Document ID: EPA-HQ-OW-2002-0010-0093
Agency: epa
Document Type: Rule
Title: Guidelines Establishing Test Procedures for the Analysis of Pollutants; Analytical Methods for Biological Pollutants in Ambient Water; Final Rule.
Posted Date: 2003-07-21T04:00Z

Monday,

July
21,
2003
Part
III
Environmental
Protection
Agency
40
CFR
Part
136
Guidelines
Establishing
Test
Procedures
for
the
Analysis
of
Pollutants;
Analytical
Methods
for
Biological
Pollutants
in
Ambient
Water;
Final
Rule
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Federal
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/
Vol.
68,
No.
139
/
Monday,
July
21,
2003
/
Rules
and
Regulations
ENVIRONMENTAL
PROTECTION
AGENCY
40
CFR
Part
136
[
FRL
 
7529
 
7]

RIN
2040
 
AD71
Guidelines
Establishing
Test
Procedures
for
the
Analysis
of
Pollutants;
Analytical
Methods
for
Biological
Pollutants
in
Ambient
Water
AGENCY:
Environmental
Protection
Agency
(
EPA).
ACTION:
Final
rule.

SUMMARY:
By
today's
action,
EPA
approves
test
methods
for
the
analysis
of
Escherichia
coli
(
E.
coli),
enterococci,
Cryptosporidium
and
Giardia
in
fresh
ambient
water
matrices.
In
addition,
EPA
approves
test
methods
for
the
analysis
of
enterococci
in
marine
ambient
water
matrices.
The
test
methods
approved
in
today's
rule
have
been
published
by
the
following
organizations:
EPA,
American
Public
Health
Association,
American
Water
Works
Association,
Water
Environment
Federation,
Association
of
Official
Analytical
Chemists
International,
and
American
Society
for
Testing
and
Materials,
or
commercial
vendors.
EPA's
approval
of
these
methods
will
help
States,
Tribes,
communities,
and
environmental
laboratories
better
assess
public
health
risks
from
microbiological
pollutants.
DATES:
This
regulation
is
effective
August
20,
2003.
The
incorporation
by
reference
of
these
methods
is
approved
by
the
Director
of
the
Federal
Register
on
August
20,
2003.
For
judicial
review
purposes,
this
final
rule
is
promulgated
as
of
1
p.
m.
(
Eastern
time)
on
August
4,
2003
as
provided
at
40
CFR
23.2.

FOR
FURTHER
INFORMATION
CONTACT:
Robin
K.
Oshiro,
Engineering
and
Analysis
Division
(
4303T),
Office
of
Science
and
Technology,
Office
of
Water,
U.
S.
Environmental
Protection
Agency,
Ariel
Rios
Building,
1200
Pennsylvania
Avenue,
NW.,
Washington,
DC
20460,
or
call
(
202)
566
 
1075
or
E­
mail
at
oshiro.
robin@
epa.
gov.

SUPPLEMENTARY
INFORMATION:

A.
Potentially
Regulated
Entities
EPA
Regions,
as
well
as
States,
Tribes,
and
Territories
authorized
to
implement
the
National
Pollutant
Discharge
Elimination
System
(
NPDES)
program,
issue
permits
to
implement
the
technology­
based
and
water
qualitybased
requirements
of
the
Clean
Water
Act
(
CWA).
Forty
five
States
and
one
Territory
are
currently
authorized
to
issue
NPDES
permits.
EPA
retains
permit
issuance
authority
in
nonauthorized
jurisdictions.
NPDES
permitting
authorities
make
a
number
of
discretionary
choices
associated
with
permit
writing,
including
the
selection
of
pollutants
to
be
measured
and,
in
many
cases,
limited
in
permits.
If
EPA
has
``
approved''
(
i.
e.,
promulgated
through
rulemaking)
standardized
testing
procedures
for
a
given
pollutant,
the
NPDES
permitting
authority
must
specify
one
of
the
approved
testing
procedures
or
an
approved
alternate
test
procedure
for
the
measurements
required
under
the
permit.
Although
EPA
is
including
test
methods
for
four
biological
pollutants
in
40
CFR
136.3,
it
recommends
their
use
for
ambient
water
quality
monitoring
only.
EPA
is
not
approving
these
test
methods
for
effluent
matrices.
Therefore,
EPA
expects
entities
operating
under
an
NPDES
permit
would
be
affected
by
the
promulgation
of
these
ambient
methods
only
where
their
permit
specifies
ambient
monitoring
requirements
for
the
specified
parameters.
EPA
developed
and
recommended
ambient
recreational
water
quality
criteria
for
E.
coli
and
enterococci
bacteria
and
is
considering
criteria
for
Cryptosporidium
and
Giardia.
The
States,
Territories,
and
Tribes
may
adopt
these
criteria
into
their
water
quality
standards
and
may
issue
water
qualitybased
permits
that
require
monitoring
for
these
pollutants
in
ambient
waters.
If
the
NPDES
permitting
authority
requires
ambient
water
monitoring
in
the
permit
for
the
specified
parameters,
dischargers
could
be
affected
by
the
standardization
of
testing
procedures
in
this
rulemaking.
Generally,
the
permitting
authority
requires
the
use
of
methods
approved
at
40
CFR
part
136
for
compliance
with
such
monitoring
requirements.
If
no
approved
methods
are
available
at
40
CFR
part
136,
then
the
permitting
authority
has
discretion
to
specify
the
use
of
suitable
methods.
In
addition,
when
a
State,
Territory,
or
authorized
Tribe
provides
certification
of
Federal
licenses
under
the
CWA
section
401,
approved
testing
procedures
generally
must
be
used
where
applicable.
Categories
and
entities
that
may
be
regulated
include:

Category
Examples
of
potentially
regulated
entities
State,
Territorial
and
Indian
Tribal
Governments.
States,
Territories,
and
Tribes
authorized
to
administer
the
NPDES
permitting
program.

Municipalities
...................................
Publicly­
owned
treatment
works
with
ambient
monitoring
requirements
for
the
specified
parameters
in
their
NPDES
permits.
Industry
...........................................
Industrial
facilities
with
ambient
monitoring
requirements
for
the
specified
parameters
in
their
NPDES
permits.

This
table
is
not
intended
to
be
exhaustive,
but
rather
provides
a
guide
for
readers
regarding
entities
likely
to
be
regulated
by
this
action.
This
table
lists
the
types
of
entities
that
EPA
is
now
aware
could
potentially
be
regulated
by
this
action.
Other
types
of
entities
not
listed
in
the
table
could
also
be
regulated.
To
determine
whether
your
facility
or
organization
is
regulated
by
this
action,
you
should
carefully
examine
the
applicability
criteria
in
parts
122
and
136
of
title
40
of
the
Code
of
Federal
Regulations.
If
you
have
questions
regarding
the
applicability
of
this
action
to
a
particular
entity,
consult
the
person
listed
in
the
preceding
FOR
FURTHER
INFORMATION
CONTACT
section.

B.
How
Can
I
Get
Copies
of
This
Document
and
Other
Related
Information?

1.
Docket.
EPA
has
established
an
official
public
docket
for
this
action
under
Docket
ID
No.
OW
 
2002
 
0010.
The
official
public
docket
consists
of
the
documents
specifically
referenced
in
this
action,
any
public
comments
received,
and
other
information
related
to
this
action.
Although
a
part
of
the
official
docket,
the
public
docket
does
not
include
Confidential
Business
Information
(
CBI)
or
other
information
whose
disclosure
is
restricted
by
statute.
The
official
public
docket
is
the
collection
of
materials
that
is
available
for
public
viewing
at
the
Water
Docket
in
the
EPA
Docket
Center,
EPA
West,
Room
B102,
1301
Constitution
Avenue,
NW.,
Washington,
DC.
The
EPA
Docket
Center
Public
Reading
Room
is
open
from
8:
30
a.
m.
to
4:
30
p.
m.
Eastern
Time,
Monday
through
Friday,
excluding
legal
holidays.
The
telephone
number
for
the
Public
Reading
Room
is
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Register
/
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68,
No.
139
/
Monday,
July
21,
2003
/
Rules
and
Regulations
202
 
566
 
1744,
and
the
telephone
number
for
the
Water
Docket
is
202
 
566
 
2426.
2.
Electronic
Access.
You
may
access
this
Federal
Register
document
electronically
through
the
EPA
Internet
under
the
Federal
Register
listings
at
http://
www.
epa.
gov/
fedrgstr/.
An
electronic
version
of
the
public
docket
is
available
through
EPA's
electronic
public
docket
and
comment
system,
EPA
Dockets.
You
may
use
EPA
Dockets
at
http://
www.
epa.
gov/
edocket/
to
view
public
comments,
access
the
index
listing
of
the
contents
of
the
official
public
docket,
and
to
access
those
documents
in
the
public
docket
that
are
available
electronically.
Once
in
the
system,
select
``
search,''
then
key
in
the
appropriate
docket
identification
number.
Although
not
all
docket
materials
may
be
available
electronically,
you
may
still
access
any
of
the
publicly
available
docket
materials
through
the
docket
facility
identified
in
section
B.
1.
3.
Copies
of
Consensus
Standards.
Copies
of
the
consensus
standards
may
be
obtained
from
the
Docket
(
see
section
B.
1.).
Copies
of
the
consensus
standards
may
also
be
obtained
from
the
following
sources,
depending
on
the
standard.
Copies
of
final
methods
published
by
American
Society
for
Testing
and
Materials
(
ASTM)
are
available
for
a
nominal
cost
through
ASTM
International,
100
Barr
Harbor
Drive,
West
Conshohocken,
PA
19428
 
2959.
Copies
of
``
Standard
Methods''
are
available
for
a
nominal
cost
from
the
American
Public
Health
Association,
1015
Fifteenth
Street,
NW.,
Washington,
DC
20005.
Copies
of
Association
of
Official
Analytical
Chemists
International
(
AOAC)
methods
are
available
for
a
nominal
cost
from
the
Association
of
Official
Analytical
Chemists
International,
481
N.
Frederick
Ave.,
Suite
500,
Gaithersburg,
MD
28077.

I.
Statutory
Authority
Today's
rule
is
promulgated
pursuant
to
the
authority
of
sections
303(
c),
304(
a),
304(
h),
and
501(
a)
of
the
Clean
Water
Act
(
CWA
or
``
the
Act''),
33
U.
S.
C.
1314(
a),
1314(
h),
1361(
a).
Section
303(
c)
of
the
Act
establishes
the
basis
for
the
current
water
quality
standards
program.
This
section
requires
EPA
to
review
and
approve
or
disapprove
Stateadopted
water
quality
standards.
Section
304(
a)
of
the
Act
requires
the
EPA
Administrator
to
develop
and
publish
water
quality
criteria
associated
with
specific
ambient
water
uses.
When
these
criteria
are
adopted
as
State
water
quality
standards
under
section
303(
c),
they
become
the
enforceable
maximum
acceptable
levels
of
pollutants
in
ambient
waters.
Section
304(
h)
of
the
Act
requires
the
EPA
Administrator
to
``
promulgate
guidelines
establishing
test
procedures
for
the
analysis
of
pollutants
that
shall
include
the
factors
which
must
be
provided
in
any
certification
pursuant
to
section
401
of
this
Act
or
permit
applications
pursuant
to
section
402
of
this
Act.''
Section
501(
a)
of
the
Act
authorizes
the
Administrator
to
``
prescribe
such
regulations
as
are
necessary
to
carry
out
his
functions
under
this
Act.''
EPA
publishes
CWA
analytical
method
regulations
at
40
CFR
part
136.

II.
Background
A.
The
Role
of
Methods
for
Biological
Pollutants
To
fulfill
the
CWA's
mandate
to
maintain
``
fishable
and
swimmable''
waters,
EPA
develops
ambient
water
quality
criteria
based
on
a
scientific
assessment
of
the
relationship
between
pollutant
concentrations
and
environmental
and
human
health
effects.
Ambient
water
refers
to
any
fresh,
marine,
or
estuarine
surface
water
used
for
recreation,
propagation
of
fish,
shellfish,
or
wildlife,
agriculture,
industry,
navigation,
or
as
source
water
for
drinking
water
facilities.
Ambient
water
quality
criteria
become
enforceable
water
quality
standards
when
adopted
by
State,
Territorial,
Tribal,
and
local
governments
and
approved
by
EPA.
For
bacterial
pollution
in
ambient
water
designated
for
recreational
use,
EPA
has
developed
water
quality
criteria
for
E.
coli
in
freshwater
and
for
enterococci
in
both
freshwater
and
marine
waters
(
51
FR
8012,
March
7,
1986).
There
are
a
number
of
zoonotic
diseases
of
concern
to
humans
(
diseases
transferred
from
animals
to
humans)
if
ambient
waters
are
contaminated
with
fecal
material
from
non­
human
animal
species.
E.
coli
species
are
a
subset
of
the
coliform
bacteria
group
that
is
part
of
the
normal
intestinal
flora
of
humans
and
animals
and
are
direct
indicators
of
fecal
contamination
from
these
sources
in
water.
Enterococci,
which
include
Enterococcus
faecalis
and
Enterococcus
faecium,
are
enteric
bacteria
used
to
indicate
fecal
contamination
and
the
possible
presence
of
pathogens
in
water.
Based
on
previous
EPA
guidance,
total
and
fecal
coliform
bacteria
are
included
in
many
water
quality
standards
as
indicators
of
bacterial
contamination
(
EPA,
1976).
More
recent
epidemiological
studies
(
Cabelli
1983,
Dufour
1984)
described
in
Ambient
Water
Quality
Criteria
for
Bacteria
 
1986
(
EPA,
1986a),
indicate
that
E.
coli
and
enterococci
show
a
direct
correlation
with
swimming­
associated
gastrointestinal
illness
rates,
while
fecal
coliforms
do
not.
As
the
concentration
of
E.
coli
and/
or
enterococci
increase(
s),
the
illness
rates
also
increase.
Thus,
using
these
indicators
as
part
of
the
bacterial
water
quality
standards
will
enhance
the
protection
of
human
health
and
the
environment.
In
addition
to
bacterial
pollution,
EPA
is
concerned
about
waterborne
parasites
and
developed
test
methods
for
Cryptosporidium
and
Giardia
in
freshwater.
These
waterborne
parasites
have
been
found
to
be
the
causative
agent
of
human
gastroenteritis
in
some
contaminated
waters
and
are
responsible
for
cases
of
severe
and
widespread
human
illness
when
present
in
drinking
water
supplies
as
a
result
of
contamination
of
source
waters.
Because
one
of
the
designated
uses
of
some
ambient
waters
may
be
use
of
the
water
body
as
a
drinking
water
source,
EPA
may
develop
ambient
water
quality
criteria
for
Cryptosporidium
and
Giardia
in
the
future.
EPA
would
expect
to
use
the
test
methods
discussed
in
this
action
to
support
these
future
criteria.
By
doing
so,
EPA
desires
to
promote
consistency
in
the
methods
used
for
these
future
criteria
to
ensure
that
the
data
collected
are
of
good
quality
and
are
comparable
for
all
freshwater.
EPA
also
wishes
to
make
these
methods
available
for
use
by
the
States
for
general
risk
assessments.
By
today's
action,
EPA
is
promulgating
test
methods
for
E.
coli,
enterococci,
Cryptosporidium,
and
Giardia
for
use
in
freshwaters,
and
enterococci
for
use
in
marine
waters.
Promulgation
of
the
bacterial
methods
supports
the
use
of
E.
coli
and
enterococci
as
indicators
of
fecal
contamination
in
addition
to
fecal
coliform
indicators
in
State,
Territorial,
Tribal,
and
local
water
quality­
based
monitoring.
States
may
use
the
test
methods
for
Cryptosporidium
and
Giardia
for
different
monitoring
purposes,
such
as
evaluating
surface
water
occurrence
of
these
organisms
and
the
associated
watershed
vulnerability
for
waterbodies
designated
as
potential
drinking
water
sources.
This
rule
provides
uniform
methodology
to
assist
State,
Territorial,
Tribal,
and
local
implementation
of
water
quality
standards,
ambient
water
monitoring
programs,
and
public
notification
programs
to
reduce
public
health
risks
posed
by
biological
pollutants
in
ambient
water.
Today's
rule
supports
several
EPA
initiatives:
The
Beaches
Environmental
Assessment
Closure
and
Health
(
BEACH)
Program,
the
Beach
Action
Plan
(
EPA
 
600
 
R
 
98
 
079),
the
Beach
Watch
Program,
the
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139
/
Monday,
July
21,
2003
/
Rules
and
Regulations
Beaches
Environmental
Monitoring
for
Public
Access
and
Community
Tracking
(
EMPACT)
Program
(
EPA
905
 
R
 
98
 
002),
and
the
Water
Quality
Criteria
and
Standards
Plan
(
EPA
 
822
 
R
 
98
 
003).
Additionally,
this
rule
is
expected
to
satisfy
requests
from
governments,
regulated
entities,
and
environmental
laboratories
that
EPA
publish
analytical
test
procedures
that
were
evaluated
through
interlaboratory
validation
for
enumerating
E.
coli,
enterococci,
Cryptosporidium,
and
Giardia
in
ambient
waters.
As
previously
noted,
EPA
developed
water
quality
criteria
for
enterococci
in
both
freshwater
and
in
marine
waters.
Today's
action
approves
methods
for
measuring
enterococci
in
both
freshwater
and
marine
waters.
EPA
has
not
developed
marine
criteria
for
E.
coli,
Cryptosporidium,
and
Giardia
because
these
pollutants
do
not
generally
survive
in
marine
conditions.
Thus,
EPA
has
not
identified
any
programmatic
need
to
promulgate
methods
for
these
pollutants
in
marine
waters.
EPA
is
aware
of
the
importance
of
having
methods
for
measuring
these
pollutants
in
wastewater
effluent.
The
Agency
does
not
currently
have
validated
methods
for
use
in
this
matrix
and
thus
was
unable
to
propose
any
such
methods
with
the
methods
for
ambient
waters.
The
Agency
is
currently
in
the
process
of
trying
to
validate
E.
coli
and
enterococci
methods
for
use
with
wastewater
effluent
and
plans
to
propose
them
by
the
end
of
2004.

B.
Summary
of
Proposed
Rule
EPA
published
a
proposed
rule
in
the
Federal
Register
on
August
30,
2001
(
66
FR
45811)
to
amend
40
CFR
part
136,
``
Guidelines
Establishing
Test
Procedures
for
the
Analysis
of
Pollutants,''
by
approving
several
analytical
test
procedures
for
enumerating
the
bacteria
Escherichia
coli
(
E.
coli)
and
enterococci
and
the
protozoans
Cryptosporidium
and
Giardia
in
ambient
water.
The
proposal
described
a
suite
of
Most
Probable
Number
(
MPN)
(
i.
e.,
multiple­
tube,
multiple­
well)
and
membrane
filter
(
MF)
methods
for
enumerating
E.
coli
and
enterococci
bacteria
in
ambient
water,
and
improved
filtration/
immunomagnetic
separation/
fluorescent
antibody
methods
for
Cryptosporidium
and
Giardia
protozoans.
These
test
methods
were
proposed
for
use
by
States,
Territories,
and
Tribes,
for
use
in
water
quality
monitoring
programs.
A
summary
of
the
major
comments
to
the
proposal
is
presented
in
Section
V.
III.
Summary
of
Final
Rule
EPA
is
approving
the
use
of
test
methods
for
E.
coli,
enterococci,
Cryptosporidium,
and
Giardia
for
ambient
fresh
water
quality
monitoring.
In
addition,
EPA
is
approving
the
use
of
test
methods
for
enterococci
for
ambient
marine
water
quality
monitoring.
Although
EPA
believes
that
these
methods
are
appropriate
for
ambient
water
quality
monitoring,
the
Agency
has
not
determined
that
these
methods
are
acceptable
for
application
to
matrices
other
than
ambient
waters.
Today's
action
promulgates
the
test
methods
described
in
the
proposed
rule
(
66
FR
45811,
August
30,
2001)
for
the
analysis
of
E.
coli,
enterococci,
Cryptosporidium,
and
Giardia
in
ambient
water.
For
E.
coli,
approved
methods
include
most
probable
number
methods
(
LTB 
EC
 
MUG,
ONPG
 
MUG)
and
membrane
filtration
methods
(
mENDO 
NA
 
MUG,
LES
 
ENDO 
NA
 
MUG,
mFC 
NA
 
MUG,
mTEC
agar,
Modified
mTEC
agar,
MI
agar,
m­
ColiBlue
24
broth).
For
enterococci,
approved
methods
include
most
probable
number
methods
(
Azide­
Dextrose/
PSE/
BHI,
MUG)
and
membrane
filtration
methods
(
mE 
EIA
agar,
mEI
agar).
For
Cryptosporidium,
EPA
approves
Methods
1622
and
1623.
For
Giardia,
EPA
approves
Method
1623.
The
proposed
rule
indicated
that
EPA
intended
to
issue
guidance
on
the
assessment
of
method
comparability
in
conjunction
with
the
final
rule.
In
the
record
for
today's
rule,
EPA
is
making
available
the
latest
version
of
the
guidance
document,
EPA
Microbiological
Alternate
Test
Procedure
(
ATP)
Protocol
for
Drinking
Water,
Ambient
Water,
and
Wastewater
Monitoring
Methods,
Guidance
(
EPA
 
821
 
B
 
03
 
004).
The
guidance
is
a
result
of
the
Agency's
desire
to
develop
a
guidance
document
to
describe
the
process
for
seeking
EPA
approval
of
alternate
test
procedures
(
ATPs)
for
microbiological
methods
or
new
microbiological
methods
for
use
in
monitoring
drinking
water,
ambient
water,
and
wastewater.
Under
EPA's
ATP
program,
any
person
may
apply
for
approval
of
the
use
of
an
ATP
or
new
method
to
test
for
a
regulated
analyte.
EPA
anticipates
that
the
standardized
ATP
procedures
described
in
the
guidance
should
generally
expedite
the
approval
of
ATPs
and
encourage
the
development
of
innovative
methods
for
compliance
monitoring
under
the
National
Pollution
Discharge
Elimination
System
(
NPDES)
permit
program.
In
addition
to
the
ATP
process,
the
guidance
describes
the
process
for
conducting
side­
by­
side
method
comparisons
and
for
conducting
quality
control
(
QC)
acceptance
criteriabased
method
studies
for
EPAdesignated
reference
methods
with
QC
acceptance
criteria.
The
guidance
document
serves
as
a
supplement
to
the
ATP
program
requirements
specified
at
40
CFR
136.4,
136.5,
and
141.27.
The
guidance
document
may
be
revised
in
the
future
based
on
comments
received
from
persons
using
the
guidance,
as
appropriate.

IV.
Changes
From
the
Proposed
Rule
A.
Revision
of
Method
Titles
To
ensure
consistency
with
other
EPA
microbiological
methods,
EPA
revised
some
of
the
EPA
methods'
titles
and
added
some
method
numbers.
The
technical
content
of
these
methods
did
not
change
from
the
versions
of
the
methods
included
in
the
proposed
rule.
Specifically,
EPA
adopted
the
following
modified
titles:
 
Method
1103.1:
Escherichia
coli
(
E.
coli)
in
Water
by
Membrane
Filtration
using
membrane­
Thermotolerant
Escherichia
coli
Agar
(
mTEC)
 
Method
1106.1:
Enterococci
in
Water
by
Membrane
Filtration
using
membrane­
Enterococcus­
Esculin
Iron
Agar
(
mE­
EIA)
 
Method
1600:
Enterococci
in
Water
by
Membrane
Filtration
using
membrane­
Enterococcus
Iron
Agar
(
mEI)
 
Method
1603:
Escherichia
coli
(
E.
coli)
in
Water
by
Membrane
Filtration
using
Modified
Membrane­
Thermotolerant
Escherichia
coli
Agar
(
Modified
mTEC)
 
Method
1604:
Total
Coliforms
and
Escherichia
coli
(
E.
coli)
in
Water
by
Membrane
Filtration
using
a
Simultaneous
Detection
Technique
(
MI
Medium)

B.
Colisure
EPA
included
this
method
in
the
proposal
because
it
anticipated
that
new
validation
data
for
ambient
waters
would
be
provided
to
the
Agency
prior
to
this
final
rule.
EPA
requested
such
data
from
the
manufacturer,
but
the
manufacturer
declined
to
conduct
the
study.
Therefore
EPA
declines
to
approve
this
method
and
did
not
include
it
in
today's
final
rule.

C.
Table
II
Protozoan
Test
Holding
Time
The
proposal
incorrectly
indicated
that
the
maximum
sample
holding
time
for
the
protozoan
tests
(
Cryptosporidium
and
Giardia)
was
72
hours.
This
has
been
changed
to
the
correct
holding
time
of
96
hours,
as
indicated
in
the
Methods,
which
were
included
in
the
docket
for
the
proposal.
The
correct
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21,
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/
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and
Regulations
holding
time
of
96
hours
is
clearly
indicated
in
the
Methods
and
can
be
found
on
page
10,
section
8.2.1
of
the
April
2001
versions
of
Method
1622
and
Method
1623.
Although
footnote
17
of
the
proposal
inaccurately
stated
the
technique
for
calculating
holding
time,
the
underlying
methods
themselves
described
this
technique
correctly.
The
footnote
has
been
corrected
to
indicate
that
holding
time
is
properly
calculated
from
the
time
of
sample
collection
to
elution
for
samples
shipped
to
the
laboratory
in
bulk
and
calculated
from
the
time
of
sample
filtration
to
elution
for
samples
filtered
in
the
field.

V.
Response
to
Major
Comments
EPA
encouraged
public
participation
in
this
rulemaking
and
requested
comments
on
the
methods
proposed
for
E.
coli,
enterococci,
Cryptosporidium,
and
Giardia.
EPA
also
requested
any
data
that
would
support
comments
on
specific
test
methods.
Fourteen
stakeholders
provided
comments
addressing
over
25
issues.
These
stakeholders
included
four
laboratories,
seven
regulatory
authorities,
and
three
industries/
industry
groups.
The
following
sections
summarize
major
comments
received
on
the
proposed
rule
and
EPA's
response.
The
complete
Response
to
Comments
document
can
be
found
in
the
Docket
for
today's
final
rule.

A.
E.
coli
and
Enterococci
Methods
for
Wastewater
Analysis
Several
commenters
requested
that
the
methods
for
E.
coli
and
enterococci
be
approved
for
the
analysis
of
wastewater
samples.
Since
these
methods
were
not
validated
in
wastewater,
they
are
not
approved
for
use
in
that
matrix.
EPA
is
in
the
process
of
validating
methods
for
the
analysis
of
E.
coli
and
enterococci
in
wastewater
and
plans
to
propose
test
methods
for
these
bacterial
indicators
by
the
end
of
2004.

B.
Cryptosporidium
and
Giardia
Methods
for
Wastewater
and
Biosolids
Analysis
Several
comments
advocated
the
use
of
EPA
Method
1622
and
1623
for
the
analysis
of
wastewater
and
biosolids
samples;
other
comments
requested
that
EPA
modify
and
approve
the
methods
for
use
in
those
matrices.
EPA
has
not
validated
these
methods
for
those
uses.
Thus
this
final
rule
applies
only
to
ambient
water.
If
EPA
develops
water
quality
criteria
for
Cryptosporidium
and
Giardia
at
a
future
time,
EPA
may
validate
EPA
Methods
1622
and/
or
1623
for
use
in
the
NPDES
Program.
C.
Limitations
of
Determinative
Technique
of
Proposed
Cryptosporidium
and
Giardia
Methods
and
Potential
for
False
Positives
Several
comments
expressed
concern
regarding
the
subjectivity
and
limitations
of
the
immunofluorescence
assay
(
IFA)­
based
determination
procedure
in
EPA
Methods
1622
and
1623
and
the
related
potential
for
false
positives.
EPA
acknowledges
that
IFA
relies
on
analyst
training
and
experience
for
reliable
results.
However,
EPA
Methods
1622
and
1623
provide
the
analyst
with
three
microscopy
tools
to
aid
in
the
identification
of
potential
target
particulates
during
microscopic
examination.
The
methods
provide
detailed,
progressive
criteria
for
determining
whether
a
particulate
is
a
Cryptosporidium
oocyst
or
a
Giardia
cyst
based
on
the
use
of
these
tools
and
include
the
use
of
immunomagnetic
separation
(
IMS)
as
the
sample
cleanup
procedure
to
minimize
the
transfer
of
non­
target
particulates
to
the
slide.
Nonetheless,
the
inherent
technical
judgement
involved
in
the
determinative
step
in
EPA
Methods
1622
and
1623,
combined
in
some
cases
with
interfering
materials
and/
or
crossreactivity
of
the
antibody
stain,
may
still
lead
to
false
positives
or
false
negatives.
Although
other
determinative
techniques
that
are
currently
under
development
have
the
promise
of
providing
less­
subjective
assessments
of
the
presence
of
Cryptosporidium
oocysts
and
Giardia
cysts
in
a
sample,
these
techniques
are
not
yet
validated
and
are
therefore
not
yet
appropriate
for
EPA
approval
for
ambient
water
monitoring.
Extensive
details
on
the
performance
of
EPA
Methods
1622
and
1623,
including
inter­
and
intralaboratory
precision
and
recovery
of
the
methods
at
multiple
laboratories
and
on
a
variety
of
ambient
water
types
(
i.
e.,
validation),
are
provided
in
the
Results
of
the
Interlaboratory
Validation
Study
of
EPA
Method
1622
(
EPA
 
821
 
R
 
01
 
027),
the
Results
of
the
Interlaboratory
Validation
Study
of
EPA
Method
1623
(
EPA
 
821
 
R
 
01
 
028)
and
the
Implementation
and
Results
of
the
Information
Collection
Rule
Supplemental
Surveys
(
EPA
 
815
 
R
 
01
 
003),
which
were
included
in
the
docket
for
the
proposal.
Given
the
robustness
of
the
validation
procedure,
the
Agency
is
confident
that
although
the
IFA
technique
requires
specialized
training,
overall,
the
methods
will
provide
for
valid
Cryptosporidium
and
Giardia
precision
and
recovery
for
use
in
ambient
waters.
D.
Application
of
Performance­
Based
Measurement
System
(
PBMS)
Concept
to
EPA
Methods
1622
and
1623
Several
commenters
recommended
that
the
performance
of
alternate
antibody
reagents
be
evaluated
for
EPA
Methods
1622
and
1623
using
a
quantitative
PBMS
approach.
EPA
agrees
with
the
comments,
and
considers
the
PBMS
Tier
2
validation
approach
described
in
Methods
1622
and
1623,
Section
9,
to
be
appropriate
for
antibody
stains
and
IMS.
However,
EPA
does
not
believe
that
the
PBMS
Tier
2
validation
approach
is
adequate
to
assess
the
comparability
of
methods
with
different
determinative
techniques,
such
as
comparing
a
polymerase
chain
reaction
(
PCR)­
based
method
to
an
IFAbased
method.
Use
of
a
different
determinative
technique
is
generally
considered
to
be
a
different
method,
rather
than
a
modified
version
of
a
method
because
it
is
usually
very
difficult
to
compare
methods
that
use
different
determinative
techniques.
For
example,
the
filtration/
IMS/
IFA
technique
employed
in
Methods
1622
and
1623
differs
considerably
from
genetic
tests
because
the
former
measures
the
infective
form
of
Cryptosporidium
and
Giardia,
while
the
latter
measures
genetic
material
(
DNA
or
RNA).
Similarly,
the
membrane
filtration
method
for
bacteria
differs
from
an
MPN
method
for
bacteria
because
the
former
is
a
direct
quantitative
method,
whereas
the
latter
employs
a
qualitative
statistical
index
rather
than
an
actual
enumeration
of
the
number
of
organisms
present
in
the
sample.
An
appropriate
approach
for
these
comparisons
would
be
to
perform
side­
by­
side
tests.
This
approach
is
outlined
in
the
draft
guidance
document,
EPA
Microbiological
Alternate
Test
Procedure
(
ATP)
Protocol
for
Drinking
Water,
Ambient
Water,
and
Wastewater
Monitoring
Methods,
Guidance
(
EPA
 
821
 
B
 
03
 
004).

VI.
Statutory
and
Executive
Order
Reviews
A.
Executive
Order
12866:
Regulatory
Planning
and
Review
Under
Executive
Order
12866
(
58
FR
51735
(
October
4,
1993)),
the
Agency
must
determine
whether
the
regulatory
action
is
``
significant''
and
therefore
subject
to
Office
of
Management
and
Budget
(
OMB)
review
and
the
requirements
of
the
Executive
Order.
The
Executive
Order
defines
``
significant
regulatory
action''
as
one
that
is
likely
to
result
in
a
rule
that
may:
(
1)
Have
an
annual
effect
on
the
economy
of
$
100
million
or
more,
or
adversely
affect
in
a
material
way
the
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economy,
a
sector
of
the
economy,
productivity,
competition,
jobs,
the
environment,
public
health
or
safety,
or
State,
local,
or
tribal
governments
or
communities;
(
2)
Create
a
serious
inconsistency
or
otherwise
interfere
with
an
action
taken
or
planned
by
another
agency;
(
3)
Materially
alter
the
budgetary
impact
of
entitlements,
grants,
user
fees,
or
loan
programs
or
the
rights
and
obligations
of
recipients
thereof;
or
(
4)
Raise
novel
legal
or
policy
issues
arising
out
of
legal
mandates,
the
President's
priorities,
or
the
principles
set
forth
in
the
Executive
Order.
It
has
been
determined
that
this
rule
is
not
a
``
significant
regulatory
action''
under
the
terms
of
Executive
Order
12866
and
is
therefore
not
subject
to
OMB
review.

B.
Paperwork
Reduction
Act
This
action
does
not
impose
an
information
collection
burden
under
the
provisions
of
the
Paperwork
Reduction
Act,
44
U.
S.
C.
3501
et
seq.
This
action
promulgates
new
test
methods
for
E.
coli,
enterococci,
Cryptosporidium,
and
Giardia
for
use
in
ambient
water
monitoring
programs.
If
the
regulating
authority
replaces
the
indicator
organism
from
fecal
coliforms
to
one
of
the
bacterial
organisms
(
E.
coli
or
enterococci)
and
the
relevant
NPDES
permit
requires
ambient
water
monitoring,
then
the
permittee
would
be
required
to
use
one
of
these
approved
methods
for
these
organisms.
Currently,
permittees
generally
are
not
required
to
monitor
for
Cryptosporidium
or
Giardia
because
EPA
has
not
developed
water
quality
criteria
for
these
protozoans.
Burden
means
that
the
total
time,
effort,
or
financial
resources
expended
by
persons
to
generate,
maintain,
retain
or
disclose
or
provide
information
to
or
for
a
Federal
agency.
This
includes
the
time
needed
to
review
instructions;
develop,
acquire,
install
and
utilize
technology
and
systems
for
the
purpose
of
collecting,
validating,
and
verifying
information,
processing
and
maintaining
information,
and
disclosing
and
providing
information;
adjust
the
existing
ways
to
comply
with
any
previously
applicable
instructions
and
requirements;
train
personnel
to
be
able
to
respond
to
a
collection
of
information;
search
data
sources;
complete
and
review
the
collection
of
information;
and
transmit
or
otherwise
disclose
the
information.
An
agency
may
not
conduct
or
sponsor,
and
a
person
is
not
required
to
respond
to
a
collection
of
information
unless
it
displays
a
currently
valid
OMB
control
number.
The
OMB
control
numbers
for
EPA's
regulations
are
listed
in
40
CFR
part
9
and
48
CFR
chapter
15.

C.
Regulatory
Flexibility
Act
The
RFA
generally
requires
an
agency
to
prepare
a
regulatory
flexibility
analysis
of
any
rule
subject
to
notice
and
comment
rulemaking
requirements
under
the
Administrative
Procedure
Act
or
any
other
statute
unless
the
agency
certifies
that
the
rule
will
not
have
a
significant
economic
impact
on
a
substantial
number
of
small
entities.
Small
entities
include
small
businesses,
small
organizations,
and
small
governmental
jurisdictions.
For
purposes
of
assessing
the
impacts
of
today's
rule
on
small
entities,
small
entity
is
defined
as:
(
1)
A
small
business
as
defined
by
the
U.
S.
Small
Business
Administration
definitions
at
13
CFR
121.201;
(
2)
a
small
governmental
jurisdiction
that
is
a
government
of
a
city,
county,
town,
school
district
or
special
district
with
a
population
of
less
than
50,000;
and
(
3)
a
small
organization
that
is
any
not­
for­
profit
enterprise
which
is
independently
owned
and
operated
and
is
not
dominant
in
its
field.
After
considering
the
economic
impacts
of
today's
final
rule
on
small
entities,
I
certify
that
this
action
will
not
have
a
significant
economic
impact
on
a
substantial
number
of
small
entities.
This
regulation
promulgates
testing
procedures
for
the
measurement
of
E.
coli
and
enterococci
bacteria,
and
Cryptosporidium
and
Giardia
protozoa
in
ambient
water.
EPA
anticipates
that
the
methods
will
be
used
by
some
State
regulatory
authorities
for
evaluating
attainment
of
water
quality
standards
or
ambient
monitoring
requirements.
EPA
NPDES
regulations
do
not
require
monitoring
of
ambient
water
conditions
in
NPDES
permits.
In
a
few
instances,
ambient
water
monitoring
requirements
may
be
included
in
an
EPA­
issued
permit
where
site­
specific
circumstances
warrant.
EPA
regulations,
do
however,
require
NPDES
permittees
to
use
EPA­
approved
test
methods
for
all
monitoring
data
reported
to
the
Agency
(
40
CFR
122.21).
Consequently,
to
the
extent
that
an
NPDES
permit
requires
monitoring
and
reporting
of
ambient
water
for
E.
coli,
enterococci,
Cryptosporidium,
or
Giardia,
EPA
approval
of
these
test
methods
arguably
may
impose
costs
on
NPDES
permit
holders,
including
small
entities.
EPA
is
unaware,
however,
of
any
EPA­
issued
NPDES
permits
that
currently
require
monitoring
of
ambient
water
for
such
pollutants.
Hence,
EPA
does
not
expect
approval
of
these
methods
to
impose
any
additional
costs
as
a
result
of
their
applicability
to
EPA
issued
permits.
As
noted
above,
EPA's
NPDES
regulations
do
not
require
monitoring
of
ambient
water
conditions.
Consequently,
to
the
extent
that
a
State
requires
such
monitoring,
those
requirements
are
imposed
under
State,
rather
than
Federal,
authority.
Because
States
have
the
discretion
not
to
require
such
monitoring,
any
increased
costs
to
small
entities
arising
from
use
of
the
methods
approved
by
EPA
today
that
are
imposed
as
a
result
of
State
law
are
not
attributable
to
this
regulation.
Nonetheless,
EPA
evaluated
these
potential
costs
to
determine
whether
EPA
approval
of
the
methods
will
have
a
significant
impact
on
a
substantial
number
of
small
entities.
As
previously
noted,
States
may
require
ambient
water
monitoring
to
evaluate
attainment
of
water
quality
standards.
A
few
States
currently
require
NPDES
permit
holders
to
monitor
ambient
waters.
Thus,
some
NPDES
permittees
are
already
testing
ambient
water
for
these
parameters.
The
impact
of
using
EPA
approved
methods
for
such
dischargers
may
represent
little
or
no
increased
burden
since,
for
these
permittees,
the
replacement
of
fecal
coliforms
with
E.
coli
or
enterococci
would
simply
require
different
methods.
The
small
entities
that
might
be
affected
by
this
rule
include
small
governmental
jurisdictions
that
have
publicly­
owned
treatment
works
(
POTWs)
and
small
businesses
with
water
quality­
based
discharge
permits.
The
average
costs
for
total
and
fecal
coliform
were
comparable
to
those
for
E.
coli
and
enterococci
($
35)
because
the
analytical
procedures
generally
employ
similar
techniques,
media,
equipment,
and
require
comparable
laboratory
time
and
effort.
Some
States
are
already
using
the
methods
for
E.
coli
and
enterococci
in
State
ambient
water
quality
monitoring
programs.
This
rule
would
formalize
current
practice
in
those
States.
Furthermore,
EPA
expects
that
any
modest
potential
increase
in
costs
for
enterococci
analyses
will
be
reduced
once
the
promulgated
methods
are
broadly
implemented
by
environmental
laboratories
and
State
water
quality
monitoring
programs.
EPA
also
reviewed
the
costs
for
testing
for
Cryptosporidium
and
Giardia.
The
costs
for
Methods
1622
and
1623
analysis
of
Cryptosporidium
and
Giardia
range
from
$
400
to
$
500
for
each
sample
(
with
matrix
spikes
being
assessed
as
individual
samples)
for
each
method.
Because
of
the
relatively
high
costs,
EPA
does
not
anticipate
that
these
test
methods
will
be
used
for
daily
or
ongoing
monitoring,
but
they
may
be
used
for
program­
specific
occurrence
assessments.

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The
purpose
of
this
rule
is
only
to
make
these
methods
available
to
States,
Tribes,
and
municipalities
that
may
want
to
use
them
for
ambient
water
monitoring.
The
costs
associated
with
Cryptosporidium
and
Giardia
analysis
would
not
be
a
Federally­
mandated
cost,
but
rather
would
emanate
from
a
State's
adoption
of
ambient
monitoring
requirements
or
other
identified
needs
such
as
evaluation
of
Best
Management
Practices
(
BMPs)
or
downstream
impacts
of
wastewater
treatment
plant
effluents
or
other
identified
needs.
The
inclusion
of
these
test
methods
in
40
CFR
136.3
is
intended
to
make
these
test
methods
available
to
States
and
others
for
use
in
water
quality
monitoring
programs.
While
monitoring
for
these
protozoans
may
be
beneficial
since
these
organisms
may
be
ingested
from
recreational
and
source
waters,
EPA
is
not
establishing
any
compliance
monitoring
requirements
for
these
pollutants.
Therefore,
EPA
believes
that
this
rule
will
not
have
a
significant
economic
impact
on
a
substantial
number
of
small
entities.

D.
Unfunded
Mandates
Reform
Act
Title
II
of
the
Unfunded
Mandates
Reform
Act
of
1995
(
UMRA),
Public
Law
104
 
4,
establishes
requirements
for
Federal
agencies
to
assess
the
effects
of
their
regulatory
actions
on
State,
Tribal,
and
local
governments
and
the
private
sector.
Under
section
202
of
the
UMRA,
EPA
generally
must
prepare
a
written
statement,
including
a
cost­
benefit
analysis,
for
proposed
and
final
rules
with
``
Federal
mandates''
that
may
result
in
expenditures
to
State,
Tribal,
and
local
governments,
in
the
aggregate,
or
to
the
private
sector,
of
$
100
million
or
more
in
any
one
year.
Before
promulgating
an
EPA
rule
for
which
a
written
statement
is
needed,
section
205
of
the
UMRA
generally
requires
EPA
to
identify
and
consider
a
reasonable
number
of
regulatory
alternatives
and
adopt
the
least
costly,
most
costeffective
or
least
burdensome
alternative
that
achieves
the
objectives
of
the
rule.
The
provisions
of
section
205
do
not
apply
when
they
are
inconsistent
with
applicable
law.
Moreover,
section
205
allows
EPA
to
adopt
an
alternative
other
than
the
least
costly,
most
cost­
effective
or
least
burdensome
alternative
if
the
Administrator
publishes
with
the
final
rule
an
explanation
of
why
that
alternative
was
not
adopted.
Before
EPA
establishes
any
regulatory
requirements
that
may
significantly
or
uniquely
affect
small
governments,
including
Tribal
governments,
it
must
have
developed
under
section
203
of
the
UMRA
a
small
government
agency
plan.
The
plan
must
provide
for
the
notification
of
potentially
affected
small
governments,
enabling
officials
of
affected
small
governments
to
have
meaningful
and
timely
input
in
the
development
of
EPA
regulatory
proposals
with
significant
Federal
intergovernmental
mandates,
and
informing,
educating,
and
advising
small
governments
on
compliance
with
the
regulatory
requirements.
EPA
has
determined
that
this
rule
does
not
contain
a
Federal
mandate
for
State,
Tribal,
and
local
governments
or
the
private
sector
that
may
result
in
expenditures
of
$
100
million
or
more
for
State,
Tribal,
and
local
governments,
in
the
aggregate,
or
the
private
sector
in
any
one
year.
This
rule
makes
available
testing
procedures
for
E.
coli,
enterococci,
Cryptosporidium,
and
Giardia
that
may
be
used
by
a
State,
Territorial,
Tribal
or
local
authority
for
compliance
with
water
quality
standards
or
ambient
monitoring
requirements
when
testing
is
otherwise
required
by
these
regulatory
authorities.
Thus,
today's
rule
is
not
subject
to
the
requirements
of
sections
202
and
205
of
the
UMRA.
EPA
has
also
determined
that
this
rule
contains
no
regulatory
requirements
that
might
significantly
or
uniquely
affect
small
governments.
As
discussed
above,
under
the
Regulatory
Flexibility
Act,
the
economic
impact
on
small
entities
is
anticipated
to
be
small.
It
would
not
significantly
affect
them
because
any
incremental
costs
incurred
are
small,
and
it
would
not
uniquely
affect
them
because
it
would
affect
entities
of
all
sizes
depending
upon
whether
testing
for
these
bacteria
or
protozoa
is
otherwise
required
by
a
regulatory
authority.
Further,
monitoring
for
small
entities
is
generally
expected
to
be
less
frequent
than
monitoring
for
larger
entities.
Thus,
today's
rule
also
is
not
subject
to
the
requirements
of
section
203
of
the
UMRA.

E.
Executive
Order
13132:
Federalism
Executive
Order
13132,
entitled
``
Federalism''
(
64
FR
43255,
August
10,
1999),
requires
EPA
to
develop
an
accountable
process
to
ensure
``
meaningful
and
timely
input
by
State
and
local
officials
in
the
development
of
regulatory
policies
that
have
federalism
implications.''
``
Policies
that
have
federalism
implications''
is
defined
in
the
Executive
Order
to
include
regulations
that
have
``
substantial
direct
effects
on
the
States,
on
the
relationship
between
the
national
government
and
the
States,
or
on
the
distribution
of
power
and
responsibilities
among
the
various
levels
of
government.''
This
final
rule
does
not
have
federalism
implications.
It
will
not
have
substantial
direct
effects
on
the
States,
on
the
relationship
between
the
national
government
and
the
States,
or
on
the
distribution
of
power
and
responsibilities
among
the
various
levels
of
government,
as
specified
in
Executive
Order
13132.
Today's
rule
promulgates
new
analytical
methods
for
conducting
analysis
of
ambient
water
for
enumeration
of
E.
coli,
enterococci,
Cryptosporidium,
or
Giardia.
EPA
does
not,
however,
require
use
of
these
methods
under
this
rule.
Thus,
Executive
Order
13132
does
not
apply
to
this
rule.
Although
Executive
Order
13132
does
not
apply
to
this
rule,
EPA
did
consult
with
representatives
of
State
and
local
governments
in
developing
the
proposed
regulation.
In
fact,
it
was
State
representatives
who
requested
that
EPA
include
test
methods
for
these
biological
pollutants
in
40
CFR
136.3
because
they
want
to
use
EPA
approved
test
methods
for
ambient
water
monitoring.
EPA
included
a
number
of
test
methods
currently
being
used
by
States
for
these
pollutants
in
today's
rulemaking.
In
the
spirit
of
Executive
Order
13132,
and
consistent
with
EPA
policy
to
promote
communications
between
EPA
and
State
and
local
governments,
EPA
specifically
solicited
comment
on
the
proposed
rule
from
State
and
local
officials.
No
significant
concerns
were
raised
by
commenters
about
these
methods.

F.
Executive
Order
13175:
Consultation
and
Coordination
With
Indian
Tribal
Governments
Executive
Order
13175,
entitled
``
Consultation
and
Coordination
with
Indian
Tribal
Governments''
(
65
FR
67249,
November
9,
2000),
requires
EPA
to
develop
an
accountable
process
to
ensure
``
meaningful
and
timely
input
by
tribal
officials
in
the
development
of
regulatory
policies
that
have
tribal
implications.''
``
Policies
that
have
tribal
implications''
is
defined
in
the
Executive
Order
to
include
regulations
that
have
``
substantial
direct
effects
on
one
or
more
Indian
tribes,
on
the
relationship
between
the
Federal
government
and
the
Indian
tribes,
or
on
the
distribution
of
power
and
responsibilities
between
the
Federal
government
and
the
Indian
tribes.''
This
final
rule
does
not
have
tribal
implications.
It
will
not
have
substantial
direct
effects
on
tribal
governments,
on
the
relationship
between
the
Federal
government
and
Indian
tribes,
or
on
the
distribution
of
power
and
responsibilities
between
the
Federal
government
and
Indian
tribes,
as
specified
in
Executive
Order
13175.
Today's
rule
promulgates
new
analytical
methods
for
conducting
analysis
of
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2003
/
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and
Regulations
ambient
water
for
enumeration
of
E.
coli,
enterococci,
Cryptosporidium,
or
Giardia.
EPA
does
not,
however,
require
use
of
these
methods
under
this
rule.
Thus,
Executive
Order
13175
does
not
apply
to
this
rule.
Moreover,
in
the
spirit
of
Executive
Order
13175,
and
consistent
with
EPA
policy
to
promote
communications
between
EPA
and
tribal
governments,
EPA
specifically
solicited
comment
on
the
proposed
rule
from
tribal
officials.
EPA
did
not
receive
comments
from
Tribal
officials.
Thus,
Executive
Order
13175
does
not
apply
to
this
rule.

G.
Executive
Order
13045:
Protection
of
Children
From
Environmental
Health
Risks
and
Safety
Risks
Executive
Order
13045
(
62
FR
19885,
April
23,
1997)
applies
to
any
rule
that:
(
1)
Is
determined
to
be
``
economically
significant''
as
defined
under
Executive
Order
12866,
and
(
2)
concerns
an
environmental
health
or
safety
risk
that
EPA
has
reason
to
believe
may
have
a
disproportionate
effect
on
children.
If
the
regulatory
action
meets
both
criteria,
the
Agency
must
evaluate
the
environmental
health
or
safety
effects
of
the
planned
rule
on
children,
and
explain
why
the
planned
regulation
is
preferable
to
other
potentially
effective
and
reasonably
feasible
alternatives
considered
by
the
Agency.
This
rule
is
not
subject
to
the
Executive
Order
because
it
is
not
``
economically
significant''
as
defined
in
Executive
Order
12866.
Further,
it
does
not
concern
an
environmental
health
or
safety
risk
that
EPA
has
reason
to
believe
may
have
a
disproportionate
effect
on
children.

H.
Executive
Order
13211:
Actions
That
Significantly
Affect
Energy
Supply,
Distribution,
or
Use
This
rule
is
not
subject
to
Executive
Order
13211,
``
Actions
Concerning
Regulations
That
Significantly
Affect
Energy
Supply,
Distribution,
or
Use''
(
66
FR
28355
(
May
22,
2001))
because
it
is
not
a
significant
regulatory
action
under
Executive
Order
12866.

I.
National
Technology
Transfer
and
Advancement
Act
As
noted
in
the
proposed
rule,
section
12(
d)
of
the
National
Technology
Transfer
and
Advancement
Act
of
1995,
(``
NTTAA''),
Public
Law
104
 
113,
section
12(
d)
(
15
U.
S.
C.
272
note),
directs
EPA
to
use
voluntary
consensus
standards
in
its
regulatory
activities
unless
to
do
so
would
be
inconsistent
with
applicable
law
or
otherwise
impractical.
Voluntary
consensus
standards
are
technical
standards
(
e.
g.,
material
specifications,
test
methods,
sampling
procedures,
and
business
practices)
that
are
developed
or
adopted
by
voluntary
consensus
standards
bodies.
The
NTTAA
directs
EPA
to
provide
Congress,
through
the
Office
of
Management
and
Budget
(
OMB),
explanations
when
the
Agency
decides
not
to
use
available
and
applicable
voluntary
consensus
standards.
This
rulemaking
involves
technical
standards.
Therefore,
the
Agency
conducted
a
search
to
identify
potentially
applicable
voluntary
consensus
standards.
EPA's
search
of
the
technical
literature
revealed
several
consensus
methods
appropriate
for
enumerating
E.
coli
and
enterococci
in
ambient
waters.
Accordingly,
methods
for
E.
coli
and
enterococci
published
by
Standard
Methods
for
the
Examination
of
Water
and
Wastewater,
ASTM,
and
AOAC­
International
are
included
for
promulgation
and
are
listed
in
Table
IA
at
the
end
of
this
document
(
see
footnotes
4,
10,
and
11,
respectively,
for
the
complete
citations).
No
voluntary
consensus
standards
were
found
for
Cryptosporidium
or
Giardia.

J.
Congressional
Review
Act
The
Congressional
Review
Act,
5
U.
S.
C.
801
et
seq.,
as
added
by
the
Small
Business
Regulatory
Enforcement
Fairness
Act
of
1996
(
SBREFA),
generally
provides
that
before
a
rule
may
take
effect,
the
agency
promulgating
the
rule
must
submit
a
rule
report,
which
includes
a
copy
of
the
rule,
to
each
House
of
the
Congress
and
to
the
Comptroller
General
of
the
United
States.
EPA
will
submit
a
report
containing
this
rule
and
other
required
information
to
the
U.
S.
Senate,
the
U.
S.
House
of
Representatives,
and
the
Comptroller
General
of
the
United
States
prior
to
publication
of
the
rule
in
the
Federal
Register.
A
major
rule
cannot
take
effect
until
60
days
after
it
is
published
in
the
Federal
Register.
This
action
is
not
a
``
major
rule''
as
defined
by
5
U.
S.
C.
804(
2).
This
rule
will
be
effective
on
August
20,
2003.

List
of
Subjects
in
40
CFR
Part
136
Environmental
protection,
Incorporation
by
reference,
Reporting
and
recordkeeping
requirements,
Water
pollution
control.

Dated:
July
11,
2003.
Linda
J.
Fisher,
Acting
Administrator.


For
the
reasons
set
out
in
the
preamble,
title
40,
chapter
I
of
the
Code
of
Federal
Regulations,
is
amended
as
follows:

PART
136
 
GUIDELINES
ESTABLISHING
TEST
PROCEDURES
FOR
THE
ANALYSIS
OF
POLLUTANTS

1.
The
authority
citation
for
part
136
continues
to
read
as
follows:

Authority:
Secs.
301,
304(
h),
307,
and
501(
a),
Pub.
L.
95
 
217,
91
Stat.
1566,
et
seq.
(
33
U.
S.
C.
1251,
et
seq.)
(
The
Federal
Water
Pollution
Control
Act
Amendments
of
1972
as
amended
by
the
Clean
Water
Act
of
1977.)


2.
Section
136.3
is
amended:


a.
In
paragraph
(
a)
by
revising
Table
IA.


b.
In
paragraph
(
b)
by
revising
references
(
10),
(
34),
(
38)
and
(
39)
and
adding
references
(
52)
through
(
62).


c.
In
Table
II
to
paragraph
(
e)
by
revising
entries
to
the
Section
labeled
``
Table
IA
 
Bacteria
Tests,''
to
read
as
follows:
*
*
*
*
*
(
a)
*
*
*

TABLE
IA.
 
LIST
OF
APPROVED
BIOLOGICAL
METHODS
Parameter
and
units
Method
1
EPA
Standard
methods
18th,
19th,
20th
Ed.
ASTM
AOAC
USGS
Other
Bacteria:
1.
Coliform
(
fecal),
number
per
100
mL.
Most
Probable
Number
(
MPN),
5
tube
3
dilution
or
p.
132
3
9221C
E
4
Membrane
filter
(
MF)
2,
single
step.
p.
124
3
9222D4
B
 
0050
 
85
5
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/
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21,
2003
/
Rules
and
Regulations
TABLE
IA.
 
LIST
OF
APPROVED
BIOLOGICAL
METHODS
 
Continued
Parameter
and
units
Method
1
EPA
Standard
methods
18th,
19th,
20th
Ed.
ASTM
AOAC
USGS
Other
2.
Coliform
(
fecal)
in
presence
of
chlorine,
number
per
100
mL.
MPN,
5
tube,
3
dilution
or
p.
132
3
9221C
E4
MF,
single
step
6
..
p.
124
3
9222D4
3.
Coliform
(
total),
number
per
100
mL.
MPN,
5
tube,
3
dilution
or
p.
114
3
9221B
4
MF2,
single
step
or
two
step.
p.
108
3
9222B4
....................
....................
B
 
0025
 
85
5
4.
Coliform
(
total),
in
presence
of
chlorine,
number
per
100
mL.
MPN,
5
tube,
3
dilution
or
p.
114
3
9221B4
MF2
with
enrichment
p.
111
3
9222(
B+
B.
5c)
4
5.
E.
coli,
number
per
100
mL
28.
MPN7,9,15,
multiple
tube,.
9221B.
1/
9221F
4,12,14
multiple
tube/
multiple
well,
9223B
4,13
....................
991.15
11
....................
Colilert
 
13,17
Colilert­
18
 
13,16,17
MF2,6,7,8,9
two
step,
or
9222B/
9222G
4,19
1103.1
20
9213D4
D5392
 
93
10
single
step
...........
1603
21
1604
22
mColiBue
24
18
6.
Fecal
streptococci
number
per
100
mL.
MPN,
5
tube,
3
dilution
p.
139
3
9230B
4
MF2,
or
................
p.
136
3
........................................
....................
B
 
0055
 
85
5
Plate
count
..........
p.
143
4
7.
Enterococci,
number
per
100
mL.
MPN7,
9
multiple
tube.
................
9230B
4
multiple
tube/
multiple
well.
................
........................................
D6503
 
99
10
....................
....................
Enterolert
 
13,23
MF2,6,7,8,9
two
step.
1106.1
24
9230C4
D5259
 
92
10
single
step,
or
......
1600
25
Plate
count
..........
p.
143
3
Protozoa:
8.

Cryptosporidium
28.
Filtration/
IMS/
FA
..
1622
26
1623
27
9.
Giardia
28
..
Filtration/
IMS/
FA
..
1623
27
Aquatic
Toxicity:
10.
Toxicity,
acute,
fresh
water
organisms
LC50,
percent
effluent.
Ceriodaphnia
dubia
acute.
2002.0
29
Daphnia
puplex
and
Daphnia
magna
acute.
2021.0
29
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139
/
Monday,
July
21,
2003
/
Rules
and
Regulations
TABLE
IA.
 
LIST
OF
APPROVED
BIOLOGICAL
METHODS
 
Continued
Parameter
and
units
Method
1
EPA
Standard
methods
18th,
19th,
20th
Ed.
ASTM
AOAC
USGS
Other
Fathead
Minnow,
Pimephales
promelas,
and
Bannerfin
shiner
Cyprinella
leedsi,
acute.
2000.0
29
Rainbow
Trout,
Oncorhynchus
mykiss,
and
brook
trout,
Salvelinus
fontinalis,
acute.
2019.0
29
11.
Toxicity,
acute,
estuarine
and
marine
organisms
of
the
Atlantic
Ocean
and
Gulf
of
Mexico,
LC50,
percent
effluent.
Mysid,
Mysidopsis
bahia,
acute.
2007.0
29
Sheepshead
Minnow
Cyprinodon
variegatus,
acute.
2004.0
29
Silverside,
Menidia
beryllina,
Menidia
menidia,
and
Menidia
peninsulae,
acute.
2006.0
29
12.
Toxicity,
chronic,
fresh
water
organisms,
NOEC
or
IC25,
percent
effluent.
Fathead
minnow,
Pimephales
promelas,
larval
survival
and
growth.
1000.0
30
Fathead
minnow,
Pimephales
promelas,
embryo
larval
survival
and
teratogenicity.
1001.0
30
Daphnia,
Ceriodaphnia
dubia,
survival
and
reproduction
1002.0
30
Green
alga,
Selenastrum
capricornutum,
growth.
1003.0
30
13.
Toxicity,
chronic,
estuarine
and
marine
organisms
of
the
Atlantic
Ocean
and
Gulf
of
Mexico,
NOEC
or
IC25,
percent
effluent.
Sheepshead
minnow
Cyprinodon
variegatus,
larval
survival
and
growth.
1004.0
31
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139
/
Monday,
July
21,
2003
/
Rules
and
Regulations
TABLE
IA.
 
LIST
OF
APPROVED
BIOLOGICAL
METHODS
 
Continued
Parameter
and
units
Method
1
EPA
Standard
methods
18th,
19th,
20th
Ed.
ASTM
AOAC
USGS
Other
Sheepshead
minnow
Cyprinodon
variegatus,
embryo
larval
survival
and
teratogenicity.
1005.0
31
Inland
silverside,
Menidia
beryllina,
larval
survival
and
growth.
1006.0
31
Mysid,
Mysidopsis
bahia,
survival,
growth,
and
fecundity
1007.0
31
Sea
urchin,
Arbacia
punctulata,
fertilization
1008.0
31
Notes
to
Table
IA:
1
The
method
must
be
specified
when
results
are
reported.
2
A
0.45
???
m
membrane
filter
(
MF)
or
other
pore
size
certified
by
the
manufacturer
to
fully
retain
organisms
to
be
cultivated
and
to
be
free
of
extractables
which
could
interfere
with
their
growth.
3
USEPA.
1978.
Microbiological
Methods
for
Monitoring
the
Environment,
Water,
and
Wastes.
Environmental
Monitoring
and
Support
Laboratory
U.
S.
Environmental
Protection
Agency,
Cincinnati,
Ohio.
EPA/
600/
8
 
78/
017.
4
APHA.
1998,
1995,
1992.
Standard
Methods
for
the
Examination
of
Water
and
Wastewater.
American
Public
Health
Association.
20th,
19th,
and
18th
Editions.
Amer.
Publ.
Hlth.
Assoc.,
Washington,
D.
C.
5
USGS.
1989.
U.
S.
Geological
Survey
Techniques
of
Water­
Resource
Investigations,
Book
5,
Laboratory
Analysis,
Chapter
A4,
Methods
for
Collection
and
Analysis
of
Aquatic
Biological
and
Microbiological
Samples,
U.
S.
Geological
Survey,
U.
S.
Department
of
Interior,
Reston,
Virginia.
6
Because
the
MF
technique
usually
yields
low
and
variable
recovery
from
chlorinated
wastewaters,
the
Most
Probable
Number
method
will
be
required
to
resolve
any
controversies.
7
Tests
must
be
conducted
to
provide
organism
enumeration
(
density).
Select
the
appropriate
configuration
of
tubes/
filtrations
and
dilutions/
volumes
to
account
for
the
quality,
character,
consistency,
and
anticipated
organism
density
of
the
water
sample.
8
When
the
MF
method
has
not
been
used
previously
to
test
ambient
waters
with
high
turbidity,
large
number
of
noncoliform
bacteria,
or
samples
that
may
contain
organisms
stressed
by
chlorine,
a
parallel
test
should
be
conducted
with
a
multiple­
tube
technique
to
demonstrate
applicability
and
comparability
of
results.
9
To
assess
the
comparability
of
results
obtained
with
individual
methods,
it
is
suggested
that
side­
by­
side
tests
be
conducted
across
seasons
of
the
year
with
the
water
samples
routinely
tested
in
accordance
with
the
most
current
Standard
Methods
for
the
Examination
of
Water
and
Wastewater
or
EPA
alternate
test
procedure
(
ATP)
guidelines.
10
ASTM.
2000,
1999,
1996.
Annual
Book
of
ASTM
Standards
 
Water
and
Environmental
Technology.
Section
11.02.
American
Society
for
Testing
and
Materials.
100
Barr
Harbor
Drive,
West
Conshohocken,
PA
19428.
11
AOAC.
1995.
Official
Methods
of
Analysis
of
AOAC
International,
16th
Edition,
Volume
I,
Chapter
17.
Association
of
Official
Analytical
Chemists
International.
481
North
Frederick
Avenue,
Suite
500,
Gaithersburg,
Maryland
20877
 
2417.
12
The
multiple­
tube
fermentation
test
is
used
in
9221B.
1.
Lactose
broth
may
be
used
in
lieu
of
lauryl
tryptose
broth
(
LTB),
if
at
least
25
parallel
tests
are
conducted
between
this
broth
and
LTB
using
the
water
samples
normally
tested,
and
this
comparison
demonstrates
that
the
false­
positive
rate
and
false­
negative
rate
for
total
coliform
using
lactose
broth
is
less
than
10
percent.
No
requirement
exists
to
run
the
completed
phase
on
10
percent
of
all
total
coliform­
positive
tubes
on
a
seasonal
basis.
13
These
tests
are
collectively
known
as
defined
enzyme
substrate
tests,
where,
for
example,
a
substrate
is
used
to
detect
the
enzyme
bglucuronidase
produced
by
E.
coli.
14
After
prior
enrichment
in
a
presumptive
medium
for
total
coliform
using
9221B.
1,
all
presumptive
tubes
or
bottles
showing
any
amount
of
gas,
growth
or
acidity
within
48
h
±
3
h
of
incubation
shall
be
submitted
to
9221F.
Commercially
available
EC
 
MUG
media
or
EC
media
supplemented
in
the
laboratory
with
50
µ
g/
mL
of
MUG
may
be
used.
15
Samples
shall
be
enumerated
by
the
multiple­
tube
or
multiple­
well
procedure.
Using
multiple­
tube
procedures,
employ
an
appropriate
tube
and
dilution
configuration
of
the
sample
as
needed
and
report
the
Most
Probable
Number
(
MPN).
Samples
tested
with
Colilert
 
may
be
enumerated
with
the
multiple­
well
procedures,
Quanti­
Tray
 
or
Quanti­
Tray
 
2000,
and
the
MPN
calculated
from
the
table
provided
by
the
manufacturer
16
Colilert­
18
 
is
an
optimized
formulation
of
the
Colilert
 
for
the
determination
of
total
coliforms
and
E.
coli
that
provides
results
within
18
h
of
incubation
at
35
°
C
rather
than
the
24
h
required
for
the
Colilert
 
test
and
is
recommended
for
marine
water
samples.
17
Descriptions
of
the
Colilert
 
,
Colilert­
18
 
,
Quanti­
Tray
 
,
and
Quanti­
Tray
 
/
2000
may
be
obtained
from
IDEXX
Laboratories,
Inc.,
One
IDEXX
Drive,
Westbrook,
Maine
04092.
18
A
description
of
the
mColiBlue24''
test,
Total
Coliforms
and
E.
coli,
is
available
from
Hach
Company,
100
Dayton
Ave.,
Ames,
IA
50010.
19
Subject
total
coliform
positive
samples
determined
by
9222B
or
other
membrane
filter
procedure
to
9222G
using
NA
 
MUG
media.
20
USEPA.
2002.
Method
1103.1:
Escherichia
coli
(
E.
coli)
In
Water
By
Membrane
Filtration
Using
membrane­
Thermotolerant
Escherichia
coli
Agar
(
mTEC).
U.
S.
Environmental
Protection
Agency,
Office
of
Water,
Washington
D.
C.
EPA
 
821
 
R
 
02
 
020.
21
USEPA.
2002.
Method
1603:
Escherichia
coli
(
E.
coli)
In
Water
By
Membrane
Filtration
Using
Modified
membrane­
Thermotolerant
Escherichia
coli
Agar
(
modified
mTEC).
U.
S.
Environmental
Protection
Agency,
Office
of
Water,
Washington
D.
C.
EPA
 
821
 
R
 
02
 
023.
22
Preparation
and
use
of
MI
agar
with
a
standard
membrane
filter
procedure
is
set
forth
in
the
article,
Brenner
et
al.
1993.
``
New
Medium
for
the
Simultaneous
Detection
of
Total
Coliform
and
Escherichia
coli
in
Water.''
Appl.
Environ.
Microbiol.
59:
3534
 
3544
and
in
USEPA.
2002.
Method
1604:
Total
Coliforms
and
Escherichia
coli
(
E.
coli)
in
Water
by
Membrane
Filtration
by
Using
a
Simultaneous
Detection
Technique
(
MI
Medium
U.
S.
Environmental
Protection
Agency,
Office
of
Water,
Washington
DC.
EPA
821
 
R
 
02
 
024.
23
A
description
of
the
Enterolert
 
test
may
be
obtained
from
IDEXX
Laboratories,
Inc.,
One
IDEXX
Drive,
Westbrook,
Maine
04092.
24
USEPA.
2002.
Method
1106.1:
Enterococci
In
Water
By
Membrane
Filtration
Using
membrane­
Enterococcus­
Esculin
Iron
Agar
(
mE­
EIA).
U.
S.
Environmental
Protection
Agency,
Office
of
Water,
Washington
DC.
EPA
 
821
 
R
 
02
 
021.
25
USEPA.
2002.
Method
1600:
Enterococci
in
Water
by
Membrane
Filtration
Using
membrane­
Enterococcus
Indoxyl­
b­
D­
Glucoside
Agar
(
mEI).
U.
S.
Environmental
Protection
Agency,
Office
of
Water,
Washington,
DC.
EPA
 
821
 
R
 
02
 
022.

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Federal
Register
/
Vol.
68,
No.
139
/
Monday,
July
21,
2003
/
Rules
and
Regulations
26
Method
1622
uses
filtration,
concentration,
immunomagnetic
separation
of
oocysts
from
captured
material,
immunofluorescence
assay
to
determine
concentrations,
and
confirmation
through
vital
dye
staining
and
differential
interference
contrast
microscopy
for
the
detection
of
Cryptosporidium.
USEPA.
2001.
Method
1622:
Cryptosporidium
in
Water
by
Filtration/
IMS/
FA.
U.
S.
Environmental
Protection
Agency,
Office
of
Water,
Washington
DC.
EPA
 
821
 
R
 
01
 
026.
27
Method
1623
uses
filtration,
concentration,
immunomagnetic
separation
of
oocysts
and
cysts
from
captured
material,
immunofluorescence
assay
to
determine
concentrations,
and
confirmation
through
vital
dye
staining
and
differential
interference
contrast
microscopy
for
the
simultaneous
detection
of
Cryptosporidium
and
Giardia
oocysts
and
cysts.
USEPA.
2001.
Method
1623.
Cryptosporidium
and
Giardia
in
Water
by
Filtration
IMS/
FA.
U.
S.
Environmental
Protection
Agency,
Office
of
Water,
Washington
DC.
EPA
 
821
 
R
 
01
 
025.
28
Recommended
for
enumeration
of
target
organism
in
ambient
water
only.
29
USEPA.
October
2002.
Methods
for
Measuring
the
Acute
Toxicity
of
Effluents
and
Receiving
Waters
to
Freshwater
and
Marine
Organisms.
Fifth
Edition.
U.
S.
Environmental
Protection
Agency,
Office
of
Water,
Washington
DC.
EPA/
821/
R
 
02/
012.
30
USEPA.
October
2002.
Short­
term
Methods
for
Estimating
the
Chronic
Toxicity
of
Effluents
and
Receiving
Waters
to
Freshwater
Organisms.
Fourth
Edition.
U.
S.
Environmental
Protection
Agency,
Office
of
Water,
Washington
DC.
EPA/
821/
R
 
02/
013.
31
USEPA.
October
2002.
Short­
term
Methods
for
Estimating
the
Chronic
Toxicity
of
Effluents
and
Receiving
Waters
to
Marine
and
Estuarine
Organisms.
Third
Edition.
U.
S.
Environmental
Protection
Agency,
Office
of
Water,
Washington
DC.
EPA/
821/
R
 
02/
014.

*
*
*
*
*
(
b)
*
*
*

REFERENCES,
SOURCES,
COSTS,
AND
TABLE
CITATIONS
*
*
*
*
*
(
10)
Annual
Book
of
ASTM
Standards,
Water,
and
Environmental
Technology,
Section
11,
Volumes
11.01
and
11.02,
1994,
1996,
1999,
and
Volume
11.02,
2000
in
40
CFR
136.3,
Tables
IA,
IB,
IC,
ID,
and
IE.
*
*
*
*
*
(
34)
USEPA.
October
2002.
Methods
for
Measuring
the
Acute
Toxicity
of
Effluents
and
Receiving
Waters
to
Freshwater
and
Marine
Organisms.
Fifth
Edition.
U.
S.
Environmental
Protection
Agency,
Office
of
Water,
Washington,
DC
EPA
821
 
R
 
02
 
012.
Available
from:
National
Technical
Information
Service,
5285
Port
Royal
Road,
Springfield,
Virginia
22161,
Pub.
No.
PB2002
 
108488.
Table
IA,
Note
29.
*
*
*
*
*
(
38)
USEPA.
October
2002.
Short­
Term
Methods
for
Measuring
the
Chronic
Toxicity
of
Effluents
and
Receiving
Waters
to
Freshwater
Organisms.
Fourth
Edition.
U.
S.
Environmental
Protection
Agency,
Office
of
Water,
Washington,
DC
EPA
 
821
 
R
 
02
 
013.
Available
from:
National
Technical
Information
Service,
5285
Port
Royal
Road,
Springfield,
Virginia
22161,
Pub.
No.
PB2002
 
108489.
Table
IA,
Note
30.
(
39)
USEPA.
October
2002.
Short­
Term
Methods
for
Measuring
the
Chronic
Toxicity
of
Effluents
and
Receiving
Waters
to
Marine
and
Estuarine
Organisms.
Third
Edition.
U.
S.
Environmental
Protection
Agency,
Office
of
Water,
Washington,
DC
EPA
 
821
 
R
 
02
 
014.
Available
from:
National
Technical
Information
Service,
5285
Port
Royal
Road,
Springfield,
Virginia
22161,
Pub.
No.
PB2002
 
108490.
Table
IA,
Note
31.
*
*
*
*
*
(
52)
IDEXX
Laboratories,
Inc.
2002.
Description
of
Colilert
 
,
Colilert­
18'',
Quanti­
Tray
 
,
Quanti­
Tray
 
/
2000,
Enterolert
 
methods
are
available
from
IDEXX
Laboratories,
Inc.,
One
Idexx
Drive,
Westbrook,
Maine
04092.
Table
IA,
Notes
17
and
23.
(
53)
Hach
Company,
Inc.
Revision
2,
1999.
Description
of
m­
ColiBlue24
 
Method,
Total
Coliforms
and
E.
coli,
is
available
from
Hach
Company,
100
Dayton
Ave.,
Ames,
IA
50010.
Table
IA,
Note
18.
(
54)
USEPA.
2002.
Method
1103.1:
Escherichia
coli
(
E.
coli)
in
Water
by
Membrane
Filtration
Using
membrane­
Thermotolerant
Escherichia
coli
Agar
(
mTEC).
U.
S.
Environmental
Protection
Agency,
Office
of
Water,
Washington
D.
C.
September
2002,
EPA
 
821
 
R
 
02
 
020.
Available
at
NTIS,
PB2003
 
100125.
Table
IA,
Note
20.
(
55)
USEPA.
2002.
Method
1106.1:
Enterococci
in
Water
by
Membrane
Filtration
Using
membrane­
Enterococcus­
Esculin
Iron
Agar
(
mEEIA
U.
S.
Environmental
Protection
Agency,
Office
of
Water,
Washington
D.
C.
September
2002,
EPA
 
821
 
R
 
02
 
021.
Available
at
NTIS,
PB2003
 
100126.
Table
IA,
Note
24.
(
56)
USEPA.
2002.
Method
1603:
Escherichia
coli
(
E.
coli)
in
Water
by
Membrane
Filtration
Using
Modified
membrane­
Thermotolerant
Escherichia
coli
Agar
(
Modified
mTEC).
U.
S.
Environmental
Protection
Agency,
Office
of
Water,
Washington,
DC
September
2002,
EPA
 
821
 
R
 
02
 
023.
Available
at
NTIS,
PB2003
 
100128.
Table
IA,
Note
21.
(
57)
Brenner
et
al.
1993.
New
Medium
for
the
Simultaneous
Detection
of
Total
Coliforms
and
Escherichia
coli
in
Water.
Appl.
Environ.
Microbiol.
59:
3534
 
3544.
Available
from
the
American
Society
for
Microbiology,
1752
N
Street
NW.,
Washington,
DC
20036.
Table
IA,
Note
22.
(
58)
USEPA.
2002.
Method
1604:
Total
Coliforms
and
Escherichia
coli
(
E.
coli)
in
Water
by
Membrane
Filtration
using
a
Simultaneous
Detection
Technique
(
MI
Medium).
U.
S.
Environmental
Protection
Agency,
Office
of
Water,
Washington
D.
C..
September
2002,
EPA
821
 
R
 
02
 
024.
Available
from
NTIS,
PB2003
 
100129.
Table
IA,
Note
22.
(
59)
USEPA.
2002.
Method
1600:
Enterococci
in
Water
by
Membrane
Filtration
using
membrane­
Enterococcus
Indoxyl­
b­
D­
Glucoside
Agar
(
mEI).
U.
S.
Environmental
Protection
Agency,
Office
of
Water,
Washington
D.
C.
September
2002,
EPA
 
821
 
R
 
02
 
022.
Available
from
NTIS,
PB2003
 
100127.
Table
IA,
Note
25.
(
60)
USEPA.
2001.
Method
1622:
Cryptosporidium
in
Water
by
Filtration/
IMS/
FA.
U.
S.
Environmental
Protection
Agency,
Office
of
Water,
Washington,
DC
April
2001,
EPA
 
821
 
R
 
01
 
026.
Available
from
NTIS,
PB2002
 
108709.
Table
IA,
Note
26.
(
61)
USEPA.
2001.
Method
1623:
Cryptosporidium
and
Giardia
in
Water
by
Filtration/
IMS/
FA.
U.
S.
Environmental
Protection
Agency,
Office
of
Water,
Washington,
DC
April
2001,
EPA
 
821
 
R
 
01
 
025.
Available
from
NTIS,
PB2002
 
108710.
Table
IA,
Note
27.
(
62)
AOAC.
1995.
Official
Methods
of
Analysis
of
AOAC
International,
16th
Edition,
Volume
I,
Chapter
17.
AOAC
International.
481
North
Frederick
Avenue,
Suite
500,
Gaithersburg,
Maryland
20877
 
2417.
Table
IA,
Note
11.
*
*
*
*
*
(
e)
*
*
*

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/
Vol.
68,
No.
139
/
Monday,
July
21,
2003
/
Rules
and
Regulations
TABLE
II.
 
REQUIRED
CONTAINERS,
PRESERVATION
TECHNIQUES,
AND
HOLDING
TIMES
Parameter
No./
name
Container
1
Preservation2,3
Maximum
holding
time
4
(
hours)

Table
IA
 
Bacteria
Tests:
1
 
5
Coliform,
total,
fecal,
and
E.
coli
......
PP,
G
.........................
Cool,
<
10
°
C,
0.008%
Na2S2O3
5
.....................
6
6
Fecal
streptococci
..................................
PP,
G
.........................
Cool,
<
10
°
0.008%
Na2S2O3
5
.........................
6
7
Enterocci
..............................................
PP,
G
.........................
Cool,
<
10
°
0.008%
Na2S2O3
5
.........................
6
Table
IA
 
Protozoa
Tests:
8
Cryptosporidium
...................................
LDPE
..........................
0
 
8
°
C
..............................................................
96
17
9
Giardia
..................................................
LDPE
..........................
0
 
8
°
C
..............................................................
96
17
*
*
*
*
*
*
*

1
Polyethylene
(
P)
or
glass
(
G).
For
bacteria,
plastic
sample
containers
must
be
made
of
sterilizable
materials
(
polypropylene
[
PP]
or
other
autoclavable
plastic).
For
protozoa,
plastic
sample
containers
must
be
made
of
low­
density
polyethylene
(
LDPE).
2
Sample
preservation
should
be
performed
immediately
upon
sample
collection.
For
composite
chemical
samples,
each
aliquot
should
be
preserved
at
the
time
of
collection.
When
use
of
an
automated
sampler
makes
it
impossible
to
preserve
each
aliquot,
then
chemical
samples
may
be
preserved
by
maintaining
at
4
°
C
until
compositing
and
sample
splitting
is
completed.
3
When
any
sample
is
to
be
shipped
by
common
carrier
or
sent
through
the
United
States
Mails,
it
must
comply
with
the
Department
of
Transportation
Hazardous
Materials
Regulations
(
49
CFR
part
172).
The
person
offering
such
material
for
transportation
is
responsible
for
ensuring
such
compliance.
For
the
preservation
requirements
of
Table
II,
the
Office
of
Hazardous
Materials,
Transportation
Bureau,
Department
of
Transportation
has
determined
that
the
Hazardous
Materials
Regulations
do
not
apply
to
the
following
materials:
Hydrochloric
acid
(
HCl)
in
water
solutions
at
concentrations
of
0.04%
by
weight
or
less
(
pH
about
1.96
or
greater);
Nitric
acid
(
HNO3)
in
water
solutions
of
0.15%
by
weight
or
less
(
pH
about
1.62
or
greater);
Sulfuric
acid
(
H2SO4)
in
water
solutions
of
concentrations
of
0.35%
by
weight
or
less
(
pH
about
1.15
or
greater);
and
Sodium
hydroxide
(
NaOH)
in
water
solutions
at
concentrations
of
0.080%
by
weight
or
less
(
pH
about
12.30
or
less).
4
Samples
should
be
analyzed
as
soon
as
possible
after
collection.
The
times
listed
are
the
maximum
times
that
samples
may
be
held
before
analyses
and
still
be
considered
valid.
Samples
may
be
held
for
longer
periods
only
if
the
permittee,
or
monitoring
laboratory,
has
data
on
file
to
show
that
for
the
specific
types
of
samples
under
study,
the
analytes
are
stable
for
the
longer
time,
and
has
received
a
variance
from
the
Regional
Administrator
under
§
136.3(
e).
Some
samples
may
not
be
stable
for
the
maximum
time
period
given
in
the
table.
A
permittee
or
monitoring
laboratory
is
obligated
to
hold
the
samples
for
a
shorter
time
if
knowledge
exists
to
show
that
this
is
necessary
to
maintain
sample
stability.
See
§
136.3(
e)
for
details.
The
term
``
analyze
immediately''
usually
means
within
15
minutes
or
less
of
sample
collection.
5
Should
only
be
used
in
the
presence
of
residual
chlorine.
*
*
*
*
*
*
*
16
Sufficient
ice
should
be
placed
with
the
samples
in
the
shipping
container
to
ensure
that
ice
is
still
present
when
samples
arrive
at
the
laboratory
However,
even
if
ice
is
present
when
the
samples
arrive,
it
is
necessary
to
immediately
measure
the
temperature
of
the
samples
and
confirm
that
the
4
°
C
temperature
maximum
has
not
been
exceeded.
In
the
isolated
cases
where
it
can
be
documented
that
this
holding
temperature
can
not
be
met,
the
permittee
can
be
given
the
option
of
on­
site
testing
or
can
request
a
variance.
The
request
for
a
variance
should
include
supportive
data
which
show
that
the
toxicity
of
the
effluent
samples
is
not
reduced
because
of
the
increased
holding
temperature.
17
Holding
time
is
calculated
from
time
of
sample
collection
to
elution
for
samples
shipped
to
the
laboratory
in
bulk
and
calculated
from
the
time
of
sample
filtration
to
elution
for
samples
filtered
in
the
field.

*
*
*
*
*
[
FR
Doc.
03
 
18155
Filed
7
 
18
 
03;
8:
45
am]

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