R.
J.;
Slifko,
T.
R.;
and
Rose
J.
B.
(
1999).
In
vitro
excystation
and
infectivity
in
mice
and
cell
culture
to
assess
chlorine
dioxide
inactivation
of
Cryptosporidium
oocysts.
Proceedings
of
the
AWWA
WQTC
Conference,
Tampa.

Rennecker,
J.
L.;
Mariñas
B.
J.;
Owens
J.
H.;
and
Rice
E.
W.
(
1999)
Inactivation
of
C.
parvum
oocysts
with
Ozone.
Water
Res.
33
(
11),
2481
­
2488.

Rochelle,
P.
A.;
Ferguson,
D.
M.;
Handojo,
T.
J.;
De
Leon,
R.;
Stewart,
M.
H.;
and
Wolfe,
R.
L.
(
1997).
An
assay
combing
cell
culture
with
reverse
transcriptase
PCR
to
detect
and
determine
the
infectivity
of
waterborne
C.
parvum.
J.
Appl.
Environ.
Microbiol.
63(
5)
2029
­
2037.

Ruffell,
K.
M;
Rennecker,
J.
L.;
and
Mariñas,
B.
J.(
2000).
Inactivation
of
C.
Parvum
oocysts
with
chlorine
dioxide.
Wat.
Res.
34
(
3),
868
­
876.
Appendix
A
­
Site
Specific
Determination
of
Contact
Time
for
Chlorine
Dioxide
and
Ozone
LT2ESWTR
Toolbox
Guidance
Manual
Proposal
Draft
June
2003
A­
10
Slifko,
T.
R.;
Friedman,
D.;
Rose,
J.
B.;
and
Jakubowski,
W.
(
1997).
An
in
vitro
method
for
detecting
infectious
Cryptosporidium
oocysts
with
cell
culture.
J.
Appl.
Environ.
Microbiol.
63(
9)
3669
­
3675.
Appendix
A
­
Site
Specific
Determination
of
Contact
Time
for
Chlorine
Dioxide
and
Ozone
LT2ESWTR
Toolbox
Guidance
Manual
Proposal
Draft
June
2003
A­
11
A.
1
Experimental
Design
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
.
.
.
.
.
.
.
.
.
.
.
.
.
A­
1
A.
2
Experimental
Procedure
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
.
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.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
A­
2
A.
2.1
Preparation
of
oocysts
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
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.
.
.
.
A­
2
A.
2.2
Source
Water
Preservation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
A­
3
A.
2.3
Experimental
Apparatus
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
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.
.
.
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.
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.
.
.
.
.
.
.
.
A­
3
A.
2.3.1
Chlorine
Dioxide
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
A­
3
A.
2.3.2
Ozone
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
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.
.
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.
.
.
.
.
.
.
.
A­
3
A.
2.4
Inactivation
experiments
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
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.
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.
.
.
.
.
.
.
A­
4
A.
2.4.1
Chlorine
Dioxide
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
A­
4
A.
2.4.2
Ozone
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
.
.
.
A­
5
A.
2.5
Sample
Processing
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
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.
.
.
A­
6
A.
2.6
Viability
Assessment
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
.
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.
.
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.
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.
.
.
A­
6
A.
3
Statistical
Analysis
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
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.
.
.
A­
8
Table
A.
1
Example
Experimental
Test
Matrix
.
.
.
.
.
.
.
.
.
.
.
.
.
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A­
2
