Document ID: EPA-HQ-OW-2002-0031-0042
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2004-02-10T05:00Z

Public
Comment
and
Responses
for
the
National
Primary
and
Secondary
Drinking
Water
Regulations:
Approval
of
Colitag
 
for
Compliance
Monitoring
of
Total
Coliforms
and
E.
coli
in
Finished
Drinking
Water
U.
S.
Environmental
Protection
Agency
Office
of
Water
Office
of
Ground
Water
and
Drinking
Water
December
2003
Docket
ID
No.
OW­
2002­
0031
2
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
01
RECOMMENDATIONS
REGARDING
EPA
APPROVAL
OF
COLITAG
 
1
ID1.001
06
 
In
conclusion,
we
feel
that
the
protection
of
public
health
is
more
than
adequately
guaranteed
at
present,
with
several
diverse
methods
currently
being
approved
for
drinking
water
compliance
testing.
We
further
feel
very
strongly
that
the
public
interest
deserves
better
than
the
inconsistent
and
haphazard
approach
exhibited
in
this
proposal,
and
that
public
health
in
fact
may
be
compromised
if
methods
were
employed
based
on
data
exhibited
herein.
We
therefore
request
that
the
US
EPA
ask
for
CPI
to
complete
a
full
ATP
Comparability
Study,
following
the
accepted
scientific
practices,
prepare
a
well­
documented
study
report
and
resubmit
the
data
to
the
US
EPA
for
public
comments
and
for
further
consideration
of
approval. 
The
approval
of
alternate
methods
for
detecting
coliform
bacteria
is
independent
of
the
number
of
existing
methods.

The
protocol
that
guided
the
Colitag
 
method
comparability
testing
is
titled
 
Protocol
for
Alternate
Test
Procedures
(
ATP)
for
Coliform
Bacteria
in
Compliance
With
Drinking
Water
Regulations, 

published
in
1995.
The
protocol
is
not
mandatory
nor
is
it
regulatory
in
nature.
Rather,
EPA
established
the
guidelines
in
the
protocol
to
encourage
the
collection
of
adequate
information
for
the
Agency s
evaluation
of
a
new
method
(
i.
e.,
to
allow
the
Agency
to
reach
a
reasoned
judgement
concerning
the
comparability
between
the
new
method
and
the
reference
methods).

Keeping
that
objective
in
mind,
EPA
notes
that
it
has
exercised
a
degree
of
flexibility
in
the
application
of
the
guidance.
While
EPA
believes
that
those
who
follow
the
protocol
guidelines
increase
the
likelihood
of
the
Agency
having
sufficient
information
on
which
to
base
an
approval
decision,
EPA
notes
that
following
the
guidelines
precisely
does
not
guarantee
method
approval.

Similarly,
deviation
from
the
guidelines
does
not
preclude
EPA
from
considering
a
method
for
approval.

EPA
considers
all
information
submitted
and,
when
there
is
a
question
or
concern
(
e.
g.,
when
there
is
a
suggestion
that
information
was
not
collected
precisely
in
accordance
with
the
guidance),
EPA
generally
considers
the
underlying
issue
that
the
protocol
was
designed
to
address.
Where
the
Agency
has
concluded
that
adequate
information
is
available
to
judge
a
particular
issue,
it
has
proceeded
with
the
evaluation
of
the
method;
this
approach
has
been
reflected
in
EPA s
past
evaluation
of
numerous
methods,
including
currently
approved
methods
for
the
measurement
of
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
3
total
coliforms
and
E.
coli.

The
commenter
requests
that
a
full,
new
ATP
study
be
done
following
accepted
scientific
practices.
A
full
ATP
comparability
study
was
in
fact,
performed.
EPA
reviewed
the
comparability
study
results
and
judged
that
the
Colitag
 
method
demonstrated
comparability
to
the
reference
methods
(
i.
e.,
the
comparability
study
did
not
identify
a
statistically­
significant
difference
in
performance
between
the
reference
methods
and
Colitag).
Specific
details
of
the
comparability
study
are
discussed
in
responses
to
other
comments.

In
summary,
EPA
believes
that
CPI s
Colitag
 
comparability
study
data
represents
an
adequate
basis
for
evaluating
the
method.
Accordingly,
it
is
not
necessary
to
perform
a
new
ATP
study,
as
the
commenter
requests.

2
ID1.002
15
 
The
original
submission
of
testing
data
in
support
of
the
application
for
EPA
approval
of
the
Colitag
 
product
was
deficient
in
numerous
areas
under
the
ATP
guidelines;

moreover,
a
number
of
questions
concerning
quality
control
of
the
supporting
data
remain
unanswered.
EPA
has
failed
to
respond
to
the
original
concerns
raised
by
independent
reviewers
and
most
recently
has
solicited
review
comments
on
only
a
portion
of
the
data
submitted
by
the
manufacturer.
In
light
of
the
fact
that
new
water
testing
products
should
be
subjected
to
rigorous
internal
and
external
review,
these
unanswered
questions
suggest
that
the
data
submitted
are
inadequate
to
support
the
contention
that
the
performance
of
the
Colitag
 
product
meets
or
exceeds
that
of
the
EPAapproved
reference
methods.
Because
it
may
be
possible
for
the
manufacturer
and/
or
the
independent
testing
laboratory
to
respond
to
these
concerns,
EPA
should
request
additional
supporting
data
and
should
resubmit
the
data
for
external
review. 
The
commenter
states
that
the
data
submitted
for
approval
of
Colitag
 
was
deficient
under
the
ATP
guidelines.
See
response
to
Comment
1
(
Docket
ID
ID1.001
Part
06)
regarding
use
of
the
ATP
protocol
as
guidance.

With
respect
to
the
concerns
expressed
by
commenters
about
quality
control,
EPA
notes
that
the
comparability
studies
were
performed
by
an
independent,
certified
drinking
water
laboratory.
As
such,
EPA
can
reasonably
expect
that
the
laboratory
has
adhered
to
QC
practices
established
for
analysis
of
drinking
water
samples.

With
respect
to
EPA s
response
to
comments
raised
previously,
EPA
notes
that
it
is
using
this
document
to
provide
an
integrated
response
to
comments
on
both
the
original
(
March
2002)
proposed
rule
and
the
December
2002
Notice
of
Data
Availability
(
NODA);
it
was
not
EPA s
intention
to
address
the
proposed­
rule
comments
via
the
NODA.
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
4
Finally,
the
data
submitted
by
CPI
to
demonstrate
the
comparability
of
Colitag
 
to
the
reference
methods
were
subjected
to
a
thorough
review
which
addressed
the
questions
from
the
commenter.
EPA
believes
that
these
data
demonstrate
comparability
between
Colitag
 
and
the
reference
methods,
and
that
further
testing
is
not
needed.

3
ID1.005
02
 
Regarding
the
recent
Federal
Register
notice
that
apparently
indicates
that
the
Colitag
 
test
will
be
approved,
this
is
a
best
premature.
First,
the
it
has
been
US
EPA
policy
that
any
method
approved
to
protect
the
public's
health
must
recover
damaged
Escherichia
coli.
(
please
see
my
letter
of
3
May
below,

with
documentation).
E.
coli
is
the
ONLY
biological
fecalspecific
indicator
for
tap
water.
How
long
E.
coli
survives
in
the
environment
has
been
central
to
the
Groundwater,
drinking
water,
and
other
rules.
To
approve
a
method
that
is
unproven
in
its
ability
to
recover
damaged
E.
coli
is
against
the
public
interest. 
Promulgation
of
the
Colitag
 
method
was
proposed
in
a
March
2002
Federal
Register
notice.
EPA
used
its
December
2002
NODA
not
to
indicate
that
Colitag
 
would
be
approved,
but
instead
to
solicit
public
comment
on
additional
Colitag
 
information
available
subsequent
to
the
March
2002
proposal;
the
December
2002
NODA
did
not
represent
final
action
on
the
Colitag
 
method.
Such
action
is
being
taken
by
EPA
via
today s
notice,
and
only
after
having
an
opportunity
to
respond
to
all
comments.

With
respect
to
the
comment
that
EPA s
policy
has
been
that
coliform
methods
must
recover
damaged
Escherichia
coli,
EPA
notes
that
its
ATP
protocol
has
recommended
that
bacteria
be
exposed
to
chlorine
so
that
there
is
an
appropriate
stressing
of
test
bacteria
prior
to
conducting
the
comparability
study
of
the
proposed
and
reference
methods.
The
goal
is
to
model
the
stress
that
bacteria
experience
in
a
water
distribution
system.
Since
knowledge
of
the
degree
of
chlorine
stress
experienced
by
bacteria
in
a
distribution
system
and
its
effect
on
bacterial
physiology
are
not
precisely
known,
the
3
to
4
log
reduction
recommendation
in
the
protocol
only
represented
a
best
estimate
at
the
time
of
the
appropriate
level
of
chlorine
stress,
and
in
past
ATP
evaluations
has
only
been
regarded
as
an
estimate
(
For
examples
of
past
method
evaluations
where
this
approach
has
been
used,

see
comparability
study
data
for
the
Colisure
and
Readycult
methods).

When
judging
the
Colitag
 
comparability
study s
test
to
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
5
determine
if
Colitag
 
could
adequately
recover
damaged
E.
coli,
EPA
experts
evaluated
the
chlorine
stress
that
was
applied
to
the
test
bacteria
and
concluded
that
the
test
bacteria
had
been
adequately
stressed,
albeit
under
a
wider
range
of
log
reduction
than
recommended
in
the
protocol.
The
rationale
for
accepting
a
wider
range
is
twofold:
(
1)
EPA
experts
believe
that
a
wider
range
(
2
to
4
logs)
may,
in
fact,
be
more
representative
of
the
wide
variety
of
chlorine
stress
that
occurs
in
realworld
conditions;
and
(
2)
in
their
experience,
a
lower
degree
of
log
reduction
(
2
to
3
logs)
may,
in
fact,
subject
the
method
being
evaluated
to
an
even
greater
challenge
than
the
original
3
to
4
log
reduction
goal.
Regarding
the
second
point,
their
reasoning
was
that
only
the
hardiest
cells
survive
at
higher
levels
of
log
reduction,

and,
therefore,
the
surviving
bacteria
in
high
log
reduction
situations
may
be
stronger
(
i.
e.,
less
stressed)

than
the
collection
of
bacteria
surviving
stress
at
lower
levels
of
log
reduction.
Therefore,
EPA
will
generally
accept
a
wider
range
of
log
reduction
(
2
to
4
logs).

4
ID1.005
04
 
Third,
there
are
many
other
methods
that
have
been
approved
by
EPA
­
why
rush
to
approve
one
that
is
suspect?
At
the
minimum,
EPA
should
defer
its
decision
to
allow
informed
public
analysis.
There
is
nothing
lost
by
doing
this
(
there
are
many
other
methods)
but
the
risk
is
great
should
this
method
fail
(
missing
a
public
health
threat).
Fourthly,
while
there
are
other
questions
regarding
the
ability
of
the
Colitag
method
to
recover
E.
coli
that
have
not
been
resolved
(
see
my
letter
of
3
May
to
EPA
below).
While
it
is
apparently
not
the
purpose
of
this
Federal
Register
notice
to
address
other
issues,
these
significant
public
health
issues
must
be
answered. 

 
It
would
be
a
tragedy
to
have
the
Colitag
method
approved
and
for
it
to
fail
in
the
field,
and
for
human
illness
to
occur.

There
is
no
reason
this
should
happen.
Therefore,
at
the
minimum
please
defer
the
final
decision
pending
sufficient
time
to
analyze
the
complete
data
set.
I
also
add
that
deferral
pending
completion
of
analysis
was
exactly
the
course
the
EPA
EPA
has
taken
the
time
it
believed
necessary
to
adequately
judge
the
method
and
notes
that
the
multiyear
review
process
for
the
Colitag
 
comparability
study
data
has
been
extensive.
EPA
has
sought,

considered,
and
responded
to
public
comments
not
only
via
the
March
2002
proposed
rule,
but
also
via
the
December
2002
NODA.
EPA
has
come
to
the
conclusion
that
the
Colitag
 
method
is
comparable
to
the
reference
methods
in
its
ability
to
initiate
growth
of
chlorine­
stressed
bacteria
and
does
not
believe
that
further
testing
is
needed.

EPA
performed
a
very
thorough
analysis
of
the
CPI
comparability
study
data
and
it
is
not
clear
what
additional
analysis
the
commenter
is
suggesting
here.

EPA
judged
that
Colitag
 
was
comparable
to
the
reference
methods
in
its
ability
to
recover
injured
E.
coli
with
the
data
submitted
because
statistical
analysis
did
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
6
took
when
the
same
question
­
the
ability
to
recover
injured
E.

coli
­
arose
regarding
the
MM0­
MUG
method.
EPA
undertook
this
re­
analysis
including
additional
studies,
and
subsequently
approved
the
method­
it
has
worked
admirably.
Conversely,

recently,
the
EPA
approved
total
coliform
methods
that
had
unacceptably
high
false­
positives,
prompting
a
re­
analysis
of
the
data.
This
should
not
happen
again. 
not
show
a
significant
difference
between
the
performance
of
the
reference
method
and
that
of
Colitag
 
.

This
situation
differs
from
the
MMO­
MUG
situation
cited
by
the
commenter
in
several
important
respects.

First,
at
the
time
that
the
MMO­
MUG
method
was
being
considered,
the
Agency
had
limited
experience
with
chromogenic/
fluorogenic
presence/
absence
methods.
As
a
result,
the
Agency
approached
the
review
of
such
methods
in
a
more
conservative
manner.

Secondly,
EPA
notes
that
at
the
time
it
was
evaluating
the
MMO­
MUG
method
the
operative
protocol
for
ATP
methods
was
quite
different
(
e.
g.,
the
protocol
at
that
time
did
not
include
log
reduction
guidelines).
Hence,

the
context
in
which
EPA
made
decisions
regarding
MMO­
MUG
differs
significantly
from
that
the
Agency
faces
today.

5
ID2.001
05
 
Finally,
as
I
have
indicated,
many
other
countries
look
to
USEPA
for
guidance.
Acceptance
of
a
new
method
based
on
incomplete
and
inferior
data
does
nothing
to
help
such
countries
nor
the
credibility
of
USEPA.
I
would
strongly
urge
you
to
look
again
at
the
data
submitted
and
to
investigate
the
other
issues
that
I
have
raised
before
"
approving"
Colitag
as
an
alternative
test
procedure. 
It
is
not
clear
from
this
comment
how
the
Colitag
 
data
is
 
incomplete
and
inferior. 
To
the
extent
this
comment
has
been
elaborated
upon
elsewhere,
EPA
has
responded
to
these
assertions.
See
also
the
responses
to
Comment
2
(
Docket
ID
I­
D1.002,
Part
15)
and
Comment
4
(
Docket
ID
I­
D1.005,
Part
04)
concerning
the
adequacy
of
the
data
and
the
extent
of
the
Agency s
evaluation.

6
I­
F.
004
08
 
Based
on
all
the
issues
identified,
we
strongly
object
the
approval
of
Colitag
for
use
under
the
Total
Coliform
Rule
as
a
presence/
absence
liquid
culture
(
LC)
method
for
detecting
total
coliforms
and
E.
coli
in
drinking
water
and
also
in
source
water.

The
Colitag
evaluation
did
not
follow
the
USATP
protocol
as
required
by
the
USEPA
to
evaluate
a
proposed
method
for
the
presence/
absence
liquid
culture
(
LC)
methods
in
microbiology.

Colitag
should
complete
a
full
ATP
Comparability
Study,

prepare
a
well­
documented
study
report
and
resubmit
the
data
to
the
USEPA
for
public
comments
and
for
further
consideration
of
approval.
The
evaluation
should
strictly
follow
Concerning
the
comment
that
the
Colitag
 
comparability
study
did
not
precisely
follow
the
ATP
protocol,
EPA
notes
that
it
has
historically
applied
a
degree
of
flexibility
in
judging
comparability
study
results
relative
to
the
protocol
guidelines.
EPA s
policy
concerning
adherence
to
the
ATP
protocol
is
explained
in
the
response
to
Comment
1
(
Docket
ID
I­
D1.001,

Part
06).
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
7
all
the
requirements
described
in
the
"
Protocol
for
Alternate
Test
Procedures
for
Coliform
Bacteria
in
Compliance
with
Drinking
Water
Regulations
 
Presence/
Absence
Liquid
Culture
Methods
for
Finished
Water"
(
Version
1.2
December
1995)
and
result
in
data
addressing
the
issues
raised
in
this
report. 

7
I­
F.
022
10
 
The
test
data
provided
indicate
that
the
USEPA
ATP
Protocol
was
not
followed,
as
noted
above.
USEPA
should
not
approve
Colitag
until
the
appropriate
ATP
comparability
studies
and
documentation
are
provided
demonstrating
that
the
method
has
been
evaluated
in
accordance
with
USEPA
ATP
Protocol.

These
studies
must
be
reviewed
by
USEPA
and
also
published
for
public
comment
prior
to
final
approval
of
the
method. 

 
In
conclusion,
it
is
very
important
for
USEPA
to
ensure
the
scientific
integrity
of
proposed
methods,
especially
for
total
coliform
and
E.
coli,
by
insisting
and
ensuring
that
the
USEPA
ATP
Protocol
is
met.
Data
and
documentation
must
be
provided
by
proposers
to
demonstrate
that
the
USEPA
ATP
Protocol
is
met. 
See
response
to
Comment
1
(
Docket
ID
I­
D1.001.
Part
06)
concerning
adherence
to
the
ATP
protocol.

02
LENGTH
OF
COMMENT
PERIOD/
RESPONSIVENESS
TO
COMMENTS
8
ID1.001
02
 
However,
we
point
out,
that
out
of
a
total
of
8
distinct
and
very
important
issues
submitted
by
various
groups
during
the
first
round
of
comments,
the
USEPA
chose
to
address
only
one
 
the
log
reduction.
It
is
our
hope
that
the
other
issues
will
be
addressed
by
the
USEPA
in
due
course. 

 
Furthermore,
no
new
data,
but
rather
a
recalculation
of
the
old
data
was
offered
in
the
form
of
a
power
point
presentation
made
by
CPI
during
a
meeting
with
the
USEPA. 

 
Given
the
extremely
limited
and
abbreviated
 
new
information 
at
hand,
we
review
the
 
old 
data
and
 
new
information 
and
offer
the
following
comments. 
See
response
to
Comment
2
(
Docket
ID
I­
D1.002,
Part
15)
concerning
the
manner
in
which
the
complete
set
of
issues
is
being
addressed.

The
December
2002
NODA
included
supplemental
data
and
a
re­
evaluation
of
previously
reported
data.
EPA
notes
that
soliciting
public
comments
on
such
via
the
NODA
process
was
the
appropriate
course
of
action.

The
 
8
distinct 
issues
mentioned
by
commenter
here
are
addressed
elsewhere
in
this
document.

9
ID1.002
03
 
Numerous
deficiencies
(
as
determined
under
the
ATP
protocol)
were
identified
in
the
original
submission
for
the
Colitag
 
product;
these
concerns
were
submitted
during
EPA s
open
solicitation
for
comments
in
April
of
2002
and
yet
the
EPA
is
responding
to
all
comments
received
regarding
the
Colitag
 
test
in
this
document.
See
also
the
responses
to
Comment
2
(
Docket
ID
I­
D1.002,
Part
15);

Comment
3
(
Docket
ID
I­
D1.005,
Part
02);
and
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
8
only
response
to
reviewer
concerns
was
to
indicate
that
 
EPA
has
received
and
is
reviewing
clarifying
information
concerning
the
evaluation
of
the
Colitag
 
test.
Thus,
EPA
is
not
taking
final
action
on
this
method
at
this
time.
EPA
will
respond
to
all
significant
comments
regarding
this
method
in
a
future
action. 

To­
date,
the
response
from
EPA
to
reviewer
comments
on
the
Colitag
 
data
has
been
limited
to
the
recomputation
of
the
log
reductions
presented
in
the
original
application
and
yet
in
the
December
2,
2002
Federal
Register
EPA
specifically
states
that
reviewer
  
comments
should
be
submitted
only
on
the
additional
information
presented
in
this
notice
and
in
Docket
No.
OW­
2002­
0031.
EPA
is
not
reconsidering
any
other
drinking
water
issue
in
this
notice
nor
will
EPA
respond
to
any
comments
on
other
issues.
Comments
should
be
limited
to
the
additional
information
described
herein
and
found
in
docket
No.
OW­
2002­
0031
and
its
applicability
to
the
Agency s
consideration
of
the
Colitag
 
method. 
In
effect,
EPA
delayed
providing
a
formal
response
to
the
initial
review
comments
and
is
now
restricting
reviewer
comment
to
only
a
small
portion
of
the
data
submitted
by
the
manufacturer.
This
lack
of
responsiveness
to
the
original
concerns
brought
up
by
the
external
reviewers
is
unacceptable;
moreover,
it
entirely
defeats
the
purpose
of
soliciting
external
review
comments
and
trivializes
the
effort
that
reviewers
invested
in
analyzing
the
data
in
the
first
place.
If
a
key
objective
of
the
ATP
is
to
promote
the
public
health
by
encouraging
the
development
of
improved
water
testing
methods,
by
all
means
EPA
must
provide
a
clear
response
to
the
concerns
submitted
by
external
reviewers
and
under
no
circumstances
should
restrict
the
review
of
data.

EPA s
decision
to
revisit
the
original
application
for
Colitag
 
is
not
justified
adequately
in
the
Federal
Register
notice
of
December
2,
2002,
especially
in
light
of
the
fact
that
EPA
failed
to
address
any
of
the
reviewer
concerns
following
the
original
application. 
Comment
8
(
Docket
ID
I­
D1.001,
Part
02)
concerning
the
response
to
the
comments
on
the
March
2002
proposal
and
the
purpose
of
the
December
2002
NODA.

10
ID1.002
07
 
Moreover,
EPA
failed
to
address
a
number
of
additional
departures
from
the
Alternate
Testing
Protocol
presented
by
external
reviewers
during
the
original
March
2002
review
of
these
data.
These
include
concerns
about
missing
data
on
See
responses
to
Comment
1
(
Docket
ID
I­
D1.001,
Part
06)
concerning
adherence
to
the
protocol,
and
Comment
2
(
Docket
ID
I­
D1.002,
Part
15)
concerning
QC
data
and
the
purpose
of
the
December
2002
NODA.
EPA
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
9
product
shelf
life,
questions
regarding
incubation
temperatures,

concerns
about
the
presentation
(
or
lack
thereof)
of
supporting
Quality
Control
data
and
inconsistencies
in
the
reporting
of
sensitivity/
specificity
data
required
under
the
ATP.
To­
date
EPA s
primary
response
to
these
concerns
has
been
to
stipulate
that
the
ATP
guidelines
are
not
legal
requirements
for
manufacturers.
As
a
consequence,
it
would
appear
that
manufacturers
are
not
compelled
to
conduct
comparability
testing
under
the
carefully
controlled
conditions
described
in
the
EPA s
own
Alternate
Testing
Protocol.
If
this
is
the
case,
by
all
means
EPA
should
disavow
the
utility
of
the
ATP
and
should
provide
alternate,
legally
binding
requirements
to
ensure
that
manufacturers
develop
and
market
products
that
are
validated
under
realistic
conditions
for
water
testing.
To
do
otherwise
is
to
provide
a
disservice
to
the
national
public
health
and
also
to
the
manufacturers
who
in
good
faith
attempt
to
follow
the
existing
USEPA
guidelines.
Finally,
failure
to
respond
completely
to
reviewer
comments
is
a
disservice
to
the
external
reviewers
themselves
who
will
not
be
keen
to
share
their
time
and/
or
expertise
in
future
evaluations
of
water
testing
data. 
may
consider
imposing
alternate
legally
binding
requirements
for
analytical
methods
in
the
future.

Today s
action,
however,
is
limited
to
approval
of
a
specific
method.

11
ID1.005
03
 
Second,
while
the
Federal
Register
notice
presents
a
cursory
summary
of
"
recalculated"
data,
there
are
not
enough
specifics
to
make
an
informed
analysis.
Is
this
not
the
purpose
of
a
public
comment
period?
While
I
respect
the
statement
that
EPA
expert(
s)
examined
the
data,
it
seems
a
mockery
of
the
process
to
provide
so
little
data
and
30
days
during
a
holiday
period
to
make
such
an
important
decision. 
See
also
the
response
to
Comment
8
(
Docket
ID
ID1.001
Part
02)
concerning
the
purpose/
scope
of
the
December
2002
NODA.
EPA
believes
that
sufficient
data
were
presented
in
the
NODA
and
the
associated
public
docket
to
conduct
an
informed
analysis,
and
for
EPA
to
make
an
informed
decision.

EPA
agreed
with
the
request
for
a
longer
comment
period
and
granted
a
2
week
extension
to
that
period.

03
EPA'S
ALTERNATE
TEST
PROCEDURES
(
ATP)
PROGRAM
03.01
Adherence
to
Specific
ATP
Guidelines
12
I­
F.
021
10
The
following
comments
are
drawn
from
review
of
the
individual
ATP
worksheets
submitted
by
the
independent
testing
facility
(
Biovir
Labs)
See
also
the
responses
to
Comment
1
(
Docket
ID
ID1.001
Part
06)
concerning
use
of
the
ATP
protocol
as
guidance
and
Comment
3
(
Docket
ID
I­
D1.005,
Part
02)

concerning
a
target
chlorine
stress
level
of
2­
4
log
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
10
 
Millbrae,
CA
#
1:
Pretreatment
of
the
seeded
test
water
with
chlorine
(~
1.0
mg/
L
for
2
minutes)
resulted
in
microbial
reductions
of
1.8­
2.1
log10.
The
experiment
failed
to
demonstrate
the
target
reduction
level
of
3­
4
log10
as
required
in
the
ATP
protocol
under
section
4.5.2.1.
Test
data
for
E.
coli
failed
to
meet
the
25%:
75%
criterion
under
the
ATP
protocol
(>
15
for
both
total
coliforms
and
E.
coli).
Should
these
test
data
be
considered
valid
if
they
failed
to
demonstrate
the
degree
of
inactivation
that
might
be
considered
an
appropriate
model
of
real
world
disinfection
conditions?
Should
the
E.
coli
data
be
considered
valid?
Only
two
dilutions
were
subjected
to
assay,
however,
the
ATP
protocol
calls
for
a
minimum
of
three
dilutions
to
bracket
the
countable
dilution.
Should
the
selected
dilution
be
considered
valid? 

 
JEA
Water
Quality:
Only
two
dilutions
were
subjected
to
assay,
however,
the
ATP
protocol
calls
for
a
minimum
of
three
dilutions
to
bracket
the
countable
dilution.
Should
the
selected
dilution
be
considered
valid? 

 
Schaumberg,
IL:
The
computed
log10
reduction
(
2
log10)
fails
to
meet
the
3­
4
log10
criterion.
Should
the
data
be
considered
valid?
Only
two
dilutions
were
subjected
to
assay,
however,
the
ATP
protocol
calls
for
a
minimum
of
three
dilutions
to
bracket
the
countable
dilution.
Should
the
selected
dilution
be
considered
valid? 

 
Watertown,
WI:
Only
two
dilutions
were
subjected
to
assay,

however,
the
ATP
protocol
calls
for
a
minimum
of
three
dilutions
to
bracket
the
countable
dilution.
Should
the
selected
dilution
be
considered
valid? 

 
Mission,
KS
#
1:
The
computed
log10
reduction
(
1.2
log10)

fails
to
meet
the
3­
4
log10
criterion.
Should
the
data
be
considered
valid?
The
E.
coli
test
data
failed
to
meet
the
25%:
75%
criterion
under
the
ATP
protocol
(
3<
5).
Should
this
data
be
considered
valid?
Only
one
dilution
was
subjected
to
reduction.

With
respect
to
comments
related
to
the
degree
of
inactivation,
EPA
experts
evaluated
these
data
and
concluded
that
the
data
were
sufficient
to
judge
the
comparability
between
the
Colitag
 
method
and
the
reference
methods.
As
stated
in
the
response
to
Comment
3
(
Docket
ID
I­
D1.005,
Part
02),
EPA
believes
that
a
wider
range
for
log
reduction
(
i.
e.,
2
to
4
logs)
is
acceptable
in
that
it
achieves
an
appropriate
degree
of
chlorine
stress.
EPA
notes
that,
per
the
data
included
in
the
December
2002
NODA,
a
log
reduction
between
2
and
4
was
achieved
in
10
of
10
total
coliform
studies
and
9
of
10
E.
coli
studies.

To
the
extent
that
the
commenter
is
basing
its
assertions
on
log
reduction
calculations
performed
in
a
manner
other
than
that
described
in
the
December
2002
NODA,

see
also
the
responses
to
Comment
24
(
Docket
ID
ID1.001
Part
03)
and
Comment
25
(
Docket
ID
I­
D1.002,

Part
04).

[
EPA
notes
that,
consistent
with
the
expression
of
the
log
reduction
goal
as
one
significant
figure,
it
has
evaluated
the
log
reduction
results
based
on
one
significant
figure
(
e.
g.,
a
1.8
log
reduction
is
viewed
as
meeting
the
2
log
reduction
goal).
EPA
notes
that
1.6
log
represents
a
97.5%
reduction,
whereas
2.0
log
represents
a
99%
reduction.
Considering
the
small
difference
in
%
reduction
associated
with
rounding,
and
considering
the
inherent
variability
in
bacterial
density
measurements
(
likely
impacting
%
reduction
and
log
reduction
precision
to
a
greater
degree
than
rounding),

the
Agency
is
satisfied
that
this
is
a
reasonable
approach.]

In
response
to
the
comment
suggesting
deficiencies
in
the
25%:
75%
split
between
positive
and
negative
results,
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
11
assay,
however,
the
ATP
protocol
calls
for
a
minimum
of
three
dilutions
to
bracket
the
countable
dilution.
Should
the
selected
dilution
be
considered
valid? 

 
Ames,
Iowa:
The
computed
log10
reduction
(
1
log10)
fails
to
meet
the
3­
4
log10
criterion
due
to
inadequate
seeding
density
(
10
cfu/
100
mL).
Should
the
data
be
considered
valid?
The
E.

coli
test
data
failed
to
meet
the
25%:
75%
criterion
under
the
ATP
protocol
(
4<
5).
Should
the
data
be
considered
valid?

Only
one
dilution
was
subjected
to
assay,
however,
the
ATP
protocol
calls
for
a
minimum
of
three
dilutions
to
bracket
the
countable
dilution.
Should
the
selected
dilution
be
considered
valid? 

 
Liberty,
MD:
The
computed
log10
reduction
(
0.7
log10)
fails
to
meet
the
3­
4
log10
criterion.
Should
the
data
be
considered
valid?
Only
one
dilution
was
subjected
to
assay,
however,
the
ATP
protocol
calls
for
a
minimum
of
three
dilutions
to
bracket
the
countable
dilution.
Should
the
selected
dilution
be
considered
valid? 

 
Salem,
OR:
The
computed
log10
reduction
(
2.1
log10)
fails
to
meet
the
3­
4
log10
criterion.
Should
the
data
be
considered
valid?
The
total
coliform
test
data
under
the
reference
method
failed
to
meet
the
25%:
75%
criterion
under
the
ATP
protocol
(
18>
15).
Should
the
data
be
considered
valid?
Only
one
dilution
was
subjected
to
assay,
however,
the
ATP
protocol
calls
for
a
minimum
of
three
dilutions
to
bracket
the
countable
dilution.
Should
the
selected
dilution
be
considered
valid? 

 
Mission,
KS#
2:
The
computed
log10
reduction
(
1.4
log10)

fails
to
meet
the
3­
4
log10
criterion.
Should
the
data
be
considered
valid?
It
appears
that
this
sample
was
collected
from
the
same
source
as
an
earlier
Kansas
submission
(
KS#
1).

Should
these
two
samples
be
considered
geographically
distinct?

Only
one
dilution
was
subjected
to
assay,
however,
the
ATP
protocol
calls
for
a
minimum
of
three
dilutions
to
bracket
the
countable
dilution.
Should
the
selected
dilution
be
considered
and
the
three­
dilution
recommendation
used
to
achieve
the
split,
EPA
first
notes
that
the
underlying
goal
of
this
split
is
to
confirm
that
very
low
numbers
of
bacteria
are
being
introduced
into
the
test
media.
Establishing
conditions
under
which
very
low
numbers
of
target
bacteria
are
expected
in
the
test
sample
(
i.
e.,
the
spiked
drinking
water
sample
used
to
innoculate
the
referencemethod
and
Colitag
 
­
method
media)
presents
a
greater
challenge
to
the
presence/
absence
methods
than
would
exist
if
high
numbers
of
bacteria
were
present
in
the
sample.
This
is
intended
to
model
conditions
potentially
found
in
distribution
systems,
where
isolated
contaminant
bacteria
could
occur.

Under
ideal
(
i.
e.,
the
most
challenging)
test
circumstances,
half
of
20
replicate
cultures
would
yield
a
positive
result
and
half
would
yield
a
negative
result
(
i.
e.,

a
 
50:
50"
split),
representative
of
a
situation
where
the
lowest
possible
numbers
of
bacteria
per
culture
were
being
inoculated.
Recognizing
the
inherently
imprecise
nature
of
microbiological
work,
and
recognizing
that
even
the
reference
methods
for
total
coliforms
and
E.

Coli
are
imperfect,
EPA
established
a
more
flexible
goal
of
a
25%:
75%
split,
such
that
when
testing
20
replicate
cultures,
5­
15
of
the
replicates
should
yield
positive
results
and
5­
15
should
yield
negative
results.
Generally,

a
25%:
75%
split
will
still
be
indicative
of
very
low
numbers
of
inoculated
bacteria
and
should
be
far
more
practical
for
laboratories
to
achieve
than
a
50:
50
split.

The
use
of
3
serial
dilutions
per
location
is
intended
to
increase
the
likelihood
that
one
of
the
dilutions
achieves
a
25%:
75%
split.

Significantly,
the
25%:
75%
split
represents
only
one
of
two
means
described
in
EPA s
ATP
protocol
to
help
ensure
that
the
bacterial
count
is
low.
The
other
means
by
which
to
judge
whether
the
bacterial
count
is
low
is
to
examine
the
post­
chlorination
bacterial
density
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
12
valid? 

 
Millbrae,
CA
#
2:
The
computed
log10
reduction
(
4.5
log10)

exceeds
the
3­
4
log10
criterion
(
likely
due
to
the
excessive
chlorine
pretreatment
of
4
mg/
L).
Should
the
data
be
considered
valid?
It
appears
that
this
sample
was
collected
from
the
same
source
as
an
earlier
California
submission
(
CA#
1).

Should
these
two
samples
be
considered
geographically
distinct?

The
total
coliform
data
under
the
reference
method
failed
to
meet
the
25%:
75%
criterion
under
the
ATP
protocol
(
17>
15).

Should
these
data
be
considered
valid?
The
experimental
data
submitted
include
either
one
or
two
dilutions
for
each
condition,
however,
the
ATP
protocol
calls
for
a
minimum
of
three
dilutions
in
order
to
bracket
the
countable
dilution.

Should
the
selected
dilution
be
considered
valid? 
determination.
Recalling
that
the
goal
(
per
section
3.1.1
of
the
ATP
protocol)
is
to
achieve
a
post­
chlorination
density
of
 
1­
10
organisms
per
100
mL
of
sample, 
EPA
notes
that
all
such
measurements
for
total
coliforms
and
E.
Coli
were,
in
fact,
between
1
and
7,
as
reflected
in
Tables
1
and
2
from
the
December
2002
NODA.

Recognizing
that
a
series
of
tests
of
both
the
reference
methods
and
the
Colitag
 
method
were
performed,
for
each
of
10
test
samples,
for
both
total
coliforms
and
for
E.
Coli,
a
total
of
40
tests
series
(
2x10x2)
was
performed.

Among
those
test
series,
32
of
40
(
80%)
were
within
the
25:
75
goal.
1
EPA
acknowledges
that,
in
the
following
cases,

deviations
from
the
25:
75
goal
occurred:

­
17
positives
for
the
reference
method
and
20
positives
for
the
Colitag
 
method
for
total
coliforms
in
sample
982305A
­
18
positives
for
the
reference
method
for
total
coliforms
in
sample
990273A
(
vs.
13
positives
for
the
Colitag
 
method)

­
16
positives
for
the
reference
method
and
17
positives
for
the
Colitag
 
method
for
E.
Coli
in
sample
982084A
­
4
positives
for
the
Colitag
 
method
for
E.
Coli
in
sample
982305A
(
vs.
6
positives
for
the
reference
method)

­
3
positives
for
the
reference
method
for
E.
Coli
in
sample
990217A
(
vs.
5
positives
for
the
Colitag
 
method)

­
4
positives
for
the
reference
method
for
E.
Coli
in
sample
990438A
(
vs.
7
positives
for
the
Colitag
 
method)

EPA
considered
the
similarity
between
the
above
reference­
method
and
Colitag
 
­
method
results.

Collectively,
these
results
are
quite
similar.
Even
on
an
1.
The
ATP
protocol
(
at
section
4.6.5)

seeks
comparison
of
test
results
wherein
an
acceptable
split
in
positive
and
negative
results
for
the
reference
method
is
achieved.

For
the
purpose
of
responding
to
comments
on
this
issue,
however,
EPA
has
assessed
results
in
which
the
25%:
75%

split
was
not
achieved
in
either
the
reference
method
or
Colitag
 
.
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
13
individual
basis,
nearly
all
of
these
dilutions
yielded
results
that
are
quite
close
when
comparing
the
performance
of
Colitag
 
and
the
reference
methods.

EPA
also
considered
the
proximity
between
these
results
and
the
25:
75
goal
articulated
in
the
ATP
protocol.

Again,
these
figures
are
very
similar;
in
only
one
instance
do
the
results
deviate
from
the
25:
75
goal
(
i.
e.,
5:
15
to
15:
5
individual
replicate
results)
by
more
than
3
individual
replicate
results.

Lastly,
EPA
considered
the
impact
of
the
above
deviations
on
the
statistical
analysis
and
concluded
that
such
individual
deviations
did
not
impact
EPA s
ability
to
perform
its
comprehensive
analysis,
since
the
 
chi
squared 
statistical
test,
used
to
determine
method
comparability,
relied
on
a
collective
data
set
from
all
samples.
[
Since
individual
test
result
deviations
from
the
25:
75
goal
were
few
(
and
small)
and
since
the
chisquared
probability
and
Fisher s
exact
two­
sided
probability
(
both
key
indicators
of
comparability
between
Colitag
 
and
the
reference
methods)
were
well
above
the
minimum
threshold
of
0.05,
this
approach
(
analysis
of
the
data
as
a
collective
set)
is
statistically
valid
and
EPA
has
confidence
in
the
conclusion
of
the
statistical
analysis
In
a
method
evaluation
where
the
individual
test
results
deviate
substantially
from
the
25:
75
goal,
and
where
the
computed
statistical
probability
is
much
closer
to
0.05,
EPA
would
not
have
that
same
confidence
The
statistical
results
are
discussed
in
greater
detail
in
the
response
to
Comment
24
(
Docket
ID
I­
D1.001,
Part
03).]

Moreover,
EPA
reminds
the
commenter
that
the
25:
75
goal
represents
only
one
of
two
means
by
which
to
judge
whether
testing
has
taken
place
using
a
very
low
bacterial
count.
Referring
to
the
other
means
by
which
to
judge
such
count
(
i.
e.,
the
post­
chlorination
density
determination),
EPA
notes
that
such
measurement
met
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
14
EPA s
goal
of
1­
10
organisms/
100mL
in
100%
of
the
cases.
Accordingly,
EPA
has
concluded
that
the
all
of
the
above
constitute
valid
results,
with
respect
to
demonstrating
that
a
low
bacterial
count
was
employed
in
the
studies.

Concerning
the
comment
regarding
the
number
of
dilutions
performed,
EPA
notes
that
where
fewer
than
3
dilutions
were
performed,
but
where
at
least
one
of
those
dilutions
yielded
reasonable
splits
between
positive
and
negative
results,
additional
dilutions
would
serve
no
useful
purpose.

In
response
to
the
comment
on
the
number
of
geographical
locations
(
in
particular,
Mission,
KS
and
Millbrae,
CA)
used
in
the
Colitag
 
comparability
study,

EPA
acknowledges
that
the
protocol
recommends
that
comparability
data
be
generated
 
from
a
single
finished
water
which
has
been
spiked
with
different
(
e.
g.,
10)

wastewaters
or
polluted
surface
waters
containing
the
target
organisms. 
The
goal
underlying
this
provision
is
to
use
 
biodiverse 
spiked
samples
(
i.
e.,
those
with
a
range
of
coliform
strains)
to
test
the
candidate
method
with
a
range
of
bacterial
metabolic
variation,
and
EPA s
protocol
recommends
that
the
wastewater/
surface
water
come
from
10
geographically
dispersed
sites.

EPA
reviewed
and
supported
CPI s
plan
to
compare
the
Colitag
 
method
to
the
reference
methods
based
on
10
samples
from
8
different
locations.
It
is
EPA s
judgment
that
this
particular
sample
set
meets
the
goal
of
achieving
substantial
biodiversity
and
represents
a
sufficient
degree
of
diversity
to
provide
the
Agency
with
confidence
that
the
Colitag
 
method
performs
adequately
over
a
wide
range
of
bacterial
strains.
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
15
It
is
helpful
to
understand
that
substantial
biodiversity
generally
exists
even
within
a
single
wastewater
sample,

and
between
multiple
wastewater
samples
from
a
single
location.
Although
it
may
be
more
obvious
that
spacial
variation
(
i.
e.,
sampling
over
multiple
locations)
achieves
an
additional
degree
of
diversity
(
i.
e.,
additional
coliform
strains),
additional
diversity
can
also
reasonably
be
expected
with
temporal
variation
(
i.
e.,
sampling
over
a
period
of
time).
Regarding
the
latter
point,
in
both
instances
in
which
samples
were
taken
from
the
same
location,
these
samples
were
collected
at
least
one
month
apart.
The
two
samples
from
Millbrae,
CA
(
98204A
and
982305A)
were
collected
on
11/
10/
98
and
12/
14/
98
and
the
two
samples
from
Mission,
KS
(
990217
and
990442A)
were
collected
on
3/
3/
99
and
4/
26/
99.

Recognizing
that
significant
biodiversity
exists
even
with
a
single
sample;
recognizing
that
substantial
spacial
variation
achieved
further
diversity
for
the
8
geographically­
dispersed
Colitag
 
samples;
and
recognizing
that
substantial
temporal
variation
achieved
further
diversity
for
the
remaining
2
samples,
EPA
concluded
that
all
10
of
the
samples
used
in
the
comparability
studies
of
Colitag
 
constitute
valid
samples,
having
met
the
goal
of
achieving
substantial
biodiversity.

Concerning
the
comment
that
the
chlorine
stress
log
reduction
used
with
Millbrae,
CA
#
2
exceeded
4
logs
of
reduction,
EPA
notes
that
the
recalculated
log
reduction
for
this
sample
was
less
than
4
in
the
data
presented
in
the
December
2002
NODA
(
i.
e.,
the
data
upon
which
EPA
is
basing
its
decision).
Specifically,
the
log
reduction
for
this
sample
was
recalculated
as
3.4.

03.01.01
Injury
level
guidelines
(
3­
4
log
goal;
recovery
rates;
alleged
math
errors;
applicability
to
natural
samples)

13
ID1.001
04
4.
 
As
shown
in
Tables
II
and
III,
per
IDEXX
calculation,
only
10%
of
samples
met
the
ATP
guidelines
for
Log10
reduction
See
responses
to
Comment
3
(
Docket
ID
I­
D1.005,
Part
02)
and
Comment
12
(
Docket
ID
I­
F.
021,
Part
10)
`
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
16
for
both
total
coliform
(#
990095A)
and
for
E.
coli
(#

990025A).
Please
note,
that
since
sample
#
982305A­
1
was
not
used
for
comparability
statistics
purposes,
it
cannot
be
used
toward
meeting
the
log
reduction
requirement
either. 
regarding
the
log
reduction
results.

Moreover,
commenter
appears
to
have
made
several
assumptions
in
calculating
(
or
asserting
an
inability
to
calculate)
individual
log
reductions.
Some
of
these
assumptions
are
either
incorrect
or
depend
upon
assertions
with
which
EPA
disagrees.
For
example,

EPA
did
not
use
prechlorination
density
data
(
i.
e.,

measurements
after
the
initial
density
determination
and
immediately
preceding
chlorination)
to
calculate
the
log
reduction.
Instead,
as
explained
in
EPA s
December
2,

2002
NODA,
the
log
reduction
was
calculated
using
the
initial
density
determination
divided
by
the
sample
dilution
factor.
Although
the
prechlorination
density
data
was
submitted
by
CPI
for
some
samples,
it
is
not
necessary
(
and
was
not
used
by
EPA)
to
calculate
the
log
reductions.

For
the
reasons
discussed
in
response
to
Comment
24
(
Docket
ID
I­
D1.001,
Part
03)
and
Attachment
1,
EPA
believes
that
the
hold
times
are
such
that
they
support
the
procedure
outlined
in
the
NODA.

Responses
concerning
specific
data
presented
in
the
commenter s
attachments
follow:

Total
Coliforms
(
Table
II):.

Sample
982084A
 
The
commenter
has
stated
that
this
sample
lacks
pre­
chlorination
density
data.
As
discussed
above,
the
fact
that
prechlorination
density
data
was
not
available
for
this
sample
does
not
invalidate
the
sample;

such
data
were
not
used
in
EPA s
analysis.

Sample
982305A­
1
 
The
commenter
suggests
that
an
inconsistent
data
set
was
used
for
the
log
reduction
calculation
and
the
statistical
analysis.
As
described
further
in
the
response
to
Comment
24
(
Docket
ID
ID1.001
Part
03),
EPA
has
repeated
its
statistical
analysis
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
17
using
the
data
from
the
1:
1000
dilution
experiments
(
sample
982305A­
1,
12/
23/
98).
As
with
the
original
analysis,
the
repeat
analysis
did
not
identify
a
statistically
significant
difference
between
the
performance
of
Colitag
 
and
the
reference
methods.

Sample
982305A­
2
­
The
commenter
has
stated
that
the
data
sheets
for
this
sample
are
missing
the
postchlorination
density.
Since,
as
described
in
the
response
to
Comment
24
(
Docket
ID
I­
D1.001,
Part
03),
EPA
has
repeated
its
statistical
analysis
excluding
the
results
from
sample
982305A­
2
(
study
results
dated
12/
29/
98)
and
substituting
the
results
from
sample
982305A­
1,
and
since
comparability
between
Colitag
 
and
the
reference
methods
continued
to
be
demonstrated,
the
missing
post­
chlorination
data
for
sample
982305A­
2
has
no
impact
on
EPA s
decision.

Sample
990025A
 
The
commenter
has
suggested
that
the
ATP
worksheets
refer
to
dilutions
of
1:
500
and
1:
2000
for
the
replicate
tests
with
total
coliforms,
yet
the
comparability
study
data
used
in
EPA s
statistical
analysis
appear
to
be
associated
with
a
1:
1000
dilution.

EPA
notes
that
it
originally
used
data
from
the
first
(
1:
500)
dilution
in
calculating
total
coliform
log
reduction.
Although
the
comparability
data
sheet
indicates
that
the
second
set
of
replicates
were
diluted
at
a
1:
1000
ratio,
EPA
believes
that
this
is
likely
a
typographical
error.
This
is
because
all
of
the
previous
references
to
dilutions
were
either
1:
500
or
1:
2000,
as
reflected
in
the
ATP
worksheet.
In
the
absence
of
a
clear
explanation
for
this
discrepancy,
EPA
believes
that
a
typographical
error
is
likely
and
that
the
dilution
figure
for
the
second
set
of
comparability
studies
should
be
1:
2000.
Since
EPA
used
the
data
associated
with
the
second
dilution
(
originally
stated
as
1:
1000,
but
presumably
1:
2000)
in
its
original
statistical
analysis,

EPA
first
recalculated
log
reduction,
using
that
data
set,
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
18
for
consistency.
(
EPA
notes
that
using
a
dilution
factor
of
1:
2000
in
the
log
reduction
calculation
instead
of
1:
1000
represents
a
more
conservative
approach;
it
yields
a
lower
log
reduction
than
would
be
obtained
using
the
1:
1000
factor).
As
described
in
the
response
to
Comment
24
(
Docket
ID
I­
D1.001,
Part
03),
that
recalculation
produced
a
log
reduction
of
3.0,
versus
the
original
log
reduction
of
3.2;
thus,
relying
on
the
figures
used
in
the
original
statistical
analysis
and
substituting
a
revised
log
reduction
(
3.0)
has
no
impact
on
EPA s
decision.
In
the
alternative,
the
comparability
study
analysis
does
not
suffer
if
the
Agency
relies
instead
upon
the
comparability
data
from
the
set
of
replicates
diluted
at
1:
500
(
i.
e.,
the
data
set
associated
with
the
originally
reported
log
reduction
of
3.2).
Although
the
reference
method
generated
slightly
more
positive
results
(
16
out
of
20)
than
ideal
to
meet
the
25%:
75%
goal
(
5
­
15
out
of
20),
this
is
not
vital
to
the
overall
analysis.
See
response
to
Comment
12
(
Docket
ID
I­
F.
021,
Part
10).

Indeed,
using
the
data
for
the
1:
500
dilution
resulted
in
no
impact
on
the
 
chi­
squared 
probability
figures
that
indicate
comparability
and,
therefore,
no
impact
on
the
conclusion
of
the
analysis.
Finally,
as
discussed
in
the
response
to
Comment
24
(
Docket
ID
I­
D1.001,
Part
03),

EPA
lacks
information
that
would
conclusively
demonstrate
appropriate
hold
times
for
four
samples;

sample
990025A
is
one
of
them.
Even
if
this
sample
were
discarded
entirely,
the
outcome
of
the
overall
comparability
analysis
(
see
response
to
Comment
24)

would
not
change.

Sample
990052A
 
Commenter
appears
to
be
using
the
prechlorination
density
data
to
calculate
the
log
reduction.
As
noted
above,
this
is
not
the
procedure
followed
by
EPA.
The
comment
in
Table
II
suggests
that
commenter
believes
that
the
log
reduction
should
be
based
on
the
dilution
factor
1:
2,500.
Using
this
dilution
factor,
and
the
associated
post­
chlorination
enumeration
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
19
data,
the
recalculated
log
reduction
remains
at
2.3
(
log
((
1,000,000/
2,500)/(
1x2))
=
2.3)
EPA
notes
that
even
if
the
commenter
had
appropriately
calculated
the
log
reduction
as
1.7,
this
would
appropriately
fit
the
2­
4
log
reduction
range
as
discussed
in
response
to
Comment
3
(
Docket
ID
I­
D1.005,
Part
02).

Sample
990438A
 
EPA
has
performed
additional
and
repeat
statistical
analyses
in
response
to
public
comments
and
has
ensured
that
the
correct
data
are
being
used.
See
also
the
response
to
Comment
33
(
Docket
ID
I­
D1.002,
Part
05).

E.
coli
(
Table
III):

Sample
982084A
 
The
commenter
has
stated
that
this
sample
lacks
pre­
chlorination
density
data.
As
discussed
above,
the
fact
that
prechlorination
density
data
was
not
available
for
this
sample
does
not
invalidate
the
sample;

such
data
were
not
used
in
EPA s
analysis.
See
also
EPA s
response
to
Comment
25
(
Docket
ID
I­
D1.002,

Part
04).

Sample
982305A­
1
 
Commenter
incorrectly
suggests
that
the
post­
chlorination
count
for
mFC
is
absent.
This
count
appears
in
section
5
on
page
2
of
the
ATP
worksheet
for
this
sample.

(
EPA
notes
that
it
is
no
longer
relying
on
the
results
from
Sample
982305A­
2.
Thus,
the
missing
data
sheet
the
commenter
refers
to
has
no
impact
on
EPA s
decision.)

Sample
990095A
 
As
reflected
in
the
December
2,
2002
NODA,
the
E.
coli
log
reduction
for
this
sample
cannot
be
determined
using
the
standard
calculation
because
the
original
density
measurement
is
not
available.
This
does
not,
however,
preclude
the
use
of
the
comparability
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
20
5.
 
Even
with
the
recalculation
per
the
tables
in
the
FR
of
12/
02/
2002,
only
50%
of
the
total
coliform
data
and
80%
of
study
results
from
the
sample
in
EPA s
analysis;
the
key
consideration
is
whether
those
results
were
generated
using
E.
coli
that
had
been
subjected
to
adequate
chlorine
stressing.
Based
on
the
following
facts,
and
absent
information
to
the
contrary,
EPA
believes
that
it
is
reasonable
to
consider
the
comparability
study
results
for
this
sample
in
its
statistical
analysis
(
i.
e.,
EPA
can
reasonably
conclude
that
the
E.
coli
used
in
the
comparability
study
were
subjected
to
adequate
chlorine
stressing):

­
100%
(
19
of
the
19)
of
the
samples
for
which
total
coliform
and
E.
coli
log
reduction
could
be
calculated
yielded
acceptable
results
(
i.
e.,
log
reductions
between
2­

4).

­
Among
the
10
total
coliform
samples
used
in
the
studies,
the
subject
sample
had
one
of
the
highest
levels
of
total
coliforms
(
at
16,000,000
CFU/
100mL).
One
can
reasonably
postulate
that
the
source
water
had
high
E.
coli
levels
as
well
(
among
the
other
9
source
waters,
E.

coli
was
measured
at
an
average
of
42%
of
the
total
coliform
measurements).

­
If
the
pre­
chlorination
sample
contained
even
moderate
E.
coli
levels,
and
since
it
is
clear
that
the
postchlorination
E.
coli
level
was
low
(
7
CFU/
100mL),
one
can
be
confident
that
a
substantial
degree
of
chlorine
stressing
occurred.
The
fact
that
a
consistent
chlorination
procedure
was
used
in
the
studies
lends
further
credence
to
this
predictive
approach.

­
The
total
coliform
log
reduction
for
this
sample,
at
2.8,

was
among
the
higher
results.

All
of
these
facts
suggest
that
the
E.
coli
in
this
sample
were
subjected
to
substantial
chlorine
stressing;
EPA
is
not
aware
of
any
facts
that
contradict
this
position.

Referring
to
the
discussion
of
EPA s
log
reduction
goal
in
the
response
to
Comment
3
(
Docket
ID
I­
D1.005,
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
21
the
E.
coli
data
satisfies
US
EPA s
chosen
interpretation
of
the
3­
4
log10
reduction
requirement,
i.
e.
rounding
up
from
2.5
onwards.
Plus,
as
stated
earlier,
the
data
selected
for
comparability
statistics
was
not
consistent
with
the
dilutions
used
for
the
Log10
reduction. 

6.
 
Data
using
ATCC
strain
25922
is
irrelevant
since
the
strain
does
not
allow
the
method
to
detect
the
diversity
of
total
coliforms
and
E.
coli
that
is
present
in
the
natural
environment.

Furthermore,
the
strain
was
used
without
the
presence
of
any
background
flora
and
background
sample
matrix.
Data
generated
from
this
type
of
experiment
does
not
prove
that
the
method
will
perform
adequately
with
natural
samples
and
therefore,
if
approved,
may
compromise
public
health.
The
US
EPA s
own
ATP
however,
if
followed,
was
designed
to
address
exactly
the
critical
natural
sample
aspects
cited
above. 
Part
02),
EPA
notes
that
such
goal
was
met
in
100%
of
the
cases
for
which
log
reduction
results
were
available
(
10
of
10
total
coliform
results
and
9
of
9
E.
coli
results).

As
discussed
above
in
the
responses
to
sample­
specific
comments
[
and
as
discussed
further
in
the
response
to
Comment
24
(
Docket
ID
I­
D1.001,
Part
03)],
EPA
has
repeated
its
statistical
analysis
using
comparability
data
and
log
reductions
associated
with
the
same
dilutions.
As
with
the
original
analysis,
the
repeat
analysis
did
not
identify
a
statistically
significant
difference
between
the
performance
of
Colitag
 
and
the
reference
methods.

The
data
presented
where
ATCC
25922
was
used
represent
additional
unsolicited
information
presented
by
CPI
together
with
re­
calculated
log
reduction
data:

the
former
information
was
not
used
in
EPA s
evaluation
of
the
Colitag
 
method.

14
ID1.004
02
 
The
data
provided
still
is
inadequate
to
determine
if
the
Colitag
product
can
in
fact
recover
chlorine
stressed
organisms.
The
ability
of
a
test
to
recover
chlorine
stressed
organisms
is
the
most
important
aspect
of
the
EPA
required
alternative
test
protocol.
The
test
principle
is
straight
forward.
A
complex
wastewater
sample
with
at
least
100,000
of
the
target
organism
/
100mL
(
E.
coli
or
total
coliforms)
is
exposed
to
a
dose
of
chlorine
for
10
to
30
minutes
which
results
in
a
3
to
4
log
reduction.
This
disinfection
stressing
protocol
is
intended
to
mimic
the
stress
experienced
by
a
naturally
occurring
organism
introduced
from
a
sanitary
sewer
cross
connection
into
a
chlorinated
public
water
supply.
The
procedure
for
preparing
stressed
organism
samples
for
use
in
the
ATP
is
clearly
outlined
by
EPA
and
is
based
on
published
literature.(
Evaluating
a
Commercially
Available
Defined
Substrate
Test
for
Recovery
of
E.
coli.
S.
C.
McCarty,
J.
H.
Standridge
and
M.
C.
Stasiak.
See
responses
to
Comment
3
(
Docket
ID
I­
D1.005,
Part
02)
and
Comment
12
(
Docket
ID
I­
F.
021,
Part
10)

concerning
recovery
of
chlorine­
stressed
bacteria.

Details
of
how
the
log
reduction
was
calculated
were
included
in
the
public
docket
and
relevant
information
on
recalculation
of
log
reductions
was
published
in
the
December
2002
NODA.

EPA
is
unclear
as
to
why
this
commenter
purports
to
lack
information
on
how
stressing
was
conducted.
This
information
was
included
in
CPI s
study
plan,
which
was
included
in
the
docket
at
proposal.
Moreover,
stressing
procedures
were
described
in
Biovir s
log
sheets
which
are
also
in
the
docket.
Similarly,
EPA
does
not
understand
why
this
commenter
purports
to
lack
information
on
the
log
reductions.
The
methodology,
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
22
JAWWA:
84:
5.
May
1992.
Pp.
91­
97)
The
tables
presented
in
the
Dec.
2nd
additional
information
still
do
not
make
it
clear
how
the
stressing
procedures
were
done
and
exactly
what
the
log
reduction
was.
The
before
and
after
disinfection
concentrations
as
measured
by
an
accepted
standard
method
must
demonstrate
at
least
a
3
log
reduction
and
result
in
a
detectable
number
of
organisms.
The
result
of
subtracting
the
base
10
log
of
the
post
disinfection
from
the
base
10
log
before
chlorination
must
be
greater
than
3.
If
this
number
is
not
greater
than
three,
the
stressing
of
the
organisms
is
insufficient.
The
numbers
presented
in
the
Dec.
2nd
tables
do
not
demonstrate
a
sufficient
log
reduction,
and
therefore
the
ATP
has
not
been
followed
and
we
have
no
data
that
tells
us
whether
or
not
the
product
is
capable
of
detecting
stressed
organisms. 

 
I
strongly
disagree
that
the
methods
and
data
presented
are
consistent
with
previous
product
approvals.
Approval
must
be
denied
until
further
evaluation
can
be
completed. 
the
individual
figures
used
in
the
calculations
and
the
results
were
published
in
the
Federal
Register
itself,
and
the
worksheets
from
which
the
individual
figures
were
derived
have
been
in
the
docket
since
approval
of
this
method
was
proposed.
Other
commenters
do
not
seem
to
have
had
the
difficulty
expressed
by
this
commenter.

EPA
disagrees
that
it
lacks
data
indicating
that
Colitag
 
is
capable
of
detecting
stressed
organisms.
As
discussed
in
response
to
Comment
3
(
Docket
ID
I­
D1.005,
Part
02),
EPA
believes
that
this
method
has
successfully
demonstrated
its
ability
to
recover
organisms
stressed
at
levels
resulting
in
2­
4
log
reductions
 
levels
which
the
Agency
believes
reflects
a
high
degree
of
stress.

With
respect
to
EPA
consistency
in
evaluating
methods,

EPA
notes
that
there
is
a
history
of
using
expert
judgement
to
supplement
the
ATP
protocol
guidance;
a
review
of
past
method
evaluations
confirms
that
the
log
reduction
guideline
and
other
guidelines
have
not
been
applied
as
rigid
requirements.
The
commenter
has
not
specified
any
decision
EPA
has
made
in
the
past
that
is
inconsistent
with
today s
decision.

15
I­
F.
004
02
1.
Colitag's
ATP
Comparability
Study
did
not
meet
the
Injury
Level
Requirements
 
As
stated
in
the
USEPA
ATP
Section
4.5.2.1
and
4.5.5,
the
chlorination
must
reduce
the
105
CFU/
100
ml
in
the
spiked
water
samples
by
3
­
4
Log10
(
i.
e.
99.9­
99.99%)
in
order
to
assure
the
presence
of
stressed
organisms.
This
is
the
most
important
criterion
for
the
USEPA
ATP
protocol
for
the
evaluation
of
a
proposed
method
to
detect
total
coliforms
and
E.
coli
in
drinking
water
as
this
is
directly
relevant
to
public
health
since
the
potability
of
drinking
water
is
assessed
by
the
presence
or
absence
of
total
coliforms
and
E.
coli
in
the
water
sample. 

 
The
comparability
data
submitted
by
CPI
International
for
the
The
commenter
states
that
the
comparability
study
data
presented
by
CPI
for
Colitag
 
does
not
meet
the
3
to
4
log
reduction
criterion
for
demonstrating
that
Colitag
 
is
able
to
recover
chlorine
stressed
organisms.
See
responses
to
Comment
3
(
Docket
ID
I­
D1.005,
Part
02)

and
Comment
12
(
Docket
ID
I­
F.
021,
Part
10)

concerning
log
reduction
and
recovery
of
chlorinestressed
bacteria,
and
the
acceptability
of
a
wider
range
of
log
reduction.

EPA
disagrees
that
 
there
was
no
documentation
on
the
injury
levels
for
.
.
.
sample
#
982305A. 
The
log
reduction
for
sample
982305A
and
the
other
samples
was
computed
based
on
the
original
density
determination
and
the
post­
chlorination
cell
counts,
as
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
23
approval
consideration
under
the
Safe
Drinking
Water
Act
did
not
meet
this
critical
requirement
(
see
Appendix)
Strikingly,

none
of
the
data
for
E.
coli
met
this
most
critical
requirement
(
the
reduction
levels
only
ranged
from
1­
2
Log10).
With
the
exception
of
two
samples
[
I.
D.
#:
990025A
&
990095A]
where
a
3.3
Log10
reduction
of
target
coliforms
was
achieved,
8/
10
samples
did
not
achieve
a
3
­
4
Log10
reduction
for
total
coliforms
(
ranged
from
0.7
to
2.1
Log10).
In
addition,
there
was
no
documentation
on
the
injury
levels
for
either
coliforms
or
E.
coli
for
sample
#
982305A.
The
average
Log10
reduction
in
cell
number
(
excluding
one
unreported
data
by
Colitag)
for
total
coliforms
was
1.89
and
for
E.
coli
was
1.51.
Clearly,

Colitag
did
not
meet
such
requirements
in
this
study
to
evaluate
its
ability
in
detecting
stressed
bacteria. 
described
in
the
December
2,
2002
NODA.
The
data
used
for
these
calculations
is
included
in
the
ATP
worksheets,
which
was
in
the
docket.

Using
the
revised
data,
the
average
log
reduction
for
total
coliforms
is
2.5
and
the
average
log
reduction
for
E.
coli
is
2.9.
EPA
surmises
that
commenter
has
either
made
errors
in
calculating
the
log
reductions
or
has
employed
a
method
other
than
the
one
described
by
the
Agency
in
the
December
2,
2002
NODA.

16
I­
F.
006
02
o
Detection
of
Escherichia
coli
(
E.
coli)
[
both
Colitag
 
and
ReadyCult]

 
To
protect
the
public's
health,
it
is
essential
that
a
procedure
accurately
detect
both
native
and
chlorine­
stressed
E.
coli.
E.

coli
is
the
indicator
of
fecal
pollution,
upon
which
rests
each
of
these
test's
protection
of
the
public
health.
In
fact
the
detection
of
E.
coli
was
considered
so
important
for
the
protection
of
public
health
that,
when
a
question
arose
regarding
the
accuracy
of
the
MMO­
MUG
test
to
detect
stressed
E.
coli,

approval
of
the
method
was
put
in
abeyance
pending
resolution.

Additional
studies
needed
to
be
conducted
by
third
parties
to
resolve
this
issue
(
see
references
1
and
2
below.
To
highlight
the
seriousness
of
this
concern,
reference
number
2
is
from
the
USEPA's
own
laboratory.).
Neither
Colitag
 
nor
ReadyCult
followed
the
procedure
and
sample
requirements
to
demonstrate
recovery
of
oxidant
stressed
E.
coli.
The
Colitag
 
product
did
not
meet
this
basic
requirement
in
80%
of
its
samples
and
the
ReadyCult
product
in
60%
of
its
samples.
This
deficit
is
particularly
disturbing
since
the
injury
protocol
is
clearly
delineated
by
USEPA
and
has
been
successfully
used,
and
described,
in
two
peer­
reviewed
papers
(
1,2).
Accordingly,
this
glaring
deficit
must
be
remedied
and
the
data
made
available
for
public
comment
prior
to
approval. 
Concerning
questions
about
accurately
detecting
native
and
chlorine
stressed
E.
coli,
whereas
such
questions
existed
in
the
case
of
MMO­
MUG
and
warranted
additional
analysis,
EPA
is
satisfied
that
its
analysis
of
the
Colitag
 
data
demonstrated
comparability
with
the
reference
methods
and
did
not
feel
that
more
testing
was
needed
for
evaluation
of
the
performance
of
Colitag
 
.

This
is
further
explained
in
the
responses
to
Comment
3
(
Docket
ID
I­
D1.005,
Part
02)
and
Comment
12
(
Docket
ID
I­
F.
021,
Part
10),
concerning
recovery
of
chlorine­
stressed
bacteria,
and
Comment
4
(
Docket
ID
ID1.005
Part
04)
concerning
the
approach
to
the
MMOMUG
review.
References:

1.
McCarty,
S.
C.,

Standridge,
J.
H.,
and
Stasiak,
M.
C.
1992.

Evaluating
a
commercially
available
definedsubstrate
test
for
recovery
of
E.
coli.
J.

American
Water
Works
Association
84(
5):
91­
97.

2.
Covert,
T.
C.,
Rice,

E.
W.,
Johnson,
S.
A.,

Berman,
D.,
Johnson,

C.
H.,
and
Mason,
P.
J.

1992.
Comparing
defined­
substrate
coliform
tests
for
the
detection
of
Escherichia
coli
in
water.
J.
American
Water
Works
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
24
Association
84(
5):
98­

104.

17
I­
F.
015
01
 
I
also
note
that
another
product,
Colitag
 
is
also
being
considered
for
regulatory
approval.
As
with
the
ReadyCult
study,
there
are
some
deviations
from
the
ATP.
In
particular,
in
the
majority
of
samples
used,
the
3­
4
log
reduction
by
disinfection
was
not
adhered
to.
As
stated
above,
this
is
a
most
important
criterion
for
deciding
on
the
suitability
of
a
method
for
assessing
the
quality
of
drinking
water.
It
is
clear
therefore
that
the
Colitag
 
product
should
not
be
approved
for
assessing
the
quality
of
drinking
water
in
the
United
States. 
See
responses
to
Comment
3
(
Docket
ID
I­
D1.005,
Part
02)
and
Comment
12
(
Docket
ID
I­
F.
021,
Part
10)

concerning
recovery
of
chlorine­
stressed
bacteria.

18
I­
F.
021
03
(
2)
 
During
comparability
testing,
it
is
critical
to
establish
that
chlorine­
injured
microorganisms
can
be
recovered
using
the
proposed
method(
s)
in
order
to
model
'
real
world'
scenarios,

with
specific
reference
to
drinking
water
distribution
systems
where
chlorination
is
practiced.
The
ATP
protocol
requires
establishment
of
3­
4
log10
reductions
of
test
microorganisms
following
chlorine
pretreatment,
however,
in
all
but
two
of
the
tests
conducted,
the
data
failed
to
meet
this
criterion
(
in
7
experiments
the
log10
reductions
were
significantly
below
this
target
level,
and
in
one
experiment
the
log10
reduction
exceeded
the
target
level
of
3­
4
log10).
If
only
20%
of
the
submitted
data
met
the
criterion
defined
under
the
ATP
protocol,
it
is
inappropriate
to
consider
the
data
set
representative
of
the
conditions
that
might
be
found
in
field
samples. 
See
responses
to
Comment
3
(
Docket
ID
I­
D1.005,
Part
02)
and
Comment
12
(
Docket
ID
I­
F.
021,
Part
10)

concerning
recovery
of
chlorine­
stressed
bacteria
stressed
to
less
than
3
to
4
logs
of
reduction.

The
commenter
also
suggests
that
the
log
reduction
of
total
coliforms
in
one
sample
(
982305A,
Millbrae,
CA)

was
4.5
in
the
proposal
of
Colitag
 
,
exceeding
the
3­
4
log
goal.
EPA
notes,
however,
that
this
number
was
recalculated
in
the
December
2002
NODA,
and
the
recalculated
number
is
3.4.

19
I­
F.
022
03
1
. 
The
injury
requirements
were
not
met
by
Colitag
 
'
s
ATP
comparability
study.
Eight
out
of
10
samples
did
not
achieve
a
3
to
4
log
reduction
for
total
coliforms,
and
none
of
the
data
for
E.
coli
met
the
3
to
4
log
reduction
requirement. 
See
responses
to
Comment
3
(
Docket
ID
I­
D1.005,
Part
02)
and
Comment
12
(
Docket
ID
I­
F.
021,
Part
10)

concerning
recovery
of
chlorine­
stressed
bacteria.
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
25
03.01.02
Sample
source
guidelines
(
spiked
samples;

10
geographically
dispersed
sites)

20
ID1.002
12
 
EPA
has
revised
the
log
reduction
following
chlorine
pretreatment
for
sample
number
990442A.
It
should
be
noted
that
this
sample
was
drawn
from
the
same
source
as
sample
number
990217A
(
Mission,
KS),
in
violation
of
ATP
section
4.1.1.
which
calls
for
samples
from
 
ten
or
more
geographically
dispersed
sites .
The
concern
regarding
two
samples
being
taken
from
Mission,
Kansas
is
addressed
in
the
response
to
Comment
12
(
Docket
ID
I­
F.
021,
Part
10).
As
explained,
EPA
concluded
that
an
acceptable
degree
of
bacterial
diversity
was
achieved
in
this
particular
case
using
the
10
samples
from
8
sites.

21
I­
F.
004
03
2.
 
Colitag
 
'
s
ATP
Comparability
Study
did
not
meet
Sample
Sources
Requirement 

 
As
stated
in
the
USEPA
ATP
protocol
section
4.1.1,
the
recommended
spiked
samples
should
be
non­
chlorinated
secondary
sewage
effluents
or
polluted
surface
waters.
And
these
samples
must
come
from
at
least
10
or
more
geographically
dispersed
sites. 

 
It
appears
that
there
were
2
sets
of
sewage
effluent
samples
that
came
from
identical
sources
(
see
Appendix).
Samples
(
I.
D
#
990442A
&
990217A)
were
from
Johnson
County
Environmental
Laboratory
[
4800
Nall,
Mission,
KS]
and
samples
(
I.
D.#
B982305A
&
982084A)
were
collected
from
City
of
Millbrae
WWTP,
CA].
This
means
that
samples
used
in
Colitag
 
'
s
ATP
study
were
ONLY
collected
from
8
different
geographically
dispersed
sites
and
the
requirements
for
sample
sources
laid
out
in
ATP
protocol
section
4.1.1
were
not
met. 
See
response
to
Comment
12
(
Docket
ID
I­
F.
021,
Part
10)
concerning
geographic
distribution
of
samples.

22
I­
F.
021
02
(
1)
 
A
major
requirement
for
comparability
testing
under
the
USEPA
Protocol
for
Alternate
Test
Procedures
for
Coliform
Bacteria
in
Compliance
with
Drinking
Water
Regulations
is
to
validate
proposed
methods
using
water
samples
collected
from
geographically
diverse
regions
of
the
U.
S.
Although
it
is
not
entirely
clear
from
the
ATP
worksheets
submitted
by
the
independent
laboratory
precisely
where
each
sample
was
collected,
it
appears
that
at
least
two
pairs
of
samples
were
collected
from
the
same
source
(
Mission,
Kansas
and
Millbrae,

California),
with
the
end
result
of
a
data
set
containing
only
8
geographically
diverse
samples.
Hence,
the
submitted
data
fails
to
comply
with
the
criteria
established
in
the
ATP
protocol. 
See
response
to
Comment
12
(
Docket
ID
I­
F.
021,
Part
10)
concerning
geographic
distribution
of
samples.
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
26
23
I­
F.
022
04
2.
 
Two
sewage
effluent
samples
were
repeat
samples.
Samples
990442A
and
990217A
were
both
from
Johnson
County
Environmental
Laboratory,
Mission,
KS.
Samples
982305A
and
982084A
were
both
from
the
City
of
Millbare,
CA.
Hence,
only
8
sites
were
actually
sampled.
The
USEPA
ATP
Protocol
calls
for
at
least
10
or
more
geographically
dispersed
sites
to
be
sampled. 
See
response
to
Comment
12
(
Docket
ID
I­
F.
021,
Part
10)
concerning
geographic
distribution
of
samples.

03.01.03
Replicate
and
data
selection
guidelines
(
ability
to
detect
1­
10
TC
or
achieve
25%/
75%
split;
replicate
analysis
on
3
dilutions;
lag
time/
inferred
bacteria
densities)

24
ID1.001
03
1.
 
Recalculation
of
Log10
reduction
by
the
US
EPA
based
on
the
dilution
factor
and
a
measurement
of
the
source
water
prior
to
dilution,
as
described
in
the
December
2,
2002
Federal
Register,
is
inappropriate.
Through
detailed
review
of
Colitag
 
 
s
ATP
worksheets,
we
found
that
in
a
number
of
samples
there
was
a
significant
lag
time
for
bacterial
density
determination
between
the
source
sample
and
the
diluted
sample
prior
to
chlorination
(
in
6
instances
per
Table
I,

boldened
for
emphasis).
Bacterial
density
and
microflora
in
the
source
samples
may
significantly
be
altered
during
this
lag
time.

In
addition,
contrary
to
standard
microbiological
laboratory
practices,
chlorination
injury
experiments
were
performed
from
the
diluted
source
samples
rather
than
from
the
original
source
samples.
Therefore,
the
 
Log10
Reduction 
calculation
for
the
pre­
chlorination
bacterial
density
should
be
based
on
the
membrane
filter
analysis
of
the
diluted
source
water
samples,

which
was
originally
reported
by
BioVir
Laboratories.
Also
per
Table
1.
(
boldened
for
emphasis),
in
one
instance
there
is
a
time
lag
between
dilution
and
chlorination,
plus
in
four
instances,

there
is
a
time
reversal
between
the
two
(
indicated
by
a
question
mark). 
With
respect
to
the
comments
concerning
 
hold
time 

between
the
initial
density
determination
and
the
postchlorination
measurement
used
to
calculate
log
reduction,
EPA
notes
that
it
has
not
established
guidelines
for
such
in
its
ATP
protocol.
The
Agency
has
not
asked
that
such
be
documented,
nor
has
it
applied
a
standard
for
such
in
previous
method
evaluations.

Further,
a
review
of
previously­
evaluated
methods
reveals
that
the
public
record
for
some
(
e.
g.,
Readycult,

Colisure)
does
not
include
sufficient
data
to
determine
hold­
time.

EPA s
presumption,
for
this
and
for
previous
method
evaluations,
is
that
the
certified
drinking
water
laboratories
performing
the
comparability
studies
will
employ
a
reasonable
hold
time.
Nonetheless,
EPA
conducted
a
thorough
review
of
the
Colitag
 
hold
times,
pursuant
to
these
comments.
In
doing
so,
EPA
considering
dates
identified
for
sample
collection;

sample
receipt
at
the
laboratory;
original
density
determination;
and
comparability
study
completion,
as
reflected
in
the
worksheets,
comparability
study
data
sheets,
and
chain
of
custody
documentation
in
the
record.
EPA
further
considered
the
chronology
and
duration
of
the
steps
associated
with
the
various
tests
performed.
Based
on
all
of
this
information,
EPA
concluded
that
a
reasonable
hold
time
could
be
documented
for
the
majority
of
the
tests,
but
that
clear
hold
times
could
not
be
determined
for
four
of
the
tests
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
27
2.
 
The
dilutions
used
for
the
comparability
data
statistics
do
not
match
the
dilutions
used
for
the
Log10
reduction
calculation
as
indicated
in
Table
II
for
total
coliforms
and
Table
III
for
E.
coli.
For
total
coliforms
this
is
the
case
in
four
instances
and
for
E.
coli
in
five
instances
(
boldened
for
emphasis
in
the
respective
tables). 
(
samples
990025A,
990052A,
990217A,
and
990273A).

See
Attachment
1
 
Description
of
CPI
Comparability
Study
Log
Sheet
Hold
Times
for
Colitag
 
 
for
information
on
how
the
hold
times
were
calculated.

EPA s
inability
to
conclude
with
certainty
that
the
hold
time
for
those
four
tests
was
reasonable
is
not
tantamount
to
concluding
that
the
hold
times
were
unreasonably
long.
Rather,
it
is
possible
that
a
reasonable
hold
time
was
achieved
for
each
of
the
aforementioned
tests.
As
noted
above,
these
samples
were
evaluated
by
a
certified
drinking
water
laboratory
and
EPA
presumes
a
reasonable
hold
time
for
all
samples.

Under
a
worst­
case
scenario,
however,
EPA
repeated
its
statistical
analysis
of
the
Colitag
 
data
set,
excluding
the
aforementioned
four
tests.
The
conclusion
(
i.
e.,
that
the
comparability
study
did
not
identify
a
statistically
significant
difference
in
performance
between
the
reference
methods
and
Colitag
 
)
did
not
change,
nor
was
the
strength
of
the
conclusion
substantially
different
with
the
limited
(
6­
test)
data
set
as
compared
to
the
full
(
10­
test)
data
set.
(
See
Attachment
2,
 
Statistical
Impact
of
Relying
on
a
6­
Test
Data
Set
Versus
a
10­
Test
Set 

for
additional
discussion.)

Accordingly,
EPA
stands
by
its
decision
to
use
the
log
reduction
data
presented
in
the
December
2002
NODA.

In
response
to
the
comment
that
the
dilutions
used
for
the
comparability
data
statistics
do
not
match
the
dilutions
used
for
the
log
reduction
calculations,
the
commenter
has
not
indicated
the
relevance
of
this
comment.
EPA
surmises
that
the
commenter
intended
to
suggest
that
the
log
reductions
presented
in
the
NODA
should
be
corrected
to
match
the
dilutions
that
were
used
for
the
comparability
studies
or
the
data
set
used
in
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
28
3.
 
Based
on
the
observations
above,
IDEXX
recalculated
the
Log10
reduction
using
the
bacterial
density
of
the
actual
diluted
source
sample
which
was
used
to
injure
the
bacteria,
rather
than
the
statistical
analysis
should
be
modified.
To
the
extent
that
such
is
the
case,
EPA
agrees
and
has
taken
steps
to
ensure
that
the
log
reduction
calculations
and
statistical
analysis
rely
on
the
same
data
set
(
i.
e,
data
associated
with
the
same
dilution).

Specifically,
the
data
sets
have
been
reviewed
and
the
statistical
analysis
has
been
repeated
by
EPA
using
logreduction
and
comparability­
study
data
associated
with
the
same
dilutions.
Under
such
approach,
total
coliform
log
reductions
remain
as
presented
in
the
NODA,
with
the
following
exceptions:
the
log
reduction
associated
with
the
1:
10,000
dilution
for
sample
982084A
would
change
from
2.1
to
1.8,
and
the
log
reduction
associated
with
the
1:
2000
dilution
for
sample
990025A
would
change
from
3.2
to
3.0.
Under
such
approach,
E.
coli
log
reductions
remain
as
presented
in
the
NODA,
with
one
exception:
the
log
reduction
associated
with
the
1:
200
dilution
for
sample
990438A
would
change
from
2.7
to
3.4.
None
of
these
changes
indicate
that
the
log
reductions
for
the
samples
analyzed
were
potentially
improper
and
none
of
these
changes
had
an
effect
on
EPA s
conclusion.

In
the
case
of
total
coliforms
for
sample
982305A,

sufficient
data
were
not
available
to
recalculate
log
reduction
for
the
data
set
associated
with
the
1:
2000
dilution
used
in
the
12/
29/
98
comparability
study
(
the
comparability
study
results
originally
used
in
EPA s
statistical
analysis);
instead,
therefore,
the
statistical
analysis
was
repeated
using
the
data
set
associated
with
the
1:
1000
dilution
used
in
the
12/
23/
98
comparability
study
(
i.
e,
the
same
data
set
used
to
calculate
the
3.4
log
reduction.)
The
conclusion
with
respect
to
reference
method/
Colitag
 
method
comparability
did
not
change.
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
29
extrapolating
from
the
original
source
sample.
In
addition,

IDEXX
used
the
bacterial
density
from
the
same
dilution
that
was
used
for
the
comparability
statistics. 
The
commenter s
recalculated
log
reduction
values
are
not
relevant
to
today s
action
because
these
calculations
were
done
differently
than
those
used
for
the
log
reduction
determinations
presented
in
the
Dec.
2,
2002
NODA
and
used
as
the
basis
for
EPA s
evaluation
of
method
comparability.
(
As
described
in
the
NODA,
the
approach
used
by
EPA
to
calculate
log
reduction
is
consistent
with
previous
method
evaluations.
It
is
also
consistent
with
EPA s
ATP
protocol.).
Also,
as
described
above,
EPA
analyzed
the
data
using
the
same
dilutions
that
were
used
in
the
comparability
study
samples,
and
found
that
these
did
not
change
EPA s
conclusions
about
the
comparability
study
results.

25
ID1.002
04
 
The
ATP
protocol
devised
by
USEPA
requires
the
testing
laboratory
to
collect
wastewater
isolates
from
geographically
diverse
regions
of
the
country
and
to
conduct
 
chlorine
injury 

experiments
to
model
pathogen
intrusion
events
that
may
occur
in
distribution
systems.
For
these
experiments,
the
independent
testing
laboratory
performed
a
series
of
enumerative
assays
on
the
wastewater
isolates.
The
initial
 
range­
finding 
assay
was
performed
to
identify
the
concentration
of
total
coliforms
and
E.
coli
in
each
wastewater
so
that
the
appropriate
dilution
could
be
identified
that
would
yield
1­
10
CFU/
100mL
following
chlorine
treatment.
The
second
assay
identified
the
densities
of
microorganisms
in
the
actual
dilution
used
for
the
chlorine
injury
experiments.
The
final
assay
was
performed
to
enumerate
test
microorganisms
following
chlorine
injury
so
that
microbial
reductions
could
be
computed.
Chlorine­
treated
samples
were
then
subjected
to
comparability
studies
using
both
the
proposed
and
reference
methods.
For
some
samples,
these
assays
were
performed
over
a
span
of
a
few
days.
For
others,
the
time
span
between
the
initial
characterization
of
the
microbial
densities
and
the
densities
following
chlorine
treatment
was
a
month
or
more.
In
its
December
2
revision
of
the
comparability
data,

EPA
relied
exclusively
upon
the
data
from
the
initial
rangefinding
assay
to
identify
the
 
pre­
chlorination 
concentration
of
test
microorganisms
rather
than
using
the
data
reported
by
the
testing
laboratory
on
the
actual,
diluted
samples
used
in
the
In
response
to
the
commenter s
suggestion
that
the
ATP
protocol
 
requires 
particular
practices,
see
response
to
Comment
1
(
Docket
ID
I­
D1.001,
Part
06).

In
response
to
commenter s
suggestion
that
the
time
lag
between
the
initial
bacterial
density
determination
and
the
dilutions
of
samples
preceding
chlorination
were
as
long
as
6
weeks,
see
response
to
Comment
24
(
Docket
ID
I­
D1.001,
Part
03)
concerning
 
hold
time 
between
initial
density
determination
and
post­
chlorination
measurement,
and
also
Attachment
1
for
specific
information
about
how
hold
times
were
determined.

The
approach
of
performing
Membrane
Filtration
analyses
on
the
spike
source
to
determine
original
density
of
target
microorganisms
and
using
this
information
to
determine
the
necessary
dilution
with
drinking
water
to
achieve
the
target
density
of
microorganisms
is
the
procedure
described
in
sections
4.1.1.2
and
4.1.1.3
of
the
ATP
protocol.
Moreover,
this
procedure
is
consistent
with
previous
method
evaluations
by
EPA.

EPA
experts
included
the
procedure
of
determining
bacterial
density
followed
by
serial
dilutions
in
the
ATP
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
30
disinfection
tests.
EPA
concluded
in
the
December
2,
2002
Federal
Register
notice
that
this
approach
was
  
sound
and
provides
consistency
with
previous
method
evaluations
by
EPA .
In
fact,
this
approach
is
inappropriate
in
that
it
assumes
that
the
microbial
densities
in
the
working
dilutions
used
for
the
chlorine
exposures
can
simply
be
extrapolated
from
the
results
of
the
initial
assay
after
accounting
for
the
dilution
factor
(
e.
g.

that
a
one
thousand­
fold
dilution
of
a
solution
of
105
bacterial
cells
will
result
in
a
solution
containing
102
cells).
This
assumption
fails
to
account
for
inaccuracies
that
may
result
from
dilution
errors
and
more
importantly
it
completely
fails
to
account
for
the
fact
that
the
initial
densities
of
test
microorganisms
were
in
numerous
instances
measured
several
weeks
earlier
than
the
actual
disinfection
experiment.
Hence,

microbial
reductions
were
computed
using
numbers
from
assays
that
were
spaced
up
to
six
weeks
apart.
This
approach
also
assumes
that
the
concentration
of
test
microorganisms
in
wastewater
is
stable
over
time.
It
should
be
noted
that
total
and
fecal
coliform
densities
in
wastewater
are
not
constant
over
time,
regardless
of
the
storage
condition.
Hence,
measurements
of
microbial
densities
that
occur
2­
6
weeks
prior
to
disinfection
treatment
are
not
valid
for
computing
the
true
response
to
disinfection.
The
revised
table
presented
in
the
December
2,

2002
Federal
Register
notice
is
a
misrepresentation
of
the
experimental
data
and
should
not
be
considered
adequate
to
support
manufacturer
claims. 
protocol
because
they
felt
that
this
was
a
sound
procedure.
The
use
of
serial
dilutions
to
achieve
a
desired
bacterial
density
is
a
standard
microbiological
practice,
and
if
done
with
care
gives
results
that
are
generally
within
the
accepted
range
of
variability
inherent
in
microbiological
enumeration
methodologies.

EPA
decided
to
use
the
methodology
for
calculating
bacterial
density
it
has
used
to
evaluate
other
analytical
methods.
Moreover,
actual
post­
dilution
enumeration
was
not
performed
for
every
sample
in
this
study.
(
See,

e.
g.,
Sample
982084A.)
The
Agency s
inability
to
employ
actual
post­
dilution
enumeration
figures
consistently
across
all
Colitag
 
samples
further
supports
EPA s
decision
to
use
figures
derived
from
an
initial
density
determination
and
a
dilution
factor.

For
the
above
reasons,
EPA
believes
that
the
December
2002
NODA
included
an
appropriate
representation
of
the
CPI
experimental
data.

See
also
response
to
Comment
24
(
Docket
ID
I­
D
1.001,
Part
03).

26
ID1.002
08
E.
coli
data
 
EPA
has
revised
the
log
reduction
following
chlorine
pretreatment
for
sample
number
982084A.
For
this
experiment
the
pre­
disinfection
concentration
of
E.
coli
was
determined
by
using
a
different
dilution
of
the
original
titer
established
from
the
initial
evaluation
of
the
wastewater
isolate
rather
than
enumerating
the
1,000­
fold
diluted
sample
used
for
the
experiments;
hence,
the
 
seed 
titer
for
the
disinfection
experiment
is
an
inferred
number
rather
than
a
direct
measurement.
The
absence
of
actual
data
for
the
diluted
solution
used
in
the
disinfection
experiment
makes
it
impossible
In
reference
to
the
comment
on
the
use
of
an
inferred
density
of
bacteria
based
on
sample
dilution
and
the
density
of
bacteria
in
the
undiluted
sample,
see
response
to
Comment
25
(
Docket
ID
I­
D
1.002
Part
04)

concerning
the
use
of
inferred
versus
measured
data
for
log
reduction
calculations.
EPA
experts
who
wrote
the
ATP
protocol
felt
that
this
was
a
reasonable
and
reliable
approach
to
determine
log
reduction.
EPA
believes
that
it
is
reasonable
to
compute
log
reduction
from
chlorine
exposure
with
this
approach.

See
response
to
Comment
12
(
Docket
ID
I­
F.
021,
Part
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
31
to
compute
the
true
log
reduction
that
resulted
from
chlorine
exposure.
Regardless,
the
microbial
reduction
for
E.
coli
in
this
experiment
failed
to
achieve
the
required
3­
4
log10
reduction. 
10)
concerning
log
reduction.

27
ID1.002
09
 
EPA
has
revised
the
log
reduction
following
chlorine
pretreatment
for
sample
number
982305A.
For
this
experiment
the
pre­
disinfection
concentration
of
E.
coli
was
determined
using
the
10,000­
fold
dilution
of
the
original
wastewater
isolate.

However,
the
1,000­
fold
dilution
of
this
isolate
provided
no
colonies
in
mFC
agar
so
the
testing
laboratory
expressed
the
concentration
as
a
detection
limit
of
 <
1.0x105
CFU .
Because
no
bacteria
were
observed
in
the
dilution
used
by
EPA
in
the
revised
data
(
Table
2),
it
is
inappropriate
to
assume
that
the
initial
concentration
of
test
microorganisms
was
identical
to
the
concentration
observed
in
the
 
original
sample 
reported
on
the
bench
sheets.
It
is
also
puzzling
that
both
enumerative
experiments
were
performed
on
the
same
day
(
12/
23/
98)
since
the
initial
experiment
(
identification
of
the
concentration
of
E.

coli
in
the
original
wastewater
isolate)
was
presumably
used
to
identify
the
specific
dilution
to
be
used
for
the
subsequent
chlorination
experiment
that
presumably
would
have
been
conducted
24
hours
later.
In
the
methods
comparability
analysis,
4
of
20
samples
were
positive
according
to
the
proposed
method;
however,
the
chi
square
tables
for
this
sample
indicate
that
6
of
20
samples
were
positive
for
E.
coli
under
the
1:
1000
dilution
used
in
the
disinfection
experiment.

This
inconsistency
must
be
explained.
Data
generated
from
sample
982305A
should
not
be
considered
in
support
of
product
approval. 
As
the
commenter
notes,
the
log
reduction
calculation
was
based
on
the
number
of
bacteria
found
in
the
original
sample.
The
1:
10,000
dilution
that
the
commenter
mentions
was
a
dilution
used
during
enumeration
of
bacteria
in
the
original
sample.
The
1:
000
dilution
that
the
commenter
mentions
was
a
dilution
that
was
made
of
the
original
sample
preceding
chlorination
for
the
purpose
of
achieving
the
desired
density
of
bacteria
for
the
chlorination
experiment.
The
measured
<
1.0
X
10
5
CFU
value
the
commenter
mentions,
from
the
1:
000
dilution
from
the
prechlorination
enumeration,
was
not
used
to
determine
prechlorination
bacterial
density,
and
instead
the
value
was
inferred
from
the
original
bacterial
density,
adjusting
for
the
dilution.
EPA
notes
the
difference
in
bacterial
numbers
observed
between
original
density
determination
of
the
sample,
where
11
colonies
were
observed
on
mFC
medium
at
a
10,000
fold
dilution,
but
no
colonies
were
observed
in
the
1000
fold
dilution
of
the
prechlorination
density
determination.
For
the
purpose
of
the
data
presented
in
the
NODA,
this
difference
is
not
relevant
because
data
were
not
used
from
the
prechlorination
density
determination.
The
1000
fold
dilution
factor
cited
in
Table
2
of
the
revised
data
that
was
associated
with
the
sample
used
for
the
chlorination
experiment
was
used,
along
with
the
initial
density
determination,
for
the
log
reduction
calculation.

As
such,
there
is
no
conflict
between
use
of
E.
coli
data
from
the
10,000
fold
dilution
of
the
original
waste
water
sample(
for
the
initial
density
determination),
and
use
of
the
subsequent
1000
fold
dilution
used
prior
to
the
chlorination
experiment.
See
also
the
response
to
Comment
25
(
Docket
ID
I­
D
1.002
Part
04)
concerning
the
use
of
calculated
bacterial
density
versus
directly
measured
bacterial
density.
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
32
In
response
to
the
commenter s
observation
that
both
the
initial
sample
density
determination
and
the
density
determination
of
the
diluted
sample
were
done
on
the
same
day
(
Dec.
23),
the
commenter
should
also
note
that
the
results
of
the
comparability
study
were
also
dated
Dec.
23.
As
discussed
in
Attachment
1,
however,
the
Colitag
 
method
takes
24
hours
to
complete
and
the
reference
methods
each
require
a
minimum
of
48
hours.

Hence,
the
dates
entered
in
the
worksheets
cannot
possibly
reflect
all
of
the
actual
dates
for
the
corresponding
steps
in
the
evaluation
of
the
Colitag
 
method.
Instead,
EPA
surmizes
that
data
may
have
been
entered
into
laboratory
notebooks
on
a
real­
time
basis
and
subsequently
transfered
to
the
ATP
worksheets,

with
the
dates
in
the
worksheets
reflecting
the
dates
of
transcription
rather
than
those
corresponding
to
the
underlying
lab
procedures.
For
more
information
concerning
the
timing
and
documentation
of
studies,
the
commenter
is
referred
to
the
response
to
Comment
24
(
Docket
ID
I­
D1.001
part
03)
and
to
Attachment
1.

The
commenter
has
observed
that
four
Colitag
 
E.
coli
replicates
were
positive
in
the
comparability
study,
but
that
six
replicates
were
positive
for
data
used
in
the
chi
squared
analysis.
The
explanation
for
this
apparent
inconsistency
is
that
two
different
comparability
experiments
were
done
for
this
sample:
one
with
four
positives;
the
other
with
six.
The
log
reduction
was
originally
reported
for
the
experiment
with
four
positive
Colitag
 
E.
coli
replicates,
but
the
chi
squared
analysis
was
run
on
the
experiment
with
six
positive
Colitag
 
E.

coli
replicates.
For
the
reasons
explained
in
the
response
to
Comment
24
(
Docket
ID
I­
D1.001,
Part
03),
EPA
repeated
the
statistical
test
with
the
data
with
four
positives,
and
found
that
there
was
no
impact
on
the
conclusion
concerning
comparability
between
the
proposed
and
reference
methods.
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
33
28
ID1.002
13
 
Again,
the
log
reduction
presented
in
Table
2
of
the
December
2
Federal
Register
notice
was
computed
using
the
assay
results
generated
from
the
initial
analysis
of
the
wastewater
isolate
[
for
sample
990442A].
Instead,
the
data
presented
on
the
bench
sheet
from
the
actual
dilution
employed
in
the
disinfection
experiment
should
have
been
used
(
1
CFU
in
10
mL
from
the
1:
1000
dilution
for
an
effective
concentration
of
10
CFU/
100
mL).
The
log
reduction
that
results
from
use
of
this
number
is
0.7
log10
rather
than
the
3.2
presented
in
Table
2.
The
bench
sheet
is
missing
several
items
and
should
be
corrected
by
the
testing
laboratory
and
resubmitted
to
external
review.
This
data
fails
the
ATP
criteria
and
should
not
be
considered
in
support
of
product
approval. 

 
EPA
has
revised
the
log
reduction
following
chlorine
pretreatment
for
sample
number
990443A.
If
the
data
from
the
actual
dilution
used
in
the
disinfection
experiments
are
used
instead
of
the
data
from
the
original
analysis
of
the
wastewater
sample,
the
log
reduction
that
would
result
is
0.7
log10
rather
than
the
3.0
log10
reported
in
Table
2.
This
level
of
inactivation
fails
to
satisfy
the
3­
4
log10
criterion
specified
in
ATP
section
4.5.2.1.
Data
generated
from
sample
990443A
should
not
be
considered
in
support
of
product
approval. 
In
response
to
the
comment
on
the
use
of
bacteria
densities
determined
from
the
initial
sample
density
determination
versus
the
measured
value
from
the
sample
after
dilution
to
the
desired
pre­
chlorination
density,
EPA
notes
that
the
ATP
protocol
provides
for
calculation
of
bacterial
density
based
on
the
dilution
figures
and
on
the
original
density
determination;
such
procedure
has
been
followed,
as
is
discussed
in
the
response
to
Comment
25
(
Docket
ID
I­
D
1.002
part
04).

While
EPA
agrees
that
some
bench
sheets
appear
to
be
missing
some
information,
none
of
this
information
is
critical
to
EPA s
evaluation
of
the
method
and
did
not
interfere
with
EPA s
ability
to
evaluate
the
test
results
in
the
statistical
analysis.

In
response
to
the
comments
suggesting
that
the
log
reductions
from
samples
990442A
and
990443A
did
not
meet
the
3
to
4
log
reduction
criteria,
see
response
to
Comment
13
(
Docket
ID
I­
D1.001,
Part
04)
and
Comment
15
(
Docket
ID
I­
F.
004,
Part
02)
concerning
EPA s
calculation
of
log
reduction
results.

29
I­
F.
004
04
3.
Colitag
 
'
s
ATP
Comparability
Study
did
not
meet
the
number
of
Replicates
and
Data
Selection
Requirements
 
These
two
criteria
are
related.
The
intent
of
these
criteria
is
to
demonstrate
the
ability
of
the
proposed
method
to
detect
1­
10
total
coliforms
and
E.
coli
per
test
volume
(
i.
e.
100
ml)
or
to
achieve
a
25%/
75%
split
in
positive
and
negative
results
in
either
direction
(
Ideally,
a
50%/
50%
split).
The
USEPA
ATP
protocol
Section
3.1.2
states
that
a
minimum
of
20
replicate
analyses
must
be
performed
by
each
method
on
the
dilution
selected
for
each
of
the
10
spiked
samples.
Replicate
analyses
on
the
three
or
more
dilutions
of
each
spiked
sample
must
be
performed
on
the
same
day
for
both
the
reference
and
proposed
method
(
i.
e.
a
total
of
at
least
60
replicates
must
be
done
for
each
method).
Therefore,
three
dilutions
will
bracket
an
ideal
The
subject
comments
outlines
concerns
that
(
1)
for
some
samples,
the
comparability
study
was
not
performed
using
three
separate
sets
of
replicates
(
i.
e.,
at
three
dilutions),
and
(
2)
the
recommended
25%/
75%

split
was
not
achieved
by
the
replicate
analysis
for
one
or
more
of
the
samples
tested.
Each
of
these
issues
is
addressed
in
detail
in
the
response
to
Comment
12
(
Docket
ID
I­
F.
021,
Part
10);
as
noted
in
that
response,

EPA
concluded
that
the
studies
met
the
underlying
goal
of
detecting
low
numbers
of
bacteria
and
produced
results
that
supported
the
Agency s
statistical
analysis.

EPA
concluded
that
the
reference
methods
and
Colitag
 
perform
comparably.
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
34
set
of
20
replicates
each
in
which
there
is
at
most
a
25%/
75%

split
(
i.
e.
5
minimum
and
15
maximum
positive
observations
out
of
20)
in
positive
&
negative
responses.
Out
of
the
60
replicates
in
the
three
dilutions,
only
one
set
of
20
is
expected
to
satisfy
the
split
and
count
toward
the
result
requirements. 

 
It
is
evident
that
in
the
study
performed
by
BioVir
Laboratories
on
Colitag
 
only
1­
2
dilutions
were
used
for
each
spiked
sample
(
in
most
cases,
only
one
dilution
was
used).
As
a
result,
much
of
the
data
is
invalid
based
on
this
25%/
75%
rule. 

 
The
USEPA
ATP
protocol
section
4.6.5
clearly
states
"
if
one
of
the
dilutions
does
not
produce
an
acceptable
split
in
positive
and
negative
results
for
the
reference
method,
the
proposer
must
return
to
the
original
spiked
sample
and
begin
again
for
the
Comparability
Study".
In
this
study,
the
data
set
for
total
coliforms
from
samples
[
I.
D.
#
B982305A
and
990273A]
and
the
data
set
for
E.
coli
from
samples
[
I.
D.
#:
982084A,

982305A,
and
990217A]
did
not
meet
the
data
selection
criteria
(
see
Appendix).
This
means
that
these
data
are
not
valid
and
do
not
meet
the
requirements
as
described
in
the
USEPA
ATP
protocol. 

30
I­
F.
006
03
o
Sensitivity
of
the
Method
to
Detect
1
CFU/
ml
[
Colitag
 
]

 
The
ATP,
and
the
U.
S.
regulations,
clearly
state
that
a
method
must
be
able
to
detect
1
CFU
of
both
total
coliforms
and
E.
coli
per
100­
ml
sample.
The
ATP
contains
a
protocol
so
that
the
applicant
can
perform
sufficient
data
points
to
demonstrate
statistically
this
required
level
of
sensitivity.
However,
the
Colitag
 
test
does
not
meet
the
basic
criteria
set
forth
in
the
ATP.
Its
data
submission
provides
insufficient
number
of
Replicates
and
Data
Selection.
It
clearly
violates
the
USEPA's
25%/
75%
rule.
The
ATP
directly
states
that
if
there
is
not
an
acceptable
split
in
the
positive
and
negative
splits
from
the
samples,
then
"
the
proposer
must
return
to
the
original
spiked
sample
and
begin
again
for
the
Comparability
Study." 
In
response
to
the
comment
that
the
Colitag
 
comparability
study
data
did
not
meet
the
25%:
75%
split
guideline,
and
therefore
did
not
demonstrate
Colitag
 
 
s
ability
to
detect
very
low
numbers
of
target
bacteria,
the
commenter
is
referred
to
the
response
to
Comment
12
(
Docket
ID
I­
F.
021,
Part
10);
as
noted
in
that
response,

EPA
concluded
that
the
studies
met
the
underlying
goal
of
detecting
low
numbers
of
bacteria
and
produced
results
that
supported
the
Agency s
statistical
analysis.

EPA
concluded
that
the
reference
methods
and
Colitag
 
perform
comparably.

EPA
is
not
aware
of
the
regulatory
requirement
the
commenter
refers
to
and
reminds
the
commenter
that
the
ATP
protocol
constitutes
guidance.
That
protocol
recommends
that
comparability
studies
be
done
using
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
35
chlorine­
stressed
bacteria
in
low
numbers
(
1­
10
CFU/
100mL);
Colitag
 
studies
met
this
goal.

31
I­
F.
021
04
(
3)
 
The
ATP
protocol
calls
for
the
performance
of
serial
dilutions
to
bracket
microbial
densities
that
can
be
compared
directly
using
robust
statistical
methods.
The
use
of
bracketed
dilutions
is
aimed
at
ensuring
that
the
presence/
absence
data
is
not
skewed
in
one
direction
(
100%
absent)
or
the
other
(
100%

present).
However,
the
independent
laboratory
failed
to
bracket
the
test
samples
using
the
required
minimum
of
3
dilutions
for
all
experiments.
The
result,
not
surprisingly,
is
that
a
number
of
the
tests
failed
to
comply
with
the
25%:
75%
'
split'
guideline
defined
under
the
ATP
protocol.
Fully
half
of
the
submitted
data
(
5
of
10
experiments)
failed
to
fall
within
these
boundaries
for
either
total
coliforms,
E.
coli,
or
both,
rendering
the
statistical
analysis
of
the
data
set
somewhat
questionable.
In
light
of
the
failure
of
the
independent
laboratory
to
comply
with
the
established
testing
guidelines,
it
is
inappropriate
to
accept
the
data
in
support
of
the
proposed
method 
In
response
to
the
comment
that
the
ATP
protocol
calls
for
serial
dilutions
to
bracket
microbial
densities
that
can
be
compared
using
robust
statistical
methods,
EPA
has
indicated
that
the
data
as
submitted
are
sufficient
to
permit
a
valid
statistical
analysis.
See
also
responses
to
Comment
12
(
Docket
ID
I­
F.
021,
Part
10)
and
Comment
29
(
Docket
ID
I­
F.
004,
Part
04)
for
additional
explanation.

32
I­
F.
022
05
3. 
The
ATP
comparability
results
did
not
meet
the
replicate
and
data
selection
requirements
of
Section
3.1.2
and
4.6,

respectively.
The
BioVir
Laboratories
data
sheets
indicated
that
only
1
to
2
dilutions
were
used
for
each
spiked
sample.
In
most
cases,
only
1
dilution
was
used.
Hence,
this
data
should
not
have
been
included
and
the
experiment
should
have
been
repeated. 
See
responses
to
Comment
12
(
Docket
ID
I­
F.
021,
Part
10)
and
Comment
29
(
Docket
ID
I­
F.
004,
Part
04)
for
additional
explanation.

03.01.04
Calculation
of
Log10/
Chi
Squared
Analysis
33
ID1.002
05
Specific
comments:

 
For
sample
number
982084A,
USEPA
revised
the
data
presented
in
the
original
application
from
the
manufacturer.

The
original
bench
sheet
for
this
sample
indicates
that
the
diluted
wastewater
used
for
the
actual
disinfection
experiment
was
not
subjected
to
enumerative
assay;
rather,
the
 
original 

sample
was
assayed
upon
receipt,
and
the
testing
laboratory
then
assumed
that
dilutions
of
1:
1,000
or
1:
10,000
would
produce
adequate
numbers
of
coliforms/
E.
coli
to
meet
the
ATP
criteria
requiring
3­
4
log10
reductions
following
chlorination
and
final
See
response
to
Comment
25
(
Docket
ID
I­
D1.002
part
04)
regarding
the
use
of
inferred
bacterial
densities.

See
response
to
Comment
24
(
Docket
ID
I­
D1.001,
Part
03)
for
a
discussion
of
a
consistent
data
set
(
i.
e.,
one
associated
with
the
same
dilution)
being
used
for
both
log­
reduction
and
comparability­
study
purposes.

See
response
to
Comment
28
(
Docket
ID
I.
D1.002,
Part
13)
concerning
incomplete
bench
sheets.
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
36
densities
of
1­
10
CFU/
100
mL.
Hence,
the
log
reduction
for
this
experiment
was
computed
using
inferred
rather
than
actual
data.
Even
in
the
case
that
this
approach
were
correct,
the
bench
sheets
indicate
that
the
final
concentration
of
test
microorganisms
post­
disinfection
was
in
excess
of
104
CFU/
mL,
more
than
a
thousand
times
greater
than
the
required
level
of
1­
10
CFU/
mL
(
the
mEndo
count
of
6
was
multiplied
by
the
dilution
factor
of
1,000
and
then
again
by
the
chlorine
solution
dilution
factor
of
2
to
produce
a
concentration
of
1.2x104
CFU/
mL).
It
may
be
that
this
computation
was
in
error,
since
the
post­
disinfection
samples
presumably
were
not
diluted
1,000­
fold
as
indicated
on
the
bench
sheet.

Nonetheless,
if
the
data
and/
or
computations
are
in
error,
the
bench
sheets
should
be
replaced
with
corrected
data
and
reviewed
again.
Also,
the
chi
square
analysis
on
the
method
comparability
study
data
following
this
disinfection
experiment
relied
upon
the
1:
10,000
dilution;
however,
the
original
and
revised
computations
for
the
log
reductions
relied
upon
the
1:
1,000
dilution.
This
inconsistency
should
be
corrected
and
the
data
reviewed
again.
Several
items
are
missing
from
the
bench
sheet
(
dates
of
assays,
initials
of
technicians,
etc.).

Data
generated
from
sample
982084A
should
not
be
considered
in
support
of
product
approval
under
ATP
guidelines. 

 
For
sample
number
982305A,
EPA
has
recomputed
the
log
reductions
for
coliform
bacteria
arising
from
chlorine
pretreatment
and
revised
the
reduction
originally
reported
by
the
manufacturer
(
4.5
log10)
to
a
lower
level
(
3.4
log10).
This
revision
is
in
error
because
of
an
incorrect
value
used
for
the
source
water
concentration
(
Msource)
of
coliform
bacteria.

Table
1
indicates
a
source
water
concentration
of
30,000,000
CFU/
100
mL
based
on
the
assay
of
the
original
wastewater
sample
received
in
the
laboratory
on
12/
16/
98.
This
value
was
See
response
to
Comment
12
(
Docket
ID
I­
F.
021,
Part
10)
concerning
the
25:
75
split
guideline
and
the
selection
of
geographically
dispersed
sites.

See
response
to
Comment
24
(
Docket
ID
I­
D1.001,
Part
03)
concerning
 
hold
time 
between
initial
density
determination
and
post­
chlorination
measurement.

See
response
to
Comment
12
(
Docket
ID
I­
F.
021,
Part
10)
concerning
log
reduction
results.

Concerning
the
comment
about
the
post
disinfection
microorganism
concentrations
for
sample
982084A
being
 
in
excess
of
10
4
CFU/
100
mL ,
the
commenter
appears
to
be
basing
such
calculation
on
an
errant
(
and
insignificant,
with
respect
to
EPA s
analysis)
note
written
on
the
bottom
of
the
subject
worksheet.
The
1:
1000
dilution
factor
was
used
by
EPA
to
calculate
the
initial
density
determination,
not
the
post­
chlorination
density,

as
suggested
by
the
commenter.
EPA
has
reviewed
its
calculations
and
confirmed
that
they
were
performed
correctly.
As
previously
discussed,
EPA
did
not
rely
on
the
log
reduction
calculations
included
in
the
worksheets.

Having
considered
all
of
the
commenter s
points
concerning
this
sample,
and
in
light
of
the
above
responses,
EPA
is
satisfied
that
it
is
appropriate
to
use
sample
982084A
in
its
analysis.

Concerning
the
comment
that
it
was
incorrect
to
change
the
log
reduction
value
for
sample
982305A
from
4.5
to
3.4,
data
on
bacterial
density
before
chlorine
stressing
were
calculated
as
described
in
sections
4.1.1.2
and
4.1.1.3
of
the
ATP
protocol.
This
method
of
calculating
of
bacterial
density
and
log
reduction
of
bacteria
due
to
chlorine
stress
is
consistent
with
EPA s
historical
approach
to
method
evaluation
and
is
the
basis
of
the
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
37
later
divided
by
the
dilution
factor
of
1,000
to
infer
the
number
of
viable
microorganisms
present
prior
to
chlorine
injury.

However,
the
testing
laboratory s
bench
sheets
indicate
that
the
actual
1,000­
fold
diluted
 
working 
solution
was
assayed,

producing
a
coliform
density
of
200,000
CFU/
100
mL.
This
value
should
be
used
to
compute
the
log
reductions
rather
than
the
inferred
value
used
by
EPA
in
the
revised
table
presented
in
the
December
2,
2002
Federal
Register
notice.
The
corrected
log
reduction
for
the
total
coliform
experiment
should
be
4.2
log10. 

 
It
is
also
unclear
how
the
comparability
data
from
this
experiment
were
used
for
the
chi
square
analysis
performed
by
the
subcontractor
providing
the
statistical
analysis.
The
bench
sheets
indicate
that
the
1,000­
fold
dilution
used
for
the
disinfection
experiments
resulted
in
20
out
of
20
positive
samples
for
the
proposed
method
and
17
out
of
20
positives
for
the
reference
method,
a
failure
of
the
 
25:
75"
rule
under
ATP
section
4.6.5,
where
greater
than
75%
of
the
test
samples
under
the
proposed
method
were
positive
(
17
of
20
positive,
for
an
actual
percentage
of
85%).
The
chi
square
analysis
indicated
that
15
of
20
samples
were
positive
under
the
proposed
method
and
17
of
20
were
positive
for
the
reference
method.
This
inconsistency
warrants
explanation.
Consequently,
data
generated
from
sample
982305A
should
not
be
considered
in
support
of
product
approval
under
ATP
guidelines.
Finally,

note
that
samples
982305A
and
982084A
both
arise
from
the
same
source
(
Millbrae,
California).
Under
ATP
guidelines
(
section
4.1.1),
wastewater
isolates
 
must
come
from
ten
or
more
geographically
dispersed
sites 
in
order
to
proceed
with
the
chlorine
injury
experiments.
One
of
these
samples
should
be
replaced
with
an
isolate
from
another
site;
the
other
should
not
be
considered
in
support
of
product
approval
under
ATP
guidelines. 

 
A
third
chlorine
injury
experiment
was
conducted
using
the
water
from
Millbrae,
California
(
presumably
from
sample
982084A
but
the
sample
number
is
missing
from
the
bench
calculations
of
log
reduction
presented
in
the
Dec.
2,

2002
Federal
Register
notice.
See
also
the
responses
to
Comment
24
(
Docket
ID
I­
D1.001,
Part
03),
Comment
13
(
Docket
ID
I­
D1.001,
Part
04)
and
Comment
25
(
Docket
ID
I­
D1.002,
Part
04).

Concerning
the
suggested
inconsistency
in
the
use
of
data
to
support
the
statistical
analysis
and
log
reduction
calculations
for
sample
982305A,
EPA
refers
to
the
responses
to
Comment
24
(
Docket
ID
I­
D1.001,
Part
03)
and
Comment
27
(
Docket
ID
I.
D.
1.002,
part
09)
and
notes
that
two
different
comparability
studies
were
done,

producing
the
two
sets
of
results.
For
consistency,
EPA
repeated
its
statistical
analysis
using
the
second
set
of
data
and
the
conclusion
concerning
comparability
did
not
change;
the
repeat
analysis
did
not
identify
a
statistically­
significant
difference
between
the
performance
of
the
reference
methods
and
Colitag
 
.

See
response
to
Comment
12
(
Docket
ID
I­
F.
021,
Part
10)
concerning
the
selection
of
geographically
dispersed
sites.

The
additional
subject
experiment
that
the
commenter
presumes
to
have
been
conducted
from
sample
982084A
was
actually
a
repeat
study
for
sample
982305A.
Log
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
38
sheets).
It
appears
that
this
experiment
was
a
repeat
of
the
total
coliform
experiment,
possibly
due
to
the
failure
of
the
previous
experiment
to
meet
the
ATP
 
25:
75"
criterion.
The
assays
from
this
experiment
resulted
in
concentrations
of
1.8x107
CFU/
100
mL
for
the
 
original 
sample
and
2.6x107
CFU/
100
mL
in
the
 
diluted 
sample.
Unfortunately,
a
page
is
missing
from
the
bench
sheets
(
the
page
including
the
results
of
the
postchlorination
assay)
so
it
is
not
possible
to
compute
the
true
log
reductions
that
occurred
following
chlorine
injury.
What
is
most
troubling
about
this
third
experiment
relates
to
the
fact
that
these
data
were
the
ones
accepted
for
the
comparability
study
and
also
used
in
the
ensuing
chi
square
analysis
(
where
15
of
20
samples
were
positive
by
the
proposed
method
and
17
of
20
were
positive
by
the
reference
method).
Hence,
EPA
used
the
results
of
the
second
experiment
on
this
sample
to
compute
the
log
reductions
but
the
results
of
the
third
experiment
to
compute
the
chi
square
analysis.
This
is
absolutely
incorrect
and
the
data
from
this
sample
are
invalid. 

 
For
sample
number
990025A,
EPA
has
recomputed
the
log
reductions
for
coliform
bacteria
arising
from
chlorine
pretreatment
and
revised
the
reduction
originally
reported
by
the
manufacturer
from
3.3
log10
to
3.2
log10.
This
revision
is
in
error
because
of
an
incorrect
value
used
for
the
source
water
concentration
(
see
previous
comment).
Table
1
indicates
a
source
water
concentration
of
11,000,000
CFU/
100
mL
based
on
an
extrapolation
of
the
bacterial
concentration
in
the
diluted
solution
used
in
the
experiments
(
this
value
was
later
divided
by
the
500­
fold
dilution
factor
to
compute
the
log
reduction
caused
by
chlorine
pretreatment).
However,
the
testing
laboratory s
bench
sheets
indicate
that
the
actual
500­
fold
and
2,000­
fold
 
working 
solutions
were
assayed,
producing
coliform
densities
of
21,000
CFU/
mL
and
6,500
CFU/
mL,
respectively.
It
is
unclear
why
EPA
has
elected
to
exclude
the
data
originally
reported
but
again
it
must
be
emphasized
that
the
initial
concentrations
of
test
microorganisms
were
inferred
rather
than
measured
to
deduce
the
initial
bacterial
concentrations
for
the
chlorine
injury
experiments.
If
the
data
from
the
500­
fold
reduction
was
computed
consistent
with
the
ATP
protocol,
and
as
described
previously
in
this
document.

As
also
discussed
above,
a
consistent
data
set
was
used
for
both
the
log
reduction
calculation
and
the
statistical
analysis
in
EPA s
reassessment
of
the
data.
EPA s
analysis
did
not
identify
a
statistically­
significant
difference
between
the
reference
methods
and
Colitag
 
.

As
the
commenter
noted,
the
comparability
study
data
from
the
experiment
used
for
log
reduction
determination
did
not
meet
the
25%:
75%
criterion,
but
this
did
not
preclude
the
use
of
this
individual
test
result
since
the
chi
squared
test
considered
results
from
all
sites
collectively.
This
issue
is
also
discussed
in
the
responses
to
Comment
12
(
Docket
ID
I­
F.
021,
Part
10)
and
Comment
29
(
Docket
ID
I­
F.
004
Part
04).

The
commenter
states
for
sample
990025A
that
the
bacterial
density
determined
after
dilution
of
the
sample,

but
before
chlorination
should
have
been
used
to
determine
log
reduction
determination
rather
than
relying
on
the
original
density
determination
and
dilution
factor
to
compute
log
reduction.
As
explained
in
the
response
to
Comment
24
(
Docket
ID
I­
D1.001,
Part
03)

and
Comment
25
(
Docket
ID
I­
D1.002,
Part
04),
the
approach
used
by
EPA
is
consistent
with
the
ATP
protocol
and
with
past
practice.
The
use
of
a
consistent
data
set
in
the
chi
squared
test
and
in
the
log
reduction
determination
for
sample
990025A
are
also
discussed
in
the
response
to
Comment
24
(
Docket
ID
I­
D
1.001
part
03).
Allowing
for
the
possibility
of
a
typographic
error
(
as
the
commenter
presumed),
EPA
used
a
dilution
factor
of
1:
2000
in
recomputing
the
total
coliform
log
reduction.
As
noted
by
the
commenter,
the
log
reduction
changed
from
3.2
to
3.0.
This
change
had
no
impact
on
EPA s
analysis
or
the
conclusion
that
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
39
dilution
is
used
to
compute
the
log
reductions,
it
would
result
in
a
3.2
log10
reduction;
if
the
2,000­
fold
dilution
were
used
instead,
the
log
reduction
would
be
3.0
log10.
Following
the
chlorine
injury
experiments,
the
method
comparability
study
was
conducted
 
albeit
ten
days
later.
Here
again,
the
chi
square
analysis
relied
upon
the
data
drawn
from
the
1,000­
fold
dilution
(
presumably
a
typographic
error?
The
disinfection
experiments
were
carried
out
at
1:
500
and
1:
2,000)
and
yet
the
disinfection
experiment
considered
by
EPA
was
drawn
from
the
experiment
where
the
sample
was
diluted
1:
500.
Hence,
the
chi
square
analysis
is
invalid
in
that
it
relied
upon
data
from
a
different
experiment.
Data
generated
from
this
sample
should
not
be
considered
in
support
of
product
approval. 

 
For
sample
number
990052A,
EPA
has
recomputed
the
log
reductions
for
coliform
bacteria
arising
from
chlorine
pretreatment
and
revised
the
reduction
originally
reported
by
the
manufacturer
from
2.0
log10
to
2.3
log10
(
see
previous
comments).
Again,
the
initial
coliform
concentrations
used
to
compute
log
reductions
are
inferred
by
assuming
a
volumetric
dilution
from
the
original
stock
concentration.
Since
the
laboratory
actually
measured
the
concentration
of
test
microorganisms
in
the
diluted
samples
used
for
chlorine
pretreatment
studies,
these
actual
numbers
should
be
used
in
place
of
the
inferred
numbers
(
as
was
done
in
the
original
submission).
The
correct
log
reduction
for
the
experiment
relying
upon
the
1:
1,250
dilution
would
be
1.9
log10,
which
fails
to
meet
the
criterion
of
3­
4
log10
required
under
section
4.5.2.1
of
the
ATP.
EPA
should
also
consider
that
its
decision
to
use
the
data
drawn
from
the
initial
range­
finding
experiment
is
inappropriate
in
light
of
the
fact
that
over
one
month
elapsed
from
the
time
that
the
initial
assay
was
conducted
and
the
date
of
the
methods
comparability
study
for
this
sample.
Hence,

EPA s
recomputation
used
two
data
points
from
assays
spaced
almost
5
weeks
apart
to
compute
the
log
reductions.
This
is
certainly
not
standard
practice
and
invalidates
the
data
generated
from
this
experiment.
Also,
the
chi
square
analysis
was
performed
using
data
from
the
1:
2,500
dilution
 
which
was
not
Colitag
 
is
an
acceptable
method.

Concerning
the
commenter s
claim
that
the
method
comparability
study
for
sample
990025A
was
conducted
10
days
after
chlorine
injury,
the
commenter
is
referred
to
the
response
to
Comment
24
(
Docket
ID
I­
D1.001,

Part
03).

The
commenter
notes
that
the
log
reduction
for
sample
990052A
was
determined
from
an
inferred
bacterial
density
derived
from
the
initial
coliform
concentration
in
the
original
sample
and
the
dilution
used
to
prepare
the
prechlorination
sample.
EPA
refers
the
commenter
to
its
response
Comment
24
(
Docket
ID
I­
D1.001,
Part
03)

and
Comment
25
(
Docket
ID
I­
D1.002,
Part
04)

concerning
calculation
of
log
reduction.
In
response
to
the
comment
suggesting
the
possibility
that
over
one
month
passed
between
the
initial
density
determination
and
chlorination,
EPA
refers
the
commenter
to
the
response
to
Comment
24
(
Docket
ID
I­
D1.001,
Part
03)

for
a
discussion
of
this
time
interval
for
sample
990052A.
Concerning
the
comment
on
the
use
of
different
dilutions
for
log
reduction
determination
and
for
the
comparability
study
and
chi
squared
analysis,

EPA
again
refers
the
commenter
to
the
response
to
Comment
24
(
Docket
ID
I­
D1.001,
Part
03).
Log
reduction
for
this
sample
was
recomputed,
for
consistency
with
the
data
set
used
in
the
statistical
analysis;
the
figure
(
2.3)
did
not
change.
Thus,
there
was
no
impact
on
EPA s
analysis.
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
40
the
consistent
with
the
1:
1,250
dilution
used
to
generate
the
data
for
the
disinfection
experiments.
Data
generated
from
sample
number
990052A
should
not
be
considered
in
support
of
product
approval
under
ATP
guidelines. 

 
For
sample
number
990095,
log
reductions
for
coliforms
were
inferred
rather
than
measured,
as
stated
for
previous
samples.

The
actual
measured
concentration
of
the
diluted
stock
coliforms
was
reported
as
72
CFU
in
a
1
mL
sample
of
the
1:
7,000
dilution.
This
number
should
be
used
as
the
initial
density
of
test
microorganisms
rather
than
the
16,000,000
CFU/
100
mL
number
reported
in
table
1
 
note
also
that
the
bench
sheet
for
this
sample
is
missing
the
derived
concentration
of
test
microorganisms.
Table
1
should
be
revised
to
reflect
the
actual,
measured
bacterial
densities
and
the
correct
log
reductions
(
3.3
log10
rather
than
2.8
log10).
In
addition,
the
independent
testing
laboratory
should
provide
the
missing
items
on
the
bench
sheet
(
microbial
concentrations,
dates
of
assays,

and
technicians
responsible
for
performing
and
documenting
assay
results).
The
log
reduction
for
coliform
bacteria
in
this
sample
was
computed
using
the
 
diluted
sample 
plate
counts
 
this
is
actually
the
correct
approach
but
is
not
the
approach
EPA
reports
using
in
the
December
2,
2002
Federal
Register
notice
to
modify
the
data
presented
in
Table
1
during
its
reconsideration
of
the
original
data
submitted
and
reviewed
in
March
of
2002.
The
revised
bench
sheet
and
table
should
be
resubmitted
for
reviewer
comment. 

 
For
sample
990217,
again
the
diluted
sample
(
1:
600)
subjected
to
chlorine
pretreatment
indicates
a
coliform
density
of
100
CFU/
100
mL.
However,
the
logarithmic
reduction
presented
in
Table
1
was
generated
using
the
bacterial
density
from
the
initial
determination
of
coliforms
in
the
stock
wastewater
isolate
(
5,000,000
CFU/
100
mL).
Therefore,
the
actual
logarithmic
determination
was
again
inferred
rather
than
measured.
If
the
actual
data
were
used
instead
(
100
CFU
per
100
mL),
the
computed
log
reduction
would
be
0.9
log10
rather
than
the
reported
2.8
log10.
This
reduction
fails
to
meet
the
ATP
The
commenter
states
that
the
log
reduction
data
from
sample
990095
was
inferred
from
the
initial
density
determination
for
the
original
sample
rather
than
directly
measuring
the
bacterial
density
in
the
diluted
sample
that
was
prepared
for
chlorination.
EPA
refers
the
commenter
to
its
responses
to
Comment
24
(
Docket
ID
I­
D1.001,
Part
03)
and
Comment
25
(
Docket
ID
ID1.002
Part
04)
concerning
calculation
of
log
reduction
for
data
as
presented
in
the
Dec.
2,
2002
Federal
Register
notice.
The
bench
sheet
for
sample
990095
contained
the
critical
information
needed
by
EPA
to
assess
the
results.
EPA
used
the
approach
reported
in
the
December
2002
NODA
to
calculate
log
reduction.

The
commenter
states
that
the
log
reduction
for
sample
990217
was
determined
by
inferring
a
bacterial
density
from
the
initial
determination
of
coliforms
in
the
stock
wastewater.
EPA
refers
the
commenter
to
its
responses
to
Comment
12
(
Docket
ID
I­
F.
021,
Part
10),
Comment
24
(
Docket
ID
I­
D1.001,
Part
03)
and
Comment
25
(
Docket
ID
I­
D1.002,
Part
04)
concerning
calculation
of
log
reduction.
In
response
to
the
comment
on
the
time
lapse
between
initial
count
of
bacteria
in
the
wastewater
sample
and
chlorination,
EPA
refers
the
commenter
to
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
41
criterion
of
3­
4
log10.
Also,
the
logic
behind
the
use
of
the
range­
finding
data
rather
than
the
actual
diluted
samples
to
compute
the
log
reductions
is
flawed
due
to
the
time
that
elapsed
between
the
two
assays
(
approximately
two
weeks
judging
by
the
dates
on
the
comparability
study
sheets).
Table
1
should
be
revised
to
reflect
the
actual,
measured
bacterial
densities
and
should
be
resubmitted
for
reviewer
comment. 

 
For
sample
990273A,
again
the
diluted
sample
(
1:
800)

subjected
to
chlorine
pretreatment
indicates
a
reported
coliform
density
of
800
CFU
per
100
mL
based
on
the
original
bench
sheets
submitted
by
the
testing
laboratory.
However,
EPA
has
used
the
coliform
density
reported
from
the
original
assay
of
the
wastewater
isolate
(
2,000,000
CFU
per
100
mL)
in
recomputing
the
log
reductions
in
Table
1
of
the
Federal
Register
notice.

The
use
of
the
inferred
data
rather
than
the
actual
data
reported
by
the
testing
laboratory
results
in
a
significant
error
(
the
true
log
reduction
should
be
1.8
log10
rather
than
the
2.3
indicated
in
Table
1.
Regardless,
the
level
of
inactivation
fails
to
meet
the
3­
4
log10
reduction
requirement
under
section
4.5.2.1
of
the
ATP.
Following
chlorine
pretreatment,
application
of
the
reference
method
resulted
in
18
of
20
positive
samples,
failing
the
 
25:
75"
rule
under
section
4.6.5
of
the
ATP.
And
again,

EPA s
use
of
the
initial
assay
data
creates
an
artifact
due
to
the
period
of
time
that
elapsed
between
this
date
and
the
date
of
the
assay
on
the
post­
chlorination
sample
(
three
weeks
later).
Note
that
the
comparability
assay
bench
sheets
indicate
that
a
dilution
of
1:
600
was
used
for
these
samples
rather
than
the
1:
800
indicated
on
the
disinfection
bench
sheets.
Which
number
is
correct?
The
log
reductions
reported
for
sample
990273A
should
not
be
considered
in
support
of
product
approval
under
ATP
guidelines. 
the
response
to
Comment
24
(
Docket
ID
I­
D1.001,
Part
03).
The
commenter
states
that
the
log
reduction
for
sample
990273A
used
an
inferred
bacterial
density
derived
from
the
original
wastewater
assay
rather
than
using
the
actual
value
determined
from
the
diluted
waste
water
spike
sample
before
chlorination.
EPA
refers
the
commenter
to
its
responses
to
Comment
24
(
Docket
ID
I­
D1.001,

Part
03)
and
Comment
25
(
Docket
ID
I­
D1.002,
Part
04)

concerning
calculation
of
log
reduction.
In
response
to
the
comment
on
the
log
reduction
criterion,
see
the
response
to
Comment
12
(
Docket
ID
I­
F.
021,
Part
10)

concerning
application
of
the
3
to
4
log
reduction
as
guidance.
Concerning
the
comment
on
the
25%:
75%

split
guideline,
EPA
refers
the
commenter
to
its
response
to
Comment
12
(
Docket
ID
I­
F.
021,
Part
10)
and
Comment
29
(
Docket
ID
I­
F.
004,
Part
04).
Concerning
the
comment
on
the
time
lapse
between
cell
counts
and
chlorination,
EPA
refers
the
commenter
to
its
response
to
Comment
12
(
Docket
ID
I­
F.
021,
Part
10)
and
Comment
24
(
Docket
ID
I­
D1.001,
Part
03).
The
commenter
observed
that
a
1:
600
dilution
was
identified
in
connection
with
the
comparability
study
results
and
a
1:
800
dilution
was
identified
in
connection
with
the
log
reduction
calculation,
per
the
disinfection
bench
sheets.

Since
one
of
these
figures
appears
to
be
an
error,
and
since
it
is
not
clear
which
figure
is
correct,
EPA
examined
the
potential
influence
of
this
difference
on
log
reduction.
EPA
did
so
by
substituting
a
600­
fold
dilution
into
the
log
reduction
calculation
in
place
of
the
800­
fold
dilution
originally
used;
doing
so
resulted
in
a
log
reduction
of
2.4
(
versus
the
2.3
log
reduction
associated
with
a
dilution
of
1:
800).
This
minor
change
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
42
 
For
sample
990438A,
again
the
log
reductions
presented
in
Table
1
of
the
Federal
Register
notice
of
December
2,
2002,
are
based
upon
an
extrapolation
of
the
initial
coliform
densities
prior
to
chlorine
treatment.
Since
the
testing
laboratory
measured
and
reported
the
actual
starting
concentrations
of
coliforms
in
the
diluted
wastewater
isolate
(
1
CFU
in
the
1:
13,000
dilution),
this
number
should
have
been
used
rather
than
the
number
reported
following
the
initial
determination
of
coliform
densities
in
the
wastewater
isolate.
The
correct
log
reduction
should
be
0.7
log10
rather
than
the
2.3
reported
in
the
revised
table;
this
reduction
fails
to
meet
the
ATP
criterion
indicated
in
section
4.5.2.1.
More
troubling
than
the
computational
errors
is
the
fact
that
the
data
used
in
the
chi
square
analysis
are
not
those
presented
in
the
bench
sheets
from
the
disinfection
experiments
and
the
ensuing
methods
comparability
study.
The
bench
sheets
indicate
a
split
of
10:
10
(
positive:
negative)
for
the
proposed
method
under
the
1:
13,000
dilution
used
for
the
disinfection
experiments;
the
reference
method
produced
a
split
of
9:
11.
And
yet
the
chi
square
tables
indicate
a
split
of
13:
7
and
9:
11
for
the
proposed
and
reference
methods,
respectively.
What
is
the
origin
of
this
data
and
how
can
it
be
reconciled
with
the
analysis
performed
to
support
the
contention
that
the
proposed
and
reference
methods
are
equivalent?
Moreover,
the
bench
sheets
indicate
dates
that
are
not
logical.
If
the
density
determination
for
the
original
sample
was
produced
on
5/
5/
99
as
indicated
on
the
bench
sheets,
how
is
it
that
the
 
subsequent 
comparability
study
was
conducted
on
4/
30/
99???
In
the
absence
of
verification
from
the
testing
laboratory,
these
data
should
be
excluded. 
in
log
reduction
has
no
impact
on
EPA s
assessment.

The
commenter
notes
that
the
log
reduction
for
sample
990438A
is
based
on
an
extrapolation
of
the
initial
coliform
densities
prior
to
chlorine
treatment.
EPA
refers
the
commenter
to
its
responses
to
Comment
24
(
Docket
ID
I­
D1.001,
Part
03)
and
Comment
25
(
Docket
ID
I­
D1.002,
Part
04)
concerning
calculation
of
log
reduction,
and
to
Comment
12
(
Docket
ID
I­
F.
021,

Part
10)
concerning
application
of
the
3
to
4
log
reduction
as
guidance.
In
response
to
the
comment
that
the
total
coliform
data
used
in
the
chi
squared
analysis
for
sample
990438A
are
not
those
presented
in
the
bench
sheets
and
ensuing
comparability
study,
EPA
has
reviewed
the
raw
data
and
confirmed
that
the
correct
values
are
10
positives:
10
negatives
for
Colitag
 
and
9
positives:
11
negatives
for
the
reference
method.
EPA
has
performed
a
number
of
repeat
analyses/
reanalyses,

as
discussed
elsewhere
in
this
document,
and
has
confirmed
that
the
correct
comparability
study
results
are
being
used.
None
of
those
analyses
identified
a
statistically
significant
difference
between
the
performance
of
the
reference
methods
and
Colitag
 
.

EPA
agrees
that
the
dates
for
the
density
determination
and
comparability
studies
documented
on
the
log
sheets
are
inconsistent
and
may
be
the
result
of
the
density
data
being
entered
on
the
log
sheet
post
data
collection.

Based
on
the
chain
of
custody
sheet
(
showing
that
the
original
sample
was
received
in
the
lab
on
4/
27/
99)
and
the
comparability
study
sheet
(
showing
Colitag
 
test
results
dated
4/
30/
99),
EPA
has
concluded
that
it
is
unlikely
that
the
samples
were
held
for
an
excessive
period.
See
also
the
response
to
Comment
24
(
Docket
ID
I­
D1.001,
Part
03)
for
additional
discussion
concerning
this
 
hold
time 
question.

Concerning
the
comment
about
spiking
sources
from
10
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
43
 
Sample
number
990442A
originates
from
the
same
source
as
sample
number
990217A
(
Mission,
KS).
Under
section
4.1.1
of
the
ATP,
spiking
sources
 
must
come
from
ten
or
more
geographically
dispersed
sites .
One
of
these
two
samples
must
be
excluded
from
consideration
in
the
study
data
and
should
not
be
considered
in
support
of
product
approval.
The
manufacturer/
independent
testing
laboratory
should
collect
at
least
one
additional
sample
and
subject
it
to
testing
under
ATP
guidelines
for
it
to
be
included
in
the
comparability
data
in
Tables
1
and
2. 

 
Following
EPA s
reexamination
of
the
original
manufacturer
data,
the
log
reductions
following
chlorine
pretreatment
of
sample
990442A
were
revised
from
1.4
log10
to
2.6
log10
(
based
upon
the
use
of
the
extrapolated
initial
concentration
of
test
microorganisms
as
previously
described).
Again,
the
actual,

measured
concentrations
of
test
microorganisms
recorded
on
the
bench
sheets
should
be
employed
to
compute
the
log
reductions.
The
coliform
density
reported
on
the
bench
sheet
for
this
sample
is
3
CFU
per
10
mL
in
the
1:
9,000
dilution.
The
correct
log
reduction
for
this
experiment
should
be
0.9
log10
which
fails
the
ATP
criteria.
In
the
absence
of
additional
clarification
and
external
review,
data
generated
from
sample
990442A
should
not
be
considered
in
support
of
product
approval. 

 
Similarly,
sample
number
990443A
was
reexamined
to
revise
the
log
reduction
originally
reported
by
the
manufacturer.
An
extrapolated
bacterial
density
was
used
to
compute
a
revised
reduction
of
2.4
log10.
This
number
should
instead
be
0.4
log10.
Regardless
of
the
computational
approach
the
reported
log
reduction
failed
to
meet
the
ATP
criterion. 
geographically
dispersed
sites,
EPA
refers
the
commenter
to
its
response
to
Comment
12
(
Docket
ID
I­
F.
021,
Part
10).

The
commenter
notes
that
the
log
reduction
for
sample
990442A
was
revised
based
on
the
use
of
log
reduction
calculations
based
on
the
initial
density
determination.

EPA
refers
the
commenter
to
its
responses
to
Comment
24
(
Docket
ID
I­
D1.001,
Part
03)
and
Comment
25
(
Docket
ID
I­
D1.002,
Part
04)
concerning
calculation
of
log
reduction,
and
to
its
response
to
Comment
12
(
Docket
ID
I­
F.
021,
Part
10)
concerning
application
of
the
3
to
4
log
reduction
as
guidance
The
commenter
notes
that
the
log
reduction
for
sample
990443A
was
revised
based
on
the
use
of
log
reduction
calculations
based
on
the
initial
density
determination.

EPA
refers
the
commenter
to
its
responses
to
Comment
24
(
Docket
ID
I­
D1.001,
Part
03)
and
Comment
25
(
Docket
ID
I­
D1.002,
Part
04)
concerning
calculation
of
log
reduction,
and
to
its
response
to
Comment
12
(
Docket
ID
I­
F.
021,
Part
10)
concerning
application
of
the
3
to
4
log
reduction
as
guidance.
The
commenter
is
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
44
referred
to
the
response
to
Comment
1
(
Docket
ID
I­
D
1.001,
Part
06)
concerning
the
use
of
the
ATP
criteria
as
guidance.

34
ID2.001
02
 
Firstly
the
data
supplied
demonstrates
that
when
the
testing
was
carried
out
using
disinfection
to
determine
if
Colitag
 
is
able
to
recover
stressed
organisms,
the
3
log
reduction
was
not
met
in
many
cases.
Furthermore,
the
method
for
calculating
a
3
log
reduction
was
incorrect.
The
correct
procedure
is
to
take
the
log
of
the
initial
number
of
bacteria
and
the
log
of
the
final
number
and
subtract
one
from
the
other.
It
would
appear
that
the
mechanism
used
for
the
Colitag
 
study
was
to
take
the
difference
between
the
initial
and
final
concentration
of
bacteria
and
use
the
log
of
that
number
to
indicate
log
reduction.
This
is
incorrect. 

 
For
example,
if
the
initial
concentration
of
bacteria
were
one
million
and
the
final
concentration
was
one
thousand,
the
correct
calculation
is
log
one
million
(
6)
minus
log
one
thousand
(
3)
equals
3.
Thus
a
three
log
reduction
has
been
achieved. 

 
If
however
the
method
used
by
CPI
is
used
one
million
minus
one
thousand
is
999000.
The
log
of
999000
is
5.999.
Might
I
suggest
that
the
data
is
re­
evaluated
and
the
correct
log
reductions
are
used.
It
is
extremely
important
that
this
part
of
the
testing
procedure
is
carried
out
and
evaluated
correctly.
A
"
near
enough"
approach
is
just
not
acceptable.
This
is
particularly
true
in
the
case
of
Colitag
 
since
the
manufacturers
claim
to
have
improved
performance
with
respect
to
recovery
of
chlorine
stressed
organisms
over
existing
methods. 
In
response
to
the
comment
suggesting
that
a
3
log
reduction
was
necessary
for
each
sample,
the
commenter
is
referred
to
the
response
to
Comment
12
(
Docket
ID
IF
021,
Part
10).
In
response
to
the
comment
suggesting
that
EPA
calculated
the
log
reduction
figures
incorrectly,

EPA
believes
that
the
log
reduction
data
were
correctly
calculated.
Referring
to
the
December
2002
NODA,

the
formula
in
the
EPA s
approach
(
log[
A/
B])
is
mathematically
equivalent
to
that
advocated
by
the
commenter.
(
logA­
logB).
Using
sample
990442A
as
an
example,
where
Msource=
1.4
x
10
7;
DF=
9000;

Mchlorinated=
2;
and
CF=
2,
Log
reduction
=

log
10
[(
1.4x10
7/
9000)/(
2x2)]=
2.6
EPA
agrees
that
it
is
important
to
correctly
calculate
log
reduction,
and
assures
the
commenter
that
the
log
reductions
have
been
correctly
calculated
as
indicated
above.

03.02
General
Comments
on
Adherence
to
ATP
Guidelines
(
e.
g.,
claim
that
protocol
must
serve
as
benchmark)

35
ID1.002
02
General
comments:

 
A
detailed
review
of
the
original
data
submitted
in
support
of
the
Colitag
 
test
for
coliforms
and
E.
coli
was
submitted
in
April,
2002.
Although
EPA
responded
to
reviewer
comments
concerning
some
of
the
products
being
considered
for
approval,
In
response
to
the
comment
on
EPA s
use
of
the
ATP
protocol
as
guidance,
see
response
to
Comment
1
(
Docket
ID
I­
D1.001,
Part
06).

See
response
to
Comment
2
(
Docket
ID
I­
D1.002,
Part
15)
concerning
the
adequacy
of
the
Colitag
 
data
and
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
45
at
that
time
EPA
elected
to
postpone
its
response
to
comments
concerning
the
data
submitted
in
support
of
the
Colitag
 
method
for
detecting
coliform
bacteria
and
E.
coli.
In
its
response
to
a
number
of
concerns
raised
by
external
reviewers
EPA
indicated
that
no
action
would
be
taken
because
  
ATP
guidelines
are
not
legal
requirements .
This
inaction
is
disappointing
due
to
the
significant
number
of
documented
failures
by
the
manufacturers
to
meet
the
criteria
stipulated
in
the
Alternate
Testing
Procedure
(
ATP)
protocol.
To
some
degree
this
decision
renders
the
ATP
a
guideline
without
purpose,
as
manufacturers
keen
to
develop
and
market
new
tools
for
potable
water
testing
are
left
with
no
standard
against
which
their
products
can
be
evaluated.
Moreover,
the
public
health
is
not
served
well
by
this
inaction,
as
products
that
fail
to
meet
numerous
criteria
stipulated
in
the
ATP
guidelines
may
nonetheless
gain
product
approval
by
EPA.
In
the
case
of
the
Colitag
 
submission,
EPA s
decision
to
reconsider
the
application
in
light
of
limited
recomputation
of
the
log
reductions
following
chlorine
injury
is
not
justified
adequately
in
light
of
the
fact
that
a
key
element
of
the
ATP
process
is
to
demonstrate
that
proposed
methods
are
better
than
or
equal
to
EPA­
approved
reference
methods
in
terms
of
sensitivity,

specificity
and
overall
applicability
for
a
given
analyte.
Failure
to
meet
a
significant
number
of
ATP
criteria
suggests
that
the
data
presented
may
not
be
adequate
to
justify
approval
for
drinking
water
testing. 
EPA s
review
of
the
data.

See
response
to
Comment
8
(
Docket
ID
I­
D1.001,
Part
02)
concerning
the
scope/
purpose
of
the
December
2002
NODA.
See
also
responses
to
Comment
2
(
Docket
ID
I­
D1.002,
Part
15)
and
Comment
3
(
Docket
ID
I­
D1.005,
Part
02)

See
response
to
Comment
14
(
Docket
ID
I­
D1.004­
Part
02)
concerning
EPA s
application
of
the
ATP
protocol
to
previous
method
evaluations.

See
response
to
Comment
12
(
Docket
ID
I­
F.
021,
Part
10)
concerning
the
comparability
between
the
reference
methods
and
Colitag
 
with
respect
to
method
sensitivity.
As
noted
in
that
response,
EPA
concluded
that
the
studies
met
the
underlying
goal
of
detecting
low
numbers
of
bacteria
method;
those
studies
produced
results
that
support
the
Agency s
decision
to
approve
the
method
for
drinking
water
monitoring.

See
response
to
Comment
40
(
Docket
ID
I­
D1.002,
Part
14)
concerning
the
acceptability
of
specificity
results.

Since
Colitag
 
detects
total
coliforms
and
E.
coli
in
drinking
water
samples
in
a
manner
comparable
to
the
reference
methods,
EPA
concluded
that
it
is
applicable
to
these
analytes.

36
ID1.002
06
 
Overall,
more
than
half
of
the
coliform
data
submitted
in
support
of
product
approval
failed
to
meet
the
ATP
criteria
for
comparability
testing.
In
the
absence
of
unambiguous
justification,
it
would
not
be
in
the
best
interest
of
the
public
health
to
accept
the
submitted
data
in
support
of
product
approval
for
coliform
testing. 
See
response
to
Comment
1
(
Docket
ID
I­
D1.001,
Part
06)
concerning
EPA s
use
of
the
ATP
protocol
as
guidance.

See
response
to
Comment
12
(
Docket
ID
I­
F.
021,
Part
10)
concerning
the
25:
75
split
guideline;
the
selection
of
geographically
dispersed
sites;
and
the
log
reduction
results.

See
response
to
Comment
24
(
Docket
ID
I­
D1.001,
Part
03)
concerning
 
hold
time 
between
initial
density
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
46
determination
and
post­
chlorination
measurement.

See
response
to
Comment
2
(
Docket
ID
I­
D1.002,
Part
15)
concerning
the
adequacy
of
the
Colitag
 
data.

Based
on
the
evaluation
of
the
comparability
data
generated
for
the
Colitag
 
method,
and
taking
into
consideration
the
public
comments
received,
EPA
has
concluded
that
the
Colitag
 
method
is
acceptable
as
an
alternative
to
the
approved
reference
methods
because
the
information
available
to
EPA
indicates
that
the
performance
of
the
Colitag
 
method
compares
favorably
to
the
approved
reference
methods.
EPA
has
assessed
the
quality
and
quantity
of
the
data
provided
by
CPI
International,
and
conducted
a
thorough
statistical
analysis
of
relevant
information,
all
of
which
was
included
in
the
public
record.
As
discussed
throughout
this
document,
each
of
the
concerns
raised
via
the
public
comment
process
has
been
addressed
to
EPA s
satisfaction.

37
I­
F.
022
01
 
USEPA
has
proposed
approval
of
two
presence/
absence
liquid
culture
methods,
Colitag
 
and
ReadyCult
Coliforms
100,

summarized
in
Table
2.
Proposed
approval
of
these
methods
is
based
on
test
results
submitted
following
the
USEPA
Protocol
for
Alternate
Test
Procedures
for
Coliform
Bacteria
in
Compliance
with
Drinking
Water
Regulations;

Presence/
Absence
Liquid
Culture
Methods
for
Finished
Waters
(
USEPA
ATP
Protocol).
As
will
be
discussed
below,
the
test
results
for
both
of
these
proposed
methods
deviated
from
the
USEPA
ATP
Protocol.
Hence,
by
proposing
these
methods
for
approval,
the
agency
is
concluding
that
the
USEPA
ATP
Protocol
need
not
be
followed
as
a
strict
protocol.
This
is
a
mistake,
and
will
cause
further
complications
for
USEPA
and
water
utilities
both
small
and
large. 

 
In
the
analytical
methods
market,
manufacturers
will
seek
to
develop
methods
that
have
advantages
over
their
competitors.

Hence,
it
is
very
important
from
a
scientific
point
of
view
to
See
response
to
Comment
1
(
Docket
ID
I­
D1.001,
Part
06)
concerning
EPA s
use
of
the
ATP
protocol
as
guidance.

See
response
to
Comment
14
(
Docket
ID
I­
D1.004,
Part
02)
concerning
EPA s
consistent
application
of
the
protocol
to
method
evaluations.

See
response
to
Comment
12
(
Docket
ID
I­
F.
021,
Part
10)
concerning
the
25:
75
split
guideline;
the
selection
of
geographically
dispersed
sites;
and
the
log
reduction
results.

See
response
to
Comment
2
(
Docket
ID
I­
D1.002,
Part
15)
concerning
the
adequacy
of
the
Colitag
 
data.
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
47
have
a
consistent
performance
benchmark,
so
that
the
quality
of
science
is
maintained
and
public
health
protected.
Furthermore,

all
manufacturers
and
methods
developers
must
have
a
standard
benchmark,
and
must
not
be
allowed
to
cut
corners
on
science
(
i.
e.,
use
less
rigorous
science)
when
their
method
is
approved.

The
USEPA
ATP
Protocol
must
serve
as
that
benchmark. 

 
A
method
developer
desiring
approval
of
a
new
procedure
must
be
required
to
follow
exactly
the
USEPA
ATP
Protocol,

otherwise
there
is
no
fair
basis
for
comparison
of
methods.

Examples
of
key
protocol
requirements
are:

 
Proposed
methods
must
demonstrate
broad
applicability.

Hence,
primary
effluent
samples
are
required
from
10
geographically
dispersed
sites
throughout
the
US.
If
this
is
not
followed,
then
the
method
cannot
be
shown
to
have
broad
applicability.

 
For
drinking
water,
recovery
of
stressed
organisms
is
particularly
important.
To
demonstrate
effective
recovery
of
injured
bacteria,
a
3
to
4
log10
reduction
in
cell
numbers
must
be
achieved
during
the
injury
test.
This
is
an
important
criterion
to
ensure
a
comparable
level
of
'
stress'
and/
or
injury
from
one
to
test
to
another.
If
a
method
cannot
detect
severely
stress
organisms
following
the
protocol,
then
it
should
not
be
approved.

 
The
USEPA
ATP
Protocol
states
that
it
is
the
intention
of
the
protocol
under
Option
B
to
achieve
a
50%/
50%
split
in
positive
and
negative
results.
But
the
protocol
allows
as
much
as
a
25%/
75%
split
in
either
direction.
If
this
is
not
met,
then
the
data
are
unacceptable,
and
the
protocol
indicates
that
the
proposer
must
begin
again. 

 
The
USEPA
ATP
Protocol
was
developed
after
extensive
deliberations
with
stakeholders
and
others
seeking
approval
of
methods
many
years
prior
to
the
March
7,
2002
notice.
This
protocol
represents
an
appropriate
"
bar"
that
all
subsequent
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
48
methods
must
clear
for
approval.
USEPA
should
make
a
firm
statement
to
methods
developers
that
the
USEPA
ATP
Protocol
must
be
followed
as
written.
Otherwise
the
"
bar"
a
propose
method
must
clear
is
inconsistent,
resulting
in
less
robust
methods
being
approved,
and
compromising
a
water
systems
ability
to
detect
contamination.
It
also
results
in
a
basic
unfairness
in
the
marketplace
to
the
developers
of
new
methods.
Together,
both
of
these
effects
will
compromise
public
health
protection. 

04
DOCUMENTATION
OF
Colitag
 
DATA
(
e.
g.,
differing
false
positive/
negative
errors
cited;
use
of
a
poster
presentation;
documentation
of
inferences)

38
ID1.002
10
 
As
explained
previously
for
the
total
coliform
data,
three
samples
were
tested
from
Millbrae,
California
(
two
samples
but
three
series
of
tests).
The
third
test
conducted
on
12/
29/
98
for
total
coliform
analysis
was
not
repeated
for
E.
coli;
however,
the
methods
comparability
data
sheets
indicate
that
the
E.
coli
assays
were
conducted
on
12/
29
(
rather
than
the
previous
week
when
the
E.
coli
disinfection
experiment
was
conducted).
It
is
unclear
where
the
data
originated
for
the
chi
square
analysis.

This
inconsistency
should
be
explained
before
the
data
is
considered
in
support
of
product
approval. 
As
the
commenter
notes,
three
series
of
comparability
study
tests
were
performed
using
samples
from
Millbrae,

CA:
two
were
associated
with
sample
982305A;
one
was
associated
with
sample
982084A.

The
statistical
evaluation
of
the
comparability
study
results
from
sample
982084A
is
based
on
the
total
coliform
and
E.
coli
data
sheets
dated
11/
11/
98.
The
chi
squared
analysis
for
total
coliforms
is
based
on
the
1:
10,000
dilution;
the
analysis
for
E.
Coli
is
based
on
the
1:
1000
dilution.
Log
reduction
for
total
coliforms,

originally
reported
as
2.1,
was
recalculated
as
1.8
based
on
the
1:
10,000
dilution
factor.
Log
reduction
for
E.
coli
remains
unchanged.

The
statistical
evaluation
of
the
comparability
study
results
for
sample
982305A
was
originally
based
on
the
total
coliform
and
E.
coli
data
sheets
dated
12/
29/
98
(
this
study
represents
a
repeat
of
experiments
associated
with
data
sheets
dated
12/
23/
98).
The
analysis
was
subsequently
repeated
using
data
from
the
12/
23/
98
studies.
The
chi
squared
analysis
for
total
coliforms
was
repeated
based
on
the
1:
1000
dilution
data;
the
analysis
for
E.
coli
was
repeated
based
on
the
1:
1000
dilution
data.
Log
reduction
for
total
coliforms
and
for
E.
coli
is
as
reported
in
the
December
2002
NODA,
and
is
based
on
the
1:
1000
dilution
data
set.
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
49
See
also
response
to
Comment
24
(
Docket
ID
I­
D1.001,

Part
03)
for
a
discussion
of
a
consistent
data
set
(
i.
e.,
one
associated
with
the
same
dilution)
being
used
for
both
log­
reduction
and
comparability­
study
purposes.

See
response
to
Comment
12
(
Docket
ID
I­
F.
021,
part
10)
concerning
the
25:
75
split
guideline.

39
ID1.002
11
 
EPA
has
revised
the
log
reduction
following
chlorine
pretreatment
for
sample
number
990025A.
For
this
experiment
the
bacterial
concentration
used
to
compute
log
reductions
following
chlorine
treatment
was
drawn
from
a
different
dilution
of
the
 
original
sample 
titration
performed
on
1/
13/
99.
However,
the
testing
laboratory
generated
a
different
bacterial
concentration
for
the
500­
fold
dilution
employed
in
the
disinfection
experiment
conducted
one
day
later
(
approximately
30%
lower).
Nonetheless,
EPA
used
the
original,
24
hour
old
titer
in
recomputing
the
log
reduction
presented
in
Table
2.
Because
the
actual
data
is
available
for
the
specific
dilution
used
in
the
experiment
(
1:
500),
this
data
should
be
used
rather
than
the
data
drawn
from
the
original
assay
of
the
wastewater
isolate
(
3.2
log10). 

 
EA
has
revised
the
log
reduction
following
chlorine
pretreatment
for
sample
number
990052A.
For
this
experiment
the
bacterial
concentration
used
to
compute
log
reductions
following
chlorine
treatment
was
drawn
from
a
different
dilution
of
the
 
original
sample 
titration
performed
on
1/
20/
99;
however,
the
disinfection
experiment
using
diluted
wastewater
was
apparently
conducted
one
month
later
(
2/
20/
99).
If
this
is
the
case,
use
of
the
one
month
old
original
titer
is
completely
inappropriate
as
the
microbial
concentration
in
the
wastewater
will
fall
appreciably
over
one
month
regardless
of
the
storage
condition.
If
the
dates
are
indeed
incorrect
on
the
bench
sheets,
and
the
true
date
of
the
experiment
is
1/
20/
99,
the
actual
E.
coli
data
indicated
for
the
1,250­
fold
dilution
of
wastewater
should
be
used
to
compute
the
pre­
disinfection
titer
of
test
microorganisms
(
1x102
CFU
In
response
to
the
comments
suggesting
that
it
was
somehow
improper
for
EPA
to
calculate
the
log
reductions
by
using
an
inferred
figure
as
described
in
the
December
2,
2002
NODA,
see
response
to
Comment
25
(
Docket
ID
I­
D1.002
part
04)
regarding
the
use
of
bacterial
densities
determined
from
the
original
density
determination.

For
an
explanation
of
the
time
between
the
initial
bacterial
density
determination
of
the
sample,
and
the
chlorination
of
the
diluted
sample
for
sample
990052A,

see
response
to
Comment
24
(
Docket
ID
I­
D1.001
Part
03).
See
also
Attachment
1.

The
commenter
suggests
that
the
post­
dilution
E.
coli
measurement
(
for
the
1250­
fold
dilution
of
wastewater)

should
be
used
as
the
predisinfection
density
to
determine
the
chlorine­
stress
log
reduction.
In
response
to
this
comment,
see
response
to
Comment
25
(
Docket
ID
I­
D1.002
part
04)
regarding
the
use
of
bacterial
densities
determined
from
the
original
density
determination.
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
50
per
mL
in
the
1:
1,250
dilution,
which
would
correspond
to
a
log
reduction
of
1.7
log10.
This
reduction
fails
to
meet
the
ATP
criterion
specified
in
section
4.5.2.1.
Further
clarification
is
needed
from
the
independent
testing
laboratory
to
verify
the
actual
date
of
experimentation.
Data
from
sample
990052A
should
not
be
used
by
EPA
in
support
of
product
approval. 

 
EPA
has
revised
the
log
reduction
following
chlorine
pretreatment
for
sample
number
990095A.
No
log
reduction
is
presented
for
E.
coli
despite
the
fact
that
the
bench
sheet
for
this
sample
indicates
mFC
counts
of
9
and
4
for
the
samples
where
1.0
mL
and
0.1
mL
were
plated,
respectively.
If
the
0.1
mL
sample
is
used,
a
viable
titer
of
400
CFU/
100
mL
can
be
computed
and
then
used
to
compute
the
log
reduction
following
chlorine
treatment.
Using
the
equation
presented
by
EPA
in
the
December
2,
2002
FR
notice
the
log
reduction
would
then
be
1.5
log10
which
fails
to
meet
the
ATP
criterion.

Also,
it
appears
that
incorrect
numbers
were
used
for
the
chi
square
analysis.
The
dilution
used
for
the
E.
coli
experiments
was
1:
3,000;
however,
the
chi
square
tables
used
the
splits
from
the
1:
7,000
dilution.
The
chi
square
should
be
recomputed
using
splits
of
14:
6
and
7:
13
rather
than
14:
6
and
12:
8.
EPA
and
the
independent
testing
laboratory
should
discuss
the
missing
data
and
present
the
corrected
bench
sheets
for
external
review
before
proceeding.
The
testing
laboratory
should
also
provide
the
missing
data
for
the
bench
sheet
(
dates
of
testing,

microbial
concentrations,
initials
of
technicians
involved
in
the
testing
and
documentation
effort).
In
the
absence
of
further
clarification,
data
generated
from
sample
990095
should
not
be
considered
in
support
of
product
approval. 
In
response
to
the
comment
suggesting
that
sample
990052A
failed
to
meet
the
3
to
4
log
reduction
criterion,
see
response
to
Comment
12
(
Docket
ID
IF
021,
Part
10)
concerning
the
log
reduction
results.

The
commenter
observed
that
no
E.
coli
log
reduction
was
presented
for
sample
990095A
in
the
December
2,

2002
NODA
although
data
on
the
density
of
bacteria
in
diluted
wastewater
used
in
the
chlorination
experiment
was
available.
The
commenter
also
mentions
that
the
log
reduction
from
sample
990095A
is
1.5
if
the
prechlorination
density
determination
is
used.

While
EPA
considered
the
approach
suggested
by
the
commenter
(
i.
e.
use
the
measured
pre­
chlorination
value)

EPA
decided
that
it
would
be
more
consistent
to
apply
the
same
methodology
for
calculating
log
reductions
to
each
sample.
This
approach
was
used
by
EPA
in
the
calculation
of
log
reduction
results
presented
in
the
December
2002
NODA.
Since
an
initial
density
determination
for
mFC
was
not
available
for
sample
990095,
the
log
reduction
could
not
be
calculated
using
EPA s
standard
approach;
thus,
log
reduction
for
this
sample
was
reported
as
NA.

The
commenter
states
that
incorrect
numbers
were
used
for
the
chi
squared
analysis.
EPA
notes
that
E.
coli
comparability
studies
were
performed
using
dilutions
of
both
1:
3000
and
1:
7000.
Data
associated
with
the
1:
7000
dilution
was
used
in
EPA s
statistical
analysis.

Since
a
log
reduction
was
not
reported
for
this
study,

there
is
no
issue
of
inconsistency
between
log
reduction
and
chi
squared
data
sets.
Thus,
EPA
stands
by
its
decision
to
use
the
1:
7000
data
set.
See
also
the
response
to
Comment
13
(
Docket
ID
I­
D1.001,
Part
04)

for
additional
detail
concerning
this
comment.
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
51
 
EPA
has
revised
the
E.
coli
log
reduction
following
chlorine
pretreatment
for
sample
number
9900217A.
Again,
the
log
reduction
following
chlorination
was
computed
using
the
concentration
of
E.
coli
assayed
in
the
wastewater
isolate
(
albeit
more
than
two
weeks
after
receiving
it!).
The
recomputed
log
reduction
presented
in
Table
2
is
2.8
log10
but
should
instead
be
1.1
log10
(
the
actual
datum
for
the
1:
800
dilution
used
in
the
disinfection
experiment
should
be
used
rather
than
the
original
titer
from
a
different
dilution
of
the
wastewater
isolate).
The
recomputed
log
reduction
fails
to
meet
the
ATP
criterion
presented
in
section
4.5.2.1.
The
bench
sheet
is
also
missing
QC
data
including
the
date
of
experimentation
and
researcher
initials.
Finally,
the
data
violate
the
25:
75
rule
in
that
17
negatives
and
3
positives
were
observed
for
E.
coli
under
the
reference
method.
Data
generated
from
sample
9900217A
should
not
be
considered
in
support
of
product
approval. 

 
EPA
has
revised
the
log
reduction
following
chlorine
pretreatment
for
sample
number
990273A.
The
recomputed
log
reduction
utilized
the
data
generated
from
the
original
enumeration
of
the
E.
coli
counts
in
the
sample
received
on
3/
19/
99.
However,
the
actual
disinfection
experiment
was
carried
out
three
weeks
later
on
4/
8/
99
and
resulted
in
a
predisinfection
concentration
of
400
CFU/
100
mL
in
the
1:
80
The
commenter
points
out
that
data
are
missing
from
the
log
sheets.
See
response
to
Comment
28
(
Docket
ID
I.
D1.002,
Part
13)
concerning
incomplete
bench
sheets.

The
missing
data
were
not
critical
to
EPA s
evaluation
of
the
method.

The
commenter
observes
that
the
log
reduction
calculation
for
sample
990217A
was
based
on
the
bacterial
concentration
in
the
initial
density
determination
of
the
original
sample.
See
response
to
Comment
25
(
Docket
ID
I­
D1.002,
Part
04)
regarding
EPA s
consistent
use
of
this
approach.
Concerning
the
comment
that
the
chlorine
stressing
of
the
bacteria
occurred
more
than
2
weeks
after
the
initial
density
determination,
this
is
addressed
in
the
response
to
Comment
24
(
Docket
ID
I­
D1.001
Part
03).
In
reference
to
the
comment
about
the
log
reduction
failing
to
meet
the
ATP
criterion
presented
in
section
4.5.2.1
of
the
ATP
protocol,
see
response
to
Comment
1
(
Docket
ID
I­
D1.001,
Part
06)
concerning
EPA s
use
of
the
ATP
protocol
as
guidance.

The
commenter
points
out
that
data
are
missing
from
the
log
sheets.
See
response
to
Comment
28
(
Docket
ID
I.
D1.002,
Part
13)
concerning
incomplete
bench
sheets.

The
commenter
points
out
that
the
comparability
study
data
for
sample
990217A
violate
the
25%:
75%
split
guideline.
See
response
to
Comment
12
(
Docket
ID
I­
F
021,
Part
10)
concerning
the
25%:
75%
split
guideline.

The
commenter
observes
that
the
log
reduction
calculation
for
sample
990273A
was
based
on
the
bacterial
concentration
in
the
initial
density
determination
of
the
original
sample.
See
response
to
Comment
25
(
Docket
ID
I­
D1.002,
Part
04)
regarding
EPA s
consistent
use
of
this
approach.
Concerning
the
comment
that
the
chlorine
stressing
of
the
bacteria
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
52
dilution.
The
correct
log
reduction
should
be
1.6
log10
which
fails
the
ATP
criterion.
Moreover,
it
would
be
incorrect
to
use
the
initial
titer
of
E.
coli
determined
upon
receipt
of
the
wastewater
isolate
because
this
sample
was
received
three
weeks
prior
to
the
actual
disinfection
experiment.
It
would
be
incorrect
to
assume
that
the
E.
coli
concentration
in
this
isolate
would
remain
stable
for
three
weeks
and
hence
any
computations
that
would
blend
three
week
old
data
with
fresh
determinations
of
chlorinated
test
microorganisms
should
be
considered
highly
suspect.
Data
generated
from
this
sample
should
not
be
considered
in
support
of
product
approval. 

 
EPA
has
revised
the
log
reduction
following
chlorine
pretreatment
for
sample
number
990438A.
As
before,
the
log
reduction
should
be
computed
using
the
actual
sample
dilution
used
for
the
chlorine
injury
experiments.
Therefore,
the
mFC
count
for
the
10
mL
sample
from
the
1:
200
dilution
(
1
CFU)

should
be
used
in
computing
the
log
reduction.
However,
it
is
unclear
from
the
bench
sheet
how
the
dilution
factors
would
be
used
because
a
different
dilution
was
employed
for
the
postchlorinated
sample
(
1:
1,000).
If
the
dilution
inconsistencies
are
typographical
errors,
then
the
true
log
reduction
would
be
0.7
log10,
which
fails
the
ATP
criterion.
The
testing
laboratory
should
provide
clarification
of
the
true
concentration
of
E.
coli
in
the
disinfected
sample
and
should
recompute
the
log
reduction
before
final
external
review
of
the
data.
Regardless,

the
initial
titration
from
the
wastewater
isolate
should
not
be
used
to
compute
the
log
reduction.
Because
the
date
of
the
E.

coli
assays
are
not
indicated
in
the
comparability
bench
sheets,
it
is
not
clear
if
the
same
inconsistency
exists
as
was
pointed
out
previously
for
the
total
coliform
tests
on
this
sample.
Without
further
clarification,
data
generated
from
sample
990438A
should
not
be
considered
in
support
of
product
approval. 
occurred
3
weeks
after
the
initial
density
determination,

this
is
addressed
in
the
response
to
Comment
24
(
Docket
ID
I­
D1.001
Part
03).
In
reference
to
the
comment
about
the
log
reduction
failing
to
meet
the
ATP
criterion
presented
in
section
4.5.2.1
of
the
ATP
protocol,
see
responses
to
Comment
1
(
Docket
ID
ID1.001
Part
06)
and
Comment
3
(
Docket
ID
I­
D1.005,

Part
02)
concerning
EPA s
use
of
the
ATP
protocol
as
guidance.

For
sample
990438A,
the
commenter
notes
that
the
log
reduction
calculation
should
be
based
on
the
prechlorination
density
of
bacteria
determined
after
the
sample
was
diluted
rather
than
using
the
initial
bacterial
density
from
the
original
sample
for
the
log
reduction
calculation.
With
regard
to
this
comment,
see
response
to
Comment
25
(
Docket
ID
I­
D1.002,
Part
04)
regarding
the
use
of
bacterial
densities
determined
from
the
original
density
determination.

Per
the
commenter s
observation
concerning
a
potential
typographical
error
in
the
dilution
values
identified
in
the
bench
sheets
for
sample
990438A,
EPA
has
reviewed
those
bench
sheets
further.
EPA
notes
that
the
dilution
factor
associated
with
the
diluted
E.
coli
sample
measurement
(
part
2
of
the
worksheet)
and
the
comparability
study
results
is
1:
200,
while
that
listed
for
the
post­
chlorination
measurement
(
part
5
of
the
worksheet)
is
1:
1000.
The
dilution
identified
in
part
6
of
the
worksheet
was
originally
listed
as
1:
1000,
but
this
was
crossed
out
and
replaced
with
1:
200
(
and
initialed
by
the
analyst).
One
of
these
figures
is
apparently
an
error,

and
it
appears
more
likely
that
1:
200
is
the
correct
value,

though
EPA
cannot
be
certain.
Accordingly,
EPA
examined
the
impact
of
the
different
figures
on
log
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
53
reduction.
EPA
did
so
by
substituting
a
200­
fold
dilution
into
the
log
reduction
calculation
in
place
of
the
1000­
fold
dilution
originally
used;
doing
so
resulted
in
a
log
reduction
of
3.4
(
versus
the
2.7
log
reduction
associated
with
a
dilution
of
1:
1000).
This
minor
change
in
log
reduction
has
no
impact
on
EPA s
assessment
of
the
method
as
both
values
are
acceptable.
See
also
the
response
to
Comment
12
(
Docket
ID
I­
F.
021,
Part
10)

concerning
the
log
reduction
criterion.

Concerning
the
comment
on
the
missing
date
on
the
comparability
study
bench
sheet
for
E.
coli
for
sample
990438A,
see
response
to
Comment
I.
D1.002,
Part
13
concerning
incomplete
bench
sheets
and
response
to
Comment
24
(
Docket
ID
I­
D1.001,
Part
03).

40
ID1.002
14
 
In
the
previous
(
April
2002)
review
of
the
data
submitted
by
CPI
a
number
of
concerns
over
the
irregular
presentation
of
false
positivity
and
false
negativity
rates
were
brought
to
EPA s
attention.
For
total
coliforms
the
false
positivity
rate
ranged
from
2%
(
Federal
Register
notice
of
March
7,
2002)
to
5%

(
08/
15/
01
memorandum
from
C.
Woodruff
submitted
as
part
of
the
docket
materials).
Although
the
upperbound
of
these
two
figures
(
5%)
may
be
considered
acceptable
by
EPA
standards,
the
inconsistency
in
the
presentation
of
the
different
rates
calls
for
verification
by
the
manufacturer,
especially
if
neither
figure
is
correct.
Similarly
the
false
positivity
rates
for
E.

coli
ranged
from
0.0%
(
Federal
Register
notice
of
March
7,

2002)
to
17.8%
(
08/
15/
01
memorandum).
Which
number
is
correct,
and
where
are
the
supporting
data
available
to
substantiate
these
claims? 
Specificity
data
are
part
of
the
data
generally
reviewed
by
EPA
for
evaluation
of
applications
for
new
ATP
tests
and,
therefore,
satisfactorily
documented
specificity
data
are
generally
submitted
as
part
of
the
ATP
evaluation.

EPA
included
a
variety
of
information
concerning
false
positive
and
false
negative
rates
(
i.
e.,
specificity)
in
the
public
record.
In
response
to
public
comments
concerning
specificity,
EPA
reviewed
that
information
and
concluded
that
the
specificity
results
best
supported
by
the
data
in
the
public
record
are
those
collected
by
the
Georgia
Department
of
Natural
Resources
(
false
positive
rates
of
5%
and
18%
for
total
coliforms
and
E.

coli,
respectively,
and
false
negative
rates
of
0%
for
both).

EPA
notes
that
additional,
and
perhaps
more
current,

specificity
information
may
be
available
through
CPI,

developers
of
Colitag
 
.
EPA
does
not
have
a
specific
standard
for
acceptable
false
positive
and
negative
rates;

however,
the
values
determined
by
the
Georgia
Department
of
Natural
Resources
are
consistent
with
the
range
of
results
produced
by
other
EPA
approved
methods.
See,
also,
response
to
Comment
41
(
ID
IF
004,
Part
05).

41
I­
F.
004
05
4.
Colitag
 
'
s
specificity
data
are
not
scientifically
valid
In
response
to
the
comment s
suggestion
that
CPI s
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
54
 
It
appears
that
CPI
submitted
several
sets
of
data
without
detailed
description
regarding
what
samples
were
used
and
how
the
experiments
were
conducted
to
determine
the
specificity
of
the
Colitag
 
medium. 

 
In
the
communication
from
Mr.
Jim
Knight
of
CPI
to
Mr.

Robert
Bordner
of
the
USEPA
dated
March
2,
1999,
it
is
claimed
that
Colitag
 
has
a
false
positive
error
of
1.33%
and
a
false
negative
error
of
0%
for
total
coliforms
and
2.2%
false
positive
error
and
1.4%
false
negative
error
for
E.
coli.
There
was
no
information
on
whether
these
data
were
derived
from
the
ATP
Comparability
Study
performed
by
BioVir
Laboratories
(
that
does
not
appear
to
be
the
case
since
there
is
no
data
in
BioVir's
raw
data
set)
or
from
testing
with
a
particular
sample
types
and
how
these
were
determined.
In
a
communication
from
Mr.
Bordner
of
the
USEPA
to
CPI
(
dated
September
15,

1998),
it
appears
that
CPI
also
submitted
two
sets
of
specificity
data
(
note:
these
data
were
collected
by
the
Georgia
Department
of
Natural
Resources
in
March
and
June,
1998).
These
two
sets
of
data
were
combined,
because
it
was
concluded
that
those
data
were
insufficient
independently
to
establish
that
the
false
positive
errors
for
Colitag
 
were
5%
(
7/
140)
for
total
coliforms
and
17.8%
(
25/
140)
for
E.
coli
and
that
the
false
negative
errors
were
0%
for
both
total
coliforms
and
E.
coli.
It
is
important
to
note
that
all
these
data
were
different
from
what
has
been
published
in
Federal
Register
(
Vol.
67,
No.
45;
March
7,
2002)
for
public
comments.
Those
data
shown
in
the
published
proposed
rule
suggest
that
Colitag
 
has
a
false
positive
error
of
2.0%
for
total
coliforms
and
2.0%
for
E.
coli
and
false
negative
rates
of
0%
for
both
total
coliforms
and
E.

coli.
The
question
is
how
these
data
were
obtained
and
what
the
relationship
is
between
this
set
of
data
and
the
submitted
data
on
Colitag
 
'
s
specificity. 

 
In
addition,
it
is
critical
to
understand
whether
the
0%
false
negative
errors
for
both
total
coliforms
and
E.
coli
as
shown
in
the
published
proposed
rule
for
Colitag
 
were
determined
by
using
samples
containing
bacteria
that
are
stressed
or
injured.
specificity
data
are
inconsistent/
incomplete
(
and
therefore
not
scientifically
valid),
EPA
has
concluded
that,
among
the
data
in
public
records,
that
collected
by
Georgia
Department
of
Natural
Resources
(
GDNR)

represents
the
most
appropriate
specificity
data
for
Colitag
 
.
GDNR
followed
an
approved
study
plan,

and
the
data
produced
by
their
study
were
reviewed
by
EPA
and
found
to
be
acceptable.
The
other
specificity
data
the
commenter
refers
to
are
not
as
well
documented,
and
EPA
is
not
relying
on
these
data
to
reach
a
decision
concerning
Colitag
 
.
See
also
the
response
to
Comment
40
(
Docket
ID
I­
D1.002,
Part
14)

concerning
the
specificity
data.

EPA
notes
that
the
target
bacteria
in
the
specificity
study
are
not
required
to
be
stressed,
because
the
specificity
determination
only
considers
correct
identification
of
target
bacteria,
and
is
separate
from
considerations
of
detecting
stressed
bacteria.
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
55
Ideally,
the
specificity
of
a
proposed
method
for
total
coliforms
and
E.
coli
is
determined
through
a
formal
USEPA
ATP
procedure.
Therefore,
false
negative
errors
of
the
proposed
method
are
determined
by
using
spiked
water
samples
that
have
been
chlorinated
to
reduce
the
target
bacteria
(
i.
e.
total
coliforms
and
E.
coli)
by
3­
4
Log10. 

 
It
is
our
position
that
these
questions
raised
here
need
to
be
addressed.
Otherwise,
Colitag
 
'
s
specificity
data
as
published
in
Federal
Register
are
not
scientifically
valid. 

42
I­
F.
021
06
Detailed
review
comments
 
Colitag
 
presence/
absence
method
for
detection
of
coliforms
and
E.
coli
Section
2:
Summary
of
Method
 
§
2.2:
It
would
be
more
appropriate
to
present
experimental
data
in
support
of
the
product
performance
claims
rather
than
to
refer
obliquely
to
a
poster
presentation
that
includes
material
that
has
not
been
processed
through
rigorous
peer
review
and
that
is
unavailable
for
review
in
the
context
of
the
USEPA
approval
process.
Is
the
material
from
the
poster
presentation
available
for
review?
If
so,
where
can
it
be
found? 

 
§
2.3:
See
comment
from
§
2.2.
Performance
claims,
including
the
comment
that
optimized
recoveries
occur
as
a
function
of
increased
pH,
should
be
accompanied
by
robust
supporting
data
(
peer
reviewed
or
other)
or
should
be
eliminated
altogether. 
The
additional
information
cited
by
the
commenter
(
poster,
performance
claims
of
optimized
recoveries
due
to
increased
pH)
is
beyond
the
scope
of
EPA s
method
evaluation
process,
and
was
not
considered
in
EPA s
decision.

43
I­
F.
021
09
Section
17:
Tables,
Diagrams,
Flow
Charts,
and
Validation
Data
 
False
pos:
The
false
positivity
rates
for
total
coliforms
and
E.
coli
computed
in
Tables
17.1
and
17.22
are
1.33%
and
2.2%,

respectively.
Rates
reported
in
the
Federal
Register
Notice
of
March
7,
2002,
are
2.0%
and
2.0%.
Is
supporting
documentation
available
to
account
for
the
differences
in
the
performance
data? 

 
False
neg:
Similarly,
the
false
negativity
rates
from
Tables
17.1
and
17.2
for
total
coliforms
and
E.
coli
are
0%
and
1.4%,
As
explained
in
the
responses
to
Comment
40
(
Docket
ID
I­
D1.002,
Part
14)
and
Comment
41
(
ID
I­
F.
004,

Part
05)
the
specificity
results
best
supported
by
the
data
in
the
public
record
are
those
collected
by
the
Georgia
Department
of
Natural
Resources.
None
of
the
alternative
figures
for
false
positive
and
false
negative
rates
were
derived
from
studies
that
followed
approved
study
plans,
were
reviewed
by
EPA
and
have
full
documentation,
in
contrast
to
the
Georgia
Department
of
Natural
Resources
data.
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
56
respectively.
The
Federal
Register
Notice
of
03/
07/
02
indicates
rates
of
0%
for
both
microbial
groups.
How
can
these
differences
be
reconciled? 

44
I­
F.
022
02
Colitag
 
 
USEPA
has
proposed
Colitag
 
,
manufactured
by
CPI
International,
for
approval
for
total
coliform
and
E.
coli.

Information
and
data
obtained
from
USEPA
regarding
the
ATP
comparability
data
for
Colitag
 
submitted
for
its
consideration
of
approval
for
use
in
detecting
total
coliforms
and
E.
coli
in
drinking
water
was
reviewed.
There
was
no
comprehensive
report
prepared
to
explain
exactly
what
was
done
in
these
studies.
A
series
of
letters
and
miscellaneous
data
sets
had
to
be
interpreted.
Hence,
it
is
unclear
whether
the
USEPA
ATP
Protocol
was
followed.
Based
on
this
information,
the
following
comments
are
offered:
EPA
reviewed
the
information
provided
for
Colitag
 
and
concluded
that
it
was
sufficient
upon
which
to
base
a
decision.
A
study
plan
was
prepared,
reviewed
and
executed,
and
study
results
were
extensively
reviewed
by
EPA.
See
also
the
response
to
Comment
2
(
Docket
ID
I­
D1.002,
Part
15).

The
report
recommended
by
the
commenter
is
beyond
the
scope
of
EPA s
method
evaluation
process.

Although
the
information
submitted
by
CPI
is
not
consistent
with
the
ATP
protocol
guidelines
in
every
respect,
it
was
sufficient
for
EPA
to
conclude
that
Colitag
 
performs
in
a
manner
comparable
to
the
reference
methods.
See
response
to
Comment
1
(
Docket
ID
I­
D1.001,
Part
06).

45
I­
F.
022
06
4.
The
Federal
Register
notice
indicates
that
the
method
has
a
false
positive
error
of
2.0
%
for
total
coliforms
and
2.0
%
for
E.

coli
and
false
negative
rates
of
0
%
for
both
total
coliforms
and
E.
coli.
However,
the
data
supporting
these
statements
could
not
be
found
in
the
docket.
The
correspondence
and
documentation
state
different
results,
and
false
positive
results
as
high
as
5
%
for
total
coliforms
and
17.8
%
for
E.
coli
have
been
reported.
The
scientific
basis
for
the
specificity
data
for
this
method
must
be
provided
for
public
comment
and
clearly
explained.
See
the
responses
to
Comment
41
(
Docket
ID
I­
F.
004,

Part
05)
and
Comment
40
(
Docket
ID
I­
D1.002,
Part
14
concerning
the
appropriateness
of
the
specificity
data.

46
I­
F.
022
08
6.
A
well
documented
report
is
needed
that
describes
the
various
studies
that
were
conducted,
which
data
was
used
in
the
analysis,
and
that
documents
how
the
USEPA
ATP
Protocol
was
followed. 
EPA
requests
certain
data
from
manufacturers
to
determine
if
a
new
coliform
method
should
be
approved
for
drinking
water
analysis.
EPA
does
not
specify
the
format
in
which
this
data
should
be
supplied.

EPA
determined
that
the
data
supplied
by
CPI
met
the
Agency s
needs
for
evaluating
Colitag
 
 
s
performance.

See
also
the
response
to
Comment
44
(
Docket
ID
IF
022,
Part
02).
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
57
05
DOCUMENTATION
OF
Colitag
 
PROCEDURES
(
safety;
instrumentation/
equipment/
supplies;
quality
control
measures;
calibration;

incubation
temperatures
and
times;
shelf
life)

47
ID1.001
05
 
7.
Data
obtained
at
44.5
C
is
irrelevant,
since
the
method
is
proposed
for
35
C
incubation. 
The
commenter
refers
to
data
included
in
the
December
2002
NODA
for
informational
purposes,
but
not
considered
by
EPA
in
its
evaluation
of
method
comparability
between
Colitag
 
and
the
reference
methods.
EPA
did
not
rely
on
this
information
in
deciding
to
approve
the
Colitag
 
method.
As
such,
the
comment
does
not
impact
the
Agency s
decision.

48
I­
F.
004
07
 
6.
Colitag
 
requires
clear
definition
of
the
starting
incubation
temperature 

 
Colitag
 
'
s
insert
[
section
11.0]
submitted
by
CPI
to
the
USEPA
claims
that
the
medium
and
samples
must
be
equilibrated
to
room
temperature
(
24­
30
°
C)
in
order
to
produce
specified
results.
As
demonstrated
in
ReadyCult's
data,
the
starting
temperature
could
have
profound
impact
on
the
accuracy
of
a
liquid
culture
method.
It
is
critical
to
clearly
define
this
starting
temperature
requirement
and
the
required
ramp
up
time
from
2­
8
°
C
to
this
starting
incubation
temperature
including
the
mechanism,
i.
e.
air­
circulated
incubator
or
water
bath. 
The
commenter
suggests
that
the
starting
temperature
of
incubation
could
have
a
profound
impact
on
the
accuracy
of
a
liquid
culture
method.
Incubation
temperature
is
generally
important
for
all
media,
not
just
Colitag
 
.
To
ensure
that
media
are
incubated
at
the
proper
temperature,
EPA
has
included
guidance
in
the
Manual
for
the
Certification
of
Laboratories
Analyzing
Drinking
Water
on
how
to
ensure
that
samples
are
incubated
at
the
proper
temperature
in
air
incubators.

EPA
requires
that
certified
drinking
water
laboratories
be
used
for
compliance
monitoring
analyses;
a
certified
laboratory
was
also
used
to
perform
the
Colitag
 
comparability
studies.
In
both
cases,
the
expectation
is
that
such
laboratories
will
follow
the
procedures
specified
in
the
aforementioned
manual.

49
I­
F.
006
04
o
 
Time
the
Test
Requires
[
both
Colitag
 
and
ReadyCult] 

 
Substantiation
of
the
required
incubation
time
is
critical
to
the
accuracy
of
the
test
method.
Neither
the
Colitag
 
nor
ReadyCult
tests
substantiate
its
incubation
time.
This
lack
of
substantiation
can
lead
to
a
lack
of
sensitivity
that
will
adversely
affect
public
health
protection.
The
ATP
clearly
requires
this
substantiation. 

 
Colitag
 
:
While
the
Colitag
 
test
states
that
it
is
to
be
incubated
for
24
hours,
plus
or
minus
2
hours,
there
is
no
information
presented
at
22
or
26
hours
incubation.
In
effect,

the
Colitag
 
test
claims
a
22
hour
incubation;
substantiation
is
The
commenter
suggests
that
substantiation
is
required
for
the
incubation
time
to
assure
the
accuracy
of
the
test
method.
The
ATP
comparability
study
was
done
for
24
hours
at
the
specified
temperature
and
yielded
results
comparable
to
the
reference
methods.
As
such,
the
acceptability
of
the
incubation
time
was
substantiated
for
Colitag
 
.

EPA
believes
that
the
+/­
2hr
range
associated
with
the
incubation
time
is
reasonable
and
is
consistent
with
methods
previously
approved,
including
the
reference
methods
for
multiple
tube
fermentation.
This
is
because
bacterial
growth
on
different
media
where
growth
is
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
58
essential. 
adequate
in
24
hours
would
not
be
expected
to
differ
to
any
substantial
extent
between
the
media
at
22
or
26
hours.
In
EPA s
experience
with
coliform
methods,

when
a
positive
result
is
visible
at
24
hours,
bacterial
growth
rates
are
such
that
the
results
are
generally
observable
at
22
hours.
Similarly,
when
bacterial
growth
is
observed
at
24
hours,
such
bacteria
continue
to
be
observable
at
26
hours.

50
I­
F.
021
05
 (
4)
No
details
are
provided
on
the
experimental
studies
conducted
in
support
of
the
contention
that
Colitag
 
can
be
stored
in
the
long
term
without
an
adverse
impact
on
product
performance.
If
these
data
are
available,
they
should
be
presented
along
with
interpretive
material
so
that
the
reagent
is
not
used
beyond
its
defined
expiration
date. 
EPA
has
not
taken
any
action
approving
or
disapproving
the
long­
term
storage
performance
of
Colitag
 
.
The
additional
information
cited
by
the
commenter
is
beyond
the
scope
of
EPA s
method
evaluation
process
and
does
not
impact
the
Agency s
decision.

51
I­
F.
021
08
Section
5:
Safety
§
5.2:
The
manufacturer
references
Standard
Methods
for
the
Examination
of
Water
and
Wastewater,
20th
edition,
but
offers
the
publication
date
of
1992.
The
20th
edition
was
published
in
1998
and
should
be
referenced
as
such.

Section
6:
Instrumentation,
Equipment
and
Supplies
§
6.3:
Handling
of
glassware
and
containment
vessels
is
practiced
according
to
guidelines
established
in
Standard
Methods.
The
guidelines
in
Standard
Methods
are
rigorous
and
include
multiple
wash
steps
(
minimum
of
6)
and
challenge
experiments
using
live
cultures
of
E.
aerogenes
to
establish
that
vessels
do
not
leach
bacteriostatic,
nutritive
or
cytotoxic
compounds
following
repeated
autoclaving.
The
manufacturer
suggests
in
this
section
that
vessels
were
washed
with
detergent,

rinsed
a
single
time
with
tap
water,
rinsed
with
reagent
water
and
then
sterilized
by
autoclaving.
If
the
rigorous
procedures
defined
in
Standard
Methods
were
in
fact
used,
then
this
practice
should
have
been
stated
explicitly.
If
other
procedures
were
followed,
such
as
those
provided
in
the
USEPA
Alternative
Testing
Protocol,
they
should
similarly
be
stated
explicitly.
The
comment
suggesting
an
error
in
the
publication
date
for
Standard
Methods
is
beyond
the
scope
of
EPA s
method
evaluation
process
and
does
not
impact
the
Agency s
decision.

The
comment
suggesting
that
more
information
should
have
been
provided
on
glassware
handling
is
beyond
the
scope
of
EPA s
method
evaluation
process
and
does
not
impact
the
Agency s
decision.
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
59
Section
7:
Reagents
and
Standards
§
7.1
The
Colitag
 
product
is
described
as
stable
when
stored
at
25­
30
°
C
away
from
light,
but
no
shelf
life
is
described.

For
how
long
can
this
reagent
be
stored?
Is
supporting
documentation
available
to
substantiate
shelf
life
stability
claims?
Section
8:
Sample
collection,
dechlorination,
preservation,

shipment
and
storage
§
8.1.1
The
description
of
sample
collection
bottles
is
inadequate;
sample
bottles
must
not
leach
cytotoxic,
nutritive
or
bacteriostatic
components
and
must
be
tested
as
such
(
ATP
section
3.4.1).

Section
9:
Quality
Control
§
9.0
An
oblique
reference
to
Standard
Methods
is
made
with
regard
to
quality
control
that
is
not
entirely
consistent
with
the
recommendations
in
this
section.
The
manufacturer
suggests
use
of
three
control
tubes,
with
the
first
used
as
a
blank,
the
second
used
as
a
'
positive'
control
and
the
third
as
a
'
negative'
control.
The
referenced
method
found
in
Standard
Methods
(
section
9223B)
includes
multiple
controls,
where
at
least
one
microorganism
is
E.
coli,
another
is
a
non­
E.
coli
representative
from
the
total
coliform
group,
and
the
third
is
a
noncoliform,
for
a
total
of
three
microbial
controls
('
negative
control'
sample
blanks
containing
no
microorganisms
would
be
considered
a
fourth
control).
Ambiguous
references
regarding
quality
control
cultures
must
by
all
means
be
removed
from
manufacturer
protocols
and
also
from
all
product
literature,
as
they
will
ultimately
confuse
the
end
user.

Section
10:
Calibration
and
Standardization
§
10.1
The
manufacturer
refers
to
section
9.0
to
define
acceptable
product
performance,
however,
the
text
in
section
EPA
has
not
taken
any
action
approving
or
disapproving
the
long­
term
storage
performance
of
Colitag
 
.
The
information
requested
by
the
commenter
regarding
appropriate
storage
time
is
beyond
the
scope
of
EPA s
method
evaluation
process
and
does
not
impact
the
Agency s
decision.

The
comment
suggesting
that
more
information
on
the
type
of
sample
collection
bottles
is
necessary
goes
beyond
the
scope
of
EPA s
method
evaluation
process
and
does
not
impact
the
Agency s
decision.

The
comment
suggesting
that
certain
quality
control
provisions
are
too
ambiguous
goes
beyond
the
scope
of
EPA s
method
evaluation
process
and
does
not
impact
the
Agency s
decision.

The
comment
suggesting
that
certain
text
regarding
inoculation
of
microorganisms
into
culture
bottles
is
inadequate
goes
beyond
the
scope
of
EPA s
method
evaluation
process
and
does
not
impact
the
Agency s
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
60
9.0
is
inadequate
in
describing
how
test
microorganisms
should
be
inoculated
into
culture
bottles.
How
much
culture
material
should
be
introduced
for
control
reactions?
A
loopful?
More?

Less?
Section
11:
Procedure
§
11.1.1
Manufacturer
protocols
indicate
that
samples
to
be
tested
must
be
equilibrated
to
room
temperature
(
24­
30
°
C)
prior
to
addition
of
Colitag
 
reagent.
How
is
this
done?
If
samples
must
be
equilibrated
to
room
temperature,
and
prewarming
of
samples
is
a
critical
variable,
should
sample
temperatures
be
measured
with
a
thermometer?
Would
the
thermometer
then
need
to
be
sterile?
Will
sample
holding
time
become
an
issue
as
the
laboratorian
waits
for
samples
to
prewarm?
These
are
nontrivial
issues,
as
typical
laboratory
practice
using
chromogenic
substrates
calls
for
addition
of
reagent,
mixing,

and
then
introduction
into
the
incubator
without
prewarming.

Section
13:
Method
Performance
Characteristics
§
13.1
False
positivity
rates
for
total
coliforms
and
E.
coli
are
reported
in
the
08/
15/
01
memorandum
from
C.
Woodruff
to
be
5.0%
and
17.8%,
respectively.
False
negativity
rates
for
total
coliforms
and
E.
coli
are
reported
to
be
0%
and
0.0%,

respectively.
However,
the
false
positivity
and
negativity
rates
reported
in
the
Federal
Register
Notice
of
March
7,
2002,
are
significantly
different
(
2%
false
positivity
for
total
coliforms
and
E.
coli,
and
0%
false
negativity
for
both
groups).
Which
numbers
are
correct?
Is
supporting
documentation
available
to
substantiate
these
numbers? 
decision.

The
commenter
suggests
that
temperature
equilibration
to
room
temperature
is
required
for
the
incubation
time
to
assure
the
accuracy
of
the
test
method.
The
procedure
for
this
is
described
in
Manual
for
the
Certification
of
Laboratories
Analyzing
Drinking
Water.

See
also
the
response
to
Comment
48
(
Docket
ID
IF
004,
Part
07)
concerning
incubation
temperature.

See
responses
to
Comment
40
(
Docket
ID
I­
D1.002,

Part
14)
and
Comment
41
(
ID
I­
F.
004,
Part
05)
concerning
the
specificity
data.

52
I­
F.
022
09
 
7.
Documentation
regarding
Colitag
 
'
s
incubation
conditions
is
needed,
as
well
as
validation
of
the
claimed
conditions
of
24
+/­
2
hours
at
35
+/­
0.5C. 
See
response
to
Comment
48
(
Docket
ID
I­
F.
004,
Part
07)
concerning
incubation
temperature
and
Comment
49
(
Docket
ID
I­
F.
006,
Part
04)
concerning
incubation
times.

06
OTHER
COMMENTS
REGARDING
Colitag
 
PERFORMANCE
(
substrates
allegedly
susceptible
to
hydrolysis;
color
of
solution;
Aeromonas
interference)
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
61
53
ID2.001
03
 
My
second
issue
relates
to
the
actual
performance
of
Colitag
 
.

As
part
of
my
company
role,
we
are
involved
in
giving
advice
to
water
companies
with
respect
to
the
use
of
alternative
technologies
as
they
become
available.
As
such
we
have
carried
out
a
limited
study
of
Colitag
 
in
comparison
to
some
other
methods.
Our
experiences
are
as
follows:

1.
Approximately
5%
of
the
Colitag
 
pieces
that
we
encountered
had
"
caked"
meaning
that
water
had
found
its
way
into
the
material.
The
substrates
used
in
these
types
of
products
are
susceptible
to
hydrolysis
and
thus
these
5%
may
have
given
false
negative
results.
They
were
not
incidentally
used
in
our
study.

2.
When
dissolved
the
Colitag
 
product
gives
a
straw
colored
solution.
After
incubation
this
makes
discrimination
between
the
original
color
and
a
positive
result
extremely
difficult,

particularly
if
the
reaction
is
weak
which
it
often
is
at
the
minimum
incubation
period.
This
can
lead
to
both
false
positive
and
false
negative
results
unless
confirmatory
steps
are
included.

3.
When
tested
with
waters
containing
beta
galactosidase
producing
Aeromonas
spp,
the
Colitag
 
product
has
absolutely
no
inhibition
of
these
organisms.
This
could
be
predicted
based
on
the
formulation
of
the
product.
Indeed
during
our
initial
studies
all
samples
which
contained
glactosidase
producing
Aeromonas
(
even
at
levels
as
low
as
1
per
milliliter)
gave
a
positive
result
irrespective
of
the
presence
of
coliforms.
Clearly
this
is
completely
unacceptable
and
does
little
to
protect
public
health.
In
small
communities
(
whether
in
the
US
or
elsewhere)

and
in
less
affluent
countries
these
type
of
results
will
lead
to
confusion
and
perhaps
unnecessary
expenditure
or
other
public
health
measures. 
The
data
in
the
public
record
are
based
on
a
comparability
study
plan
developed
based
on
the
ATP
protocol;
reviewed
and
approved
by
EPA;
and
executed
by
an
independent,
certified
laboratory.
While
EPA
appreciates
the
commenter s
insight
based
on
their
 
limited
study, 
the
Agency
does
not
have
adequate
information
to
assess
the
quality
of
the
data
from
such
study.
Accordingly,
EPA
will
rely
on
the
results
of
the
more
complete,
aforementioned
comparability
study.

Commenter s
concerns
regarding
 
caking 
and
 
straw
colored 
solution
are
beyond
the
scope
of
EPA s
method
evaluation
process
and
do
not
impact
the
Agency s
decision.

EPA
further
notes
the
 
caking 
and
the
 
straw
color 

suggested
by
the
commenter
have
not
otherwise
been
identified
as
issues,
nor
do
they
appear
to
have
impacted
the
successful
performance
of
the
aforementioned
comparability
studies.
The
potential
false
negative
problem
suggested
by
the
commenter
was
not
observed
in
the
comparability
study
results.

EPA
acknowledges
that
Aeromonas
occurs
commonly
in
ambient
waters.
EPA
further
notes
that
potential
interference
by
Aeromonas
is
not
unique
to
the
Colitag
 
method;
at
least
one
coliform
method
previously
approved
by
EPA
has
had
the
same
potential
for
this
interference.
Based
on
the
judgement
of
EPA s
microbiology
experts,
however,
and
based
on
the
false
positive
and
false
negative
data
discussed
in
the
response
to
Comment
40
(
Docket
ID
I­
D1.002,
Part
14),
EPA
determined
that
this
was
not
a
significant
concern;
the
Colitag
 
method
performance
was
comparable
to
that
of
the
reference
methods,
as
documented
by
the
results
of
the
comparability
study.
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
62
54
I­
F.
021
07
 
Section
4:
Interferences
§
4.1
The
manufacturer
reports
no
known
interferences
in
drinking
water
or
source
water
that
may
interfere
with
product
performance,
however,
no
supporting
data
are
presented
to
substantiate
this
claim.
Is
it
appropriate
to
accept
this
contention
in
the
absence
of
supporting
data? 
The
issue
of
potential
interferences
raised
by
the
commenter
is
generally
outside
the
scope
of
EPA s
method
evaluation
because
EPA
experts
who
evaluate
alternative
coliform
methods
generally
have
not
included
interferences
in
the
factors
to
be
considered.
Moreover,

EPA
notes
that
the
Agency
did
not
observe
evidence
of
any
interferences
in
its
evaluation
of
the
study
results.

07
SOURCE
WATER
DATA
(
lacks
data
to
support
approval
for
source
water
applications)

55
I­
F.
004
06
 
5.
Colitag
 
lacks
sufficient
data
for
source
water
Colitag
 
lacks
sufficient
data
to
support
its
proposed
approval
for
source
water
application.
Only
a
limited
amount
of
data
(
23
data
points)
collected
from
Oct.
1994
to
April
24,
1995
may
be
relevant
for
Colitag
 
'
s
approval
for
source
water
application.

And
there
is
no
detailed
description
regarding
how
the
study
was
performed
and
how
the
data
were
interpreted
(
no
statistical
analysis
of
data).
Also,
the
comparative
evaluation
of
a
proposed
method
against
a
reference
method
for
source
water
application
needs
to
be
performed
in
a
quantitative
manner
in
order
to
accurately
assess
the
equivalency
between
these
two
methods.

It
is
not
clear
whether
the
specificity
data
presented
in
Tables
17.1
and
17.2
represent
the
specificity
of
Colitag
 
in
drinking
water
or
source
water.
This
is
again
due
to
the
lack
of
a
welldocumented
report
to
describe
what
types
of
samples
were
used
to
derive
these
data.
If
the
specificity
data
presented
in
Tables
17.1
and
17.2
were
part
of
the
data
derived
from
the
study
from
Oct.
1994
to
April
24,
1995
and
these
data
were
used
to
determine
the
specificity
of
Colitag
 
for
drinking
water
application
then
this
is
scientifically
inappropriate.
The
specificity
of
a
proposed
method
for
drinking
water
application
should
be
determined
using
a
drinking
water
matrix,
preferably
following
the
USEPA
ATP
protocol
and
for
source
water
application
should
be
derived
from
testing
with
source
water
samples.

Based
on
the
aspects
mentioned
above,
we
believe
that
Approval
of
Colitag
 
is
only
being
sought
for
drinking
water,
not
source
water.
Therefore,
data
for
source
water
was
not
necessary
for
EPA
to
evaluate
Colitag
 
 
s
applicability
for
drinking
water.

The
Georgia
Department
of
Natural
Resources
specificity
study
was
completed
using
drinking
water
spiked
with
target
bacteria.
EPA
experts
evaluated
the
specificity
data
presented
for
Colitag
 
,
and
found
it
acceptable
to
support
the
use
of
Colitag
 
in
drinking
water
applications
because
the
Colitag
 
satisfactorily
detected
the
target
bacteria
and
did
so
in
a
manner
comparable
to
the
reference
methods.
EPA
experts
concluded
that
this
experiment
satisfactorily
determined
Colitag
 
 
s
specificity
for
drinking
water.
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
63
Colitag
 
should
not
be
approved
for
detecting
total
coliforms
and
E.
coli
in
source
water. 

56
I­
F.
022
07
 
5.
Data
is
insufficient
to
support
approval
of
Colitag
 
for
source
water
monitoring.
Details
concerning
how
the
1994
to
1995
study
was
performed
and
how
the
data
were
interpreted
was
not
found
in
the
docket
and
must
be
provided
for
public
comment. 
Approval
of
Colitag
 
is
only
being
sought
for
drinking
water,
not
source
water.
See
also
the
response
to
Comment
55
(
Docket
ID
I­
F.
004,
Part
06).

08
OTHER
COMMENTS
57
ID1.003
02
 
I/
we
have
some
information
about
Japanese
situations
per
docket
ID
OW­
2002­
0031
that
was
the
US
EPA s
request
for
additional
information.

JWWA(
Japan
Water
Worker s
Association)
has
finished
the
comparison
study
about
new
­
formation
mediums
(
for
the
detection
kit
of
total­
coliforms
and
E.
coli
in
drinking
water)

,2(
more)
years
before.
At
that
time,
MMO­
MUG
medium
(
Colilert)
and
LB­
BGLB
medium
were
used
for
the
standard
medium.

After
this
comparison
study,
they
published
a
new
edition
of
 
Standard
Test
Methods
for
Drinking
Water 
at
Oug.
23,2001.

And
they
approved
new
3­
mediums
on
this
book.

Also,
this
book
is
the
only
one
standard
procedure
that
was
published
in
Japan.

The
new
3
formulations
were
following.

1.
ONPG­
MUG
medium
with
IPTG
(
named
 
Colitag
 
 
)

2.
Xgal­
MUG
medium
(
named
 
Fluorocult
LMX­
Broth )

3.
Xgal­
MUG
medium
with
Pyruvic
Acid
(
named
 
EC­
Blue )

The
summary
of
the
comparison
study s
results
(
the
details
were
explained
on
this
book)
was
following.

1.
From
 
the
comparison
test
of
the
sensitivity
study
using
standard
strain ,
there
has
no
problems
(
about
the
sensitivity).
EPA
appreciates
the
comment,
however
the
data
presented
from
Colitag
 
 
s
comparability
study
,
and
included
in
the
public
record,
is
being
used
as
the
basis
for
EPA s
judgement
on
the
acceptability
of
Colitag
 
as
an
alternate
drinking
water
coliform
method.
None
of
the
information
presented
by
this
comment
suggest
that
Colitag
 
is
inappropriate
for
the
purposes
described
in
today s
rule;
rather,
they
point
to
the
apparently
successful
use
of
Colitag
 
in
Japan.
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
64
And
also,
has
no
problems
about
 
the
influence
of
the
coexistence
strains .

2.
From
 
the
comparison
test
of
the
sensitivity
study
suing
standard
strain
that
was
treated
by
Chlorine ,
Colitag
 
and
(
Xgal­
MUG
with
Pyruvic
Acid)>=
MMO­
MUG>
Xgal­
MUG
3.
From
 
the
comparison
test
of
the
sensitivity
study
using
source
water
for
drinking
water
(
15
surface
water,
4
lake
water
and
4
underground
water:
those
were
sampled
from
23
sections
(
the
whole
country
of
Japan))
treated
by
Chlorine ,

Colitag
 
(
cultivate:
36'
C,
24hr.)>

MMOMUG
cultivate:
36'
C,
24hr).>=(
Xgal­
MUG
with
Pyruvic
Acid:
cultivate:
36'
C,
24hr.)>=
Xgal­

MUG(
cultivate:
36'
C,
48hr.)>=
LB­
BGLB>
Xgal­

MUG(
cultivate:
36'
C,
24hr.)

4.
From
 
the
comparison
test
of
the
sensitivity
study
using
source
water
for
drinking
water
(
15
surface
water,
4
lake
water
and
4
underground
water:
those
were
sampled
from
23
sections
(
the
whole
country
of
Japan))
no­
treated
(
raw)
water ,
(
Xgal­

MUG
with
Pyruvic
Acid:
cultivate:
36'
C,
24hr.)>=
Colitag
 
(
cultivate:
36'
C,
24hr).>
Xga
l­
MUG(
cultivate:
36'
C,
48hr.)>=

MMOMUG
cultivate:
36'
C,
24hr).>
Xgal­

MUG(
cultivate:
36'
C,
24hr.)>=
LB­
BGLB
If
you
want
to
know
more
details,
please
check
this
book
on
p.

641­
626
and
guide
book
on
p.
841­
843.

After
the
publishing
of
this
new
edition
of
 
Standard
Test
Methods
for
Drinking­
Water 
at
Oug.
23,2001,
a
few
ministry
agreed
to
usage
of
those
new
mediums
for
the
quality
test
of
the
drinking
water
and
source
water
for
drinking.

Anyhow,
depending
of
this
time s
approval
study
and
the
publishing
of
this
new
edition,
testing
laboratory s
workers
can
extend
of
there s
choice
for
these
kinds
of
test.

Lastly,
Colitag
 
and
the
other
new(
2)
mediums
were
used
on
many
testing
laboratory
(
official
and
public
lab),
in
allover
Comment
No.
Docket
ID
Docket
Part
Comment
Response
Notes
65
Japan,
alaready.
And
extend
the
marker
share
instead
of
MMOMUG
medium
and
LB­
BGLB.

Because,
those
mediums
were
formed
with
the
well­
known
chemicals
those
were
confirmed
about
safety
and
usefulness.

And,
the
usefulness
about
there s
performance
was
recognized
by
JWWA.

[
P.
S.]

If
EPA
or
someone
has
some
safety
information
and
truecomponent
and/
or
MSDS
about
 
Solanium ,
please
let
me
know
about
this. 

58
ID2.001
04
 
An
additional
note,
a
colleague
attempted
to
purchase
more
Colitag
 
to
extend
our
study
but
was
told
that
it
would
not
be
available
until
after
the
deadline
for
submission
of
comments
to
EPA.
I
find
that
statement
worrying
and
wonder
about
the
rationale
for
it!! 
This
issue
is
neither
related
to
the
scope
of
the
March
2002
proposal
nor
the
December
2002
NODA.
This
comment
has
no
bearing
on
EPA s
decision
to
approve
the
Colitag
 
method.

59
ID2.002
02
 
In
an
evaluation
of
Colitag
 
vs.
Colilert,
using
mEndo
and
mTEC
as
control
methods,
I
found
Colitag
 
have
a
19%

failure
rate
in
recovery
and
identification
of
both
E.
coli
and
total
coliform.
I
have
attached
my
data
file
for
your
examination. 
The
data
in
the
public
record
is
based
on
a
comparability
study
plan
developed
based
on
the
ATP
protocol;

reviewed
and
approved
by
EPA;
and
executed
by
an
independent,
certified
laboratory.
While
EPA
appreciates
the
commenter s
insight
based
on
their
evaluation,
the
Agency
will
rely
on
the
results
of
the
more
complete,
aforementioned
comparability
study.

The
Agency
does
not
have
adequate
information
to
assess
the
quality
of
the
data
from
the
evaluation
the
commenter
cites,
nor
does
EPA
know
the
details
of
how
the
commenter s
evaluation
was
performed.
EPA
notes
that
the
method
comparability
study
data
did
not
show
evidence
of
the
suggested
failure
rate,
since
the
results
of
the
Colitag
 
comparability
study
were
not
statistically
different
from
those
of
the
reference
methods.
See
also
the
responses
to
Comment
40
(
Docket
ID
I­
D1.002,

Part
14)
and
Comment
41
(
Docket
ID
I­
F.
004,
Part
05)

concerning
specificity
data.
66
67
Notes
I­
F:
comments
from
the
March
2002
proposed
rule
I­
D:
comments
from
the
December
2002
NODA
Contents
01
RECOMMENDATIONS
REGARDING
EPA
APPROVAL
OF
Colitag
 
,
page
2
02
LENGTH
OF
COMMENT
PERIOD/
RESPONSIVENESS
TO
COMMENTS,
page
7
03
EPA'S
ALTERNATE
TEST
PROCEDURES
(
ATP)
PROGRAM,
page
9
03.01
Adherence
to
Specific
ATP
Guidelines,
page
9
03.01.01
Injury
level
guidelines(
3­
4
log
goal;
recovery
rates;
alleged
math
errors;
applicability
to
natural
samples),
page
15
03.01.02
Sample
source
guidelines
(
spiked
samples;

10
geographically
dispersed
sites),
page
24
03.01.03
Replicate
and
data
selection
guidelines
(
ability
to
detect
1­
10
TC
or
achieve
25%/
75%
split;
replicate
analysis
on
3
dilutions;
lag
time/
inferred
bacteria
densities),
page
25
03.01.04
Calculation
of
Log10/
Chi
Squared
Analysis,
page
34
03.02
General
Comments
on
Adherence
to
ATP
Guidelines
(
e.
g.,
claim
that
protocol
must
serve
as
benchmark),
page
43
04
DOCUMENTATION
OF
Colitag
 
DATA
(
e.
g.,
differing
false
positive/
negative
errors
cited;
use
of
a
poster
presentation;
documentation
of
inferences),
page
46
05
DOCUMENTATION
OF
Colitag
 
PROCEDURES
(
safety;
instrumentation/
equipment/
supplies;
quality
control
measures;
calibration;
incubation
temperatures
and
times;

shelf
life),
page
55
06
OTHER
COMMENTS
REGARDING
Colitag
 
PERFORMANCE
(
substrates
allegedly
susceptible
to
hydrolysis;
color
of
solution;
Aeromonas
interference),
page
59
07
SOURCE
WATER
DATA
(
alleged
lack
of
data
to
support
approval
for
source
water
applications),
page
60
08
OTHER
COMMENTS,
page
61