svEPA
United States
Environmental Protection
Agency
SOURCE WATER MONITORING GUIDANCE
MANUAL FOR PUBLIC WATER SYSTEMS
FOR THE FINAL LONG TERM 2 ENHANCED
SURFACE WATER TREATMENT RULE
Office of Water (4601M)
EPA815-R06-005
February 2006
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U.S. Environmental Protection Agency
Off ice of Water (4601M)
1200 Pennsylvania Avenue NW
Washington DC 20460
EPA815-R-06-005
http://www.epa.gov/safewater/disinfection/lt2/compliance.html
February 2006
Printed on Recycled Paper
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Disclaimer
The Standards and Risk Management Division, of the Office of Ground Water and Drinking Water, has
reviewed and approved this guidance for publication. Neither the United States Government nor any of its
employees, contractors, or their employees make any warranty, expressed or implied, or assumes any
legal liability or responsibility for any third party's use of or the results of such use of any information,
apparatus, product, or process discussed in this report, or represents that its use by such party would not
infringe on privately owned rights. This guidance is not a substitute for applicable legal
requirements, nor is it a regulation itself. Mention of trade names or commercial products does not
constitute endorsement or recommendation for use.
Contact for technical inquiries regarding Method 1622 or 1623 and laboratory approval:
Carrie Moulton
U.S. Environmental Protection Agency
Office of Ground Water and Drinking Water
Technical Support Center, MC 140
26 West Martin Luther King Drive
Cincinnati, OH 45268
513-569-7919 phone
513-569-7191 fax
moulton.carrie@epa.gov
Contact for technical inquires regarding sampling and analysis for E. coli:
Jennifer Best
U.S. Environmental Protection Agency
Office of Ground Water and Drinking Water
Technical Support Center, MC 140
26 West Martin Luther King Drive
Cincinnati, OH 45268
513-569-7012 phone
513-569-7191 fax
best.jennifer@epa.gov
All other inquiries should be addressed to
Sean Conley
U.S. Environmental Protection Agency
Office of Ground Water and Drinking Water
Mail Code 4607M
USEPA Headquarters, EPA East
1200 Pennsylvania Ave., NW
Washington, DC 20460
202-564-1781 phone
202-564-3767 fax
conlev. sean@epa. gov
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TABLE OF CONTENTS
Section 1: General Requirements 1
1.1 Introduction 1
1.2 Schedules 1-3: Large Systems and Wholesalers (as applicable) 2
1.2.1 Wholesale Systems and Combined Distribution Systems 3
1.3 Schedule4- Small Systems 8
1.4 Consecutive Systems 11
1.5 Monitoring Avoidance 11
1.6 Reports and Notices 11
1.7 Questions? 13
Section 2: Contracting for Laboratory Services 15
2.1 Defining Your Needs and Developing a Contract 15
2.1.1 Client Information 16
2.1.2 Sample Information 16
2.1.3 Sampling Schedules 19
2.1.4 Analytical Methodology for Cryptosporidium 19
2.1.5 Analytical Methodology for E. coli 20
2.1.6 Data Deliverables and Other Contract Issues 20
2.2 Developing a Bid Sheet 24
2.3 Soliciting the Contract 25
2.3.1 Approved Laboratories for Cryptosporidium 25
2.3.2 Certified Laboratories for E. coli 26
2.3.3 Primary and Backup Laboratory Contracts 26
2.4 Evaluating Bids 26
2.4.1 Identifying Responsive Bidders 27
2.4.2 References 27
2.5 Communicating with the Laboratory 27
Section 3: Sampling Location and Schedule 29
3.1 Sample Collection Location and Description 29
3.1.1 Plants That Do Not Have a Sampling Tap Located Prior to Any Treatment 30
3.1.2 Plants That Use Different Water Sources at the Same Time 30
3.1.3 Plants That Use Presedimentation 31
3.1.4 Plants That Use Raw Water Off-Stream Storage 31
3.1.5 Plants That Use Bank Filtration 31
3.1.6 Plants That Use Ground Water Under the Direct Influence of Surface Water 31
3.1.7 Submitting Sample Location Descriptions 31
3.2 Source Water Sampling Schedule 32
3.2.1 Part-Year Plants 32
3.2.2 Sample Collection Problems 32
3.2.3 Changing the Sampling Schedule 33
Section 4: Collecting and Shipping Source Water Samples 35
4.1 Sample Collection Guidance 35
4.1.1 Sample Collection Documentation 35
4.1.2 Cryptosporidium Sample Collection 36
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4.1.3 Matrix Spike Sample Collection 38
4.1.4 E. coli Sample Collection 39
4.2 Sample Temperatures 40
4.3 Measuring Turbidity 41
4.3.1 Measuring Sample Turbidity During LT2 Monitoring 42
4.3.2 General Quality Control for Turbidity Measurements 42
Section 5: Grandfathering Cryptosporidium Data 43
5.1 Intent to Grandfather 43
5.2 Requirements for Grandfathered Cryptosporidium Data 43
5.2.1 Sample Collection Location and Volume 43
5.2.2 Sample Collection Frequency and Schedule 44
5.2.3 Cryptosporidium Analytical Methods for Grandfathered Data 44
5.2.4 Cryptosporidium Laboratories for Grandfathered Data 45
5.2.5 E. coli and Turbidity Measurements 45
5.3 Checklists for Grandfathering Cryptosporidium Data 45
5.4 Reporting Grandfathered Data 45
5.4.1 Grandfathered Data Package Contents 46
5.5 Rejected or Missing Cryptosporidium Data 48
Section 6: Cryptosporidium Data: Use, Recording, Submitting and Review 49
6.1 Use of Cryptosporidium Data 49
6.1.1 Determining Bin Classification -Filtered Systems 49
6.1.2 Determining Mean Cryptosporidium Levels - Unfiltered Systems 50
6.1.3 Cryptosporidium Matrix Spike Data 50
6.2 Cryptosporidium Data Recording at the Laboratory 51
6.2.1 LT2 Sample Collection Form 51
6.2.2 Method 1622/1623 Bench Sheet 51
6.2.3 Method 1622/1623 Cryptosporidium Slide Examination Form 51
6.3 Submitting Cryptosporidium Data through the LT2/Stage 2 Data Collection and
Tracking System (DCTS) 52
6.3.1 Data Entry/Upload 54
6.3.2 PWS Data Review 54
6.3.3 EPA/State Review 55
6.3.4 Archiving Examination Results 55
6.4 (Optional) Review and Validation of Raw Cryptosporidium Data 55
6.4.1 Data Completeness Check 55
6.4.2 Evaluation of Data Against Method Quality Control Requirements 56
6.4.3 Calculation Verification 56
6.4.4 Data Archiving Requirements 58
Section 7:E. coliData: Use, Recording, Submitting and Review 59
7.1 Use of E. coli Data 59
7.2 E. coli Data Recording at the Laboratory 59
7.2.1 Sample Identification Information 60
7.2.2 Primary Data 60
7.2.3 Sample Processing and Quality Control Information 60
7.2.4 Sample Results 61
7.3 Submitting E. coli Data through the LT2/Stage 2 Data Collection and
Tracking System (DCTS) 61
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7.3.1 Data Entry/Upload 61
7.3.2 PWS Data Review 62
7.3.3 EPA/State Review 62
7.4 (Optional) Reviewing and Validating RawE1. coli Data 62
7.4.1 Data Completeness Check 62
7.4.2 Evaluation of Data Against Method Quality Control Requirements 63
7.4.3 Calculation Verification 64
7.4.4 Data Archiving Requirements 69
Section 8: References 71
February 2006
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TABLES
Table 1 -1. Summary of LT2 Rule Monitoring Requirements (Initial Round) 4
Table 1 -2. SCHEDULE 1: Timeline for required monitoring (Initial Round) 5
Table 1-3. SCHEDULE 2: Timeline for required monitoring (Initial Round) 6
Table 1 -4. SCHEDULE 3: Timeline for required monitoring (Initial Round) 7
Table 1-5. SCHEDULE 4: Timeline for required monitoring (Initial Round) 9
Table 1-5. SCHEDULE 4: Timeline for required monitoring (Initial Round) (continued)... 10
Table 1-6. Submitting Reports and Notices 12
Table 4-1. Minimum Data Elements to Record During Sample Collection 36
Table 4-2. Contacts for Filters Approved for Using EPA Method 1622/1623 37
Table 6-1. Bin Classifications for Filtered PWSs 49
Table 6-2. LT2/Stage 2 Data Collection and Tracking System (DCTS) Data Entry,
Review, and Transfer Process 53
Table 7-1. Approved^, co//Methods for LT2 Rule 60
Table 7-2. Examples of Different Combinations of Positive Tubes 69
IV
February 2006
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APPENDICES
Appendix A Intent to Provide Maximum Treatment- Example Notice
Appendix B Cost Estimate for Bulk Water Sample Analysis
Appendix C Cost Estimate for Field-Filtered Sample Analysis
Appendix D Sampling Location Worksheet
Appendix E LT2 Sample Collection Location Schematics
Appendix F LT2 Sample Collection Form
Appendix G Envirochek™ Field Filtration for Cryptosporidium
Appendix H Filta-Max® Field Filtration for Cryptosporidium
Appendix I Collecting Bulk Water Samples for Laboratory Filtration and Cryptosporidium Analysis
Appendix J Collecting Source Water Samples for E. coll Analysis
Appendix K Intent to Grandfather- Example Notice
Appendix L Recommended Checklist for Beginning Grandfathered Cryptosporidium Monitoring
Appendix M Grandfathered Cryptosporidium Data Package Report Checklist
February 2006
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ACRONYMS
CPU
CNFG
DAPI
DCTS
DIG
EPA
FA
FITC
GWUDI
ICR
IDC
IESWTR
IFA
IMS
IPMC
IPR
IPX
L
LT2 Rule
LT2ESWTR
mL
MPC
MPN
MS
MS/MSD
NA-MUG
NELAC
nm
NPDWR
NTU
OPR
OPT
PBMS
PT
PWS
QA
QAP
QC
RSD
SDWA
SOP
TNTC
uv
Colony-forming unit
Confluent growth
4, 6-diamidino-2-phenylindole
LT2/Stage 2 Data Collection and Tracking System
Differential interference contrast
United States Environmental Protection Agency
Fluorescence assay
Fluorescein isothiocyanate
Ground water under the direct influence [of surface water]
Information Collection Rule
Initial demonstration of capability
Interim Enhanced Surface Water Treatment Rule
Immunofluorescence assay
Immunomagnetic separation
Information Processing and Management Center
Initial precision and recovery
Initial proficiency testing
Liter
Long Term 2 Enhanced Surface Water Treatment Rule
Long Term 2 Enhanced Surface Water Treatment Rule
Milliliter
Magnetic particle concentrator
Most probable number
Matrix spike
Matrix spike/matrix spike duplicate
Micrometer
Nutrient agar (NA) with 4-methylumbelliferyl-beta-D-glucuronide (MUG)
National Environmental Laboratory Accreditation Conference
Nanometer
National Primary Drinking Water Regulations
Nephelometric turbidity unit
Ongoing precision and recovery
Ongoing proficiency testing
Performance-based measurement system
Proficiency testing
Public water system
Quality assurance
Quality assurance plan
Quality control
Relative standard deviation
Safe Drinking Water Act
Standard operating procedure
Too numerous to count
Ultraviolet
VI
February 2006
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SECTION 1: GENERAL REQUIREMENTS
1.1 Introduction
The Long Term 2 Enhanced Surface Water Treatment Rule (LT2 Rule) (Reference 8.1) requires public
water systems (PWSs) that use surface water or ground water under the direct influence (GWUDI) of
surface water to monitor their source water (influent water prior to treatment plant) for Cryptosporidium
and/or E. coli, and turbidity for a limited period [40 CFR § 141.701]. In support of the monitoring
requirements specified by the rule, three documents have been developed to provide guidance on
monitoring and reporting data under the LT2 Rule to the affected PWSs and the laboratories that support
them:
• Source Water Monitoring Guidance Manual for Public Water Systems for the Long Term 2 Enhanced
Surface Water Treatment Rule (LT2 Rule) (this document). This guidance manual for PWSs affected
by the rule provides information on laboratory contracting, sample collection procedures, and data
evaluation and interpretation. This guidance manual also provides information on grandfathering
requirements for Cryptosporidium and E. coli data.
• Microbial Laboratory Guidance Manual for the Long Term 2 Enhanced Surface Water Treatment
Rule (LT2 Rule). This manual provides Cryptosporidium and E. coli laboratories analyzing samples in
support of the LT2 Rule with guidance and detailed procedures for all aspects of microbial analyses
under the rule to maximize data quality and consistency.
• Users' Manual for the LT2/Stage 2 Data Collection and Tracking System (DCTS). This manual
provides PWSs and laboratories with instructions on using the DCTS for entry, review, and approval
of electronic data and provides instructions for States and EPA for generating tracking reports.
These manuals, an Online Microscopy Training Module, and other guidance and information are
available at http://www.epa.gov/safewater/disinfection/lt2/compliance.html
This guidance document is provided to help implement the LT2 Rule. This guidance document does not,
however, substitute for the LT2 Rule or the analytical methods approved for use under the rule. The
material presented here is intended solely for guidance and does not alter any regulatory or analytical
method requirements.
This manual provides guidance on the following aspects of the LT2 Rule:
• Section 1: Overview of the rule's monitoring requirements
• Section 2: Establishing a Cryptosporidium laboratory contract
• Section 3: Sampling location and schedule guidance
Section 4: Guidance on collecting and shipping LT2 monitoring samples
Section 5: Guidance on submitting historical data ("grandfathering")
Section 6: Understanding Cryptosporidium data and analyses
• Section 7: Understanding E. coli data and analyses
February 2006
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Section 1: General Requirements
The LT2 Rule is a National Primary Drinking Water Regulation that requires monitoring, reporting, and
public notification for all PWSs that use surface water or GWUDI sources. The LT2 Rule was developed
to improve the control of microbial pathogens, including specifically the protozoan Cryptosporidium, in
drinking water and to address risk trade-offs with disinfection byproducts.
The LT2 Rule requires PWSs that use surface water or GWUDI to monitor their source water (influent
water prior to treatment plant) for Cryptosporidium, and/or E. coli, and turbidity [40 CFR § 141.701 (a)
and (c-h)]. Monitoring requirements vary by PWS size (large or small), treatment type (filtered or
unfiltered) and selling relationship (wholesaler or non-wholesaler). Monitoring results will be used to
determine whether additional treatment is required at PWSs and to refine the relationship established
between Cryptosporidium and E. coli levels in source water. All PWSs that must comply with the
requirements of the LT2 Rule must initiate monitoring according to the schedule in Table 1-1 [40 CFR §
141.701(c)]. A second round of monitoring is also required for all PWSs after the initial round of
monitoring described in this guidance [40 CFR § 141.701(c)].
Every PWS that is subject to the LT2 Rule should receive a letter from EPA or the state with information
on the LT2 Rule and a determination of source water monitoring requirements and schedule (schedule 1,
2, 3, or 4). Systems that EPA or the state anticipates are on schedules 1 and 2 should receive a letter in
February 2006. Systems that EPA or the state anticipates are on schedules 3 and 4 should receive a letter
in July 2006. States determined your requirements and schedule based on their records on your population
served and connections to other systems. You should make sure the schedule determination in the letter is
consistent with your system size, source water type, and buying/selling relationships with other PWSs
before proceeding. If you have questions about your schedule and requirements, please discuss them with
your point of contact for the LT2 listed at http://www.epa. gov/safewater/disinfection/lt2/compliance .html.
1.2 Schedules 1-3: Large Systems and Wholesalers (as applicable)
Large systems (those serving at least 10,000 people) affected by the LT2 Rule include both filtered and
unfiltered PWSs.
A large, filtered system in the LT2 Rule is a system that
Uses surface water or GWUDI
Serves at least 10,000 people
Provides filtration or is unfiltered, but required to install filtration because the system no longer
meets all filtration avoidance criteria
Large filtered PWSs and wholesalers (as applicable - see section 1.2.1) are required to conduct
initial source water monitoring that includes sampling for Cryptosporidium, E. coli, and
turbidity at least once per month for two years [40 CFR § 141.701(a)(l)].
A large, unfiltered system in the LT2 Rule is a system that:
Uses surface water or ground GWUDI
Serves at least 10,000 people
Does not currently provide filtration and meets all filtration avoidance criteria
2 February 2006
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Section 1: General Requirements
Large unfiltered PWSs and wholesalers (as applicable - see section 1.2.1) are required to
conduct initial source water monitoring that includes sampling for only Cryptosporidium at least
once per month for two years [40 CFR § 141.701(a)(2)].
All of the Cryptosporidium sampling requirements and guidance discussed in this document apply equally
to both filtered and unfiltered PWSs, unless otherwise stated. However, the E. coll and turbidity guidance
in this document does not apply to unfiltered PWSs.
The steps suggested and required for LT2 Rule compliance for large PWSs on the first three schedules
and the timelines for these steps are summarized in Tables 1-2 through 1-4. PWSs that will be submitting
previously collected data ("grandfathering") should also consult Section 5 of this guidance. Details on the
use of the Cryptosporidium and E. coll data collected under the LT2 Rule are provided in Sections 6
and 7.
1.2.1 Wholesale Systems and Combined Distribution Systems
A wholesale system is a public water system that treats source water as necessary to produce finished
water and then delivers some or all of that finished water to another public water system [40 CFR §
141.2]. Wholesale systems must comply with the requirements based on the population of the largest
system in the combined distribution system [40 CFR § 141.700(b)(l)].
A combined distribution system is the interconnected distribution system consisting of the distribution
systems of wholesale systems and the consecutive systems that receive finished water [40 CFR § 141.2].
As described in Section 1.1, States have made determinations of wholesalers, combined distribution
systems and monitoring requirements and schedules based on information available regarding
buying/selling relationships and interconnection types (permanent, seasonal or emergency) and their
usage. If you have questions about your requirements or combined distribution system status, contact
your point of contact listed at http://www.epa.gov/safewater/disinfection/lt2/compliance.html or send an
email to stage2mdbp@epa.gov.
February 2006
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Section 1: General Requirements
Table 1-1. Summary of LT2 Rule Monitoring Requirements (Initial Round)
SCHEDULE
SCHEDULE 1*.
Large systems
serving >1 00,000
SCHEDULE 2*
Large systems
serving >50,000
and <1 00, 000
SCHEDULE 3*
Large systems
serving >1 0,000
and<50,000
SCHEDULE 4*:
Small filtered
systems (serving
fewer than 10,000)
Small unfiltered
systems (serving
fewer than 10,000)
and
Small filtered
systems exceeding
E. coli trigger
levels d or that
elect to proceed
directly to
Cryptosporidium
monitoring or that
fail to conduct E.
coli monitoring
Monitoring
begins
October 1,
2006
April 1, 2007
April 1, 2008
October 1,
2008
April 1,2010
Monitoring
duration
2 years a
2 years a
2 years a
1 yeara'd
Monitoring parameters and sample frequency
requirements
Cryptosporidium
Minimum
1 sample/month b
Minimum
1 sample/month b
Minimum
1 sample/month b
See next row
1 year b, 2 Cryptosporidium
samples per month,
or
2 years e, 1 Cryptosporidium
sample per month
E. coli
Minimum
1 sample/month °
Minimum
1 sample/month °
Minimum
1 sample/month °
Every two weeks
N/A
Turbidity
Minimum
1 sample/month °
Minimum
1 sample/month °
Minimum
1 sample/month °
N/A
N/A
* Wholesale systems must comply with the requirements based on the population of the largest system in
the combined distribution system [40 CFR § 141.700(b)(1)]
PWSs may be eligible to use (grandfather) data collected prior to the applicable monitoring start date if certain requirements are
met (see Section 5) [40 CFR § 141.707(a)(1)]
b PWSs monitoring for Cryptosporidium may collect more than one sample per month if sampling is evenly spaced over the
monitoring period [40 CFR § 141.701(a)(7)]
0 Unfiltered systems serving >10,000 are not required to perform E. coli or turbidity monitoring but to conduct source water
monitoring that includes only Cryptosporidium sampling [40 CFR § 141.701 (a)(2)]
d Filtered systems serving fewer than 10,000 people must monitor for Cryptosporidium only if their E. coli annual mean
concentrations is greater than 10 £. co///100 ml for systems using lakes/reservoirs or is greater than 50 £. co///100 ml for
systems using flowing streams [40 CFR § 141.701 (a)(4)]
e Small systems collecting one sample per month for 2 years are still required, where applicable, to meet the treatment technique
implementation deadlines in 40 CFR § 141.713 (c). The same treatment compliance dates apply to the PWS regardless of which
Cryptosporidium sampling frequency is used (i.e., selecting the 2 year Cryptosporidium sampling frequency does not extend
Cryptosporidium treatment compliance deadlines).
N/A = Not applicable. No monitoring required.
February 2006
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Section 1: General Requirements
Table 1-2. SCHEDULE 1: Timeline for required monitoring (Initial Round)
- Large Systems serving at least 100,000 and
- Systems that sell water and are part of a network of systems (a combined distribution
system) with its largest system serving 100,000 or more persons
Event
Establish contract with a Cryptosporidium
laboratory approved under EPA's Lab QA
Program (see Section 2.3.1) (Reference 8.2)
Verify that the laboratory you plan to use to
perform E. coli analyses under LT2 is certified
under the drinking water laboratory certification
program to perform a similar technique to the £.
coli method that has been selected for use a
(See Section 2.3.2)
Verify that the party who will measure turbidity has
been approved by the State a
Work with your Cryptosporidium laboratory to
establish a mutually acceptable sampling
schedule (see Section 2.1.3 and Section 3.2)
Submit sampling schedule (see Section 3.2) or
submit notice of intent to provide full treatment as
defined in 40 CFR § 141.701(d) (see Section 1.5)
Submit sampling location and source water
monitoring description (see Section 3.1)
Submit notice of intent to grandfather
(if applicable) (see Section 5) b'c
Submit grandfathered Cryptosporidium data
package (if applicable) (see Section 5) b'c
Collect monitoring samples c (see Section 4)
Submit monitoring results c (see Sections 6.3 and
7.3)
Timeline
As soon as possible, recommended by
June 2006
Required- No later than July 1, 2006
Required (if grandfathering) - No later
than July 1, 2006
Required (if grandfathering) - No later
than December 1, 2006
Required- No later than the month
beginning October 1, 2006
Required- No later than 10 days after
the end of the first month following the
month that the sample was collected
(approximately 40 to 70 days after
sample collection, depending on when
during the month the sample is
collected)
Duration
N/A- single event
At least once per
month for 2 yearsd
a Not applicable to large, unfiltered systems because these systems are not required to monitor for E. coli or turbidity [40 CFR §
141.701(a)(2)]
b PWSs with fewer than two years of grandfathered data at the time of LT2 Rule promulgation, or that have at least two years of
grandfathered data but intend to conduct monitoring under the LT2 Rule, must also submit a sampling schedule [40 CFR §
141.702(a) and 40 CFR § 141.707(f)(1)]
0 PWSs may be eligible to use historical (grandfathered) data in lieu of monitoring requirements if certain quality assurance and
quality control criteria are met (see Section 5) [40 CFR § 141.707]
d PWSs monitoring for Cryptosporidium may collect more than one sample per month if sampling is evenly spaced over the
monitoring period [40 CFR § 141.701(a)(7)]
N/A = Not applicable
February 2006
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Section 1: General Requirements
Table 1-3. SCHEDULE 2: Timeline for required monitoring (Initial Round)
- Large Systems serving > 50,000 and <100,000, and
- Systems that sell water and are part of a network of systems (a combined distribution
system) with its largest system serving 50,000-99,999 persons
Event
Establish contract with a Cryptosporidium
laboratory Approved under EPA's Lab QA
Program (see Section 2.3.1)
Verify that the laboratory you plan to use to
perform E. coli analyses under LT2 is certified
under the drinking water laboratory certification
program to perform a similar technique to the £.
coli method that has been selected for use a (see
Section 2.3.2)
Verify that the party who will measure turbidity has
been approved by the State a
Work with your Cryptosporidium laboratory to
establish a mutually acceptable sampling
schedule (see Section 2.1.3 and Section 3.2)
Submit sampling schedule (see Section 3.2) or
submit notice of intent to provide full treatment as
defined in §141.701 (d) (see Section 1.5)
Submit sampling location and source water
monitoring description (see Section 3.1)
Submit notice of intent to grandfather
(if applicable) (see Section 5) b'c
Submit grandfathered Cryptosporidium data
package (if applicable) (see Section 5) b'c
Collect monitoring samples c (see Section 4)
Submit monitoring results c (see Sections 6.3 and
7.3)
Timeline
As soon as possible, recommended by
December 2006
Required- No later than January 1,
2007
Required (if grandfathering) - No later
than January 1, 2007
Required (if grandfathering) - No later
than June 1, 2007
Required- No later than the month
beginning April 1, 2007
Required- No later than 10 days after
the end of the first month following the
month that the sample was collected
(approximately 40 to 70 days after
sample collection, depending on when
during the month the sample is
collected)
Duration
N/A- single event
At least once per
month for 2 yearsd
a Not applicable to large, unfiltered systems because these systems are not required to monitor for E. coli or turbidity [40 CFR §
141.701(a)(2)]
b PWSs with fewer than two years of grandfathered data at the time of LT2 Rule promulgation, or that have at least two years of
grandfathered data but intend to conduct monitoring under the LT2 Rule, must also submit a sampling schedule [40 CFR §
141.702(a) and 40 CFR § 141.707(f)(1)]
0 PWSs may be eligible to use historical (grandfathered) data in lieu of monitoring requirements if certain quality assurance and
quality control criteria are met (see Section 5) [40 CFR § 141.707]
d PWSs monitoring for Cryptosporidium may collect more than one sample per month if sampling is evenly spaced over the
monitoring period [40 CFR § 141.701(a)(7)]
N/A = Not applicable
February 2006
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Section 1: General Requirements
Table 1-4. SCHEDULE 3: Timeline for required monitoring (Initial Round)
- Large Systems serving > 10,000 and <50,000, and
- Systems that sell water and are part of a network of systems (a combined distribution
system) with its largest system serving 10,000-49,999 persons
Event
Establish contract with a Cryptosporidium
laboratory Approved under EPA's Lab QA
Program (see Section 2.3.1)
Verify that the laboratory you plan to use to
perform E. coli analyses under LT2 is certified
under the drinking water laboratory certification
program to perform a similar technique to the £.
coli method that has been selected for use a (see
Section 2.3.2)
Verify that the party who will measure turbidity has
been approved by the State a
Work with your Cryptosporidium laboratory to
establish a mutually acceptable sampling
schedule (see Section 2.1.3 and Section 3.2)
Submit sampling schedule (see Section 3.2) or
submit notice of intent to provide full treatment as
defined in § 141.701 (d) (see Section 1.5)
Submit sampling location and source water
monitoring description (see Section 3.1)
Submit notice of intent to grandfather (if
applicable) (see Section 5) b'c
Submit grandfathered Cryptosporidium data
package (if applicable) (see Section 5) b'c
Collect monitoring samples c (see Section 4)
Submit monitoring results c (see Sections 6.3 and
7.3)
Timeline
Recommended by December 2007
Required- No later than January 1,
2008
Required (if grandfathering) - No later
than January 1, 2008
Required (if grandfathering) - No later
than June 1, 2008
Required- No later than the month
beginning April 1, 2008
Required- No later than 10 days after
the end of the first month following the
month that the sample was collected
(approximately 40 to 70 days after
sample collection, depending on when
during the month the sample is
collected)
Duration
N/A- single event
At least once per
month for 2 yearsd
Not applicable to large, unfiltered systems because these systems are not required to monitor for E. coli or turbidity [40 CFR §
141.701(a)(2)]
b PWSs with fewer than two years of grandfathered data at the time of LT2 Rule promulgation, or that have at least two years of
grandfathered data but intend to conduct monitoring under the LT2 Rule, must also submit a sampling schedule [40 CFR §
141.702(a) and 40 CFR § 141.707(f)(1)]
0 PWSs may be eligible to use historical (grandfathered) data in lieu of monitoring requirements if certain quality assurance and
quality control criteria are met (see Section 5) [40 CFR § 141.707]
d PWSs monitoring for Cryptosporidium may collect more than one sample per month if sampling is evenly spaced over the
monitoring period [40 CFR § 141.701(a)(7)]
N/A = Not applicable
February 2006
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Section 1: General Requirements
1.3 Schedule 4 - Small Systems
Small systems (those serving fewer than 10,000 people) affected by the LT2 Rule include both filtered
and unfiltered systems.
A small Filtered system under the LT2 Rule is a system that
• Uses surface water or ground water under the direct influence of surface water
• Serves fewer than 10,000 people
• Provides filtration, or is unfiltered but required to install filtration because the system no longer meets
all filtration avoidance criteria.
Small, filtered systems are required to conduct initial source water monitoring for E. coli biweekly
as an indicator of Cryptosporidium. Those small filtered systems that exceed E. coli trigger levels,
those that opt to proceed directly to Cryptosporidium monitoring without collecting E. coli data, and
those that fail to conduct E. coli monitoring must conduct monitoring for Cryptosporidium. [40 CFR
§ 141.701(a)(3)].
A small unfiltered system under the LT2 Rule is a system that
• Uses surface water or ground water under the direct influence of surface water
• Serves fewer than 10,000 people
• Does not currently provide filtration and meets all filtration avoidance criteria
Small, unfiltered systems are required to conduct initial source water monitoring that includes
Cryptosporidium sampling only (small unfiltered systems do not conduct E. coli indicator
monitoring). [40 CFR § 141.701(a)(6)].
The steps required for LT2 Rule compliance for small PWSs and the schedule for these steps are
summarized in Table 1-5.
February 2006
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Section 1: General Requirements
Table 1-5. SCHEDULE 4: Timeline for required monitoring (Initial Round)
- Small systems serving <10,000 persons, and
- Systems that sell water and are part of a network of systems (a combined distribution
system) with its largest system serving <10,000 persons
Filtered Systems - E. coli monitoring
Event
Verify that the laboratory that will perform your
£ coli analyses under the LT2 Rule is certified
under the drinking water laboratory certification
program to perform the analytical method you
plan to use (See Section 2.3.2)
Submit sampling schedule (see Section 3.2) or
submit notice of intent to provide full treatment
as defined in § 141.701 (d) (see Section 1.5)
or submit notice of intent to avoid £ coli
monitoring by monitoring for Cryptosporidium
Submit sampling location and source water
monitoring description
Submit notice of intent to grandfather (if
applicable) (see Section 5) d'e
Submit grandfathered £. coli data package (if
applicable) (see Section 5) d'e
Collect £ coli samples e (See Section 4)
Submit £ coli monitoring results (See Section
7.3)
Timeline
Recommended by June, 2008
Required- No later than July 1, 2008
Required (if grandfathering) - No later
than July 1,2008 d'e
Required (if grandfathering) - No later
than December 1, 2008 d'e
Required— No later than the month
beginning October 1, 2008
Required- No later than 10 days after the
end of the first month following the month
that the sample was collected
(approximately 40 to 70 days after sample
collection)
Duration
N/A - single event
One year (one
sample every two
weeks) b
At least once per
month for one year
Table 1-5 continued on the following page
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Section 1: General Requirements
Table 1-5. SCHEDULE 4: Timeline for required monitoring (Initial Round) (continued)
Unfiltered Systems and
Filtered Systems that Exceed £. co//Trigger Levels3 or
that Elect to Bypass £. co// Monitoring and Proceed Directly to Cryptosporidium Monitoring or
that fail to conduct E. co// monitoring
Establish contract with a Cryptosporidium
laboratory approved under EPA's Lab QA
Program
Work with your Cryptosporidium laboratory to
establish a mutually acceptable sampling
schedule
Submit sampling schedule (see Section 3.2) or
submit notice of intent to provide full treatment
as defined in § 141.701 (d) (see Section 1.5)
Submit sampling location and source water
monitoring description
Submit notice of intent to grandfather (if
applicable) (see Section 5) d'e
Submit grandfathered Cryptosporidium data
package (if applicable) (see Section 5) d'e
Recommended by December, 2009
Required- No later than January 1, 2010
No later than January 1, 2010 '
No later than June 1, 2010'
N/A - single event
Collect Cryptosporidium samples
Required- No later than the month
beginning April 1, 2010
One year (two
samples per
month)b or two
years (one sample
per month)c
Submit Cryptosporidium monitoring results
Required- No later than 10 days after the
end of the first month following the month
that the sample was collected
(approximately 40 to 70 days after sample
collection)
At least once per
month for one year
a Small filtered systems are required to monitor for Cryptosporidium, beginning six months after completion of £. co// monitoring if
the £. co//annual mean concentration exceeds 10 £. co///100 ml for systems using lakes/reservoirs or exceeds 50 £. co///100 ml
for systems using flowing streams
b PWSs may sample more frequently if the sampling frequency is evenly spaced over the monitoring period [40 CFR §
141.701(a)(7)]
0 Small systems collecting one sample per month for 2 years are still required, where applicable, to meet the treatment technique
implementation deadlines in 40 CFR § 141.713 (c). The same treatment compliance dates apply to the PWS regardless of which
Cryptosporidium sampling frequency is used (i.e., selecting the 2 year Cryptosporidium sampling frequency does not extend
Cryptosporidium treatment compliance deadlines).11 PWSs may be eligible to use historical (grandfathered) data in lieu of
monitoring requirements if certain quality assurance and quality control criteria are met (see Section 5) [40 CFR § 141.707]
e Small systems with less than a complete set of grandfathered data, or that intend to conduct additional monitoring beyond the
required minimum under the LT2 Rule, must also submit a sampling schedule [40 CFR § 141.702(a) and 40 CFR § 141.707(f)(1)]
N/A = Not applicable
10
February 2006
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Section 1: General Requirements
1.4 Consecutive Systems
A consecutive system is a public water system that receives some or all of its finished water from one or
more wholesale systems. Delivery may be through a direct connection or through the distribution system
of one or more consecutive systems [40 CFR § 141.2].
Consecutive systems are not exempt from the requirements of the LT2 Rule. However, consecutive
systems may receive water that a wholesale system has monitored, and treated if required, to comply with
the LT2 Rule. In this case, the consecutive system is not required to conduct additional monitoring or
install additional treatment on that water under the requirements of the LT2 Rule.
1.5 Monitoring Avoidance
Filtered systems are not required to conduct source water monitoring under the LT2 Rule if the system
will provide a total of at least 5.5-log of treatment for Cryptosporidium, equivalent to meeting the
treatment requirements of Bin 4 in the LT2 Rule [40 CFR § 141.701(d)(l) and 40 CFR § 141.711].
Unfiltered systems are not required to conduct source water monitoring under the LT2 Rule if the system
will provide a total of at least 3-log Cryptosporidium inactivation, equivalent to meeting the treatment
requirements for unfiltered systems with a mean Cryptosporidium concentration of greater than 0.01
oocysts/L in 40 CFR § 141.712 [40 CFR § 141.701(d)(2)].
If a system chooses to provide the level of treatment as described above, rather than start source water
monitoring, the system must submit written notification (Appendix A) no later than the date the system is
otherwise required to submit a sampling schedule for monitoring. Alternatively, a system may choose to
stop sampling at any point after it has initiated monitoring if it provides written notification that it will
provide this level of treatment.
Systems that are considering this option to avoid monitoring, or that believe that they already meet
the requirements to avoid monitoring, should consult with their point of contact as listed on the
contact list on the LT2 web site at http://www.epa.gov/safewater/disinfection/lt2/compliance.html.
Systems should review with the point of contact the microbial toolbox credits provided in the LT2 Rule,
and the PWS' existing treatment capabilities and any credits already determined by the State, to be sure of
the treatment capabilities needed to fulfill the LT2 Rule requirements. PWSs must install and operate
technologies to provide this level of treatment by the applicable treatment compliance date in 40 CFR §
141.713 [40 CFR § 141.701(d)(3)].
1.6 Reports and Notices
For the Initial Round of monitoring, the required reports and notifications for PWSs on Schedules 1-3
should be submitted through the options listed below in Table 1-6. Reporting procedures for PWSs on
Schedule 4 will be provided at a later date.
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Section 1: General Requirements
Table 1-6.
Submitting Reports and Notices
Report/Notice
Sampling Schedule *
Sample Location Description
Intent to Provide Maximum Treatment
Intent to Grandfather Data
Grandfathered Data
Grandfathered Data Supporting
Documentation
Initial Round Monitoring Data *
Options for submitting reports and notices
E mail
/
/
/
/
Hardcopy
/
/
/
/
/
DOTS
/
/
/
* Sampling schedules and Initial Round monitoring data must be submitted through the DCTS unless EPA or
State approve an alternative means [40 CFR §141.702(a)(2) and 40 CFR § 141.706(b)]. If the schedule is
not submitted through the DCTS by the required date, you may not be able to use the DCTS to input your
schedule and should call your point-of-contact as indicated on the contact list at
http://www.epa.gov/safewater/disinfection/lt2/compliance.html for assistance).
Depending on the option chosen, reports and notices should be submitted as follows:
Via email to: stage2mdbp@epa.gov
Hardcopies mailed or faxed to:
LT2ESWTR and Stage 2 DBPR
P.O. Box 98
Dayton, OH 45401
Fax: (937)586-6557
Submissions through the LT2/Stage 2 Data Collection and Tracking System (DCTS):
Access the DCTS through the LT2 web site at http://www.epa.gov/safewater/disinfection/lt2
12
February 2006
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Section 1: General Requirements
1.7 Questions?
In some cases, EPA will be your main point of contact during the first phases of the source water
monitoring. In other cases, your state will be your main point of contact. To identify your point of contact
for LT2 source water monitoring, visit EPA's website at
http://www.epa.gov/safewater/disinfection/lt2/compliance.html.
If after reading this guidance you still have questions, please submit them as follows:
• Submit them to your point-of-contact as indicated on the contact list at
http://www.epa.gov/safewater/disinfection/lt2/compliance.html.
Or
• Send them via email to stage2mdbp@epa.gov.
Or
• Call the Safe Drinking Water Hotline at 1 -800-426-4791
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14 February 2006
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SECTION 2: CONTRACTING FOR LABORATORY SERVICES
Although many public water systems (PWSs) have established procedures and policies governing the
purchase of services and supplies, these procedures may not lend themselves to the purchase of analytical
services. This section provides a basic framework for addressing the technical and contractual issues
associated with purchasing laboratory services for the LT2 Rule, awarding contracts, and working with
contract laboratories. In several instances a separate laboratory will be necessary for Cryptosporidium
analysis from the laboratory performing E. coli analysis. Many Cryptosporidium laboratories do not
perform E. coli analysis, and PWSs will need to have their coliform laboratory perform the E. coli
analysis, or contract with a different laboratory.
Successfully contracting for laboratory services for LT2 Rule monitoring relies on the following steps:
Step 1: Define the scope of your analytical requirements to develop a detailed contract
Step 2: Develop a standardized bid sheet/cost estimate (Appendices B and C)
Step 3: Solicit approved/certified laboratories
Step 4: Award contracts to a primary laboratory(ies) and a backup laboratory(ies)
Step 5: Work closely with your laboratory(ies) before monitoring begins and maintain communications
throughout monitoring
These general steps, and details on the activities associated with each, are discussed in Sections 2.1
through 2.5. Whether you contract with one laboratory for both Cryptosporidium and E. coli analyses or
separate laboratories, the same general procedures apply.
Remember: you must use an approved laboratory for Cryptosporidium analysis and a certified laboratory
for E. coli analysis, as described in Section 2.3 below [40 CFR § 141.705].
2.1 Defining Your Needs and Developing a Contract
The first step in developing an analytical services contract for analyses for LT2 Rule monitoring is
identifying the "who, " "what, " "when, " and "how " of the project for your system (the "why" is the LT2
Rule itself). A well-written contract will address each of these issues, as well as the administrative issues,
such as laboratory payments and adjustments.
The best way to ensure that you get the data you need for LT2 Rule monitoring within the required time
period is to specify your requirements in detail in the contract. A well-written contract can minimize or
eliminate many common problems in procuring analytical services, and enable you to collect reliable and
timely results.
Recommendations on the factors to consider in defining the scope of the services you need, and the
information you should be sure to include in your contract, are provided below.
15 February 2006
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Section 2: Contracting for Laboratory Services
2.1.1 Client Information
"Who " defines your PWS to the laboratories that you would like to submit bids for the project. Will you
be contracting for laboratory services for a single plant or will this contract require analyses to fulfill
monitoring requirements for multiple plants in a system?
Clearly identify in your contract the name and public water system identification number (PWSID)
of your PWS, as well as the name(s) and identification number of the facility(ies) for which samples
need to be analyzed. This information ultimately will be used to identify your samples in the
LT2/Stage 2 Data Collection and Tracking System (DCTS); the laboratory(ies) you use for sample
analyses will need to know this information. (Alternately, you can provide this information after
award to the awarded laboratory(ies) only.)
2.1.2 Sample Information
"What" describes the samples to be analyzed. As noted in Sections 2.1.2.1 through 2.1.2.5, this
encompasses a variety of factors, each of which should be evaluated and defined before you develop your
contract.
2.1.2.1 Number of Samples
What is the total number of samples the laboratory(ies) will need to analyze? Will your PWS monitor
once per month or twice or more per month? This total includes not only routine monitoring samples
(field (monitoring) samples), but also the Cryptosporidium matrix spike (MS) samples that are required at
a frequency of 1 per 20 field (monitoring) samples. Field (monitoring) samples and MS samples are
considered "billable" samples (sample analyses for which the laboratory will be paid their per-sample
cost).
Internal laboratory quality control (QC) samples, such as method blanks and ongoing precision and
recovery (OPR) samples should be considered "unbillable" samples—sample analyses that are required,
but apply to multiple PWS clients. Rather than charging clients for these samples directly, laboratories
typically will distribute the costs of these samples across billable samples.
If your PWS has not sampled for Cryptosporidium or worked with the lab previously, you should
consider adding a couple of practice samples to make sure all sampling systems are functioning
properly before starting required monitoring.
If a sample is collected and sent to the laboratory, but cannot be submitted under the LT2 Rule because of
a problem unrelated to laboratory performance (such as shipping delays that violate the sample holding
time), your PWS will be required to collect a replacement sample for Cryptosporidium analysis (see
Section 3.2.2 for details). You should add to the contract, as an option to be exercised at your direction in
such an event, additional sample analyses as replacement samples. Appendices B and C provide a
worksheet to estimate the number of samples including matrix spike, practice and replacement samples.
16 February 2006
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Section 2: Contracting for Laboratory Services
Clearly indicate in your Cryptosporidium contract the total number of: (1) field (monitoring) samples
and (2) MS samples that the laboratory will be required to analyze. Be sure to include additional
optional sample analyses that can be exercised if replacement samples are required due to
problems unrelated to laboratory performance. Adding practice samples is also a good idea.
2.1.2.2 Type of Samples
Will your PWS collect and ship bulk water samples to the laboratory for filtration and processing or will
your PWS filter samples onsite and ship the filter to the laboratory? Shipping and analytical costs are
likely to be lower if you filter your samples onsite, but you will need to purchase or rent sample filtration
equipment (see Section 4.1 for details) and have staff trained to use the required procedures or pay for the
laboratory or another firm to perform these tasks.
Clearly specify in the contract whether the laboratory will receive bulk water samples or filtered
samples. If filtered samples will be sent, indicate which filter you will use (see Section 2.1.4.2).
If you will be filtering onsite, and will be using your own equipment to filter the samples, you can
purchase filters directly from the vendor or through your Cryptosporidium contract laboratory. Be sure to
indicate clearly in the contract whether filters should be included in the laboratory's' bid price.
(Additional information on filtering samples onsite and purchasing filters is provided in Section 4.1.2).
Matrix spike samples must be shipped to the lab as 10 L bulk samples [40 CFR § 141.704(a)(2)].
If your PWS will be purchasing filters directly, specify this in the contract, so the laboratory knows
not to include this in their per-sample price. Be sure to purchase extra filters if the source water to
be sampled is highly turbid. PWSs with highly turbid source water must filter as much volume as
two filters can accommodate before clogging.
For E. coll, all samples should be collected in 125 mL or 250 mL bottles and shipped to the laboratory for
analysis.
2.1.2.3 Anticipated Sample Volume
The LT2 Rule [40 CFR § 141.704(a)(l)] requires that at least 10 L be analyzed for each sample for
Cryptosporidium (with some exceptions - see Section 4.2). Will your PWS collect 10 L samples or collect
higher-volume samples, such as 50 L samples? If your PWS will be shipping bulk water samples to the
laboratory, greater sample volumes will result in higher shipping costs and will likely result in higher
analytical costs. If your PWS will be filtering samples on-site and shipping filters to the laboratory, the
sample volume should not affect shipping or analytical costs, but the greater sample volumes filtered may
result in higher packed pellet volume and multiple subsamples (Section 2.1.2.4, below).
Clearly indicate in the contract the volume you anticipate collecting for each Cryptosporidium
sample.
17 February 2006
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Section 2: Contracting for Laboratory Services
For E. coll samples, the sample volume collected should be at least 100 mL.
Clearly indicate in your contract for Cryptosporidium analyses that different sample prices are
needed for: (1) full sample analyses, (2) subsample analyses, and (3) extra filters and the cost of
analysis of the extra filters.
2.1.2.4Subsamples and Filter Clogs
Additional steps are required at the laboratory for samples that generate more than 0.5 mL of packed
pellet volume or samples that clog before 10 L have been filtered. Specifically, the laboratory will need to
process two or more "subsamples" through the method or process two filters to meet LT2 Rule sample
volume analysis requirements [40 CFR § 141.704(a)(l)]. If you know that the source water(s) to be
monitored by your PWS are sometimes characterized by turbidity levels containing excessive particulates
that interfere with filtration or sample purification, you will need to consider that some of your samples
may need to be processed as multiple subsamples or may require two filters. One option for estimating the
number of subsamples is to send the laboratory a 10L sample (either bulk or filtered) during a peak
turbidity event for concentration by the lab. Alternatively, you may want to allow for the possibility that
some samples may require multiple analyses. By including subsample costs in the original contract
(which would apply only if you encountered this issue), you will avoid changes to the contract on short
notice if subsamples are required during monitoring.
For E. coli, if the laboratory uses a membrane filter method and experiences clogging of the
membrane filter, smaller volumes should be filtered or another method should be chosen.
2.1.2.5 Extra Services
Will any additional services be required of the laboratory outside of actual sample analyses? Possible
services include:
Sampling kit rental or purchase for on-site filtration
Sample shipping containers
• Sample archiving for Cryptosporidium
• Training for sample collection personnel
• Extra analytical time for challenging water matrices
Some of these services may be included in the sample analysis cost by some laboratories. Defining the
specific services your PWS will need, and specifying these services clearly in the contract will enable the
laboratories to better assess whether the requested services are included in their routine costs or are extra,
and respond accordingly.
Clearly specify in your contract any services required in addition to routine sample analysis.
18 February 2006
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Section 2: Contracting for Laboratory Services
2.1.3 Sampling Schedules
Table 1-1 and Section 1 of this guidance provide information regarding the minimum required sampling
frequency for PWSs. As described in Section 2.1.2.1 earlier, you should consider the minimum
requirements for your PWS and determine if you will sample more often.
If at all possible, do not establish a firm sampling schedule with specific dates at this point. Most of the
laboratories available to perform Cryptosporidium analyses have multiple PWS clients and need to evenly
distribute their sample load within each week and across weeks in a month to meet holding time
requirements. Rather than dictating a sample collection schedule to the laboratory-and potentially
discouraging laboratories from bidding on the work or risk violating holding times during monitoring-
work with the awarded laboratory to establish a schedule that will comply with LT2 Rule requirements
and is mutually acceptable to your PWS and the laboratory. Section 3 provides information on the
sampling schedule that must be submitted prior to monitoring.
Indicate in your contract the month that you plan to begin monitoring and how frequently you will
monitor.. If possible, do not specify actual sample collection dates and days during the week; work
with the awarded laboratory to establish a schedule that meets your needs and does not cause
problems for the laboratory.
2.1.4 Analytical Methodology for Cryptosporidium
"How " describes the analytical method that the laboratory will use. This involves two sets of options for
Cryptosporidium: which method to use (EPA Method 1622 or EPA Method 1623) and which filter to use,
regardless of method. It also refers to the QC requirements that must be met during sample processing and
analysis (Reference 8.3 and 8.4).
2.1.4.1 EPA Method 1622 Versus EPA Method 1623
Will your PWS monitor for Cryptosporidium only or Cryptosporidium and Giardial Method 1623
targets both Cryptosporidium and Giardia. Method 1622 is identical but reagents are specific to only
Cryptosporidium. The LT2 rule does not require monitoring for Giardia, and only Cryptosporidium data
need to be submitted. However, most laboratories analyze samples for both Cryptosporidium and
Giardia using EPA Method 1623 because Giardia serves as a good internal control, provides extra
information to the PWS and may be offered at a less expensive price than Cryptosporidium-on\y due to
QA/QC considerations. The method should be agreed upon by the laboratory and the PWS, and specified
in the contract. Some labs may not offer 1622 analysis if they don't have sufficient clients requesting it.
2.1.4.2 Filter Options
Unless you have a reason for specifying a particular filter, leave this up to the laboratory in your contract.
If your PWS has experience monitoring for Cryptosporidium and has a filter preference, you will need to
indicate this to the laboratories interested in bidding on the project, as not all laboratories are approved by
EPA through the Lab QA Program to perform all versions of the methods.
19 February 2006
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Section 2: Contracting for Laboratory Services
If your PWS has experience with Cryptosporidium sampling and would like analyses performed
using a specific filter, clearly indicate this in the contract. Otherwise, do not specify a filter type.
2.1.4.3 Quality Control Requirements
Although EPA Methods 1622 and 1623 (Reference 8.3 and 8.4) specify the QC requirements that must be
met during performance of the method, your contract should reiterate that all of the QC requirements for
the method must be met at the required frequency during processing and analysis of your samples. As
noted earlier in Section 2.1.2.1, the costs for the method blank, ongoing precision and recovery, and
staining control tests should be distributed by the laboratory across the cost of sample analysis.
Reiterate in the contract that method blanks, ongoing precision and recovery tests, and staining
controls must be performed at the frequency required in the method, and that all holding times
must be met.
2.1.5 Analytical Methodology for E. coli
The choice of method may depend on what methods your laboratory is certified to perform and the
quality of your source water. Unless you have a reason to select a particular method, it is best to allow the
laboratory to determine the E. coli method.
None of the QC requirements should be billable, but rather should be distributed by the contract
laboratory across the cost of monitoring samples for all of their clients.
2.1.6 Data Deliverables and Other Contract Issues
In addition to the "who, " "what, " "when, " and "how " questions that should be addressed by the
contract, you also should provide details on data delivery, adjustments for lateness, and sample reanalysis
cost issues. These issues are discussed in Sections 2.1.6.1 through 2.1.6.5.
2.1.6.1 Data Submission
EPA has developed the web-based LT2/Stage 2 Data Collection and Tracking System (DCTS) to allow
laboratories to report data to PWSs electronically and allow PWSs to verify the data electronically before
submitting the monitoring results to EPA [40 CFR § 141.706]. This reporting process is summarized in
Section 6.3 for Cryptosporidium data and Section 7.3 for E. coli and turbidity data, and discussed in detail
in the Users' Manual for the LT2/Stage 2 Data Collection and Tracking System (DCTS). The laboratory,
at a minimum, should submit the results for each monitoring sample to you electronically. (Although your
PWS also could enter these data into the DCTS, based on hardcopy results from the laboratory, this is
strongly discouraged, as the potential for error increases when personnel unfamiliar with the generation of
the data for a sample enter these data into the DCTS.)
20 February 2006
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Section 2: Contracting for Laboratory Services
Clearly indicate in your contract that the laboratory is required to enter monitoring results for your
samples into the LT2/Stage 2 Data Collection and Tracking System. Specify that all laboratory
data must be recorded on appropriate laboratory bench sheets.
2.1.6.2 Hardcopy Data Deliverables
Note: If you do not intend to review all of the raw data generated by the laboratory, this section is not
relevant and can be ignored.
If your PWS intends to review all of the raw data associated with your LT2 samples (discussed in Section
6), you should request copies of the forms used by the laboratory to record sample measurements, sample
processing times, and sample examination results, as well as information on the QC samples associated
with your monitoring sample. (Original data forms should stay at the laboratory; copies can be sent to the
PWS. If bench sheets, etc., are requested on a monthly basis, the PWS may expect additional charges
from the laboratory)
PWSs that want to review raw data for each Cryptosporidium analysis should request the following:
• Monitoring sample identification information
Monitoring sample result, in oocysts/L
Laboratory quality control batch associated with the sample
• ID number and result for the ongoing precision and recovery (OPR) sample analyzed for this QC
batch
ID number and result for the method blank sample analyzed for this QC batch
• LT2 sample collection form initiated by your utility and completed with sample receipt information
by the laboratory
Method 1622/1623 Bench Sheet with raw data associated with the monitoring sample (and MS
sample, if applicable)
• Method 1622/1623 Cryptosporidium Slide Examination Form with raw data for the monitoring
sample (and MS sample, if applicable)
Laboratory comments. If the laboratory provided comments on the sample analyses or results that
require follow-up, contact the laboratory to discuss, if necessary. Comments may include any
applicable data qualifiers. The following is a list of potential data qualifiers:
The recovery for the associated ongoing precision and recovery (OPR) sample did not meet
method requirements
Oocysts were detected in the method blank
Positive and negative staining controls were not acceptable or not examined
Method holding times were not met
Sample arrived at the laboratory in unacceptable condition
21 February 2006
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Section 2: Contracting for Laboratory Services
PWSs that want to review the raw data for each E. coll analysis should request the following:
• Monitoring sample identification information
• Monitoring sample result, in E. co///100 mL
E. coli Method Bench Sheet with raw data for the monitoring sample
Laboratory comments. If the laboratory provided comments on the samples analyses or results that
require follow-up, contact the laboratory to discuss, if necessary. Comment may include any
applicable data qualifiers. See section 7.4.1 of this manual for a list of possible data qualifiers for E.
coli analysis.
If you want the laboratory to submit hardcopy results (this should not be requested unless you
intend to review all of the raw data), clearly indicate in your contract the materials that are required.
You may also choose to request a hard copy of only the summary results.
2.1.6.3 Data Turnaround Requirements
Under the LT2 Rule, PWSs are required to submit data no later than 10 days after the end of the first
month following the month when the sample is collected (this is approximately 40 to 70 days after sample
collection, depending on when during the month the sample is collected ) [40 CFR § 141.706(a)]. For
example, if a sample is collected on March 17, data must be submitted no later than May 10.
The turnaround requirement for the laboratory should be shorter to provide your PWS time to review the
data before the submission deadline to EPA. The required data turnaround should be stated clearly in the
contract. This turnaround time should be expressed in calendar days (not working days), and should start
from the sample collection date. The data turnaround time calculations should consider the day that the
sample is collected "day zero," and the following day as "day one." (Data turnaround times in analytical
contracts typically start from the receipt of the sample at the laboratory, but calculating it from the sample
collection date is more logical in this case because the LT2 Rule's data submission requirements are
based on sample collection date.)
If the data turnaround time starts from sample collection, rather than sample receipt by the laboratory, this
turnaround should accommodate the potential for shipping delays that will be outside of the laboratory's
control. The maximum shipping time is 4 days, including delays. This includes up to 4 days between
sample collection and initiation of the elution step, which effectively is the maximum time for any
shipping delay, as samples received more than 4 days after collection will not be valid and cannot be
submitted through the DCTS. As a general rule, the data turnaround time should be less than 15 days.
Using the 15 days allowed for sample analysis by the methods (plus additional time to compile the data
package and mail the results, if hardcopies are required) as the shortest realistic turnaround time,
determine when you will actually need the results. The same turnaround time can be specified for both
submission of electronic data and receipt of hardcopy materials.
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Section 2: Contracting for Laboratory Services
2.1.6.4 Liquidated Damages and Penalties
You should consider including penalty or damage clauses in your contract as incentives to preclude
laboratories from submitting data late or performing analyses improperly. Due to the nature of the
services provided, assessing actual damages caused by improperly performed analyses is often difficult.
Liquidated damages often are used in analytical services contracts in lieu of actual damages. Liquidated
damages typically specify that, if the laboratory fails to deliver the data specified in the deliverables
section of the contract, or fails to perform the services within the specified data turnaround time, the
laboratory will pay a fixed, agreed, price to compensate the organization to whom the services should
have been delivered. For example, some EPA contracts for analytical services specify that the laboratory
will pay, as fixed, agreed, and liquidated damages, 2 percent of the analysis price per calendar day of
delay, to a maximum reduction of 50 percent of the analysis price.
If liquidated damages or penalties are involved, they should (1) be based in terms of cost by each late day,
(2) be strong enough to discourage late delivery, and (3) be reasonable enough that they will not
discourage laboratories from bidding. The contract should specify that the laboratory will not be charged
with liquidated damages when the delay in delivery or performance arises out of causes beyond the
control and without the fault or negligence of the laboratory. It also may be necessary to limit damages to
a certain dollar value or scope.
Other types of damages that should be considered and may be included in the contract include costs for
resampling and administrative costs associated with the evaluation and processing of unacceptable data
(data that do not meet the requirements specified in the contract or the QC requirements specified in the
analytical method).
*& Clearly indicate in your contract whether liquidated damages will be applied to late data or other
problems, how these liquidated damages are calculated, and the limits and conditions associated
with the damages.
2.1.6.5 Re-Analysis Costs
Every laboratory periodically produces data that are associated with unacceptable QC data or are invalid
for other reasons. The contract should stipulate that the laboratory will reanalyze samples at no cost to
your PWS if the problems are due to laboratory error. If the problems are due to an error outside of the
laboratory's control (such as the laboratory's rejection of a Cryptosporidium sample received at > 20°C
that results in resampling by your PWS), the laboratory should not be responsible for the additional costs
that may result.
Clearly indicate in your contract when the laboratory would be required to bear the costs of sample
re-analysis and when these costs will be borne by your PWS.
The contract also should state that you have the right to inspect the results, and if they do not meet the
requirements in the contract, you have the right to reject the data, returning them to the laboratory without
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Section 2: Contracting for Laboratory Services
payment. Rejection of data should be based on sound technical review of the results. It also obligates you
to make no use of those results without making some payment to the laboratory.
Clearly indicate in your contract that your PWS has the right to inspect results and reject the
results if they do not meet contract requirements.
2.2 Developing a Bid Sheet
After all project requirements have been established, you should develop a bid sheet to accompany the
analytical requirements summary during the solicitation. The bid sheet allows laboratories to submit bids
in the same format, making bid evaluations easier, and also helps to clarify the project. Development and
use of a bid sheet is recommended regardless of whether your PWS solicits the project competitively to
multiple laboratories, or is simply requesting a quote from a laboratory you already know you will be
using, as it provides a very clear vehicle for submitting and evaluating costs.
Bid sheets for analytical services typically are formatted as a table, with costs in the columns and
descriptions of services and supplies heading the rows (Appendices B and C).
The bid sheet should include the following information:
• Project identifier (e.g. "LT2 Monitoring Sample Analyses for [PWS name and/or facility name]")
• Space for laboratory identification information (for when they submit their bid)
Day, date, and time (including time zone) of the bid deadline
PWS information (contact and mailing address, fax number, phone number, and/or email address)
Estimated award date
• Laboratory period of performance (includes the period of time during which the laboratory is obliged
to resolve issues associated with analysis of the samples—generally 6 months after shipment of last
sample)
Data turnaround time (time from sample collection to reporting results)
Bid validity period (period of time during which bid prices are considered valid—generally 45 days
after the bid deadline; if the project is awarded after the period you specify, you must contact bidding
laboratories to determine whether their bid is still valid, or needs to be revised)
• A summary of the analytical requirements:
Method (e.g., Cryptosporidium and Giardia by EPA Method 1623)
E. coli method of choice (if needed)
Filter preference, if any. (This should not be specified unless your PWS has experience with
Cryptosporidium and a basis for requesting the use of a specific filter; if you know that you will
be field filtering using a specific filter and shipping this to the laboratory, you should specify this)
Whether samples will be shipped as filtered samples or bulk water samples for Cryptosporidium
Sample volume for Cryptosporidium (e.g., 10 L, 50 L)
Total number of field (monitoring) samples to be analyzed, plus extra, in case of replacement samples
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Section 2: Contracting for Laboratory Services
• Two optional "practice" samples
• Total number of MS samples to be analyzed for Cryptosporidium, at least 1 for every 20 samples.
• Total number of potential subsamples to be analyzed for Cryptosporidium (expressed as "Up to [no.]
subsamples" so you are not committing to this - just leaving it as an option)
The number generally should not exceed four per sample
If you have high-turbidity water, you may need to specify up to four subsamples for all of your
field (monitoring) and MS samples
If you have a low-turbidity water, you should specify a minimal number, just in case the need
arises
(These costs would not be incurred unless subsamples actually have to be analyzed)
• Total number of potential extra filters for Cryptosporidium (in case one or more samples clog during
LT2 Rule monitoring):
If you will be shipping bulk samples to the laboratory, express this as "Up to [no.] extra
filters/elutions"
If you will be filtering samples in the field, but receiving filters from the laboratory, express this
as "Up to [no.] extra filters"
(These costs would not be incurred unless more than one filter actually has to be used)
Columns for laboratories to enter per-analysis and total costs
• Costs for cubitainers or carboys, if you would like the laboratory to provide this
• Cost of sampling apparatus, if you would like the laboratory to provide this
• Cost of shipping supplies to PWS, if applicable
2.3 Soliciting the Contract
Procedures for soliciting and awarding contracts to perform analytical services can vary, depending upon
the scope of the project and purchasing requirements within the organization that is issuing the contract.
At one end of the spectrum are contracts that are awarded after placing a single phone call and obtaining a
quote from a single laboratory. The opposite end of the spectrum are contracts awarded after a
competitive solicitation and bidding process involving the distribution of a detailed project description
and a formal bid sheet via fax or mail.
2.3.1 Approved Laboratories for Cryptosporidium
Regardless of whether you will be soliciting the project to multiple laboratories or working with a single
laboratory (although a backup laboratory is strongly recommended—see below), you must limit your
laboratories to only those approved by EPA through the Laboratory Quality Assurance Evaluation
Program for Analysis of Cryptosporidium Under the Safe Drinking Water Act (Laboratory QA Program)
or approved for Cryptosporidium by an equivalent State approval program [40 CFR § 141.705(a)].
However, at the time of publication of this guidance document there were no equivalent State programs
for approval of Cryptosporidium laboratories. Information on the Laboratory QA program and a list of
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Section 2: Contracting for Laboratory Services
approved laboratories are posted at http://www.epa.gov/safewater/disinfection/lt2/lab home.html. The
Laboratory QA Program is also described in detail in the Microbial Laboratory Guidance Manual for the
Long Term 2 Enhanced Surface Water Treatment Rule (LT2 Rule) that is also available at
http://www.epa.gov/safewater/disinfection/lt2/lab home.html.
Briefly, the objectives of the program are to evaluate laboratories' competency to measure reliably for the
occurrence of Cryptosporidium in surface water using EPA Method 1622/1623. Each laboratory
participating in the program is required to complete the following steps to be qualified through this
program:
Participate in an on-site evaluation of their technical, data management, and quality assurance
procedures
Acceptably perform proficiency testing on blind samples every 4 months
2.3.2 Certified Laboratories for E. coli
The PWS must choose a laboratory for E. coli analysis that is certified by EPA, the National
Environmental Laboratory Accreditation Conference (NELAC) or the State for total coliform or fecal
coliform analysis under 40 CFR § 141.74. The laboratory must use the same technique for E. coli
analysis as the technique for which they are certified to perform total or fecal coliform analyses
under 40 CFR § 141.74. For a list of certified laboratories, the PWS may refer to their State's drinking
water laboratory certification web page (if available) or contact their State drinking water laboratory
certification officer. A list of E. coli methods that certified laboratories can perform under the LT2 Rule is
provided in Table 7-1. EPA notes that this approach differs from the approach typically used in its
Laboratory Certification program in that the latter program is based on certification for the specific
method (not simply the same technique) being used in compliance monitoring.
2.3.3 Primary and Backup Laboratory Contracts
Because a laboratory's approval status could change during the LT2 Rule monitoring period, you should
plan to award a primary contract and a backup contract. If no performance problems or other problems are
encountered during the LT2 Rule monitoring period by the laboratory awarded the primary contract, then
this laboratory would provide uninterrupted sample analysis support for the entire monitoring period.
However, if the laboratory encountered performance problems and was disapproved, or was otherwise
unable to meet contract requirements, your PWS could switch sample analyses to the backup laboratory
under the contract you established with this laboratory before monitoring began. You may discuss the
award of primary and backup contracts with the laboratories in the contract solicitation.
2.4 Evaluating Bids
After the laboratories have received the solicitation and submitted their bids, you may evaluate the bids to
identify the laboratory that will be awarded the analytical services contract. Specific procedures for
evaluating bids may vary, depending upon the requirements of your organization, but the bid evaluation
process generally entails evaluation and comparison of each laboratory's proposed cost and capability to
meet the analysis requirements.
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Section 2: Contracting for Laboratory Services
2.4.1 Identifying Responsive Bidders
You may consult your legal department or purchasing department to identify any applicable requirements
for evaluating competitive bids. Review all bids and recalculate subtotals and totals to ensure that the
bidding laboratories did not make any mathematical errors. In addition, you may want to verify that there
are no unacceptable contingencies associated with any of the bids. Either eliminate from consideration
bids from laboratories that bid with contingencies, or contact the laboratory(ies) to discuss the bid and
verify that the laboratory cannot perform the specified services.
Of the remaining (responsive) bids, identify the lowest bidder (or the laboratory that best meets your
requirements) to award the primary contract and a second bidder to award the backup contract. If
additional assessments of a laboratory's performance or responsibility are needed, you may want to
contact references.
2.4.2 References
If you have not worked with a particular laboratory before and would like to verify that the laboratory will
meet your needs throughout the monitoring period, you can ask the laboratory to provide contacts and
phone numbers of utility or government clients for whom the laboratory has performed Cryptosporidium
or E. coli sample analyses or other comparable services.
Questions to ask the references include:
• Did the laboratory provide data by the required due date?
Were the data provided by the laboratory of acceptable quality and compliant with contract
requirements and in an easy to understand format?
• Were laboratory personnel easy to work with when problems arose during all phases of the project,
including sample scheduling, sample analysis, and data review? If problems were noted during data
review, was the laboratory prompt and responsive in addressing your concerns?
Do you have any reservations in recommending this laboratory?
2.5 Communicating with the Laboratory
After the analytical services contract is awarded, request laboratory contact information for the following
roles, and provide the laboratory with PWS contacts for the same roles:
A technical contact for analytical questions or problems
A sample control contact for shipping delays on the PWS end and sample receipt problems on the
laboratory end
An administrative contact for invoicing and payment
Maintaining communications with the laboratory is critical to identifying and resolving problems quickly
and minimizing the need for resampling and reshipments. At a minimum, notify the laboratory of sample
shipments the day you ship and confirm that the laboratory received the sample on time and in acceptable
condition. You also can consider contacting the laboratory each week before you sample to verify that
they know to expect samples.
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Section 2: Contracting for Laboratory Services
Although most communications are typically conducted over the phone, these communications also can
be conducted via email, which has the added benefit of providing your PWS and the laboratory with a
written record of sample receipt confirmations, problem notifications, and problem resolutions.
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SECTION 3: SAMPLING LOCATION AND SCHEDULE
Public Water Systems (PWSs) required to monitor under the LT2 Rule must meet specific requirements
regarding where and when to sample, and must submit a sample location description and sample
schedule. As noted previously, monitoring requirements for each system size and the schedule for each
stage of monitoring are described in Table 1-1. This section provides guidance for meeting the
requirements regarding submission of sampling location descriptions and sampling schedules as required
by the rule.
3.1 Sample Collection Location and Description
LT2 Rule monitoring is intended to assess the mean Cryptosporidium level in the influent to drinking
water plants that treat surface water or ground water under the direct influence (GWUDI) of surface
water.
PWSs are required to collect source water samples for the LT2 Rule from each plant intake prior to
chemical treatment, unless the State approves the system to collect a source water sample after chemical
treatment [40 CFR § 141.703(b)(2)]. To grant this approval, the State must determine that collecting a
sample prior to chemical treatment is not feasible for the system and that the chemical treatment is
unlikely to have a significant adverse effect on the analysis of the sample [40 CFR § 141.703(b)]. PWSs
that recycle filter backwash water must collect source water samples prior to the point of filter backwash
water addition [40 CFR § 141.703(c)]. All Cryptosporidium, E. coll, and turbidity source water samples
collected under LT2 Rule requirements must be collected prior to chemical treatment [40 CFR §
141.703(b)] and should be collected from the same sampling location.
Generally, monitoring is required for each plant that treats a surface water or GWUDI source. However,
where multiple plants receive all of their water from the same influent (e.g., multiple plants draw water
from the same pipe or intake) the State may approve one set of monitoring results to be used to satisfy the
requirements for all plants [40 CFR § 141.703(a)].
PWSs must submit a description of their sampling location(s) at the same time as the sampling schedule
required under this rule [40 CFR § 141.703(f)]. This description must address the position of the sampling
location in relation to the system's water source(s) and treatment processes, including pretreatment, points
of chemical treatment, and filter backwash water recycle.
Appendix D is provided as a worksheet to assist PWSs in describing proper sampling locations. Some of
the information from Appendix D should be submitted with each sample and will be useful to know
when determining sample location, such as
• Public Water System Identification (PWSID)
• Water system facility identification. This is the ID, usually assigned by the State, which uniquely
identifies a water system facility. In the case of the LT2 Rule, this is an individual water treatment
plant. If there is no State-generated or other number provided by the PWS, the DCTS will
autogenerate one. The PWS can change this number in the DCTS if desired.
• Source water sample collection point number. This is a number, usually assigned by the State,
which uniquely identifies a point within a water system facility from which the sample is collected.
The DCTS uses this number to track sample results. If there is no State-generated or other number
provided by the PWS, the DCTS will autogenerate one. The PWS can change this number in the
DCTS if desired.
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Section 3: Sampling Location and Schedule
Appendix E provides 9 example sample location schematics to illustrate the correct sample collection
location. A blank sample location schematic is also provided. A completed worksheet (Appendix D)
accompanied by a sampling location schematic from Appendix E (a marked up version of an example
schematic or a blank schematic (figure 10)) may be submitted to meet the sampling location description
requirement.
Additional guidance on sampling at plants is provided below, in Sections 3.1.1 through 3.1.6.
3.1.1 Plants That Do Not Have a Sampling Tap Located Prior to Any Treatment
Plants in this situation may pursue one of the following options:
• Manually collect source water samples as close to the intake as is feasible, at a similar depth and
distance from shore.
• Install a sampling tap prior to treatment.
• Collect a source water sample after chemical treatment only if it is determined by the State that
collecting a sample prior to chemical treatment is not feasible and that the chemical treatment is
unlikely to have a significant adverse effect on the analysis of the sample [40 CFR § 141.703(b)(2)].
3.1.2 Plants That Use Different Water Sources at the Same Time
This includes multiple surface water sources and blended surface water and ground water sources. The
use of multiple sources during monitoring must be consistent with routine operational practice.
Plants in this situation should pursue one of the following options:
• If there is a sampling tap after the sources are combined but prior to treatment, the sample must be
collected from the tap [40 CFR § 141.703(e)(l)].
• If a sampling tap located after the sources are combined but prior to treatment is not available, PWSs
must collect samples at each source near the intake on the same day and must use one of the
following options for sample analysis [40 CFR § 141.703(e)(2)]:
Compositing: Samples may be collected manually at each source near the intake on the same day
and composited into one sample. The volume of sample from each source should be weighted
according to the proportion of that source used by the plant. For example, if a plant has two
sources and 75 percent of the drinking water is from Source A and 25 percent is from Source B,
then for a 10 L sample, 7.5 L would be collected from Source A and combined with 2.5 L
collected from Source B. Compositing of samples should reflect plant operation at the time the
sample is collected and may change during the monitoring period.
Weighted Average: Separate samples may be collected manually at each source near the intake
on the same day and analyzed independently. The individual results must then be used to
calculate a weighted average of the analysis results. The weighted average must be calculated by
multiplying the analysis result for each source by the fraction of the source contribution to total
plant flow at the time the samples were collected, and then summing these values. For example, if
a plant has two sources and 75 percent of the drinking water is from Source A and 25 percent is
from Source B, then one sample would be collected from each source and analyzed
independently. If the concentration of oocysts for the sample from Source A was 5 oocysts/L and
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Section 3: Sampling Location and Schedule
the concentration of the sample from Source B was 1 oocyst/L, the final result for the plant for
this sampling event would be 4 oocysts/L ([5 oocysts/L x 0.75] + [1 oocyst/L x 0.25]).
3.1.3 Plants That Use Presedimentation
PWSs may collect samples after presedimentation if no chemical treatment (for example, coagulation) is
used in or prior to the pre sedimentation basin. If chemical pretreatment is used, PWSs must collect
samples prior to presedimentation or get permission from the State to sample after chemical treatment as
described in Section 3.1. Use of presedimentation basins during monitoring should be consistent with
routine operational practice and should be documented. However, if samples are collected after (i.e.,
downstream of) the presedimentation process, the PWS will not be eligible for Cryptosporidium treatment
credit for the presedimentation process. In this case, the removal achieved by the presedimentation
process will be reflected in the monitoring results and bin classification. PWSs that collect samples prior
to the presedimentation basins may be eligible for additional treatment credit as described in 40 CFR §
141.717.
3.1.4 Plants That Use Raw Water Off-Stream Storage
For these plants, source water samples should be collected after the off-stream storage reservoir. Use of
off-stream storage during monitoring should be consistent with routine operational practice and should be
documented.
3.1.5 Plants That Use Bank Filtration
The correct sampling location for PWSs with plants using bank filtration differs depending on whether
the bank filtered water is treated by subsequent filtration for compliance with the Interim Enhanced
Surface Water Treatment Rule (IESWTR) [40 CFR § 141.703(d)].
• PWSs that receive Cryptosporidium treatment credit for bank filtration towards compliance with the
IESWTR or the Long TermlEnhanced Surface Water Treatment Rule, as applicable, must take source
water samples in the surface water prior to bank filtration [40 CFR § 141.703(d)(l)].
• PWSs that use bank filtration as pretreatment to a filtration plant must collect source water samples
from the well (i.e., after bank filtration). Use of bank filtration during monitoring must be consistent
with routine operational practice and should be documented. PWSs collecting samples after a bank
filtration process may not receive treatment credit for the bank filtration under 40 CFR §
141.717(c)[40 CFR § 141.703(d)(2)].
3.1.6 Plants That Use Ground Water Under the Direct Influence of Surface Water
PWSs using a source of ground water under the direct influence of surface water must collect source
water samples from the ground water (e.g., the well) unless they received credit for bank filtration as
described above.
3.1.7 Submitting Sample Location Descriptions
Sample location descriptions will not be considered confidential business information and are
subject to the Freedom of Information Act. Therefore, the submission should not contain
information that poses a security risk to the system. The worksheet provided in Appendix D and the
schematics provided in Appendix E are generic enough that they should not pose a security risk.
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Section 3: Sampling Location and Schedule
PWSs must submit the sampling location descriptions by the deadlines indicated in Tables 1-2 through
1-5. The sampling location descriptions should be submitted as directed in Section 1.6 of this manual.
3.2 Source Water Sampling Schedule
PWSs required to conduct source water monitoring must submit a sampling schedule that specifies the
calendar dates when the system will collect each required sample. PWSs are required to collect samples in
accordance with the schedule, which should be established and agreed to by the PWS and the laboratory
prior to submission. PWSs should contact the laboratory as early as possible to establish a mutually
acceptable sampling schedule if they want to be sure that the laboratory can analyze samples on the
desired dates. PWSs may collect samples more frequently than required (e.g., twice-per-month, weekly),
provided the samples are evenly spaced throughout the monitoring period [40 CFR § 141.701 (a)(7)].
PWSs are required to collect samples within two days before or after the dates indicated in their sampling
schedules [40 CFR § 141.702(b)].
The schedule should be entered using the scheduler function within the LT2/Stage 2 Data Collection and
Tracking System (DCTS). Details on the use of the scheduler are provided in the Users' Manual for the
LT2/Stage 2 Data Collection and Tracking System (DCTS). The DCTS can be accessed at
http://www.epa.gov/safewater/disinfection/lt2/. PWSs that are unable to submit their schedules
electronically should contact their point of contact as indicated on the contact list at
http://www.epa.gov/safewater/disinfection/lt2/compliance.html. at least one month prior
to the deadline for submission.
3.2.1 Part-Year Plants
Water treatment plants that use surface water or ground water under the direct influence (GWUDI), but
are operated only part of the year should follow these requirements [40 CFR § 141.701(e)]:
• PWSs must sample their source water only during the months that the plant operates, unless the State
specifies another monitoring period based on plant operating practices.
• PWSs with plants that operate less than six months per year and that monitor for Cryptosporidium
must collect at least six Cryptosporidium (plus E. coll and turbidity, if applicable) samples per year
during each of the 2 years of monitoring. Samples must be evenly spaced throughout the monitoring
period when the plant is operating.
3.2.2 Sample Collection Problems
Permissible exceptions to collecting samples on the dates specified in the sampling schedule are noted as
follows:
If extreme conditions or situations exist that may pose danger to the sampler, or which are unforeseen
or cannot be avoided and which cause the system to be unable to sample in the required time frame,
the PWS must sample as close to the scheduled date as feasible, unless the State approves an
alternative sampling date. The PWS must submit an explanation for the delayed sampling date to
EPA/the State concurrent with the shipment of the samples to the laboratory [40 CFR §
141.702(b)(l)]. Sample results and explanations should be submitted through the LT2/Stage 2 Data
Collection and Tracking System.
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Section 3: Sampling Location and Schedule
• If a PWS is unable to report a valid analytical result for a scheduled sampling date due to equipment
failure, loss of our damage to the sample, failure to comply with the analytical method requirements,
including the quality control requirements in 40 CFR § 141.704 or the failure of an approved
laboratory to analyze the sample, the PWS must collect a replacement sample. The PWS must collect
the replacement sample not later than 21 days after receiving information that an analytical result
cannot be reported for the scheduled date, unless the PWS demonstrates that collecting a replacement
sample within this time frame is not feasible or EPA/the State approves an alternative resampling
date. The PWS must submit an explanation for the delayed sampling date to EPA/the State concurrent
with the shipment of the sample to the laboratory [40 CFR § 141.702(b)(2)].
Alternative sample collection dates should be timed so as not to coincide with another scheduled
Cryptosporidium sample collection date.
Monitoring results with sample collection dates that do not comply with the schedule entered into the
DCTS by the PWS before monitoring began will be flagged. If EPA approves the explanations for the
missed sampling dates, the DCTS will automatically update the schedule to permit the analytical results
to be submitted through the DCTS without qualification. The Users' Guide for the LT2/Stage 2 Data
Collection and Tracking System provides more information on the procedures for reporting results in
these circumstances.
3.2.3 Changing the Sampling Schedule
Before the sampling schedule deadline, if EPA or the State has not already approved the sampling
schedule, the PWS can access the scheduler module of the DCTS any time to make changes to the
schedule submitted. After the schedule has been approved, or after the deadline to submit the schedule,
changes to the schedule can only be made through contacting your point of contact as indicated on the
contact list at http://www.epa.gov/safewater/disinfection/lt2/compliance.html
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34 February 2006
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SECTION 4: COLLECTING AND SHIPPING
SOURCE WATER SAMPLES
4.1 Sample Collection Guidance
During each of the scheduled sampling events, several actions should be performed in addition to
collecting the sample. Guidance and procedures for each of these sample collection activities is provided
in Sections 4.1.1 - 4.1.5, below.
4.1.1 Sample Collection Documentation
PWSs should be prepared to record the information in Table 4-1 during sample collection to link the
monitoring result to the plant, meet the data reporting requirements of the LT2 Rule, provide the
laboratory with the information needed to meet holding times, and to provide information that will be
used to refine the relationship between Cryptosporidium and E. coli levels.
For samples that are shipped offsite, this information should be documented on an LT2 sample collection
form (Appendix F), or similar form provided by your contract laboratory. For samples analyzed onsite by
your utility's laboratory, this information can be documented in a sampling log book or other standard
form used by your utility; the LT2 sample collection form can also be used.
Sample collection personnel must select from four source water types on the LT2 sample collection form:
• Flowing stream (defined under the LT2 Rule as "a course of running water flowing in a definite
channel")
• Reservoir/lake (defined under the LT2 Rule as "a natural or man made basin or hollow on the Earth's
surface in which water collects or is stored that may or may not have a current or single direction of
flow")
Ground water under the direct influence (GWUDI) of surface water, with flowing stream as the
nearest surface water body
• GWUDI with reservoir/lake as the nearest surface water body
The source water type should be selected based on the type of source water that accounts for the majority
of the surface water used as source water at the time of sample collection. For example, if the plant uses a
mix of approximately 55 percent reservoir/lake water and 45 percent flowing/stream water, the
"reservoir/lake" option should be circled on the LT2 sample collection form.
For GWUDI systems, the selection of source water type is based on the type of surface water that is the
nearest surface water body; if no surface water body is nearby, the PWS must select GWUDI with
lake/reservoir [40 CFR § 141.701(a)(4)(iv)].
Cryptosporidium sample collection procedures are discussed in Section 4.1.2; E. coli sample collection
procedures and turbidity measurement procedures are discussed in Section 4.1.4 and 4.4, respectively.
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Section 4: Collecting and Shipping Source Water Samples
Table 4-1. Minimum Data Elements to Record During Sample Collection
Sampling Information
PWS name
Public Water System Identification
(PWSID) number3
Facility name
Facility ID a
Sample collection point name
Sample collection point ID
Source water type b
Requested analysis
Sample collection date a
Sample collection time (start and
stop time for field-filtered samples) c
Meter readings (for field-filtered
samples only)
Source water temperature
Turbidity d
Required
/
/
/
/
/
Recommended
/
/
/
/
/
/
/
/
a The combination of these elements constitute the unique sample identifier for LT2 monitoring samples
b This information must be recorded with the E. coli sample collection information.
0 Not required, but important for calculating holding time.
d This information must be recorded with the E. coli sample collection information for filtered PWSs serving >10,000 people.
4.1.2 Cryptosporidium Sample Collection
Several options are available to the PWS in collecting untreated surface water samples for
Cryptosporidium analysis, including the following:
• On-site filtration of water samples using the Pall Gelman Envirochek™ or Envirochek™ HV capsule
filter. A detailed protocol for filtering samples onsite from pressurized and unpressurized sources is
provided as Appendix G.
On-site filtration of water samples using the IDEXX Filta-Max® foam filter. A detailed protocol for
filtering samples onsite from pressurized and unpressurized sources is provided as Appendix H.
Collection of bulk water samples for shipment to the laboratory for filtration and analysis. A detailed
protocol for collecting, packing, and shipping bulk samples is provided as Appendix I.
The sample must be eluted from the filter within 96 hours of sample collection, regardless of the
procedure used to collect Cryptosporidium samples, per EPA Method 1622/1623 [40 CFR § 141.704];
additional information on the methods is provided in Section 8.2 of this manual. If this holding time is
violated, the sample will be considered non-compliant and the laboratory will reject the sample. Your
PWS will be required to recollect and reship the sample [40 CFR § 141.702(b)(2)].
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Section 4: Collecting and Shipping Source Water Samples
LT2 Rule requirement: Each sample must meet EPA Method 1622/1623 requirements, which include
holding time requirements [40 CFR § 141.704(a)].
4.1.2.1 Purchasing Filters
If one of the field filtration options is used, you may want to consider purchasing filters in bulk from the
manufacturer (or the manufacturer's local distributor), as opposed to purchasing the filters from your
Cryptosporidium contract laboratory as part of the sampling kit. The sampling kit contains tubing, flow
meter and flow control valve etc. see appendices G and H for list of materials and equipment needed for
sampling. This approach provides your PWS with a ready supply of extra filters onsite in case a filter
clogs during a sampling event. Plants wishing to explore this option should call one of the contacts in
Table 4-2.
Table 4-2.
Contacts for Filters Approved for Using EPA Method 1622/1623
Pall Life Sciences
(Envirochek™ and Envirochek™ HV capsule filters)
IDEXX
(Filta-Max® foam filters)
www.pall.com/qelman
600 South Wagner Road
Ann Arbor, Ml 48103
Sales:
Phone: (800) 521-1520 ext.2
Fax: (734)913-6114
Technical Support:
Phone: (800) 521-1520 ext.3
Fax: (734)913-6114
www.idexx.com
Sales:
Phone: (800) 321-0207 ext.1
Fax: (207) 856-0630
Technical Support:
Phone: (800) 321-0207 ext.2
Fax: (207) 856-0630
E-mail: water@idexx.com
Your PWS can also purchase and assemble the entire sampling kit and maintain this kit on site, rather
than shipping it back and forth between the Cryptosporidium laboratory and the plant. If the filters you
use have associated shelf lives and storage conditions, ensure that the filters are stored according to the
manufacturers' directions and are not used past the specified shelf life.
The components and part numbers for the sampling kit are specified in the individual protocols for each
filter. If the sampling kit is maintained onsite by the utility, the utility should use disposable materials
wherever possible to mitigate the risk of cross-contamination between samples or sampling events, and
should disinfect the non-disposable sampling equipment between uses.
Sampling kit cleaning may consist of the following:
• Cleaning equipment with warm detergent solution and exposing to hypochlorite solution (5 percent
solution of fresh bleach and water) for at least 30 minutes at room temperature
• Rinsing the equipment with reagent water and placing the equipment in an area free of potential
Cryptosporidium contamination until dry
4.1.2.2 Cryptosporidium Sample Volumes
Under the LT2 Rule Cryptosporidium sample volume requirements [40 CFR § 141.704(a)(l)], PWSs are
required to analyze, at a minimum, either:
• 10 L of sample, or
2 mL of packed pellet volume, or
37
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Section 4: Collecting and Shipping Source Water Samples
• As much volume as two filters can accommodate before clogging (this condition applies only to
filters that have been approved by EPA for nationwide use with EPA Method 1622/1623—the Pall
Gelman Envirochek™ and Envirochek™ HV filters, or the IDEXX FiltaMax® foam filter).
The LT2 Rule sample volume analysis requirement of 10 L (rather than 10.0 or 10.00 L) accommodates
the potential for imprecisely filled sample containers or filters. Therefore, sample volumes of 9.5 L and
higher would meet the rule requirements, and PWSs may analyze volumes larger than 10 L. Larger
volumes analyzed should increase analytical sensitivity (detection limit) and representativeness, provided
method performance is acceptable. EPA encourages PWSs to analyze similar sample volumes throughout
the monitoring period. However, data sets including different samples volumes will be accepted provided
the system meets the minimum sample volume requirements noted above.
PWSs with turbid source waters containing excessive particulates that interfere with filtration or sample
purification are more likely to clog filters than PWSs with lower turbidity waters. If your PWS
encounters highly turbid water regularly, or variable water quality that clogs the filter unpredictably, you
should routinely bring two filters to the sampling point for each sampling event:
• If the water quality allows a full 10 L to be filtered without clogging, your PWS can simply ship the
filter to the laboratory and save the remaining materials for subsequent events.
If the first filter clogs after 5 L or more have been filtered, your PWS should be able to filter the
remaining volume through the second filter and ship both filters to the laboratory for processing.
If two filters clog, be sure to document the volume passed through each filter. Although more expensive
to ship, utilities likely to clog filters may want to consider sending the laboratory a 10 L bulk water
sample for processing with centrirugation instead of filtration.
4.1.3 Matrix Spike Sample Collection
In addition to routine monitoring samples, matrix spike (MS) samples are also required by the LT2 Rule
[40 CFR § 141.704(a)(2)]. An extra bulk water sample must be collected so that the laboratory can spike
it with Cryptosporidium oocysts and filter it in the laboratory [40 CFR § 141.704(a)(2)(i)] to assess
recovery in your PWS' source water matrix. Section 9.1.8 of EPA Method 1622/1623 specifies that MS
samples be analyzed at a frequency of 1 MS per 20 routine monitoring samples.
For all PWSs, the first MS sample should be collected and analyzed during the first sampling event under
the monitoring program, per Section 9.1.8 of EPA Method 1622/1623. If it is not possible to analyze an
MS sample for the first sampling event (due to laboratory capacity or schedule, for example), the first MS
sample should be analyzed as soon as possible to identify potential method performance issues with the
matrix. The laboratory and the PWS may evaluate the MS recoveries, as well as other attributes of sample
processing and examination with that particular site, and work together to determine whether sample
filtration and processing procedures are working acceptably, or need to be re-evaluated. Matrix spike
samples may be analyzed more frequently than one every 20 field (monitoring) samples to better
characterize method performance in the matrix.
The MS frequency specified in EPA Method 1622/1623 (one at the first sampling event and one for every
20 field (monitoring) sample thereafter) translates to the following, for each plant category:
• For large PWSs that perform monthly monitoring for 2 years (resulting in 24 monitoring samples),
two MS samples will need to be collected and analyzed
• For large PWSs that perform semi-monthly monitoring for 2 years (resulting in 48 samples), a
minimum of three MS samples will need to be collected and analyzed
• For large PWSs that perform weekly monitoring for 2 years (resulting in 104 samples), a minimum of
six MS samples will need to be collected and analyzed
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Section 4: Collecting and Shipping Source Water Samples
• For small PWSs that are triggered into Cryptosporidium monitoring and collect semi-monthly
samples for 1 year or monthly samples for 2 years (resulting in 24 samples), two MS samples must be
collected and analyzed
As specified in Section 9.5 of EPA Method 1622/1623, the MS sample volume analyzed must be within
10 percent of the volume analyzed for the associated monitoring sample. If the volume of the MS sample
is greater than 10 L, the system is permitted to filter all but 10 L of the MS sample in the field, and ship
the filtered sample and the remaining 10 L of source water to the laboratory to have the laboratory spike
the remaining 10 L of water and filter it through the filter used to collect the balance of the sample in the
field [40 CFR § 141.704(a)(2)(ii)].
Utilities should split their sample stream and collect the monitoring sample volume and MS sample
volume simultaneously:
The sample stream should be split using flow controllers on both sides of the split to regulate the
pressure difference between the side being subjected to filtration (resulting in higher pressure) and the
side flowing into a bulk sample container
• If splitting the sample stream is not practical, the utility should collect the MS sample immediately
before or after the monitoring sample
MS sample results will not be used to adjust Cryptosporidium recoveries at any individual source water;
the MS results will be used collectively to assess overall recovery and variability for EPA Method
1622/1623 in source water. No resampling will be necessary for MS samples that do not meet Method
1622/1623 recovery guidelines.
LT2 Rule requirements: Each sample must meet EPA Method 1622/1623 requirements [40 CFR §
141.704(a)], which include the following: (1) The MS and field (monitoring) sample must be collected
from the same sampling location by splitting the sample stream or collecting the samples sequentially
(method Section 9.5.1). (2) The volume of the MS sample analyzed must be within 10% of the volume
of the field (monitoring) sample analyzed (method Section 9.5.1). (3) The MS and field (monitoring)
sample must be analyzed by the same procedure (method Section 9.5.1).
4.1.4 E. co/i Sample Collection
For most large PWSs, E. coll analyses will be conducted onsite and samples will not be shipped.
However, many small PWSs will collect E. coll samples and ship them offsite for analysis. Regardless of
whether the samples are analyzed by the utility's own laboratory or by a commercial laboratory,
laboratories analyzing E. coll samples for the LT2 Rule must use an E. coll method approved for use
under the rule and must be certified for total coliform or fecal coliform analysis that uses the same
technique as the E. coll method the laboratory plans to use for LT2 Rule monitoring [40 CFR §
141.704(b) and 141.705(b)]. Approved E. coll methods and the drinking water certification program are
provided in the Mlcrobial Laboratory Guidance Manual for the Long Term 2 Enhanced Surface Water
Treatment Rule (LT2 Rule). Information on these methods is also provided in Table 7.1 of this document.
PWSs monitoring for E. coll under the LT2 Rule should collect and analyze at least 100 mL of sample to
ensure sufficient volume for sample analysis. If spillage or leakage occurs during shipment, the sample
may have become contaminated, and the sample should not be analyzed (see Section 7.4.1). Collect E.
coll samples in sterile, non-toxic, plastic, or glass containers with a leak-proof lid. The capacity of sample
containers should be at least 120 mL (4 oz.) or 250 mL (8 oz.) to allow for sufficient sample volume and
at least a 1-inch head space to facilitate mixing of the sample by shaking prior to analysis. A detailed
protocol for collecting source water samples for E. coll analysis, as well as packing and shipping guidance
for utilities that transport samples offsite for analysis, is provided as Appendix J.
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Section 4: Collecting and Shipping Source Water Samples
EPA encourages laboratories to analyze samples as soon as possible after collection. E. coll samples must
be analyzed within 30 hours of sample collection [40 CFR § 141.704(b)(l)]. If the State determines that it
is not feasible for the sample to analyze the sample within the 30-hour holding time, the State may
authorize the holding time to be extended to 48 hours but only for the Colilert method. The holding time
can be extended to 48 hours only when authorized by the State, and is done on a case-by-case basis [40
CFR § 141.704(b)(2)]. Note: This is a longer time period than currently permitted for analysis of samples
under the Surface Water Treatment Rule.
E. coll samples should be maintained between 0°C and 10°C by storing in a refrigerator or in a cooler
with wet ice, blue ice, or gel packs, etc. [40 CFR § 141.704(b)(3)]. Additional guidance on monitoring
sample temperature is available in Section 4.2 of this manual.
4.2 Sample Temperatures
Source water samples are dynamic environments and, depending on sample constituents and
environmental conditions, Cryptosporidium oocysts present in a sample can degrade and E. coll present in
a sample can grow or die off, biasing analytical results. To reduce biological activity and preserve the
state of source water between collection and analysis, samples should be chilled. However, freezing is
also a concern for Cryptosporidium filters and 120 mL or 250 mL E. coll samples that are shipped offsite
with coolant materials, such as wet ice, blue ice, or gel packs. Samples can freeze under these conditions
if not packed properly.
Upon receipt, the laboratory must record the sample temperature and reject Cryptosporidium samples that
are received at >20°C or frozen, or Cryptosporidium samples that the laboratory has determined exceeded
20°C or froze during shipment [Section 8.1.3 of EPA Method 1622/1623]. E. coll samples that are
received at >10°C or frozen, or E. coll samples that the laboratory has determined exceeded 10 °C or
froze during shipment, must also be rejected.
After receipt, Cryptosporidium samples must be stored at the laboratory between 1 °C and 10°C, and not
frozen, until processed (Section 8.1.3 of EPA Method 1622/1623) [40 CFR § 141.704(a)]. E. coll
samples must be stored at the laboratory between 0°C and 10°C, and not frozen, until processed [40 CFR
§ 141.704(b)(3)].
The following steps should help maintain acceptable temperatures:
• If Cryptosporidium and E. coll samples are collected early in the day, chill samples by storing in a
refrigerator between 1 °C and 10°C or pre-icing the sample in a cooler. If the sample is pre-iced
before shipping, replace with fresh ice immediately before shipment.
• If Cryptosporidium samples are collected later in the day, these samples may be chilled overnight in a
refrigerator between 1 °C and 10°C. Overnight refrigeration is recommended for bulk water
Cryptosporidium samples that will be shipped offsite, as this minimizes the potential for water
samples collected during the summer to melt the ice in which they are packed and arrive at the
laboratory at >20°C.
The sample collection procedures in Sections 4.1.2 through 4.1.4 and Appendices G, H, I, and J provide
sample packing procedures for the various ways of collecting E. coll and Cryptosporidium samples, as
well as guidance on packing samples to maintain appropriate temperatures. Utility personnel should
follow these procedures to ensure that samples remain at acceptable temperatures during shipment.
Several options are available to measure sample temperature upon receipt at the laboratory and, in some
cases, during shipment:
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Section 4: Collecting and Shipping Source Water Samples
• Temperature sample. One option, for Cryptosporidium filtered samples (not for 10 L bulk samples)
and E. coli 120 and 250 mL samples, is for the PWS to fill a small, inexpensive sample bottle with
water and pack this "temperature sample" next to the field (monitoring) sample. The temperature of
this extra sample volume is measured upon receipt to estimate the temperature of the field
(monitoring) sample. Temperature sample bottles are not appropriate for use with 10 L bulk samples
because of the potential effect that the difference in sample volume may have in temperature
equilibration in the sample cooler. Example product: Cole Farmer cat. no. C-06252-20 or equivalent.
• Temperature vial. A similar option is to use a thermometer that is securely housed in a liquid-filled
vial. Temperature vials are not appropriate for use with 10 L bulk samples for the reasons stated
above. Unlike temperature samples, the laboratory does not need to perform an additional step to
monitor the temperature of the vial upon receipt, but instead just reads the thermometer. Example
product: Eagle-Picher Sentry Temperature Vial 3TR-40CS-F or 3TR-40CS or equivalent.
• iButton. Another option for measuring the sample temperature during shipment and upon receipt is a
Thermocron® iButton. An iButton is a small, waterproof device that contains a computer chip to
record temperature at different time intervals. The information is then downloaded from the iButton
onto a computer. The iButton should be placed in a temperature sample in the cooler rather than
placed directly in the cooler, where it may be affected by close contact with the coolant. Again, this
option is not appropriate for use with 10 L bulk samples. Example product: Thermocron® iButtons
or equivalent.
All temperature measurement devices should be calibrated routinely, where possible, to ensure accurate
measurements. See the U.S. EPAMctnuctlfor the Certification of Laboratories Analyzing Drinking Water
(Reference 8.5) for more information.
4.3 Measuring Turbidity
Filtered PWSs collecting samples for Cryptosporidium analysis must measure the turbidity of the source
water at the time of Cryptosporidium and E. coli sample collection during LT2 Rule monitoring [40 CFR
§ 141.701(a)]. Turbidity must be measured by a party approved by the State [40 CFR § 141.705(c)] using
approved methods for turbidity measurement [40 CFR § 141.74(a)(l) and 40 CFR § 141.704(c)]. These
methods include:
• Method 2130B, published in Standard Methods for the Examination of Water and Waste-water (19th or
20th Edition) (Reference 8.6)
• GLI Method 2, "Turbidity," November 2, 1992, Great Lakes Instruments, Inc., 8855 North 55th
Street, Milwaukee, Wisconsin 53223
• EPA Method 180.1, "Methods for the Determination of Inorganic Substances in Environmental
Samples," EPA/600/R-93/100, August 1993; available atNTIS, PB94-121811
Hach FilterTrak Method 10133; a description of the Hach FilterTrak Method 10133, "Determination
of Turbidity by Laser Nephelometry," January 2000, Revision 2.0, can be obtained from: Hach Co.,
P.O. Box 389, Loveland, Colorado 80539-0389. Phone: 800-227-4224
Many States have designated certified operators and professional engineers as approved parties for
turbidity measurement. If you are not sure about your State's requirements, please contact your State
drinking water program representative.
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Section 4: Collecting and Shipping Source Water Samples
4.3.1 Measuring Sample Turbidity During LT2 Monitoring
When measuring turbidity, cuvettes should be clear, colorless glass or plastic. The tube should be kept
clean, both inside and out, to provide accurate readings. If a sample tube is scratched, it must be
discarded.
• Measuring Sample Turbidity Using SM 21 SOB. Measure turbidity immediately after sample
collection to prevent temperature changes, particle flocculation, and sedimentation from changing
sample characteristics. Shake sample well before pouring into cuvette. Gently agitate to remove air
bubbles from the inside of the sample before pouring the sample into cell. Wait until all the air
bubbles disappear and remove all moisture from the outside of the sample cell before placing it into
the instrument. If fogging occurs, warm the sample cell in a warm water bath for a short time, then re-
agitate the sample before placing it in the turbidimeter. Read turbidity directly from instrument
display. Note: Measurements should be within the calibration range.
• Measuring Sample Turbidity Using GLI Method 2 or Revised EPA Method 180.1. Different
procedures should be followed, depending on the turbidity of the sample:
For turbidities estimated to be less than 40 NTU. Shake the sample thoroughly to disperse the
solids. After waiting for the air bubbles to disappear pour the sample into the turbidimeter tube
and read directly from the instrument scale.
For turbidities estimated to be greater than 40 NTU. Dilute the sample with turbidity-free water
and compute the turbidity with the dilution factor included.
4.3.2 General Quality Control for Turbidity Measurements
Utilities performing environmental sample measurements must be approved by the State (or EPA Region,
for States that do not have primacy) under the drinking water laboratory certification program [40 CFR §
141.705(c)]. Each utility laboratory should operate a formal quality control (QC) program and maintain
performance records that define the quality of the data generated. Two types of calibration are
recommended for turbidity measurements:
• A primary suspension standard. The primary suspension standard should be used to calibrate the
turbidimeter initially and at least every four months in order to prevent instrument drift. The
calibration should be documented. The standards should be replaced when they exceed the expiration
date. Acceptable primary suspensions include Formazin (recipes for preparation can be found in EPA
Method 180.1 and Standard Method 2130B), AMCO CLEAR (available from GFS Chemicals), and
Hach StablCal Stabilized Formazin Standards (available from Hach Company). Please note that
Formazin standards are relatively unstable, particularly at low concentrations. Therefore, dilutions
used for calibration need to be prepared on the day they will be used. Stock solutions may be stable
for a month (at 400 NTU) to one year (at 4000 NTU). Consult an approved method for more
information.
• A secondary suspension standard. The secondary suspension standard is used daily to check the
calibration of the instrument. The calibration should be documented, and should not vary by more
than 10% from the initial calibration values (if they do vary by more than 10%, the system should be
recalibrated so that performance is acceptable). The standards should be replaced when they exceed
the expiration date. Acceptable secondary standards include all primary standards, or other materials
that are suggested by instrument manufacturers - such as sealed sample cells filled with a labeled
suspension or metal oxide particulates in a polymer gel, or a turbid glass cube. The purpose of the
secondary standard is to provide a quick check of calibration. The secondary standards should have a
fixed turbidity that does not vary from use to use.
42 February 2006
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SECTION 5: GRANDFATHERING CRYPTOSPORIDIUM DATA
"Grandfathered" Cryptosporidium data are results generated before monitoring begins as required under
the LT2 Rule and that a PWS intends to use in determining its bin classification (Section 6.1.1) or mean
Cryptosporidium level under the rule. Grandfathered data may be used in lieu of, or in addition to, results
generated for the LT2 Rule initial round of monitoring [40 CFR § 141.707]. This section of the manual is
designed to assist PWSs in producing and reporting grandfathered data that should be equivalent to the
data collected during LT2 Rule initial round of monitoring and therefore eligible for use in bin
classification or establishing a mean Cryptosporidium level.
5.1 Intent to Grandfather
If your PWS will submit grandfathered data, you must first report that you intend to submit previously
collected monitoring results (Appendix K). This report must specify the number of previously collected
results that you will submit, the dates of the first and last sample, and whether your PWS will collect
additional source water monitoring samples to meet the requirements of [40 CFR 141.701(a)]. PWSs
must report this information no later than the date the PWS is required to submit a schedule for sampling
under the LT2 Rule, as described in Tables 1-2 through 1-5 [40 CFR § 141.707(f)(l)]. The information
must be submitted as indicated in Section 1.6 of this manual.
If your PWS plans to collect samples in addition to the grandfathered data, you must also submit a
monitoring schedule as specified in Tables 1-2 through 1-5 and Section 3.2 of this guidance manual.
5.2 Requirements for Grandfathered Cryptosporidium Data
To be eligible for grandfathering, previously collected Cryptosporidium monitoring must meet the
requirements in section 141.707 of the LT2 rule and the State must approve of the grandfathering. If the
previously collected data does not meet requirements, it may not be approved. Appendix L provides a
checklist for PWSs that plan to begin collecting data for grandfathering. The remainder of this section
provides information on the grandfathering requirements.
5.2.1 Sample Collection Location and Volume
The sampling location for grandfathered samples must meet the same location requirements as those that
apply to new sampling conducted under the LT2 Rule [40 CFR § 141.707(d)]. These sample location
criteria are described in Section 3.1 of this guidance. The samples must be representative of a plant's
source water(s) and the source water(s) must not have changed since the samples were collected [40 CFR
§ 141.707(f)(2)(m)].
PWSs must provide a description of the sampling location that addresses the position of the sampling
location in relation to the water source(s) and treatment processes, including points of chemical addition
and filter backwash recycle [40 CFR § 141.707(f)(2)(iii)]. PWSs may submit Appendix D (Sample
Location Worksheet) and one of the schematics in Appendix E with the grandfathered date package to
meet this requirement.
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Section 5: Grandfathering Cryptosporidium data
Samples collected for grandfathering must meet the same volume analysis requirements as for LT2 Rule
monitoring [40 CFR § 141.707(c)(2)]. These requirements are described in Section 4.2 of this manual.
5.2.2 Sample Collection Frequency and Schedule
Cryptosporidium samples submitted for grandfathering must have been collected no less frequently than
each calendar month on a regular schedule, beginning no earlier than January 1999 (when EPA Method
1622 was first released as an interlaboratory-validated method) [40 CFR § 141.707(e)]. Sample collection
intervals may vary for the conditions described in Section 3.2.2 of this manual. The PWS must provide
documentation of the conditions that caused the variability in sampling when reporting the grandfathered
results.
Before beginning to monitor PWSs should develop a schedule listing the calendar date on which each
Cryptosporidium sample will be collected, and include this schedule when submitting the grandfathered
data package. If your PWS has been monitoring without an established sampling schedule, you should
develop a schedule that summarizes previously collected sampling and specifies the dates for the
collection of remaining samples. PWSs should collect remaining samples within 2 days before or after the
dates indicated in their sampling schedules unless they can document the conditions in Section 3.2.2 of
this manual as indicated above. PWSs that will collect additional samples on or after the applicable
beginning date for required monitoring [40 CFR § 141.701(c) and Section 1 of this manual] should
submit a schedule for the additional samples so that the laboratory can submit sample results through the
DCTS.
5.2.3 Cryptosporidium Analytical Methods for Grandfathered Data
The LT2 rules requires methods 1622 or 1623 for analysis of grandfathered Cryptosporidium monitoring
[40 CFR § 141.707(c)(l)]. The procedures in EPA Methods 1622/1623 are performance-based and allow
for modifications, if the laboratory was approved to use the modified procedures under the Lab QA
Program discussed in Section 5.2.3. The following are acceptable versions of Methods 1622 and 1623 for
grandfathered monitoring data collected before the LT2 Rule effective date of March 6, 2006:
• Method 1623: Cryptosporidium and Giardia in Water by Filtration/IMS/FA. U.S. Environmental
Protection Agency, EPA-815-R-05-002. 2005.
• Method 1622: Cryptosporidium in Water by Filtration/IMS/FA. U.S. Environmental Protection
Agency, EPA-815-R-05-001. 2005.
• Method 1623: Cryptosporidium and Giardia in Water by Filtration/IMS/FA. U.S. Environmental
Protection Agency, Office of Water. 2003. (Note: This was the proposed version of the 2005 method,
which was available via the EPA website)
• Method 1622: Cryptosporidium in Water by Filtration/IMS/FA. U.S. Environmental Protection
Agency, Office of Water. 2003. (Note: This was the proposed version of the 2005 method, which was
available via the EPA website)
• Method 1623: Cryptosporidium and Giardia in Water by Filtration/IMS/FA. U.S. Environmental
Protection Agency, Office of Water. EPA-821-R-01-025. 2001.
• Method 1622: Cryptosporidium in Water by Filtration/IMS/FA. U.S. Environmental Protection
Agency, Office of Water. EPA-821-R-01-026. 2001.
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Section 5: Grandfathering Cryptosporidium data
• Method 1623: Cryptosporidium and Giardia in Water by Filtration/IMS/FA. U.S. Environmental
Protection Agency, Office of Water. EPA-821-R-99-006. 1999.
• Method 1622: Cryptosporidium in Water by Filtration/IMS/FA. U.S. Environmental Protection
Agency, Office of Water. EPA-821-R-99-001. 1999.
On and after March 6, 2006 (the effective date of the LT2 Rule), laboratories should use the 2005 version
of the methods or the PWS may run the risk of the State rejecting the data.
5.2.4 Cryptosporidium Laboratories for Grandfathered Data
EPA has established the Laboratory Quality Assurance Evaluation Program for the Analysis of
Cryptosporidium in Water (Lab QA Program) to approve laboratories to perform Cryptosporidium
analyses under the LT2 Rule. EPA recognizes that some PWSs could begin generating grandfathered
Cryptosporidium data prior to when the Lab QA Program is fully implemented (e.g., before EPA is able
to evaluate all laboratories that will participate in the program). Consequently, PWSs should ensure that
their grandfathered Cryptosporidium samples are analyzed by laboratories that are approved or will be
seeking approval under the Lab QA Program. Data from samples analyzed by laboratories that do not
meet the criteria for approval under the LT2 Rule may not be accepted for grandfathering. Information
about the Lab QA program and a list of currently approved laboratories can be found at
http://www.epa.gov/safewater/disinfection/lt2/lab_home.html.
5.2.5 E. co/i and Turbidity Measurements
Filtered PWSs may grandfather Cryptosporidium samples to meet the requirements of § 141.701(a) when
the system does not have corresponding E. coli and turbidity samples [40 CFR § 141.707(a)(2)].
However, EPA continues to recommend that filtered PWSs conducting early (i.e., grandfathered)
Cryptosporidium monitoring also collect and analyze E. coli samples with each Cryptosporidium sample
and measure turbidity during each sampling event.
E. coli samples analyzed for grandfathering must meet the analytical method and approved laboratory
requirements in §§ 141.704 and 141.705 of the LT2 rule, which are described in Section 7 of this
guidance.
5.3 Checklists for Grandfathering Cryptosporidium Data
To help PWSs interested in using grandfathered Cryptosporidium data (or in monitoring before your
scheduled start date), two checklists have been developed. The "Checklist for Beginning Grandfathered
Cryptosporidium Monitoring" (Appendix L) is designed to be used by PWSs to check their intended
monitoring approach before proceeding with monitoring. The "Grandfathered Cryptosporidium Data
Submittal Report" (Appendix M) is designed to be used by PWSs to check their data package when
submitting grandfathered monitoring data for review.
5.4 Reporting Grandfathered Data
PWSs must submit previously collected monitoring results for grandfathering along with all required
documentation described below no later than two months after the date the system is required to start
monitoring. [40 CFR § 141.707(f)(2)]. The documentation may be submitted with the notice of intent to
45 February 2006
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Section 5: Grandfathering Cryptosporidium data
Grandfather (Section 5.1) which is required no later than 3 months prior to the date the system is required
to start monitoring. Section 1.6 of this guidance provides information regarding where to send
grandfathered data packages. Appendix M contains a checklist that PWSs can use to assure that their
grandfathered data package is complete. The information to be submitted is described below.
5.4.1 Grandfathered Data Package Contents
The grandfathered data package should include the following [40 CFR § 114.707(f)(2)]:
• A signed cover letter from the PWS certifying that the data represent the plant's current source water
and that all source water Cryptosporidium monitoring results generated during the time period
beginning with the first reported result and ending with the final reported result are included in the
package (See Section 5.3.1.1 for details)
• Sample collection schedule (recommended)
• Sampling location description
Where applicable, documentation addressing the dates and reason(s) for re-sampling, as well as the
use of presedimentation, off-stream storage, or bank filtration during monitoring
• A list of Cryptosporidium field (monitoring) and MS samples submitted in the data package (see
Section 5.3.1.1, below, for details), identified by sample ID and collection date
• Cryptosporidium sample results for all field (monitoring) and MS samples (see Section 5.3.1.2,
below, for details) and
• Documentation that all method-required quality control requirements were acceptable for every
Cryptosporidium field (monitoring) and MS sample submitted with the package (see Section 5.3.1.3,
below, for details).
5.4.1.1 Sample Results to be Reported
PWSs that conduct monitoring for grandfathering must submit results for all source water
Cryptosporidium samples generated during the time period beginning with the first reported result and
ending with the final reported result [40 CFR 14.707(f)(2)(ii)]. This will include all samples that were:
• Collected from the sampling location designated for LT2 Rule monitoring
Not spiked
Analyzed using the laboratory's routine process for Method 1622/1623 analyses, including analytical
technique and QA/QC.
For example, if your PWS monitored monthly, but collected additional samples periodically from the
same monitoring location and analyzed them using the same procedure as for the routine monitoring
samples, you must include these results with your grandfathered data.
Sample results generated after the last sample result in the PWS's data package are considered outside the
PWS's LT2 Rule monitoring period and need not be submitted to EPA for LT2 Rule binning purposes.
However, these results may be subject to reporting requirements under other federal or State regulations.
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Section 5: Grandfathering Cryptosporidium data
The grandfathered data set must also include matrix spike (MS) samples at the required frequency, one
MS sample for every 20 field (monitoring) samples. The requirements for analysis of MS samples are the
same for LT2 Rule monitoring and for grandfathered data [40 CFR § 141.707(f)(2)(i)]. These
requirements, and guidance on MS sample collection, are discussed in Section 4.1.3 of this manual.
5.4.1.2 Data Elements to be Reported for Each Sample Result
The following data elements, at a minimum, must be submitted for each Cryptosporidium monitoring
sample and MS sample [40 CFR § 141.707(f)(2)(i)]:
• PWS ID
• Facility ID
Sample collection date
Sample type (field (monitoring) or MS)
• Sample volume filtered (L), to nearest % L
• Was 100% of filtered volume examined?
• Number of oocysts counted
• For matrix spike samples, PWSs must also report the sample volume spiked and estimated number of
oocysts spiked. These data are not required for field (monitoring) samples.
• For samples in which less than 10 L is filtered or less than 100% of the sample volume is examined,
PWSs must also report the number of filters used and the packed pellet volume.
• For samples in which less than 100% of sample volume is examined, PWSs must also report the
volume of resuspended concentrate and volume of this resuspension processed through
immunomagnetic separation.
EPA recommends that these data elements be reported by submitting a completed sample collection form,
laboratory bench sheet, and Cryptosporidium slide examination form for each sample. Example bench
sheets and Crypto Slide Examination Forms are provided as attachments in the Microbictl Laboratory
Guidance Manual for the Long Term 2 Enhanced Surface Water Treatment Rule (LT2 Rule), available for
download from http://www.epa. gov/safewater/disinfection/lt/compliance .html. Sample documentation
forms that are different from these examples, but that contain the minimum required data elements listed
above, may be acceptable.
5.4.1.3 Supporting Quality Control Information
The grandfathered data package must include a signed letter from the laboratory certifying that all
method-required quality control elements (including sample temperature upon receipt, ongoing precision
and recovery (OPR) and method blank results, holding times, and positive and negative staining controls)
were performed at the required frequency, and were acceptable for every monitoring and MS sample
submitted with the package. The letter may include a list of the applicable monitoring and MS samples,
and the corresponding OPR and method blank sample ID for each. [40 CFR § 141.707(f)(2)(iv)].
Alternately, the PWS may include the bench sheet and Cryptosporidium slide examination form (or
comparable detailed data reporting forms) for each OPR and method blank sample associated with the
field (monitoring) and MS samples in the grandfathered data package. If this option is selected, the letter
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Section 5: Grandfathering Cryptosporidium data
from the laboratory should certify that sample temperature upon receipt, holding times, and positive and
negative staining controls were acceptable for all samples. (The letter is not necessary if detailed data
reporting forms containing this information are submitted for the field (monitoring) and MS sample
results.)
5.5 Rejected or Missing Cryptosporidium Data
The State may approve grandfathering of previously collected data where there are time gaps in the
sampling frequency if the system conducts additional monitoring as specified by the State to ensure that
the data used to comply with the initial source water monitoring requirements of 40 CFR § 141.701(a) are
seasonally representative and unbiased [40 CFR § 141.707(e)(l)].
If some of the data that a PWS submits are rejected because they do not meet the requirements for
grandfathering, the PWS must conduct additional monitoring to replace rejected data on a schedule that
the State approves. The PWS is not required to begin this additional monitoring until two months after
notification that data has been rejected and additional sampling is necessary [40 CFR § 141.707(h)].
If the State determines that a previously collected data set was generated during source water conditions
that were not normal for the PWS, such as a drought, the State may disapprove the data. Alternatively,
the State may approve the previously collected data if the system reports additional source water
monitoring, as determined by the State, to ensure that the data set represents average source water
conditions forthe PWS [40 CFR § 141.707(g)(l)].
If the Cryptosporidium sampling frequency varies from month to month, and the monitoring results are
approved, the monthly averaging procedure in 40 CFR § 141.710(b)(5) or 40 CFR § 141.712(a)(3), as
applicable, must be used to calculate the bin classification for filtered PWSs or the mean Cryptosporidium
concentration for unfiltered PWSs [40 CFR § 141.707(e)(2)]. This procedure is described in Section
6.1.1.1 of this manual.
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SECTION 6: CRYPTOSPORIDIUM DATA:
USE, RECORDING, SUBMITTING AND REVIEW
When Cryptosporidium samples are processed and analyzed by the laboratory, data on sample
measurements, sample processing times, and slide examination results are recorded at the laboratory on
the bench sheet and reported to the PWS through the LT2/Stage 2 Data Collection and Tracking System
(section 6.3). This data may also be reported to the PWS via hardcopy forms as indicated in the contract
agreement. This section provides an overview of the data recording and reporting processes and describes
the significance of the examination results reported by the laboratory. This information is discussed in
more detail in the Microbial Laboratory Guidance Manual for the Long Term 2 Enhanced Surface Water
Treatment Rule (LT2 Rule).
6.1 Use of Cryptosporidium Data
Two types of Cryptosporidium data are collected under the LT2 Rule: Cryptosporidium occurrence data
from the analysis of monitoring (field) samples, and method performance data from the analysis of matrix
spike (MS) samples. The use of occurrence data from monitoring samples is discussed in Sections 6.1.1
and 6.1.2; the use of method performance data from MS samples is discussed in Section 6.1.3.
6.1.1 Determining Bin Classification - Filtered Systems
Filtered PWSs monitoring for Cryptosporidium will use the concentration of Cryptosporidium oocysts in
source water samples analyzed under the LT2 Rule to calculate a mean Cryptosporidium bin
concentration for each treatment plant for which monitoring was required. The mean Cryptosporidium
bin concentration is then used to classify each treatment plant into a treatment requirements "bin" [40
CFR § 141.710]. These bin classifications are provided in Table 6-1 for filtered PWSs. The treatment bin
classifications established for filtered PWSs are used to determine whether additional treatment is needed.
PWSs in Bin 1 are not required to implement additional treatment; PWSs in Bins 2-4 will be required to
implement increasing levels of treatment [40 CFR § 141.711].
Table 6-1. Bin Classifications for Filtered PWSs
Cryptosporidium Bin Concentration
Cryptosporidium < 0.075 oocysts/L
0.075 oocysts/L < Cryptosporidium < 1.0 oocyst/L
1.0 oocyst/L < Cryptosporidium < 3.0 oocysts/L
Cryptosporidium > 3.0 oocysts/L
PWSs that serve fewer than 1 0,000 people and NOT
required to monitor for Cryptosporidium a
Bin Classification
Bin 1
Bin 2
Bin 3
Bin 4
Bin 1
Filtered PWSs serving fewer than 10,000 people are not required to monitor for Cryptosporidium if they monitor for E. coli and
demonstrate a mean concentration of E. coli less than or equal to 10 £. co///100 mL for lake/reservoir sources or 50 £. co///100
mL for flowing stream sources or do not exceed an alternative State-approved indicator trigger [40 CFR § 141.701 (a)(4) and §
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_ Section 6: Cryptosporidium Data: Use, Recording, Submitting and Review
6.1.1.1 Calculating Bin Concentrations
The method used to average individual sample concentrations to determine a PWS's bin classification
depends on the number of samples collected and the length of the sampling period. For filtered PWSs
monitoring for Cryptosporidium under the LT2 Rule, bin concentrations must be calculated as follows:
• For PWSs that collect at least 48 samples during the required monitoring period, the Cryptosporidium
bin concentration is equal to the arithmetic mean of all sample concentrations [40 CFR §
For PWSs that collect at least 24 samples, but not more than 47 samples, during the required
monitoring period, the Cryptosporidium bin concentration is equal to the highest arithmetic mean of
all sample concentrations in any 12 consecutive months in the monitoring period [40 CFR §
141.710(b)(2)]
For PWSs serving fewer than 10,000 people and monitoring Cryptosporidium for only one year (i.e.,
collect 24 samples in 12 months), the bin concentration is equal to the arithmetic mean of all sample
concentrations [40 CFR § 141.710(b)(3)]
For PWSs with plants operating only part of the year that monitor fewer than 12 months per year, the
bin concentration is equal to the highest arithmetic mean of all sample concentrations during any year
of Cryptosporidium monitoring [40 CFR § 141.710(b)(4)]
For filtered PWSs in which sampling frequency varies, PWSs must first calculate a monthly average
for each month of monitoring. The PWS must then use these monthly average concentrations, rather
than individual sample concentrations, in the applicable calculation for bin classification [40 CFR §
The Cryptosporidium bin concentration is then used with the table above to determine the bin
classification.
6.1.2 Determining Mean Cryptosporidium Levels - Unfiltered Systems
The level of Cryptosporidium inactivation unfiltered PWSs must provide is based on mean
Cryptosporidium levels. Unfiltered PWSs with a mean Cryptosporidium level of <0.01 oocysts/L must
provide at least 2-log Cryptosporidium inactivation. If the mean Cryptosporidium level is >0. 01 oocysts/L,
the unfiltered system must provide at least 3 -log Cryptosporidium inactivation [40 CFR § 141.712(b)].
Unfiltered PWSs must calculate the arithmetic mean of all Cryptosporidium samples concentrations [40
CFR § 141.712(a)(l)]. For unfiltered PWSs in which sampling frequency varies, PWSs must first
calculate a monthly average for each month of monitoring. PWSs must then use these monthly average
concentrations, rather than individual sample concentrations, in the calculation of the mean
Cryptosporidium level [40 CFR § 141.712(a)(3)].
6.1.3 Cryptosporidium Matrix Spike Data
During LT2 Rule Cryptosporidium monitoring, PWSs are required to collect one matrix spike (MS)
sample for every 20 monitoring samples from their source water starting with the first sampling event or
as soon as possible, per the requirements in EPA Methods 1622/1623 (Section 9.1.8, per EPA Method
1622/1623). For PWSs that perform monthly Cryptosporidium monitoring for two years and collect 24
monitoring samples and for small PWSs that are triggered into Cryptosporidium monitoring and collect
24 monitoring samples, two MS samples will be analyzed. For large PWSs that perform semimonthly or
more frequent monitoring for two years and collect 48 or more samples, a minimum of three MS samples
will be analyzed.
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Section 6: Cryptosporidium Data: Use, Recording, Submitting and Review
Although MS sample results will not be used to adjust Cryptosporidium recoveries at any individual
source water, the results will be used collectively to assess overall recovery and variability for EPA
Method 1622/1623 in source water.
6.2 Cryptosporidium Data Recording at the Laboratory
The Cryptosporidium laboratory may record LT2 Rule monitoring data using the following standardized
forms:
6.2.1 LT2 Sample Collection Form
This form (an example of which is provided as Appendix F) is initiated at the plant upon sample
collection and is completed at the laboratory. The following information is recorded on this form by the
Cryptosporidium laboratory:
• Date and time of sample receipt
Laboratory personnel receiving the sample
Sample temperature upon receipt
• Sample condition upon receipt
Although none of this information is entered into the LT2/Stage 2 Data Collection and Tracking System,
it provides documentation for the utility, the laboratory, and EPA or State officials on sample receipt
information relevant to LT2 Rule requirements regarding sample temperatures and sample holding times.
6.2.2 Method 1622/1623 Bench Sheet
The laboratory uses the bench sheet to record all information associated with sample processing, up to,
but not including, sample examination. Information on filtration (if performed in the laboratory), elution,
concentration, immunomagnetic separation, and sample staining are documented on this form. These data
include:
Dates and times for all steps associated with method-required holding times
All primary measurements used to calculate sample volume analyzed, if less than 100% of the
volume filtered was analyzed. This information includes the following:
The volume of the sample after the concentrate (packed pellet) has been resuspended
The volume of this resuspended concentrate that was actually analyzed
(These two values are used to calculate the percent of the sample volume analyzed, if less than
100% of the volume filtered was analyzed.)
Filter clog and packed pellet information, which should be provided to demonstrate compliance with
LT2 Rule sample analysis requirements if less than 10 L was analyzed
• Cryptosporidium spiking information for OPR and MS samples
Analyst names or initials for each step
• Reagent and filter lot information
6.2.3 Method 1622/1623 Cryptosporidium Slide Examination Form
The laboratory uses the slide examination form to document detailed information on slide examination.
This information includes the following:
• Date and time the examination was completed
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Section 6: Cryptosporidium Data: Use, Recording, Submitting and Review
Positive and negative staining control results
Detailed information on the characteristics of each object on the slide that the analyst determined was
a Cryptosporidium oocyst, including the following:
Size of the oocyst
Shape of the oocyst
Whether the DAPI stain applied to the sample was negative or whether it was positive with
intense blue staining or revealed the presence of nuclei, and, if so, how many were observed by
the analyst
Whether during DIC examination the analyst observed empty or amorphous oocysts or oocysts
with internal structures
6.3 Submitting Cryptosporidium Data through the LT2/Stage 2 Data
Collection and Tracking System (DCTS)
During the LT2 Rule, laboratories report Cryptosporidium data through the LT2/Stage 2 Data Collection
and Tracking System (DCTS), where it is also available to their PWS clients. The DCTS is a web-based
application that allows laboratory users to enter or upload data, then electronically "release" the data to
the PWS for review, approval, and submission to EPA and the State. Although ownership of the data
resides with the PWS throughout this process, the DCTS increases the ease and efficiency of the data
entry and transfer process from one party to another by transferring the ability to access the data from the
laboratory to the PWS to EPA and the State, and ensuring that data cannot be viewed or changed by
unauthorized parties.
A summary of the data entry, review, and transfer process through the DCTS is provided below in Table
6-2 and in Sections 6.3.1 through 6.3.3. More detailed information about the DCTS is provided in the
Users' Manual for the LT2/Stage 2 Data Collection and Tracking System, including detailed information
on the PWS and Laboratory user registration process. Information on the DCTS and a downloadable
Users' Manual are available at http://www.epa.gov/safewater/disinfection/lt2.
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Section 6: Cryptosporidium Data: Use, Recording, Submitting and Review
Table 6-2. LT2/Stage 2 Data Collection and Tracking System (DCTS) Data Entry,
Review, and Transfer Process
Laboratory actions
• Laboratory posts analytical results to the DCTS
• DCTS reduces data and checks for completeness and compliance with LT2 Rule requirements
• Laboratory Principal Analyst confirms that data meets quality control requirements
• Laboratory "releases" results electronically to the PWS for review
• Laboratory user cannot edit data after it is released to the PWS
ro
T3
o
o
ro
PWS actions
(0
PWS reviews electronic data through the DCTS
b
PWS cannot edit data - only review data and either return to laboratory to resolve errors or submit to
EPA 6
T3
PWS "releases" data back to the laboratory if it has questions <
• If no questions, PWS submits data to EPA as "approved" or "contested" (indicating that samples have
been correctly analyzed, but that the PWS contends that they are not valid for use in LT2 binning)
• If the PWS does not review the sample result by the deadline for submitting it to EPA (no later than 10
days after the end of the first month following the month when the sample was collected) the sample
result status in the DCTS is automatically changed to "approved - not reviewed" to prevent a monitoring
violation report from generating.
EPA and State actions
• EPA and State users cannot edit data - only review data
• EPA and State review data through the DCTS and approve results where appropriate
• Contested results
If EPA/the State rejects the PWS explanation for the contested sample, the sample is marked
"EPA approved" in the DCTS
If EPA/the State accepts the PWS explanation for the contested sample, the sample is invalidated
and the PWS must resample
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Section 6: Cryptosporidium Data: Use, Recording, Submitting and Review
6.3.1 Data Entry/Upload
The analyst or another laboratory staff member enters a subset of the data recorded at the bench (Section
6.2) into the DCTS, either by entering the data using web forms or by uploading data in XML format.
This information includes the following:
• PWS ID
• Facility ID
Sample collection date
Sample type (field (monitoring) or MS)
• Sample volume filtered (L), to nearest % L
• Was 100% of filtered volume examined?
Number of oocysts counted
• For samples in which less than 10 L is filtered or less than 100% of the sample volume is examined,
the laboratory also must enter or upload the number of filters used and the packed pellet volume.
For samples in which less than 100% of sample volume is examined, the laboratory also must report
the volume of resuspended concentrate and volume of this resuspension processed through
immunomagnetic separation.
• For matrix spike samples, the laboratory also must report the sample volume spiked and estimated
number of oocysts. These data are not required for field (monitoring) samples.
By entering Cryptosporidium data into the DCTS, the laboratory acknowledges that the following QC
requirements were met: all holding times, sample condition on receipt, results of associated method blank,
OPR, and positive and negative staining controls.
After the information has been entered or uploaded into the DCTS, it will reduce the data to yield final
sample results, in oocysts/L, verify that LT2 Rule Cryptosporidium sample volume analysis requirements
were met for samples in which less than 10 L were analyzed (see Section 4.2), and calculate MS
recoveries.
The laboratory's Principal Analyst under the Lab QA Program is responsible for verifying the quality and
accuracy of all sample results in the laboratory. The principal analyst or other senior laboratory
representative (e.g., laboratory manager or QA officer) should review and approve the results before they
are submitted to the PWS for review. If inaccuracies or other problems are identified, the Principal
Analyst discusses the sample information with the analyst or data entry staff and resolves the issues
before the data are approved for PWS review.
If no inaccuracies or other issues are identified, the laboratory approves the reported data for "release" to
the PWS for review (EPA does not receive the data at this point). When the data are approved, the rights
to the data are transferred electronically by the DCTS to the PWS, and the data can no longer be changed
by the laboratory.
6.3.2 PWS Data Review
After the laboratory has released Cryptosporidium data electronically to the PWS using the DCTS, the
PWS will review the results. The PWS user cannot edit the data, but if the PWS has an issue with the
sample result, such as if the PWS believes that the collection date is incorrect, the PWS can release the
results back to the laboratory for issue resolution. In addition to noting in the DCTS the reason for the
return of the data to the laboratory, the PWS should contact the laboratory to discuss the issue.
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Section 6: Cryptosporidium Data: Use, Recording, Submitting and Review
If the PWS determines that they have no issues with the data, the PWS releases the results to EPA (and
the State, if applicable) as "approved" results. If the PWS believes that the data are not valid for LT2
binning purposes, the PWS can release the results to EPA and the State as "contested." Contested samples
are those that have been correctly analyzed, but that a PWS contends are not valid for use in LT2 binning
and has submitted to EPA for evaluation. To contest the data, the PWS must indicate in the DCTS its
reason for contesting the results. If EPA or the State concurs with the contesting, the data will be
invalidated and the PWS will be able to work with EPA or the State to reschedule a sample. If EPA or
the State believe the sample result is valid, the result will be approved and must be included in the binning
calculation.
6.3.3 EPA/State Review
After the PWS has released the results as approved or contested, they are available to EPA and State users
to review through the DCTS. EPA and State users cannot edit the data.
6.3.4 Archiving Examination Results
Although not required, laboratories also may archive slides and/or take photographs of slides to maintain
for clients. Slides should be stored in a humid chamber in the dark at 1 °C to 10°C. An alternative
mounting medium may be used, which might preserve slides longer. Details are provided in the Microbial
Laboratory Guidance Manual for the Long Term 2 Enhanced Surface Water Treatment Rule.
6.4 (Optional) Review and Validation of Raw Cryptosporidium Data
If your PWS plans to review the raw data generated by the laboratory, you should request from the
laboratory the hardcopy data needed to verify the electronic results (see Section 2.1.6). This step is not
required. However, for those PWSs interested in taking this extra step, Sections 6.4.1 through 6.4.3
provide guidance on how to review and validate hardcopy data and verify accuracy.
6.4.1 Data Completeness Check
Upon receipt of the hardcopy sample results for a monitoring sample, verify that the laboratory has
submitted the following information:
• Monitoring sample identification information
• Monitoring sample result, in oocysts/L
• Laboratory quality control batch associated with the sample
Result for the ongoing precision and recovery (OPR) sample analyzed for this QC batch
Result for the method blank sample analyzed for this QC batch
LT2 sample collection form initiated by your utility and completed with sample receipt information
by the laboratory
Method 1622/1623 Bench Sheet with raw data associated with the monitoring sample (and MS
sample, if applicable)
• Method 1622/1623 Cryptosporidium Slide Examination Form with raw data for the monitoring
sample (and MS sample, if applicable)
• Laboratory comments. If the laboratory provided comments on the sample analyses or results that
require follow-up, contact the laboratory to discuss, if necessary. Comments may include any
applicable data qualifiers. The following is a list of potential data qualifiers:
The recovery for the associated ongoing precision and recovery (OPR) sample did not meet
method requirements
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Section 6: Cryptosporidium Data: Use, Recording, Submitting and Review
Oocysts were detected in the method blank
Positive and negative staining controls were not acceptable or not examined
Method holding times were not met
Sample arrived at the laboratory in unacceptable condition
Any of the above data qualifiers would result in the sample being considered invalid for LT2 use and the
laboratory should not report the results for the sample to EPA. The PWS may be required to resample.
If forms are missing, incomplete, or incorrect, contact the laboratory immediately to discuss and request
resubmission of the missing forms and/or spreadsheets.
6.4.2 Evaluation of Data Against Method Quality Control Requirements
To verify that the laboratory analyzed your monitoring sample within the analytical controls specified by
the method, check the following information:
• Sample condition upon receipt. Verify on the completed LT2 sample collection form that your
Cryptosporidium sample was received in acceptable condition (not leaking, etc.), and at a temperature
<20°C, but not frozen.
Method blank. Verify that the laboratory analyzed a method blank with the monitoring sample's QC
batch and confirm that the method blank did not contain any oocysts.
• Ongoing precision and recovery sample. Verify that the laboratory analyzed an OPR sample with
the monitoring sample's QC batch and that the OPR sample recovery was between 11% and 100%, as
required by EPA Methods 1622 and 1623.
Holding times. Using the sample collection date and time on the LT2 data collection form and the
dates and times of the method steps recorded by the laboratory on the Method 1622/1623 bench sheet
and slide examination form for the monitoring sample, verify the following:
The laboratory began elution no more than 96 hours from sample collection
The laboratory performed the elution, concentration, purification, and slide preparation
(application of the purified sample to the slide) within one working day (the date of the elution
step should be the same as the date of the slide preparation step)
The laboratory stained the slide within 72 hours of application of the purified sample to the slide
(the date and time next to the slide staining step should be no more than 72 hours later than the
date and time next to the slide preparation step)
The laboratory examined the slide within 168 hours (seven days) of staining (the examination
date should be no more than seven days from the slide staining date)
Positive and negative staining controls. Based on the information at the top of the Method
1622/1623 Cryptosporidium Slide Examination Form, verify that the laboratory performed positive
and negative staining controls, and that the results of these controls were acceptable.
6.4.3 Calculation Verification
The laboratory does not directly report the final concentration of oocysts/L in the sample to EPA. Instead,
they report a series of primary measurements that are used by the LT2 Data Collection System to
automatically calculate the final concentration. The volume filtered, the total volume of resuspended
concentrate, and the volume transferred to IMS are used to determine the volume analyzed. The
laboratory also records the total count of oocysts detected, which is divided by the volume analyzed to
determine the final concentration of oocysts/L. Although the final results are automatically calculated by
the LT2 Data Collection System using the primary measurements supplied by the laboratory, the plant
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Section 6: Cryptosporidium Data: Use, Recording, Submitting and Review
still may wish to verify them. Guidance on recalculating and verifying final results using primary
measurements is provided below.
6.4.3.1 Field (monitoring) Sample Calculations
To calculate the concentration of Cryptosporidium in your field (monitoring) sample, reported as
oocysts/L, the following information is needed:
• Number of oocysts detected in the sample (recorded as a primary measurement from the examination
results form)
Volume analyzed
Using these two data elements, the final concentration is calculated as:
oocysts detected in the sample
final concentration =
volume analyzed (L)
If 100% of the sample volume filtered is examined, then the volume analyzed equals the volume filtered.
This applies whether one filter or more than one filter was used; if more than one filter was used, and all
of the volume filtered through the multiple filters is processed through the remainder of the method, then
the volume examined is simply the sum of the volumes filtered through each of the filters used.
If less than 100% of the volume filtered was processed through the remainder of the method, then
additional calculations are needed to determine the volume analyzed. This is discussed below.
Determining Volume Analyzed when Less than 100% of Sample Was Examined
When less than 100% of the sample filtered is processed through the remainder of the method and
examined (such as when the volume filtered yields greater than 2 mL of packed pellet volume after
centrifugation), then the volume analyzed must be determined using the following equations to determine
the percentage of the sample that was examined.
total volume of resuspended concentrate transferred to IMS (see Section 6.2.2)
percent examined =
total volume of resuspended concentrate produced
volume analyzed (L) = percent examined * sample volume filtered
Determining the Volume of Resuspended Concentrate to Use for Packed Pellets > 0.5 mL
Packed pellets with a volume greater than 0.5 mL must be divided into subsamples. Use the formula
below to determine the total volume of resuspension required in the centrifuge tube before separating the
concentrate into two or more subsamples and transferring to IMS.
pellet volume (ml) after centrifugation
total volume of resuspended concentrate (ml) required = x 5 mL
0.5 mL
Example. A 10 L field (monitoring) sample was filtered and processed, producing a packed pellet
volume of 2.7 mL. The laboratory transferred 20 mL of the 27 mL total resuspended
concentrate to IMS and examination. The laboratory detected 20 oocysts during examination.
The following calculations were performed to determine the volume analyzed and final
concentration.
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Section 6: Cryptosporidium Data: Use, Recording, Submitting and Review
2.7 mL
total volume of resuspended concentrate (ml) required = * 5 mL = 27 mL
0.5 mL
20 mL
percent examined = = 0.74 (74%)
volume analyzed (L) = 0.74 x 10 L = 7.4 L
20 oocysts
final concentration (oocysts/L) = = 2.7 oocysts/L
7.4 L
6.4.3.2 Matrix Spike Sample Calculations
For matrix spike (MS) samples, the laboratory records all of the same information that is recorded for
field (monitoring) samples, in addition to information specific to matrix spike samples. The sample
volume spiked and the estimated number of oocysts spiked into the sample are used to calculate the
concentration of spiked organisms in the sample. To correct for background concentration, the number of
oocysts detected in the unspiked field (monitoring) sample is subtracted from the number of oocysts
detected in the MS sample.
To determine the percent recovery for a matrix spike (MS) sample, the following information is needed:
The number of oocysts detected in the MS sample
The true value of the oocysts spiked into the MS sample
• The number of oocysts detected in the unspiked field (monitoring) sample (to correct for background
concentration)
oocysts counted in MS sample - oocysts counted in unspiked field (monitoring)
percent recovery = sample x 100%
oocysts spiked into MS sample
This calculation assumes that the same sample volume was examined for both the field (monitoring) and
MS samples. If the sample volumes examined are different, calculate the number of oocysts per L for
both the field (monitoring) and MS samples before calculating percent recovery.
6.4.4 Data Archiving Requirements
The PWS is required to keep all original, hardcopies of monitoring results including quality control data
associated with LT2 sample analyses (both initial and second round of monitoring) for 3 years after bin
classification for filtered PWSs or determination of mean Cryptosporidium level for unfiltered PWSs [40
CFR § 141.722(a)]. Although it is the PWS's responsibility to meet LT2 Rule data storage requirements
for compliance monitoring samples, the PWS may contract this work to the laboratory.
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SECTION 7: E. cou DATA:
USE, RECORDING, SUBMITTING AND REVIEW
When E. coll samples are processed and analyzed by the laboratory, data on sample results and sample
processing times are recorded at the laboratory and reported to the PWS through the LT2/Stage 2 Data
Collection and Tracking System (DCTS). This section provides an overview of the data recording and
reporting processes and provides guidance on how to review the data you receive from the laboratory.
Sample analysis and data review are also described in more detail in the Microbial Laboratory Guidance
Manual for the Long Term 2 Enhanced Surface Water Treatment Rule (LT2 Rule).
7.1 Use of E. coll Data
E. coll data are being collected by large filtered PWSs and applicable wholesalers during LT2 Rule
monitoring to confirm or refine the E. coll levels that trigger Cryptosporidium monitoring by small,
filtered PWSs. E. coll data are being collected by small filtered PWSs because only those small filtered
PWSs with mean E. coll levels that exceed the trigger level will be required to monitor for
Cryptosporidium to determine bin placement [40 CFR § 141.701 (a)(4)]. Based on the data from the
Information Collection Rule (ICR) and Information Collection Rule Supplemental Survey (ICRSS), the
E. coll trigger levels were set at a mean of 50 E. co///100 mL for flowing stream-type source waters and
10 E. colll 100 mL for reservoir/lake source waters. The E. coll and turbidity data from large PWS
monitoring will be reviewed and, if appropriate, guidance on alternate indicator trigger values will be
issued to States prior to when small PWSs begin monitoring. States are allowed to approve alternative
approaches to indicator monitoring for small PWSs [40 CFR § 141.701(a)(5)].
Approved methods for E. coll analysis under the LT2 Rule are listed below in Table 7-1.
7.2 E. coll Data Recording at the Laboratory
The laboratories performing E. coll analyses during the LT2 Rule should record the following general
types of information (For further detail see section 4.6 of Microbial Laboratory Guidance Manual for the
Long Term 2 Enhanced Surface Water Treatment Rule):
• Sample identification information
All primary measurements used to calculate the final E. coll concentration for each sample
The incubation start and read times for each method to verify that method requirements were met
The name of the analyst performing the sample analysis
• Quality control (QC) analysis results (e.g., positive/negative controls, blanks, etc.)
59 February 2006
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Section 7: E. coli Data: Use, Recording, Submitting and Review
Table 7-1. Approved E. coli Methods for LT2 Rule
E. coli Methods Approved for
LT2 Rule
Standard Methods
9221 B. 1/9221 F (LTB-EC-MUG)
Standard Methods 9223B
(Colilert®/Colilert-18®)
Standard Methods 9222B/9222G1
(mEndo/LES-Endo-NA-MUG)
Standard Methods 9222D/9222G
(mFC-NA-MUG)
Standard Methods 921 3D/
EPA Method 1103.1 (mTEC)
EPA Method 1603 Modified
mTEC
EPA Method 1604 Ml medium1
m-ColiBlue24® Broth 1
Method Format
multiple tube fermentation/
most probable number
multiple tube/multiple well
membrane filtration, two step
membrane filtration, two step
membrane filtration, one step
membrane filtration, one step
membrane filtration, one step
membrane filtration, one step
Method Citation
Standard Methods for the
Examination of Water and
Wastewater (Reference 8.6 )2
Standard Methods for the
Examination of Water and
Wastewater (Reference 8.6)2;
IDEXX Laboratories, Inc.
(Reference 8.8)
Standard Methods for the
Examination of Water and
Wastewater (Reference 8.6 )2
Standard Methods for the
Examination of Water and
Wastewater (Reference 8.6)2
EPA Method 1103.1 (Reference
8.9); Standard Methods for the
Examination of Water and
Wastewater (Reference 8.6)2
EPA Method 1603 (Reference
8.10)
EPA Method 1604 (Reference
8.11)
Hach Company (Reference 8.12)
' If high levels of non-E. coli total coliforms interfere with the ability to accurately enumerate E. coli despite additional dilutions, an
alternate method should be used (i.e., SM 9222D/9222G, SM 9213D/EPA Method 1103.1, EPA Method 1603, SM 9221 B.1/9221 F,
and SM 9223B)
218th, 19th, or 20th Editions of Standard Methods for the Examination of Water and Wastewater may be used.
7.2.1 Sample Identification Information
Sample identification information is used to track the sample through sample collection, analysis, and
data reporting. At a minimum, the laboratory records the sample ID, the target parameter (E. coli), and the
method being used (e.g., Membrane Filtration: SM 9222D/SM 9222G).
7.2.2 Primary Data
The laboratory should record all primary measurements needed to calculate the final concentration of E.
coli per 100 mL. Primary measurements for membrane filtration methods will include the volumes
filtered and the plate counts for each volume filtered. The multiple-well and multiple-tube formats will
include the volumes or dilutions of samples analyzed and the number of positive wells or tubes per each
volume analyzed.
7.2.3 Sample Processing and Quality Control Information
The laboratory should record information on the bench sheet on how they processed and analyzed the
sample, including incubation start/end date and times, and the analyst performing each step of the method.
The lot numbers of reagents, media, and materials used to process the sample and the results of QC
analyses should be recorded in a media log book or QC checklist. In addition to being used to resolve
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Section 7: E. coli Data: Use, Recording, Submitting and Review
questions regarding validity of results, this information may be used by the laboratory to determine the
source of any problems the laboratory is having with method performance.
7.2.4 Sample Results
The final E. coli concentration for field (monitoring) samples will be reported as CPU/100 mL or
MPN/100 mL depending on the method used for analysis. If no E. coli are detected in the sample, the
detection limit based on the volume of sample analyzed may be reported (e.g., <1 CPU /100 mL or <1.8
MPN/100 mL) or a zero for purposes of the DCTS.
7.3 Submitting E. coli Data through the LT2/Stage 2 Data Collection
and Tracking System (DCTS)
During the LT2 Rule, laboratories report E. coli data electronically through EPA's LT2/Stage 2 Data
Collection and Tracking System (DCTS) to the PWS staff responsible for approving and submitting
monitoring results to EPA. The DCTS is a web-based application that allows laboratory users to enter or
upload data, then electronically "release" the data to the appropriate PWS staff for review, approval, and
submission to EPA and the State. Although ownership of the data resides with the PWS throughout this
process, the DCTS increases the ease and efficiency of the data entry and transfer process from one party
to another by transferring the ability to access the data from the laboratory to the PWS to EPA and the
State, and ensuring that data cannot be viewed or changed by unauthorized parties. A summary of the data
entry, review, and transfer process through the Data Collection System for both Cryptosporidium and E.
coli samples is provided in Table 6-2, in Section 6.3 of this manual.
The data reporting process is summarized below, in Sections 7.3.1 through 7.3.3, and discussed in detail
in the Users' Manual for the LT 2/Stage 2 Data Collection and Tracking System. The Users' Manual also
provides detailed information on the laboratory registration process. Information on the DCTS and a
downloadable Users' Manual are available at http://www.epa.gov/safewater/disinfection/lt2.
7.3.1 Data Entry/Upload
The analyst or another laboratory staff member enters a subset of the data recorded at the bench (Section
7.2) into the DCTS either by entering the data using web forms or by uploading data in XML format. This
required information includes the following [40 CFR § 141.706 (e)(2)]:
• PWS ID
• Facility ID
• Sample collection date
• Analytical method number
• Method type
Source water type (provided by PWS on sample collection form)
Turbidity result (provided by PWS on sample collection form) (if applicable)
• E. co///100 mL (see note below)
Note: The laboratory may then enter the final result for the sample that was calculated at the laboratory,
or may enter the primary measurements recorded at the bench and have the DCTS automatically calculate
the final sample concentration. Because this information is specific to method type (membrane filtration,
multiple tube fermentation, 51-well, and 97-well), the system provides different entry screens for each
method type. The laboratory staff entering the data should verify that all holding times and other QC
specifications were met.
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Section 7: E. coli Data: Use, Recording, Submitting and Review
The laboratory's official contact is responsible for verifying the quality and accuracy of all sample results
in the laboratory, and should review and approve the results before they are submitted to the PWS for
review. If inaccuracies or other problems are identified, the official contact discusses the sample
information with the analyst or data entry staff and resolves the issues before the data are approved for
PWS review.
If no inaccuracies or other issues are identified, the laboratory's official contact approves the data for
"release" to the PWS for review (EPA does not receive the data at this point). When the data are
approved, the rights to the data are transferred electronically by the DCTS to the PWS, and the data can
no longer be changed by the laboratory.
7.3.2 PWS Data Review
After the laboratory has released E. coli data electronically to the PWS using the DCTS, the PWS will
review the results. The PWS user cannot edit the data, but if the PWS has an issue with the sample result,
such as if the PWS believes that the collection date is incorrect, the PWS can release the results back to
the laboratory for issue resolution. In addition to noting in the DCTS the reason for the return of the data
to the laboratory, the PWS should contact the laboratory to discuss the issue.
If the PWS determines that they have no issues with the data, the PWS releases the results to EPA (and
the State, if applicable) as "approved" results. If the PWS believes that the data are not valid for
calculating mean E. coli level, the PWS can release the results to EPA and the State as "contested."
Contested samples are those that have been correctly analyzed, but that a PWS contends are not valid for
use in calculating mean E. coli level and has submitted to EPA for evaluation. To contest the data, the
PWS must indicate in the DCTS its reason for contesting the results. If EPA or the State concurs with the
contesting, the data will be invalidated and the PWS will be able to work with EPA or the State to
reschedule a sample. If EPA or the State believe the sample result is valid, the result will be approved
and must be included in calculating the mean E. coli level.
7.3.3 EPA/State Review
After the PWS has released the results as approved or contested, they are available to EPA and State users
to review through the DCTS. EPA and State users cannot edit the data.
7.4 (Optional) Reviewing and Validating Raw E. coli Data
If the PWS staff responsible for submitting data to EPA plans to review the raw data generated by the
laboratory, the original, hardcopy records (whether generated by an in-house laboratory or a contract
laboratory) should be compared to the electronic results. However, this step is not required. Sections 7.4.2
through 7.4.3 provide guidance on how to review and validate the hardcopy data and verify accuracy.
7.4.1 Data Completeness Check
Upon receipt of hardcopy sample results for a monitoring sample, verify that the following information is
included on the applicable forms:
• Monitoring sample identification information
• Monitoring sample result, in E. coli/'100 mL
Laboratory quality control checklist (or other verification from the laboratory that all QC
specifications were met)
LT2 sample collection form initiated at the time of sample collection and completed with sample
receipt information by the laboratory
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Section 7: E. coli Data: Use, Recording, Submitting and Review
• E. coli Method Bench Sheet completed by the laboratory with primary sample processing and
analysis data associated with the monitoring sample
Laboratory comments. If the laboratory provided comments on the sample analyses or results that
require follow-up, contact the laboratory to discuss, if necessary. Comments may include any applicable
data qualifiers. The following is a list of potential data qualifiers:
Sample arrived at the laboratory in unacceptable condition (e.g., leaking)
Sample holding time exceeded
• Sample holding temperature not within acceptable range
Unacceptable blank sample result
Unacceptable positive or negative culture control result
• Media sterility checks were not acceptable
• Method incubation times or temperatures were not within acceptable ranges
• Membrane filtration: Too much sediment on the filter
• Membrane filtration: Confluent growth of non-target organism (CNFG)
• Membrane filtration: Colonies too numerous to count (TNTC)
Membrane filtration: Pre- or post- filtration series sterility check not acceptable (e.g., contamination
with E. coli or other organisms)
Quanti-Tray® was damaged or leaked
Sample was not distributed to all wells in Quanti-Tray®
• All rows of tubes were not inoculated
• Presumptive positive tubes were not transferred into the appropriate confirmatory medium
Any of the above data qualifiers would result in the sample being considered invalid for LT2 use, and the
results for the sample should not be entered into the LT2/Stage 2 Data Collection and Tracking System
(DCTS). If the laboratory enters the results into the DCTS, the PWS should not submit the results to EPA.
If forms are missing, incomplete, or incorrect, contact the laboratory immediately to discuss and request
resubmission of the missing forms and/or spreadsheets.
7.4.2 Evaluation of Data Against Method Quality Control Requirements
To verify that the laboratory analyzed your monitoring sample within the analytical controls specified by
the method, check the following information:
• Sample condition upon receipt. If the sample was shipped to the laboratory, verify on the completed
LT2 sample collection form that your sample was received in acceptable condition (e.g., not leaking,
etc.), and at a temperature between 0°C and 10°C, but should not be frozen.
QC samples associated with field (monitoring) samples. The frequency of analysis of quality
control samples including method blanks, positive and negative controls, etc. varies according to
63 February 2006
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Section 7: E. coli Data: Use, Recording, Submitting and Review
method requirements and specifications of the certifying authority. Verify that the required QC
samples were analyzed with the field (monitoring) sample.
• Holding time. Using the sample collection date and time on the LT2 data collection form and the
date and time of the first method step recorded by the laboratory on the E. coli method bench sheet,
verify that the laboratory began sample analysis within 30 hours of sample collection (or 48 hours
with State approval if using Colilert).
7.4.3 Calculation Verification
Method-specific data which should be recorded for each of the individual method types as well as
standardized calculations for each method type are discussed in Sections 7.4.3.1 through 7.4.3.4.
7.4.3.1 Calculations for Determining the E. coli Concentration Using the Colilert® Quanti-
Tray 2000® (97-well)
A. Select appropriate dilution to yield countable results. If multiple dilutions are used, the tray
exhibiting positive wells in the 40% to 80% range (39 to 78 total positive large and small wells)
should be used to determine MPN value.
Note: The analytical result can be automatically calculated using the LT2/Stage 2 Data
Collection and Tracking System. See Section 7.3.1 for additional information.
B. Determine MPN. Using the number of positive wells from the appropriate dilution, identify the
corresponding MPN/100 mL in the table provided by the vendor. Using the number at the
intersection of large positive wells and small positive wells, identify the corresponding MPN/100
mL in the table provided by the vendor. Large well values are located in the left column; small
well values are located in the top row. For example, if a 100 mL sample was analyzed, and there
were 29 large positive wells and five small positive wells, the corresponding MPN would be 49.6
MPN/100 mL.
C. Adjust for dilution factor. Because the MPN/100 mL values in the table are based on 100 mL
samples, the MPN value should be adjusted if less than 100 mL of sample volume was analyzed.
Use the following calculation to adjust the MPN to account for the dilution:
100
Analytical Results = MPN Value x
mL of sample analyzed
Example:
Volume analyzed =10 mL of sample (in 90 mL of dilution water)
Large wells positive = 39
Small wells positive = 5
The MPN value calculated based on the intersection of 39 and 5 in the table.
MPN = 81.3
100
Analytical result = 81.3 x = 813 £. coli MPN/100 mL
10
7.4.3.2 Calculations for Determining the E. coli Concentration Using the Colilert® Quanti-
Tray51®(51-well)
A. Select appropriate dilution. If multiple dilutions are used, the tray exhibiting 80% positive
wells (41 positive wells) should be used to determine MPN value.
Note: The analytical result can be automatically calculated using the LT2 Data Collection
System. See Section 7.3.1 for additional information.
64 February 2006
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Section 7: E. coli Data: Use, Recording, Submitting and Review
B. Determine MPN. Using the number of positive wells from the appropriate dilution, identify the
corresponding MPN/100 mL in the table provided by the vendor. For example, if a 100 mL
sample was analyzed, and there were 41 positive wells, the corresponding MPN would be 83.1
MPN/lOOmL
C. Adjust for dilution factor. Because the MPN/100 mL values in the table are based on 100 mL
samples, the MPN value should be adjusted if less than 100 mL of sample volume was analyzed.
Use the following calculation to adjust the MPN to account for the dilution:
100
MPN value x = £. coli MPN/100 mL
mL sample analyzed
Example:
Volume analyzed (mL) = 10 mL (in 90 mL of dilution water)
Number of positive wells = 41
MPN = 83.1
The analytical result is calculated as follows:
100
83.1 x = 831 E. coli MPN/100 mL
10
7.4.3.3 Calculations for Determining the E. coli Concentration Using Membrane Filter Data
(adapted from Reference 8.6)
A. E. coli counts should be determined from the volume(s) filtered that yielded 20 to 80 E. coli
colonies (20-60 for mFC-NA-MUG), and not more than 200 total colonies per plate. (Guidance
for samples that do not yield countable plates is provided in Sections E and F)
Note: The analytical result can be automatically calculated using the LT2/Stage 2 Data
Collection and Tracking System. See Section 7.3.1 for additional information.
B. If there are greater than 200 colonies per membrane, even for the lowest dilution, the result is
recorded as "too numerous to count" (TNTC). These results cannot be reported for LT2
monitoring, as they cannot be used for the required data analyses. During the next sampling
event, analyze an additional, lower dilution volume (the highest dilution volume may be omitted)
unless conditions were unusual (e.g., heavy rains, flooding, etc.) during the sampling event
yielding TNTC for all dilutions.
C. If colonies are not sufficiently distinct for accurate counting, the result is recorded as "confluent
growth" (CNFG). To prevent CNFG from occurring, smaller sample aliquots should be filtered.
For example, if sample volumes of 100, 10, 1, and 0.1 mL are analyzed and even the 0.1 mL plate
results in CNFG, then potentially 0.01 mL should be analyzed during the next sampling event.
For sample volumes less than 1 mL, serial dilutions should be used, and 1 mL volumes of the
dilutions should be filtered. The 100 mL volume can be eliminated.
Note: If growth is due to high levels of total coliforms but low E. coli then another method should
be chosen for analyses that does not rely on total coliform determination prior to or simultaneous
with E. coli determination.
Note: Results that are TNTC or CNFG are not appropriate for LT2 microbial data analysis,
and cannot be entered into the LT2/Stage 2 Data Collection and Tracking System.
65 February 2006
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Section 7: E. coli Data: Use, Recording, Submitting and Review
D. Using the E. coli counts from the appropriate dilution, E. coli CPU/100 mL is calculated based on
the following equation:
E. coli CPU
Example 1:
100
= E. co//CFU/100mL
mL sample filtered
Filter 1 volume = 100 mL
Filter 2 volume = 10 mL
Filter 3 volume = 1.0 mL
Filter 4 volume = 0.1 mL
CPU = TNTC
CPU = 40
CPU = 9
CFU = 0
Using the guidance on countable colonies in Step A, the counts from the 10 mL plate will
be used to calculate the E. coli concentration for the sample:
40 E. coli CPU
100
10 mL
= 400E. coli CPU/100 mL
If no E. coli colonies are present, the detection limit (i.e., 1 CPU per volume filtered) is calculated
and reported per 100 mL (see example below).
Example 2:
Filter 1 volume (mL) = 100 mL
Filter 2 volume (mL) = 10 mL
Filter 3 volume (mL) = 1.0 mL
Detection limit =
lOOmL
Largest volume filtered
CFU = 0
CFU = 0
CFU = 0
= E. co//CFU/100mL
100 mL
100 mL
= <\E. co///100mL
Example 3:
Filter 1 volume (mL) = 100 mL
Filter 2 volume (mL) = 10 mL
Filter 3 volume (mL) = 1.0 mL
Calculation ofE. coli/WQ mL:
CPU = Lab accident, no data available
CFU = 0
CFU = 0
100 mL
10 mL
= <10E. coli CPU /100 mL
If there are no filters with E. coli counts in the 20-80 colony range (20-60 for mFC-NA-
MUG), sum the E. coli counts on all filters, divide by the volume filtered and report as
number per 100 mL.
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Section 7: E. coli Data: Use, Recording, Submitting and Review
Example 4:
Filter 1 volume (mL) = 50 mL CPU =15
Filter 2 volume (mL) = 25 mL CPU = 6
Filter 3 volume (mL) = 10 mL CPU = 0
The analytical result is calculated as:
100
(15 + 6 + 0) x = 25 E. coli CFU/100 mL
(50+25+10)
Example 5:
Filter 1 volume (mL) = 50 mL CPU =105
Filter 2 volume (mL) = 25 mL CPU = 92
Filter 3 volume (mL) = 10 mL CPU = 85
The analytical result is calculated as:
100
(105 + 92 + 85) x = 332 E. coli CFU/100 mL
(50 + 25 + 10)
Example 6:
Filter 1 volume (mL) = 100 mL CPU = 82
Filter 2 volume (mL) = 10 mL CPU = 18
Filter 3 volume (mL) = 1.0 mL CPU = 0
The analytical result is calculated as:
100
(82 + 18 + 0) x = 90 E. coli CFU/100 mL
(100 + 10+1)
Example 7:
Filter 1 volume (mL) = 50 mL CPU = TNTC
Filter 2 volume (mL) = 25 mL CPU = TNTC
Filter 3 volume (mL) = 10 mL CPU = 83
The analytical result is calculated as:
100
83 x = 830 E. coli CFU/100 mL
10
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Section 7: E. coli Data: Use, Recording, Submitting and Review
7.4.3.4 Calculation of £. coli Concentrations Using Multiple-Tube Methods
The guidance and examples for determining E. coli concentrations using multiple-tube methods are based
on the revision of Standard Methods 9221C included in the 2001 Supplement to the 20th Edition of
Standard Methods (Reference 8.13), approved by the Standard Methods Committee in 1999.
Note: The analytical result can be automatically calculated using the LT2 Data Collection
System. See Section 7.3.1 for additional information.
A. For each sample volume (e.g., 10, 1, 0.1, and 0.01 mL or additional sample volumes as
necessary), determine the number of positive tubes out of five.
B. A dilution refers to the volume of original sample that was inoculated into each series of tubes.
Only three of the dilution series will be used to estimate the MPN. The three selected dilutions are
called significant dilutions and are selected according to the following criteria. Examples of
significant dilution selections are provided in Table 7-2, below.
Choose the highest dilution (the most dilute, with the least amount of sample) giving positive
results in all five tubes inoculated and the two succeeding higher (more dilute) dilutions.
(Table 7-2, Example A).
If the lowest dilution (least dilute) tested has fewer than five tubes with positive results, select
it and the two next succeeding higher dilutions (Table 7-2, Examples B and C).
When a positive result occurs in a dilution higher (more dilute) than the three significant
dilutions selected according to the rules above, change the selection to the lowest dilution
(least dilute) that has less than five positive results and the next two higher dilutions (more
dilute) (Table 7-2, Example D).
When the selection rules above have left unselected any higher dilutions (more dilute) with
positive results, add those higher-dilution positive results to the results for the highest
selected dilution (Table 7-2, Example E).
If there were not enough higher dilutions tested to select three dilutions, then select the next
lower dilution (Table 7-2, Example F).
C. MPN values must be adjusted based on the significant dilutions series selected above. Because
the MPN/100 mL values in the table are based on the 10 mL, 1 mL, and 0.1 mL dilutions, per
method requirements, the MPN value must be adjusted if these are not the significant dilutions
selected. Use the following calculation to adjust the MPN when the 10 mL, 1 mL, and 0.1 mL
dilutions are not the significant dilutions selected:
MPN value
Analytical result = = E. coli MPN/100 mL
# of mL in middle dilution
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Section 7: E. coli Data: Use, Recording, Submitting and Review
Table 7-2. Examples of Different Combinations of Positive Tubes (Significant Dilution Results
Are in Bold and Underlined)
Example
A
B
C
D
E
F
Least d i 1 ute Most d i 1 ute
(Lowest) (Highest)
10
mL
5
4
0
5
5
5
1 mL
5
5
0
4
4
5
0.1
mL
1
1
1
4
4
5
0.01
mL
0
0
0
1
0
5
0.001
mL
0
0
0
0
1
2
Combination
of positives
5-1-0
4-5-1
0-0-1
4-4-1
4-4-1
5-5-2
MPN Index from
Standard Methods
33
48
1.8
40
40
540
£. co/;7100 mL
(after adjustment)
330
48
1.8
400
400
54,000
Example A:
The significant dilution series for the 5-1-0 combination of positives includes the
1 mL, 0.1 mL, and 0.01 mL dilutions. Because the 10 mL, 1 mL, and 0.1 mL
dilutions were not selected, an adjustment is necessary to account for the
dilutions selected:
Analytical result =
33
Example B:
Example C:
Examples D and E:
0.1
= 330E. coli /100mL
Because the 10 mL, 1 mL, and 0.1 mL dilutions are the significant dilutions, no
adjustment is necessary and the result is 48 E. co//'/100 mL.
Because the 10 mL, 1 mL, and 0.1 mL dilutions are the significant dilutions, no
adjustment is necessary and the result is 1.8 E. co//'/100 mL.
The significant dilution series for the 4-4-1 combination of positives includes the
1 mL, 0.1 mL, and 0.01 mL dilutions. Because the 10 mL, 1 mL, and 0.1 mL
dilutions were not selected, an adjustment is necessary to account for the
dilutions selected:
Analytical result =
40
0.1
= 400£. coli /100mL
Example F: The significant dilution series for the 5-5-2 combination of positives includes the 0.1 mL,
0.01 mL and 0.001 mL dilutions. Because the 10 mL, 1 mL, and 0.1 mL dilutions were
not selected, an adjustment is necessary to account for the dilutions selected:
Analytical result =
540
0.01
= 54,000 £. coli /100ml
7.4.4 Data Archiving Requirements
The PWS is required to keep all original, hardcopy quality control data associated with LT2 sample
analyses (both initial and second round of monitoring) for 3 years after bin classification or determination
of the mean Cryptosporidium level [40 CFR § 141.722(a)]. Although it is the PWS's responsibility to
meet LT2 Rule data storage requirements for compliance monitoring samples, the PWS may contract this
work to the laboratory.
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SECTION 8: REFERENCES
8.1 USEPA. 2006. National Primary Drinking Water Regulations: Long Term 2 Enhanced Surface
Water Treatment Rule. 40 CFR parts 9, 141, and 142. Supporting guidance documents are
available at: http://www.epa.gov/safewater/disinfection/lt2/.
8.2 USEPA. 2005. Laboratory Quality Assurance Evaluation Program for Analysis of
Cryptosporidium under the Safe Drinking Water Act; Agency Information Collection: Proposed
Collection; Comment Request. Federal Register: June 3, 2005. 70 FR 32607.
8.3 USEPA. 2005. Method 1622: Cryptosporidium in Water by Filtration/IMS/FA. U.S.
Environmental Protection Agency, Office of Water, Washington, D.C. EPA-815-R-05-001.
Document is available for download at: http://www.epa.gov/safewater/disinfection/lt2/.
8.4 USEPA. 2005. Method 1623: Cryptosporidium and Giardia in Water by Filtration/IMS/FA. U.S.
Environmental Protection Agency, Office of Water, Washington, D.C. EPA-815-R-05-002.
Document is available for download at: http://www.epa.gov/safewater/disinfection/lt2/.
8.5 USEPA 2005. Manual for the Certification of Laboratories Analyzing Drinking Water; Criteria
and Procedures; Quality Assurance. Fifth Edition. EPA 815-R-05-004. Office of Ground Water
and Drinking Water, U.S. Environmental Protection Agency, 26 Martin Luther King Drive,
Cincinnati, OH 45268.
8.6 American Public Health Associated. 1998. Standard Methods for the Examination of Water and
Wastewater; 20th Edition. American Public Health Association, Washington D.C. Standard
Methods may be ordered from: American Water Works Association Bookstore, 6666 West
Quincy Avenue, Denver, CO 80235.
8.7 Connell, Kevin, et al. 2000. ICRSS - Building a Better Protozoa Data Set, J. AWWA. 92(10): 30
43.
8.8 IDEXX Laboratories, Inc., Description of Colilert®, Colilert-18®, Quanti-Tray®, Quanti-
Tray®/2000, and Colisure™ methods may be obtained from: IDEXX Laboratories, Inc., One
IDEXX Drive, Westbrook, Maine 04092.
8.9 USEPA. 2002. Method 1103.1: Escherichia coli in Water by Membrane Filtration Using
membrane-Thermotolerant&c/7enc/7/a coll Agar (mTEC). U.S. Environmental Protection
Agency, Office of Water, Washington, D.C. EPA-821-R-02-020.
8.10 USEPA. 2002. Method 1603: Escherichia coli (E. coli) in Water by Membrane Filtration Using
Modified membrane-Thermotolerant&c/7enc/7/a coli agar (Modified mTEC). U.S.
Environmental Protection Agency, Office of Water, Washington, D.C. EPA-821-R-02-023.
8.11 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. EPA-821-R-02-024.
71 February 2006
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Section 8: References
8.12 Hach Company, Inc. m-ColiBlue24 Method is available from: Hach Company, P.O. Box 389,
Loveland, CO 80539
8.13 2001 Supplement to the 20th Edition of Standard Methods 9221 C: Explanation of Bacterial
Density. This supplement is available for download at http://www.techstreet.com/cgi-
bin/detail?product id=923645.
8.14 USEPA. 2005. Microbial Laboratory Guidance Manual for the Long Term 2 Enhanced Surface
Water Treatment Rule (LT2 Rule). This manual is available for download from
http://www.epa.gov/safewater/disinfection/lt2/compliance.html
72 February 2006
-------
Appendix A
Intent to Provide Maximum Treatment
Example Notice
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Long Term 2 Enhanced Surface Water Treatment Rule
Intent to Provide Maximum Treatment - Example Notice
Public Water Systems (PWSs) who choose to provide the maximum level of treatment for
Cryptosporidium applicable to their plant type rather than start source water monitoring must
submit written notification no later than the date the PWS is otherwise required to submit a
sampling schedule for monitoring. Alternatively, a PWS may choose to stop sampling at any
point after it has initiated monitoring if it provides written notification that it will provide this level
of treatment. This form is an example of the necessary written notification. PWSs must install
and operate technologies to provide this level of treatment by their applicable treatment
compliance date.
PWS Information
PWS Name:
PWS ID:
PWS Address:
Email Address:
Water Treatment Plant Name:
Water System Facility ID:
Type of system that will be providing maximum treatment instead of conducting monitoring:
D Filtered system providing a total of at least 5.5-log of treatment for Cryptosporidium [40 CFR part
141.701 (d)(1)].
D Unfiltered system providing a total of at least 3-log Cryptosporidium inactivation [40 CFR part
141.701 (d)(2)].
Planned date of treatment compliance:
Planned treatment to achieve compliance:
D Yes D No
D Yes D No
I understand the treatment requirements that my PWS is required to meet and am
aware of the deadline for providing the treatment.
I have discussed these requirements with a State or U.S. EPA representative
Signature:
Name (print)
Date:
Phone:
This notice may be submitted using one of the following options:
• As an email attachment sent to stage2mdbp@epa.gov
• By mail or fax to the following: LT2ESWTR and Stage 2 DBPR
P.O. Box 98
Dayton, OH 45401
FAX: (937)-586-6557
A-1
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Appendix B
Cost Estimate for Bulk Water Sample Analysis
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LT2 Monitoring Bulk Water Sample Analysis for [PWS name and/or facility name]
PWS required fie\d:\_
For further information on this bid sheet, refer to Section 2.2 of the Source Water Monitoring Guidance Manual for Public Water Systems
Laboratory name:
Laboratory address:
Laboratory contact:
Phone/fax/email:
Submit bid to:
PWS name:
PWS address:
PWS contact:
Phone/fax/email:
Bid deadline (day, date, time (including time zone)):
Estimated award date:
Bid validity period:
Laboratory period of performance:
Results turnaround time:
Extra Services (if applicable):
Costs for Cryptosporidium-On\y or CryptosporidiumlGiardia Analysis
Sample
PWS requests bid for Cryptosporidium analysis using Method 1 623*
[Specify sample volume if other than 1 0 L]
Bulk water samples - full analysis
Matrix spike samples
Practice samples
Potential replacement samples
Estimated subsamples**
Equipment
Cubitainers/Carboys***
Sampling if conducted by the laboratory or subcontractor (including turbidity)
Shipping****
Shipment of cubitainer/carboy to PWS
Shipment of collected samples to laboratory
Number of
plants
Number of
plants
Number of
plants
Samples
required per
plant
Up to [no.]
Up to [no.]
Equipment
required per
plant
Shipments per
plant
(A)
Total
samples
Total
equipment
Total
shipments
(B)
Cost per
sample
Cost per
unit
Cost per
shipment
(AxB)
Total
cost
Total
cost
Total
cost
Total
Costs for E. co// analysis
Sample
PWS requests bid for E.coli analysis using [Specify method]
Field sample analysis
Equipment
Sample collection bottles
Shipping****
Shipment of sample collection bottles to PWS
Shipment of collected samples to laboratory
Number of
plants
Units required
per plant**
(A)
Total
units
(B)
Cost per
unit
(AxB)
Total
cost
Total
^laboratories may require special schedule and increased QA costs if 1622 is requested by the PWS.
**IMS, staining, and examination of each 0.5-mL portion of a sample concentrate that exceeds 0.5 ml_ packed pellet volume; include number for field, matrix
spike, practice, and replacement samples
***AII cubitainers/carboys required for field samples, matrix spike samples, practice samples, and potential replacement samples (may be purchased directly from supplier)
****Shipment cost of replacement equipment and samples should also be discussed and decided with laboratory
B-1
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Appendix C
Cost Estimate for Field-Filtered Sample Analysis
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LT2 Monitoring Field-Filtered Sample Analysis for [PWS name and/or facility name]
Laboratory name:
Laboratory address:
Laboratory contact:
Phone/fax/email:
Submit bid to:
PWS name:
PWS address:
PWS contact:
Phone/fax/email:
Bid deadline (day, date, time (including time zone)):
Estimated award date:
Bid validity period:
Laboratory period of performance:
Results turnaround time:
Extra Services (if applicable):
Costs for Cryptosporidium-On\y or CryptosporidiumlGiardia Analysis
Sample
PWS requests bid for Cryptosporidium analysis using Method 1623*
[Specify sample volume if other than 1 0 L]
Field-filtered samples - full analysis
Matrix spike samples (bulk sample)
Practice samples
Potential replacement samples
Estimated subsamples**
Equipment
Filters [Envirochek™, Envirochek™ HV or Filta-Max®]***(extra if clogging expected)
Sampling apparatus for rental or purchase (used during monitoring period)
Cubitainer/Carboy (for use with each matrix spike)
Sampling if conducted by the laboratory or subcontractor (including turbidity)
Shipping
Shipment of filters to PWS***
Shipment of cubitainer/carboy (for matrix spike) to PWS
Shipment of filter apparatus to PWS
Shipment of cubitainer/carboy (for matrix spike) to laboratory
Shipment of collected samples to laboratory***
Number of
plants
Number of
plants
Number of
plants
Samples
required per
plant
Up to [no.]
Up to [no.]
Equipment
required per
plant
Shipments per
plant
(A)
Total
samples
Total
equipment
Total
shipments
(B)
Cost per
sample
Cost per
unit
Cost per
shipment
(AxB)
Total
cost
Total
cost
Total
cost
Total
Costs for E. co// analysis
Sample
PWS requests bid for E.coli analysis using [Specify method]
Field sample analysis
Equipment
Sample collection bottles
Shipping****
Shipment of sample collection bottles to PWS
Shipment of collected samples to laboratory
Number of
plants
Units required
per plant**
(A)
Total
units
(B)
Cost per
unit
(AxB)
Total
cost
Total
^Laboratories may require special schedule and increased QA costs if 1622 is requested by the PWS.
**IMS, staining, and examination of each 0.5-mL portion of a sample concentrate that exceeds 0.5 ml_ packed pellet volume; include number for field, matrix
spike, practice, and replacement samples
***AII filters required for field samples, matrix spike samples, practice samples, and potential replacement samples (may be purchased directly from supplier)
****Shipment cost of replacement equipment should be discussed and decided with laboratory p *
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Appendix D
Sampling Location Worksheet
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Long Term 2 Enhanced Surface Water Treatment Rule
Sampling Location Worksheet
Public Water System (PWS) Name:
Water Treatment Plant Name:
PWS ID:
Water System Facility ID:
1. Source Name
2. Source Type
Flowing stream, Lake/Reservoir, or
GWUDI
3. Source Water Sampling
Location Provide State assigned
number
4. Usage
All year, Part-year, or Emergency
(Describe conditions, constraints,
months in operation)
5. Proportion of typical average
daily flow
6. Pretreatment Practices
Presedimentation, Bank filtration, or
Off-stream storage
7. Recycling Practices (if
applicable)
Description and return flow location
8. Chemical Pretreatment
(Indicate location on plant
schematic)
9. Sample Compositing Procedure
(if applicable) Blended sample tap,
Composite sample, or Weighted
Use additional sheets or reverse side to provide more information
D-1
-------
Additional comments (Indicate item number from table on reverse side)
D-2
-------
Appendix E
LT2 Sample Collection Location Schematics
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Figure Description
1 Sample tap before chemical treatment and backwash water recycle (if applicable)
2 Multiple sources: sample tap after two combined sources
3 Multiple sources: two (or more) sources to be composited
4 Multiple plants with the same influent
5 Bank filtration
6 Ground water under the direct influence of surface water
7 Presedimentation basin
8 Raw water off-stream storage
9 Mixed source water: ground water and surface water sources
10 Blank schematic for submission to EPA
E-1
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Figure 1. Sample Tap before Chemical Treatment
and Backwash Water Recycle (if applicable)
Raw water
chemical building
Water treatment plant
Raw water intake
LT2 Rule: Systems must collect source water
samples prior to chemical treatment [40 CFR §
141.703(b)(1)], unless approved by the State to
collect source water samples after chemical
treatment [40 CFR § 141703(b)(2)]. Systems that
recycle filter backwash water must collect source
water samples prior to the point of filter backwash
water addition [40 CFR § 141.703(c)].
Backwash water
recycle (if applicable)
Backwash water
lagoon
Chemical
addition
Backwash water
recycle
(if applicable)
-------
Figure 2. Multiple Sources: Sample Tap after Two Combined Sources
If a sample tap is available where the
sources are combined prior to
treatment, the sample must be
collected from the tap [40 CFR §
141.703(e)(1)]*
LT2 Rule: Systems must collect source water
samples prior to chemical treatment [40 CFR §
141.703(b)(1)], unless approved by the State to
collect source water samples after chemical
treatment [40 CFR § 141703(b)(2)]. Systems that
recycle filter backwash water must collect source
water samples prior to the point of filter backwash
water addition [40 CFR § 141.703(c)].
Water treatment plant
Raw water
blending station
Systems unable to sample from
this combined source location
should refer to Figure 3.
Backwash water
recycle (if applicable)
Backwash water
lagoon
Multiple Alternating Sources:
Systems that use multiple alternating sources
(seasonal or due to water quality changes) must
sample from the source water in use on the designated
sampling date following normal operating practices
[40 CFR §141.703(e)].
E-3
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Figure 3. Multiple Sources: Two (or More) Sources to be Composited
Manually
collect^
sample <
75%
. of flow
If a sampling tap where the sources are combined
prior to treatment is not available, systems must
collect samples at each source near the intake on
the same day and follow the options discussed
below [40 CFR § 141.703(e)(2)].
Manually
| collect
sample
OPTION 1 (Recommended Option):
Collect samples manually at each source near
the intake on the same day and composite
them into one sample to be analyzed. The
volume of sample from each source must
reflect its proportion of the total plant flow at
the time the samples were collected [40 CFR
§141.703(e)(2)(i)].
75%
Source water
"A"
25%
Source water
"B"
To be analyzed
LT2 Rule: Systems must collect source water samples prior to chemical
treatment [40 CFR § 141.703(b)(1)], unless approved by the State to collect
source water samples after chemical treatment [40 CFR § 141.703(b)(2)].
Systems that recycle filter backwash water must collect source water samples
prior to the point of filter backwash water addition [40 CFR § 141.703(c)].
Water treatment plant
Raw water
blending station
Backwash water
recycle (if applicable)
Backwash water
lagoon
OPTION 2:
Collect samples manually at each source near the intake on the same
day and analyze each independently, then calculate a weighted
average of the analysis results. This is done by multiplying the result
for each source by the percentage of its contribution to the total plant
flow at the time the samples were collected, and then summing these
values [40 CFR § 141.703(e)(2)(ii)].
Analyzed
Multiply result by
percentage of
contribution to total
flow
Source
water
"B"
E-4
Analyzed
Multiply result by
percentage of
contribution to total
flow
Sum together all
source water
results for total
value
-------
Figure 4. Multiple Plants with the Same Influent
Raw water
chemical building
LT2 Rule: Systems must collect source water samples
priorto chemical treatment [40 CFR § 141.703(b)(1)],
unless approved by the State to collect source water
samples after chemical treatment [40 CFR §
141.703(b)(2)]. Systems that recycle filter backwash water
must collect source water samples prior to the point of filter
backwash water addition [40 CFR § 141.703(c)].
Water treatment plant "A"
Raw water intake
Where multiple plants draw from the same influent,
such as the same pipe or intake, the State may
approve one set of monitoring results to be used to
satisfy the requirements for all plants [40 CFR §
141.703(a)].
Water treatment plant "B
Chemical
addition
E-5
To plant "B"
-------
Figure 5. Bank Filtration
The correct sampling location for systems using bank filtration differs depending
on whether the bank filtered water is treated by subsequent filtration:
Scenario 1: Systems that receive Cryptosporidium
treatment credit for bank filtration must collect
source water samples in the surface water prior to
bank filtration [40 CFR § 141703(d)(1)].*
Raw water
chemical building
Manually
collect 4
sample
7
Flowing
stream
Groundwater
Chemical
addition
Water treatment plant
Minimal surface
water mixing with
ground water
Backwash water
recycle (if applicable)
Backwash water
lagoon
Bank filtration cross section
Scenario 2: Systems using bank filtered water that is treated by subsequent
filtration must collect source water samples from the well source (i.e., after bank
filtration) but before any other treatment.** Use of bank filtration during
monitoring should be consistent with routine operational practice. Systems
collecting samples after a bank filtration process may not receive
Cryptosporidium treatment credit for the bank filtration [40 CFR § 141703(d)(2)].
Raw water
chemical building
Water treatment plant
Backwash water
recycle (if applicable)
Backwash water
lagoon
Bank filtration cross section
* Refers to systems using bank filtration to meet Cryptosporidium removal requirements of the
Interim Enhanced Surface Water Treatment Rule (IESWTR) or Long Term 1 ESWTR under 40 CFR
§ 141.173(b)or40CFR§ 141.522(a).
** Refers to systems where bank filtration serves as pretreatment to a filtration plant.
E-6
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Figure 6. Ground Water Under the Direct Influence of Surface Water
(GWUDI)
Systems that use ground water under the
direct influence of surface water (GWUDI)
must collect source water samples from
the well source unless they received
Cryptosporidium treatment credit for bank
filtration (see Figure 5).
LT2 Rule: Systems must collect source water
samples prior to chemical treatment [40 CFR §
141.703(b)(1)], unless approved by the State to
collect source water samples after chemical
treatment [40 CFR § 141703(b)(2)]. Systems that
recycle filter backwash water must collect source
water samples prior to the point of filter backwash
water addition [40 CFR § 141.703(c)].
Raw water
chemical building
LT2
sample
point
Water treatment plant
Backwash water
recycle (if applicable)
GWUDI cross section
Backwash water
lagoon
E-7
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Figure 7. Presedimentation Basin
Scenario 1:
Systems using a presedimentation basin with chemical addition should collect source water
samples prior to chemical treatment, unless approved by the State to collect source water
samples after chemical treatment. Systems that recycle filter backwash water must collect source
water samples prior to the point of filter backwash water addition [40 CFR § 141.703(c)].
Raw water
chemical building
Presed.
basin
Water treatment plant
Backwash water
recycle (if applicable)
Backwash water lagoon
Scenario 2:
Systems without chemical addition prior to or in a presedimentation basin, or that have been
approved by the State to collect source water samples after chemical treatment, may sample
after the presedimentation basin but will not receive any treatment credit for presedimentation.
Presed.
basin
Water treatment plant
Raw water intake
Sampling here will result in
no Cryptosporidium
treatment credit
E-8
-------
Figure 8. Raw Water Off-Stream Storage
Raw water
chemical building
LT2 Rule: Systems must collect source water
samples prior to chemical treatment [40 CFR §
141.703(b)(1)], unless approved by the State to
collect source water samples after chemical
treatment [40 CFR § 141703(b)(2)]. Systems that
recycle filter backwash water must collect source
water samples prior to the point of filter backwash
water addition [40 CFR § 141.703(c)].
Water treatment plant
Source water samples should be
collected after the off-stream storage
reservoir. Use of off-stream storage
during monitoring should be consistent
with routine operational practice.
Backwash water
recycle (if applicable)
Backwash water
lagoon
E-9
-------
Figure 9. Mixed Source Water: Ground Water and Surface Water Sources
Scenario 1 :
Raw water
chemical building
Water treatment plant
LT2
sample
point
Raw water intake
Well
Groundwater
Ground water well
cross section
If a sample tap is available
where the sources are combined
prior to treatment, systems must
collect samples from the tap [40
CFR§ 141.703(e)(1)]. Systems
unable to sample from this
combined source location should
refer to Figure 3.
Backwash water
recycle (if applicable)
Backwash water
lagoon
Scenario 2:
Raw water intake
Raw water
chemical building
Water treatment plant
LT2 Rule: Systems must collect source
water samples prior to chemical
treatment [40 CFR § 141.703(b)(1)],
unless approved by the State to collect
source water samples after chemical
treatment [40 CFR § 141.703(b)(2)].
Systems that recycle filter backwash
water must collect source water samples
prior to the point of filter backwash water
addition [40 CFR § 141.703(c)].
Systems that use ground water pumped
directly to the distribution system should
collect samples only from the surface
water source and prior to backwash water
or chemical addition.
Backwash water
lagoon
Well
To
-distribution—K
system
Ground water sources are not required
to be monitored under the LT2 Rule.
No sample is needed from this source.
Ground water well
cross section
E-10
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Figure 10. Blank Schematic for Submission to EPA
Public Water System (PWS) name:
PWS ID:
Water treatment plant name:
Water system facility ID:
Indicate the following on the diagram that best represents your facility
type (if applicable'):
1. LT2 sampling location
2. Points of chemical treatment prior to the treatment plant
3. Filter backwash water addition
4. Pretreatment processes (e.g., presedimentation basins, bank filtration)
5. Multiple source waters (show by adding additional sources)
Water treatment plant
E-11
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Appendix F
LT2 Sample Collection Form
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Long Term 2 Enhanced Surface Water Treatment Rule
Sample Collection Form
Utility Information
PWS name:
PWS address:
Sampler name:
Shipping Information For Lab Use
Lab name: Date received:
Time received:
Lab address:
Received by:
Sample temperature
on receipt:
Sample
Date shipped: condition
Tracking number: on rece'Pt:
Only
Sample Identification Information (the combination of bolded items are used to identify the sample under LT2)
Sample ID (optional):
PWS ID:
Facility ID:
Facility name:
Sample collection point ID:
Sample collection point name:
Sample collection date:
Source water type3 (circle one): Flowing stream (FS) Reservoir/lake (RL)
GWUDIb-FS GWUDIb-RL
Requested analysis (circle one): Cryptosporidium field sample Cryptosporidium matrix spike
£. co/;
Sample Collection Information
Cryptosporidium E. coli
If M ™eter;eadin? , . Sample collection time:
(field-filtered samples only): K
Final meter reading _ ..... ,..-,.. .,
(field-filtered samples only): Turbldlty (NTU):
Sample collection time
(or start time, if field filtering) :
Sample collection stop time
(field-filtered samples only):
Source water temperature:
Additional comments:
Sampler signature: Date:
a The source water type should be selected based on the type of source water that accounts for the majority of the
surface water used as source water at the time of sample collection
b Ground water under the direct influence of surface water
F-1
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Appendix G
Envirochek™ Field Filtration for Cryptosporidium
-------
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Procedure for Field-Filtering Samples for Cryptosporidium Analysis
Using the Pall Life Sciences
Envirochek™ or Envirochek™ HV Capsules
1.0 Materials
The following materials should be available before collecting sample:
nD Several pairs of new, powder-free latex gloves
nD Sample collection form (Appendix F)
nD Pall Life Sciences Envirochek™ or Envirochek™ HV capsule (recommend that two capsules be
kept on hand in case the first one clogs prior to filtering 10 L)
nD Sanitary >30 L carboy (if sample is not from a pressurized source)
nD Stopwatch
nD Sample label
nD Cooler, approximately 16-quart
nD Temperature monitoring device (e.g., thermometer vial, Thermochron™ iButton, or equivalent)
(if measuring temperature during shipment)
nD Two large plastic trash bags
nD One 8-lb bag of ice or gel ice packs
nD Three gallon size ziplock bags
nD Strapping tape
nD Two self-adhesive plastic airbill sleeves
nD Airbill for shipment
The following items may be purchased as a unit for sampling from a vendor or laboratory supplying
sampling apparatus for field filtering Cryptosporidium samples:
nD Pump (if sample is not from a pressurized source) - Electric centrifugal or Electric peristaltic
pump, or any equivalent pump that can create a flow of approximately 2 L/min
nD Two 0.5-in. X 0.375-in. barbed reducing connectors
nD Five lengths of clean 12.7-mm (0.5-in.) internal-diameter clear, vinyl, laboratory tubing
nD Five pairs of hose clamps fit to tubing
nD One 0.5-in. x 3-in. nipple
nD One coupling to fit 0.5-in. internal diameter tubing
nD One roll teflon tape
nD One 0.5-in. x 0.5-in. x 0.25-in. tee
nD Six 0.5-in. barbed male adapters
nD One garden hose barbed adapter
nD Pressure regulator
G-1 February 2006
-------
nD Pressure gauge (capsule's maximum operating pressure, Envirochek™ - 30 PSID, Envirochek™
HV - 60 PSID)
nD Water meter (flow totalizer)
nD Flow rate meter with valve or flow control valve (capsule's maximum flow rate, Envirochek™ -
2 L/min, Envirochek™ HV - 4 L/min)
2.0 Collecting the Sample
If the sample will be collected from a pressurized source, use the sample collection procedures in Section
2.1. If the sample will be collected from an unpressurized source, use the sample collection procedures in
Section 2.2.
2.1 Sample Filtration from a Pressurized Source
2.1.1 Before connecting the sampling system to the tap or source, turn on the tap and allow the water to
flow for 2 to 3 minutes until the temperature has stabilized or until any debris that has
accumulated in the source water lines has cleared and the turbidity in the water becomes visibly
uniform. Turn off the tap.
2.1.2 Put on a pair of powder-free latex gloves to prevent contamination from outside sources. Any
contamination inside the sampling apparatus may bias the final results.
2.1.3 Determine the pressure of the water source with the pressure gauge.
2.1.4 Assemble the sampling system, minus the Envirochek™ /Envirochek™ HV capsule. In place of
the Envirochek™ /Envirochek™ HV capsule, insert a 0.5-in. barbed connector between the outlet
tubing from the sample valve or the pressure regulator or gauge and the inlet tubing of the flow
totalizer, flow meter or control valve. For high pressure (>30 PSIG for Envirochek™ capsule or
>60 PSIG for Envirochek™ HV capsule) sites, the sampling system should be assembled in the
following order, as shown in Figure 1 below:
nD Reinforced influent tubing
nD Pressure regulator
nD Pressure gauge
nD Reinforced inlet tubing
nD Envirochek™ /Envirochek™ HV capsule
nD Reinforced outlet tubing
nD Flow totalizer (mechanical or graduated collection device)
nD Flow rate meter with valve or flow control valve
nD Effluent tubing to drain
G-2 February 2006
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Figure 1.Sample System Setup for Collecting Cryptosporidium Samples from a Pressurized Source
( >30 PSIG for Envirochek™ capsule or >60 PSIG for Envirochek™ HV capsule)
/P&
Outlet tubing
lit
Flow rate meter*
with valve
Effluent tubing
\
(Valve)
DIRECTION OF FLOW
Flow control
valve may be
used in place of
flow rate meter
For low pressure (1 to 30 PSIG for Envirochek™ capsule or 1 to 60 PSIG for Envirochek™ HV capsule)
sites, the sampling system should be assembled at the sample port valve in the following order, as shown
in Figure 2 below :
n Influent tubing
D Envirochek™ /Envirochek™ HV capsule
n Outlet tubing
n Flow rate meter with valve or flow control valve
n Flow totalizer (mechanical or graduated collection device)
n Effluent tubing to drain
Figure 2.Sample System Setup for Collecting Cryptosporidium Samples from a Pressurized Source
(1 - 30 PSIG for Envirochek™ capsule or 1 - 60 PSIG for Envirochek™ HV capsule)
Flow rate meter* -
with valve
Flow totalizer
Effluent tubing
Influent
tubing
Outlet
Envirochek™ capsule tubing
DIRECTION OF FLOW
Flow control
valve may be
used in place of
flow rate meter
2.1.5 Connect the sampling system, with the connector in place of the Envirochek™ /Envirochek™
HV capsule, to the pressurized water system, using appropriate fittings and clamps.
G-3
February 2006
-------
2.1.6 Slowly turn the tap to fully open. Allow a minimum of 20 L to flush the system. During this
period, perform the following steps:
2.1.6.1 Measuring the flow rate with the flow rate meter or the flow totalizer and a stopwatch,
adjust the flow rate to approximately 2 L/min (approximately 0.5 gpm) for the
Envirochek™ capsule or 4 L/min (approximately 1.0 gpm) for the Envirochek™ HV
capsule. If a flow control valve at the appropriate flow rate is used, no adjustment of the
flow rate is necessary. Using the pressure regulator, adjust the pressure to a maximum of
30 PSIG if using the Envirochek™ or 60 PSIG if using the Envirochek™ HV. Observe
the system for leaks and take the necessary corrective action if any are present.
2.1.6.2 Record the following information on the sample collection form (Appendix F):
%> Public water system (PWS) name
^ PWS address
%> Sampler name
%> Sample ID (optional)
•% Public Water System Identification (PWS ID) number
%> Public Water System facility ID number
%> Facility name
%> Sample collection point ID
%> Sample collection point name
%> Sample collection date
%> Source water type (optional [but required for E. coll sample forms])
%> Requested analysis (circle Cryptosporidium field sample for routine monitoring
sample; circle both "Cryptosporidium field sample" and "Cryptosporidium
matrix spike" sample if you are sending an additional sample with the monitoring
sample for matrix spike analysis)
2.1.6.3 After the assembly has been flushed, measure the turbidity of the source water and any
optional water quality parameters such as temperature, and/or pH.
2.1.7 Turn off the water at the sample port valve when the flow rate has been adjusted and the system
has been flushed.
2.1.8 Record the following information on the capsule label with a waterproof pen:
^ PWS ID
%> Facility name
%> Date of sample collection
2.1.9 Record the following information on the sample collection form:
%> Initial meter reading
2.1.10 Remove the connector and in its place, install the Envirochek™ /Envirochek™ HV capsule in
line, securing the inlet and outlet ends with the appropriate fittings/clamps.
G-4 February 2006
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Note! Retain the vinyl caps provided with the Envirochek™ /Envirochek™ HV capsule. These
caps will be needed to seal the capsule for shipment.
2.1.11 Slowly turn on the pressurized water source. Adjust the flow rate to approximately 2 L/min for
Envirochek™ capsule or 4 L/min for Envirochek™ HV capsule, if necessary. Record the
following information on the capsule label or sample collection form:
•% Start time
2.1.12 Vent the residual air in the capsule using the bleed valve by turning it counter-clockwise. When
the capsule is full of water, close the bleed valve.
2.1.13 Monitor the water meter. When the targeted volume (actual sample volumes will be selected by
the utility, but volumes are typically 10 L [2.64 gal] to 50 L [13.2 gal] and preferred to be
consistent between sampling events for each source) has passed through the Envirochek™
/Envirochek™ HV capsule, shut off the water source. Allow the pressure to decrease until the
water stops.
2.1.14 Record the following information on the capsule label and/or sample collection form:
%> Stop time (when the water was shut off)
%> Final meter reading
%> Comments to laboratory, if needed
2.1.15 With the capsule inlet pointed up, loosen the outlet end of the Envirochek™ /Envirochek™ HV
capsule and allow water to drain as much as possible. Water drainage from the capsule through
the outlet is acceptable, as the sample has passed through the membrane. Opening the bleed valve
during the draining will speed the process. Be sure to close the valve when finished.
2.1.16 Disconnect the inlet end of the Envirochek™ /Envirochek™ HV capsule, making sure not to spill
any of the water remaining in the capsule through the inlet port. This water is part of your sample.
Capsule may be shipped with or without residual water.
2.1.17 Seal the inlet of the capsule with the vinyl end cap that was previously saved.
2.1.18 Seal the outlet of the capsule with the vinyl end cap that was previously saved. Place the
Envirochek™ /Envirochek™ HV capsule in a plastic ziplock bag for shipment.
2.1.19 Immediately following sample collection, place the bag containing the capsule in a refrigerator to
chill prior to packing the shipping cooler for shipment. If no refrigerator is available, and the
sample will not be shipped for several hours, place the bag in the shipping cooler with ice to chill.
Replace the ice with fresh ice before shipping.
Note! Method 1622/1623 requires that the temperature of the sample upon arrival at the
laboratory must be <20°C (but not frozen), and the laboratory must begin sample
processing within 96 hours of sample collection. If the sample temperature and holding
time requirements are not met, then the sample is invalid and must be recollected.
G-5 February 2006
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2.2 Sample Filtration Using an Unpressurized Source
2.2.1 Put on a pair of powder-free latex gloves to prevent contamination from outside sources. Any
contamination inside the sampling apparatus may bias the final results.
2.2.2 If sampling from a source of unlimited volume, it may be desirable to pre-flush the sampling
system. Assemble the sampling system, minus the Envirochek™ /Envirochek™ HV capsule. In
place of the Envirochek™ /Envirochek™ HV capsule, insert a 0.5-in. coupling to connect the
influent tubing to the inlet tubing of the pump. The sampling system should be assembled in the
following order, as shown in Figure 3 below:
D Influent tubing
D Envirochek™ /Envirochek™ HV capsule
D Outlet tubing
D Centrifugal or peristaltic pump
D Tubing
D Flow rate meter with valve or flow control valve
D Flow totalizer (mechanical or graduated collection device)
D Effluent tubing to drain
Figure 3. Sample System Setup for Collecting Cryptosporidium Samples from an
Unpressurized Source
Sample
Envirochek
\
Influent
tubing
™ capsule
i
— *• hn [Q
/ m
Flow rate meter* ' 1
with valve \ j
^j^J/ '-'-' ISLLJ —
/ SJf-^
Outlet centrifugal
tubing pumpu
w
DIRECTION OF FLOW
Flow totalizer Efl
*QJ (Valve)
^^~^\ Flow control
^^£N\ y valve may be
(J^-JU/ used in place of
^5^ f'ow rate meter
luent tubing
When assembling sample chain, make sure that it is as airtight as possible in order to create a vacuum. To
accomplish this, make sure that clamps are used at each connection and that rubber washers are inserted
into the hose connections on the inlet and outlet ends of the centrifugal pump.
2.2.3 Place the inlet end of the inlet tubing into the sample source, away from any walls, bottom, or
other environmental surfaces.
2.2.4 Turn on the pump and allow a minimum of 20 L to flush the system. If sampling source water
from a carboy, continuously refill the carboy as necessary to flush the system. As a
recommendation, the carboy should not be removed from the sampling chain and should be
refilled using a separate container. Observe the system for leaks and take the necessary corrective
action if any are present. During this period, perform the following steps:
G-6
February 2006
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2.2.4.1 Measuring flow rate with the flow rate meter or the flow totalizer and a stopwatch, adjust
the flow rate to approximately 2 L/min (approximately 0.5 gpm) for the Envirochek™
capsule or 4 L/min (approximately 1.0 gpm) for the Envirochek™ HV capsule by varying
the pump speed or adjusting the valve (if pump is not variable speed). If a flow control
valve at the appropriate flow rate is used, no adjustment of the flow rate is necessary.
2.2.4.2 Record the following information on the sample collection form:
%> Public water system (PWS) name
^ PWS address
%> Sampler name
%> Sample ID (optional)
•% Public Water System Identification (PWS ID) number
%> Public Water System facility ID number
%> Facility name
%> Sample collection point ID
%> Sample collection point name
%> Sample collection date
%> Source water type (optional [but required for E. coll sample forms])
%> Requested analysis (circle Cryptosporidium field sample for routine monitoring
sample; circle both "Cryptosporidium field sample" and "Cryptosporidium
matrix spike" sample if you are sending an additional sample with the monitoring
sample for matrix spike analysis)
2.2.5 Turn off the pump when the flow rate has been adjusted and the assembly has been flushed.
2.2.6 Record the following information on the capsule label:
^ PWS ID
%> Facility name
%> Date of sample collection
2.2.7 Record the following information on the sample collection form:
%> Initial meter reading
2.2.8 Install the Envirochek™ /Envirochek™ HV capsule in line, securing the inlet and outlet ends
with the appropriate fittings/clamps.
Note! Retain the vinyl caps provided with the Envirochek™ /Envirochek™ HV capsule. These
caps will be needed to seal the capsule for shipment.
2.2.9 Refill the carboy with the sample to be filtered. As a recommendation, the carboy should not be
removed from the sampling assembly and should be refilled using a separate container.
G-7 February 2006
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2.2.10 Turn on the pump. Adjust the flow rate to approximately 2 L/min for the Envirochek™ capsule or
4 L/min for the Envirochek™ HV capsule, if necessary. Record the following information on the
capsule label and/or sample collection form:
•% Start time
2.2.11 Monitor the water meter continuously refilling the carboy as needed. When the targeted volume
(actual sample volumes are selected by the utility, but volumes are typically 10 L [2.64 gal] to 50
L [13.2 gal] and preferred to be consistent between sampling events for each source) is reached,
remove tubing from the carboy and then shut off the pump.
2.2.12 Record the following information on the capsule label and/or sample collection form:
%> Stop time (when the pump was shut off)
%> Final meter reading
%> Comments to laboratory, if needed
2.2.13 With the capsule inlet pointed up, loosen the outlet end of the Envirochek™ /Envirochek™ HV
capsule and allow water to drain as much as possible. Water drainage from the capsule through
the outlet is acceptable, as the sample has passed through the membrane. Opening the bleed valve
during the draining will speed the process. Be sure to close the valve when finished.
2.2.14 Disconnect the inlet end of the Envirochek™ /Envirochek™ HV capsule, making sure not to spill
any of the water remaining in the capsule through the inlet port. This water is part of your sample.
Capsule may be shipped with or without residual water.
2.2.15 Seal the inlet of the capsule with the vinyl end cap that was saved previously.
2.2.16 Seal the outlet of the capsule with the vinyl end cap that was saved previously. Place the
Envirochek™ /Envirochek™ HV capsule in a plastic ziplock bag for shipment.
2.2.17 Immediately following sample collection, place the bag containing the capsule in a refrigerator to
chill prior to packing the shipping cooler for shipment. If no refrigerator is available, and the
sample will not be shipped for several hours, place the bag in the shipping cooler with ice to chill.
Replace the ice before shipping.
Note! Method 1622/1623 requires that the temperature of the sample upon arrival at the
laboratory must be <20°C (but not frozen), and the laboratory must begin sample
processing within 96 hours of sample collection. If the sample temperature and holding
time requirements are not met, then the sample is invalid and must be recollected.
3.0 Packing the Sample
3.1 Insert two large plastic trash bags into the shipping cooler to create a double liner. Immediately
before packing the cooler, create two 4-pound bags of ice in two separate ziplock bags. To
prevent leaks place each ice pack into an additional ziplock bag. Gel packs or blue ice may be
used instead of wet ice, as long as the sample is maintained in the appropriate temperature range.
Seal the ziplock bags, expelling as much air as possible, and secure top with tape.
Note! Shipping companies may delay sample shipments if leakage occurs. Double liners and
ziplock bags around ice will prevent leakage and delays.
G-8 February 2006
-------
3.2 Place the bag containing the capsule into the shipping container. Wrap the capsule in bubble wrap
to prevent freezing. Inflated, empty sample bags can be placed between the capsule and the ice
packs to prevent the sample from freezing.
3.3 If you will be monitoring sample temperature during shipment, place in the cooler the
temperature monitoring device (e.g., extra sample bottle for measuring sample temperature upon
receipt at the laboratory, thermometer vial, or Thermochron™ iButton). Seal each liner bag by
twisting top of bag and tying in a knot.
3.4 Peel the backing off one of the plastic airbill sleeves and attach the sleeve to the inside of the
cooler lid.
%> Sign and date the sample collection form.
Fold the completed sample collection form, and place it inside the plastic sleeve.
3.5 Close the cooler lid, seal the horizontal joints with strapping tape, and secure the lid with tape by
taping the cooler at each end, perpendicular to the seal.
Note! Shipping companies may delay sample shipments if leakage occurs. Be sure to seal the
cooler joints.
3.6 Peel the backing off of the second airbill sleeve and attach the sleeve to the outside of the cooler
lid. Complete the shipping airbill with the laboratory address, billing information, sample weight,
and shipping service. Remove the shipper's copy of the airbill, and place the remainder of the
airbill inside the plastic sleeve.
4.0 Shipping and Tracking
4.1 Ship samples on the day of collection and use a reliable shipping service for overnight delivery. If
samples are not shipped the day of collection, the sample must be maintained between 1°C and
10°C (but not frozen) by chilling in a refrigerator or cooler filled with ice.
4.2 Contact the laboratory to notify them of the sample shipment. Request that the laboratory contact
you the next day if the sample is not received.
4.3 Using the airbill number on the shipper's copy of the airbill, track the sample shipment using the
shipping company's web page or by contacting the shipping company over the phone.
4.4 If problems are encountered with the shipment, communicate with the shipping company to
resolve, and update the laboratory regarding the status of the shipment.
G-9 February 2006
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Appendix H
Filta-Max® Field Filtration for Cryptosporidium
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Procedure for Field-Filtering Samples for Cryptosporidium Analysis
Using IDEXX Filta-Max® Filters
1.0 Materials
The following materials should be available before collecting your sample:
nD Several pairs of new, powder-free latex gloves
nD Sample collection form (Appendix F)
nD Filta-Max® foam filter module (IDEXX, cat. number, FMC 10603) with housing (IDEXX, cat.
number, FMC 10504)
nD Sanitary >30 L carboy (if sample is not from a pressurized source)
nD Stopwatch
nD Sample number label
nD Cooler, approximately 16-quart
nD Temperature monitoring device (e.g., thermometer vial, Thermochron™ iButton, or equivalent)
(if measuring temperature during shipment)
nD Two large plastic trash bags
nD One 8-lb bag of ice or gel ice packs
nD Three gallon size ziplock bags
nD Strapping tape
nD Two self-adhesive plastic airbill sleeves
nD Airbill for shipment
The following items may be purchased as a unit for sampling from a vendor or laboratory supplying
sampling apparatus for field filtering Cryptosporidium samples:
nD Electric peristaltic pump (if sample is not from a pressurized source)
nD Two 0.5-in. x 0.375-in. barbed reducing connectors
nD Five lengths of clean 12.7-mm (0.5-in.) internal-diameter clear, vinyl, laboratory tubing
nD Five pairs of hose clamps fit to tubing
nD One 0.5-in. x 3-in. nipple (if sample line pressure is >120PSIG)
nD One coupling to fit 0.5-in. internal diameter tubing
nD One roll teflon tape
nD One 0.5-in. x 0.5-in. x 0.25-in. tee (if sample line pressure is >120 PSIG)
nD Six 0.5-in. barbed male adapters
nD One garden hose barbed adapter
nD Pressure regulator (if sample line pressure is >120PSIG)
nD Pressure gauge (filter's maximum operating pressure 120 PSIG)
H-1 February 2006
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nD Water meter (flow totalizer)
nD Flow rate meter or flow control valve (filter's maximum flow rate is 3-4 L/min)
2.0 Collecting the Sample
If the sample will be collected from a pressurized source, use the sample collection procedures in Section
2.1. If the sample will be collected from an unpressurized source, use the sample collection procedures in
Section 2.2.
2.1 Sample Filtration from a Pressurized Source
2.1.1 Before connecting the sampling system to the tap or source, turn on the tap and allow the water to
flow for 2 to 3 minutes until the temperature has stabilized or until any debris that has
accumulated in the source water lines has cleared and the turbidity in the water becomes visibly
uniform. Turn off the tap.
2.1.2 Put on a pair of powder-free latex gloves to prevent contamination from outside sources. Any
contamination inside the sampling apparatus may bias the final results.
2.1.3 Determine the pressure of the water supply using a pressure gauge.
2.1.4 Assemble the sampling system, minus the Filta-Max® filter. For high pressure (>120 PSIG) sites,
the sampling system should be assembled at the sample port valve in the following order, as
shown in Figure 1 below:
nD Reinforced influent tubing
nD Pressure regulator
nD Pressure gauge
nD Reinforced inlet tubing
nD Filta-Max® filter housing, with the direction of flow depicted with an arrow
nD Reinforce outlet tubing
nD Flow totalizer (mechanical or graduated collection device)
nD Flow rate meter with valve or flow control valve
nD Effluent tubing to drain
H-2 February 2006
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Figure 1. Sample System Setup for Collecting Cryptosporidium Samples from a Pressurized
Source (>120 PSIG)
ft
Outlet tubing
Flow rate meter*—
with valve
Influent
tubing
Pressure Inlet tubing I
regulator Filta-Max®
filter housing
Flow totalizer
DIRECTION OF FLOW
Effluent tubing
Flow control
valve may be
used in place of
flow rate meter
For a low-pressure (7.5 to 120 PSIG) site, the sampling system should be assembled at the sample port
valve in the following order, as shown in Figure 2 below:
n Influent tubing
n Filta-Max® filter housing, with the direction of flow depicted with an arrow
n Outlet tubing
n Flow rate meter with valve or flow control valve
n Flow totalizer (mechanical or graduated collection device)
n Effluent tubing to drain
Figure 2. Sample System Setup for Collecting Cryptosporidium Samples from a Pressurized
Source (7.5-120 PSIG)
Flow rate meter*-
with valve
Influent
tubing
Filta-Max® filter housing
Outlet
tubing
DIRECTION OF FLOW
/
Flow totalizer
-(Valve)
Flow control
valve may be
used in place of
flow rate meter
Effluent tubing
H-3
February 2006
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2.1.5 Connect the sampling system, with an empty Filta-Max® filter housing, to the pressurized water
system. Verify that the filter housing is installed so that the end closest to the screw top cap is the
inlet and the opposite end is the outlet.
2.1.6 Slowly turn the tap to fully open. Allow a minimum of 20 L to flush the system. During this
period, perform the following steps:
2.1.6.1 Measuring the flow rate with the flow rate meter or the flow totalizer and a stopwatch,
adjust the flow rate to approximately 3-4 L/min (approximately 0.8 - 1 gpm). If a flow
control valve at the appropriate flow rate is used, no adjustment of the flow rate is
necessary. At high pressure sites, using the pressure regulator, adjust the pressure to a
maximum of 120 PSIG. Observe system for leaks and take the necessary corrective
action if any are present. A differential pressure of 7.5 PSI is required to create flow
through the filter. The recommended differential pressure to produce the flow rate of 3 to
4 L/min is 75 PSI. Do not exceed the maximum operating pressure of 120 PSIG.
2.1.6.2 Record the following information on the sample collection form (Appendix F):
%> Public water system (PWS) name
^ PWS address
%> Sampler name
%> Sample ID (optional)
•% Public Water System Identification (PWS ID) number
%> Public Water System facility ID number
%> Facility name
%> Sample collection point ID
%> Sample collection point name
%> Sample collection date
%> Source water type (optional [but required for E. coll sample forms])
%> Requested analysis (circle Cryptosporidium field sample for routine monitoring
sample; circle both "Cryptosporidium field sample" and "Cryptosporidium
matrix spike" sample if you are sending an additional sample with the monitoring
sample for matrix spike analysis)
2.1.6.3 After the system has been flushed, measure the turbidity and any optional water quality
parameters such as temperature and/or pH.
2.1.7 Turn off the water at the sample port valve when the flow rate has been adjusted and the system
has been flushed.
2.1.8 Record the following information on the sample collection form (Appendix F):
%> Initial meter reading
2.1.9 Install the Filta-Max® filter into the housing and secure the housing cap by hand tightening.
Apply gentle pressure to create a seal between the module and the "O" rings in the base and lid of
the housing. Excessive tightening is not necessary, and may shorten the life of the "O" rings. A
H-4 February 2006
-------
light application of vacuum grease may be used to lubricate the "O" rings, but too much grease
will produce a negative effect.
Note! Retain the rubber stoppers provided with the filter housing. These stoppers will be
needed to seal the housing for shipment.
2.1.10 Slowly turn on the pressurized water source. Adjust flow to within 3 to 4 L/min, if necessary.
%> Record start time on the sample collection form.
2.1.11 Monitor the water meter. When the targeted volume (actual sample volumes will be selected by
the utility but volumes are typically 10 L [2.64 gal] or 50 L [13.2 gal] and preferred to be
consistent between sampling events for each source) has passed through the Filta-Max® filter,
shut off the water source. Allow the pressure to decrease until the water stops.
2.1.12 Record the following information on the sample collection form (Appendix F):
%> Stop time (when the water was shut off)
%> Final meter reading
%> Comments to laboratory, if needed
2.1.13 Disconnect the inlet end of the filter housing, making sure not to spill any of the water remaining
in the housing. This water is part of your sample. Disconnect the outlet end of the filter housing.
2.1.14 The filter can either be shipped in the filter housing or removed from the filter housing and
shipped alone.
2.1.15 If the filter will be shipped without the housing, open the housing while wearing a fresh pair of
gloves and dump the filter and the water remaining in the housing into a ziplock bag. Place this
bag inside a second ziplock bag and seal.
2.1.16 If the filter will be shipped in the housing, seal the inlet and outlet of the housing with the rubber
stoppers that were previously saved. Place the filter housing containing the filter in a plastic
ziplock bag for shipment. Ensure that there is residual water in the housing before shipping.
2.1.17 Place a label on the outer ziplock bag containing the filter and using a waterproof pen record the
following information:
^ PWS ID
%> Facility name
%> Date of sample collection
2.1.18 Immediately following sample collection, place the bag containing the filter (with or without
filter housing) in a refrigerator to chill prior to packing the shipping cooler for shipment. If no
refrigerator is available, and the sample will not be shipped for several hours, place the bag in the
shipping cooler with ice to chill, and replace the ice before shipping.
Note! Method 1622/1623 requires that the temperature of the sample upon arrival at the
laboratory must be <20°C (but not frozen), and the laboratory must begin sample
H-5 February 2006
-------
processing within 96 hours of sample collection. If the sample temperature and holding
time requirements are not met, then the sample is invalid and must be recollected.
2.2 Sample Filtration from an Unpressurized Source
2.2.1 Put on a pair of powder-free latex gloves to prevent contamination from outside sources. Any
contamination inside of the sampling apparatus may bias the final results.
2.2.2 If sampling from a source of unlimited volume, it may be desirable to pre-flush the sampling
system. Assemble the sampling system, minus the Filta-Max® filter. Verify that the filter housing
is installed so that the end closest to the screw top cap is the inlet and the opposite end is the
outlet. The sampling system should be assembled in the following order, as shown in Figure 3
below:
n Influent tubing
n Peristaltic pump
n Inlet tubing
n Filta-Max® filter housing, with the direction of flow depicted with an arrow
n Outlet tubing
n Flow rate meter with valve or flow control valve
n Flow totalizer (mechanical or graduated container)
n Effluent tubing to drain
Figure 3. Sample System Setup for Collecting Cryptosporidium Samples from an Unpressurized
Source
Sample
Flow rate
meter*
Peristaltic pump
-L
Influent tubing
p w'm v
-------
recommendation, the carboy should not be removed from the sampling assembly and should be
refilled using a separate container. Observe the system for leaks and take the necessary corrective
action if any are present. During this period, perform the following steps:
2.2.4.1 Measuring the flow rate with the flow rate meter or the flow totalizer and a stopwatch,
adjust the flow rate to approximately 3-4 L/min (approximately 0.8-1 gpm) by varying
the pump speed or adjusting the valve (if pump is not variable speed). If a flow control
valve at the appropriate flow rate is used, no adjustment of the flow rate is necessary.
2.2.4.2 Record the following information on the sample collection form:
%> Public water system (PWS) name
^ PWS address
%> Sampler name
%> Sample ID (optional)
•% Public Water System Identification (PWS ID) number
%> Public Water System facility ID number
%> Facility name
%> Sample collection point ID
%> Sample collection point name
%> Sample collection date
%> Source water type (optional [but required for E. coll sample forms])
%> Requested analysis (circle Cryptosporidium field sample for routine monitoring
sample; circle both "Cryptosporidium field sample" and "Cryptosporidium
matrix spike" sample if you are sending an additional sample with the monitoring
sample for matrix spike analysis)
2.2.5 Turn off the pump when the flow rate has been adjusted and the assembly has been flushed.
Following flushing, or if flushing is not performed, proceed with the following steps:
2.2.6 Record the following information on the sample collection form:
%> Initial meter reading
2.2.7 Install the Filta-Max® filter into the housing and secure the housing cap by hand tightening.
Apply gentle pressure to create a seal between the module and the "O" rings in the base and lid of
the housing. Excessive tightening is not necessary, and may shorten the life of the "O" rings. A
light application of vacuum grease may be used to lubricate the "O" rings, but too much grease
will produce a negative effect.
Note! Retain the rubber stoppers provided with the filter housing. These rubber stoppers will be
needed to seal the housing for shipment.
2.2.8 Turn on the peristaltic pump. Adjust the flow rate to approximately 3-4 L/min, if necessary.
Record the following on the sample collection form:
•% Start time
H-7 February 2006
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2.2.9 Monitor the water meter continuously refilling the carboy as needed. When the targeted volume
(actual sample volumes will be selected by the utility, but volumes are typically 10 L [2.64 gal] to
50 L [13.2 gal] and preferred to be consistent between sampling events for each source) is
reached, remove tubing from the carboy. Turn off the pump and allow the pressure to decrease
until the water stops.
2.2.10 Record the following information on the sample collection form:
%> Stop time (when the pump was shut off)
%> Final meter reading or measured total volume sample
%> Comments to laboratory, if needed
2.2.11 Disconnect the inlet end of the filter housing, making sure not to spill any of the water remaining
in the capsule. This water is part of your sample. Disconnect the outlet end of the filter housing.
2.2.12 The filter can either be shipped in the filter housing or removed from the filter housing and
shipped alone.
2.2.13 If the filter will be shipped without the housing, open the housing while wearing a fresh pair of
gloves and dump the filter and the water remaining in the housing into a ziplock bag. Place this
bag inside a second ziplock bag and seal.
2.2.14 If the filter will be shipped in the housing, seal the inlet and outlet of the housing with the rubber
stoppers that were saved previously. Place the filter housing containing the filter in a plastic
ziplock bag for shipment. Ensure that there is residual water in the housing before shipping.
2.2.15 Place a label on the outer ziplock bag containing the filter and using a waterproof pen record the
following information:
^ PWS ID
%> Facility name
%> Date of sample collection
2.2.16 Immediately following sample collection, place the bag containing the filter housing in a
refrigerator to chill prior to packing the shipping cooler for shipment. If no refrigerator is
available, and the sample will not be shipped for several hours, place the bag in the shipping
cooler with ice to chill. Replace the ice before shipping.
Note! Method 1622/1623 requires that the temperature of the sample upon arrival at the
laboratory must be <20°C (but not frozen), and the laboratory must begin sample
processing within 96 hours of sample collection. If the sample temperature and holding
time requirements are not met, then the sample is invalid and must be recollected.
3.0 Packing the Sample
3.1 Insert two large plastic trash bags into the shipping cooler to create a double liner. Immediately
before packing the cooler, create two 4-lb packs of ice in two separate ziplock bags. To prevent
leaks place each ice pack into an additional ziplock bag. Gel packs or blue ice may be used
H-8 February 2006
-------
instead of wet ice, as long as the sample is maintained in the appropriate temperature range. Seal
the ziplock bag, expelling as much air as possible, and secure top with tape.
Note! Shipping companies may delay sample shipments if leakage occurs. Double liners and
ziplock bags around ice will prevent leakage and delays.
3.2 Place the bag containing the filter, or filter within housing, into the shipping container. Wrap the
filter in bubble wrap to prevent freezing. Inflated, empty sample bags can be placed between the
filter housing and the ice packs to prevent the sample from freezing.
3.3 If you will be monitoring sample temperature during shipment, place in the cooler the
temperature monitoring device (e.g., extra sample bottle for measuring sample temperature upon
receipt at the laboratory, thermometer vial, or Thermochron™ iButton). Seal each liner bag by
twisting top of bag and tying in a knot.
3.4 Peel the backing off one of the plastic airbill sleeves and attach the sleeve to the inside of the
cooler lid.
%> Sign and date the sample collection form
Fold the completed sample collection form, and place it inside the plastic sleeve.
3.5 Close the cooler lid, seal the horizontal joints with strapping tape, and secure the lid with tape by
taping the cooler at each end, perpendicular to the seal.
Note! Shipping companies may delay sample shipments if leakage occurs. Be sure to seal the
cooler joints.
3.6 Peel the backing off of the second airbill sleeve and attach the sleeve to the outside of the cooler
lid. Complete the shipping airbill with the laboratory address, billing information, sample weight,
and shipping service. Remove the shipper's copy of the airbill, and place the remainder of the
airbill inside the plastic sleeve.
4.0 Shipping and Tracking
4.1 Ship samples on the day of collection and use a reliable shipping service for overnight delivery. If
samples are not shipped the day of collection, the sample must be maintained between 1°C and
10°C (but not frozen) by chilling in a refrigerator or cooler filled with ice.
4.2 Contact the laboratory to notify them of the sample shipment. Request that the laboratory contact
you the next day if the sample is not received.
4.3 Using the airbill number on the shipper's copy of the airbill, track the sample shipment using the
shipping company's web page or by contacting the shipping company over the phone.
4.4 If problems are encountered with the shipment, communicate with the shipping company to
resolve, and update the laboratory regarding the status of the shipment.
H-9 February 2006
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Appendix I
Collecting Bulk Water Samples for
Laboratory Filtration and Cryptosporidium Analysis
Recommended Procedure
-------
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Recommended Procedure for Collecting Bulk Water Samples for
Laboratory Filtration and Cryptospordium Analysis
1.0 Materials
The following materials should be available before collecting samples:
nD Several pairs of new, powder-free latex gloves
nD Sample collection form (Appendix F)
nD Sanitary 10 L cubitainer
nD Sample number label
nD Waterproof pen
nD Cooler, approximately 34-quart
nD Two large plastic trash bags
nD Two 8-lb. bags of ice, or the equivalent number of ice packs sufficient to maintain the sample
between 1°C and 10°C
n Four gallon size ziplock bags
n Strapping tape
n Two self-adhesive plastic airbill sleeves
n Airbill for shipment
2.0 Collecting the Sample
2.1 Put on a pair of powder-free latex gloves.
2.2 If sampling from a pressurized sample tap, turn on the influent tap and flush the system by allowing
source water to flow for 2 to 3 minutes or until the temperature has stabilized and any debris that
has accumulated has cleared or the turbidity in the water becomes visibly uniform.
2.3 While the system is flushing or if you are collecting a sample directly from the source water, record
the following information on the sample collection form (Appendix F):
%> Public water system (PWS) name
^ PWS address
%> Sampler name
%> Sample ID (optional)
•% Public Water System Identification (PWS ID) number
%> Public Water System facility ID number
%> Facility name
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%> Sample collection point ID
%> Sample collection point name
%> Sample collection date
%> Source water type (optional [but required for E. coll sample forms])
%> Requested analysis (circle Cryptosporidium field sample for routine monitoring sample; circle
both "Cryptosporidium field sample" and "Cryptosporidium matrix spike" sample if you are
sending an additional sample with the monitoring sample for matrix spike analysis)
2.4 After the system has been flushed, measure the turbidity of the source water and any optional water
quality parameters such as temperature, and/or pH.
2.5 Fill the 10 L (or multiple) cubitainer(s). If both a field sample and a matrix spike sample are being
collected, fill one immediately after the other. Record the following information on the sample
collection form:
%> Sample collection time
%> Comments to laboratory, if needed
2.6 Immediately following sample collection, tighten the cubitainer cap(s) and place the cubitainer(s) in
a refrigerator to chill to <10°C prior to packing the shipping cooler for overnight shipment. If no
refrigerator is available, place the cubitainer(s) in the shipping cooler with ice to chill, and replace
the ice with fresh ice before shipping.
Note! Method 1622/1623 requires that the temperature of the sample upon arrival at the
laboratory must be <20°C (but not frozen), and the laboratory must have time to process
the sample before 96 hours elapses after sample collection. Source water samples that are
collected above 10°C, should be chilled before shipment. If the sample temperature and
holding time requirements are not met, then the sample is invalid and must be recollected.
3.0 Packing the Sample
3.1 Insert two large plastic trash bags into the shipping cooler to create a double liner. Immediately
before packing the cooler, place a fresh 8-lb bag of ice into each of the two plastic, ziplock bags. To
prevent leaks place each ice pack into an additional ziplock bag. Seal each ziplock bag, expelling as
much air as possible, and secure top with tape.
Note! Shipping companies may delay sample shipments if leakage occurs. Double liners and
ziplock bags around ice will prevent leakage and delays.
3.2 Place the chilled cubitainer upright into the center of the lined cooler. Place the two ice packs into
the cooler, one on each side of the cubitainer.
3.3 If you will be monitoring sample temperature during shipment, place in the cooler the temperature
monitoring device (e.g., extra sample bottle for measuring sample temperature upon receipt at the
laboratory, thermometer vial, or Thermochron™ iButton). Seal each liner bag by twisting top of
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bag and tying in a knot.
3.4 Peel the backing off one of the plastic airbill sleeves and attach the sleeve to the inside of the cooler
lid.
%> Sign and date the sample collection form.
Fold the completed sample collection form, and place it inside the plastic sleeve.
3.5 Close the cooler lid, seal the horizontal joints with strapping tape, and secure the lid with tape by
taping the cooler at each end, perpendicular to the seal.
Note! Shipping companies may delay sample shipments if leakage occurs. Be sure to seal the
cooler joints.
3.6 Peel the backing off of the second airbill sleeve and attach the sleeve to the outside of the cooler lid.
Complete the shipping airbill with the laboratory address, billing information, sample weight, and
shipping service. Remove the shipper's copy of the airbill, and place the remainder of the airbill
inside the plastic sleeve.
4.0 Shipping and Tracking
4.1 Ship sample after it has chilled to between 1°C and 10°C and use a reliable shipping service for
overnight delivery. If samples are not shipped the day of collection, the sample must be maintained
between 1°C and 10°C by chilling in a refrigerator or cooler filled with ice.
4.2 Contact the laboratory to notify them of the sample shipment. Request that the laboratory contact
you the next day if the sample is not received.
4.3 Using the airbill number on the shipper's copy of the airbill, track the sample shipment using the
shipping company's web page or by contacting the shipping company over the phone.
4.4 If problems are encountered with the shipment, communicate with the shipping company to resolve,
and update the laboratory regarding the status of the shipment.
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Appendix J
Collecting Source Water Samples
for E. coll Analyses -
Recommended Procedure
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Recommended Procedure for Collecting Source Water Samples
forE. coll Analyses
1.0 Materials
The following materials should be available before collecting samples:
nD Several pairs of new, powder-free latex gloves (optional)
nD Sterile, non-toxic, glass or plastic container with a leak-proof lid. Container should be capable of
holding 120-mL or 250-mL with ample headspace to facilitate mixing of sample by shaking prior
to analysis
nD Sample number label
The following additional materials may be needed if the sample will be shipped off-site for analysis:
nD Sample collection form
nD Gallon size zippered plastic bag
nD Cooler, approximately 9-quart
nD Two large plastic trash bags
nD One 8-lb. bag of ice or gel ice packs
nD Strapping tape
nD Bubble wrap
nD Two self-adhesive plastic airbill sleeves
nD Airbill for shipment
nD Temperature monitoring device (thermometer vial, or Thermochron™ iButton) (if measuring
temperature during shipment)
2.0 Collecting the Sample
2.1 Record the sample number, sample location, samplers name, observations, and sampling date and
time in a sampling log book if the sample will be analyzed on-site.
If the sample will be shipped off-site, record the following information on the sample collection
form (Appendix F):
%> Public water system (PWS) name
^ PWS address
%> Sampler name
%> Sample ID (optional)
•% Public Water System Identification (PWSID) number
%> Public Water System facility ID number
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%> Facility name
%> Sample collection point ID
%> Sample collection point name
%> Sample collection date
%> Source water type
%> Requested analysis (circle "E. coir)
2.2 Water taps used for sampling should be free of aerators, strainers, hose attachments, mixing type
faucets, and purification devices. The service line should be cleared before sampling by
maintaining a steady water flow for at least two minutes (until the water changes temperature).
Please note: Pre-rinsing the sample containers with sample is prohibited when collecting E. coll
samples.
2.3 Adjust the flow of water out of the tap or hose so the water will not splash out when it is collected
into the sample container.
2.4 If there is not an inline tap that allows for the sampling of source water prior to treatment,
samples should be collected as close to the intake as possible from either land or boat. Source
water samples should be collected close to the surface using a grab sampling technique. Samples
may be collected manually by direct submersion of the bottle into the water or by using a grab
sampling device, as simple as a metal pole with an adjustable clamp at one end that holds the
sampling bottle in place.
2.5 Using aseptic technique (i.e., sanitize tap, do not touch the inside of the sample container, etc.),
fill the E. coll sample container, leaving at least 1 inch of head space, if possible. Volume
collected will depend on anticipated organism density. It is recommended that utilities collect at
least 100 mLs. Do not expose an opened container any longer than necessary. Record the system
name, sampler's name, sample number, date and time of sample collection, sample location, and
analysis requested on the sample container.
2.6 Immediately following sample collection, tighten the sample container lid. If the sample will be
shipped off-site for analysis, and will not be shipped for several hours, place the sample container
upright in a refrigerator to maintain the sample at a temperature between 1°C and 10°C, but not
freezing, prior to shipment. If a refrigerator is not available, wrap the sample with insulation such
as bubble wrap or paper towels (to prevent freezing), place the sample in a ziplock bag, and place
the bag containing the sample in the shipping cooler with wet ice or ice packs. Replace with fresh
ice or ice packs immediately prior to shipment.
3.0 Suggested Packaging of the Sample (Applicable to Samples Shipped Off-Site for
Analysis)
3.1 Insert two large plastic trash bags into the shipping cooler to create a double liner. Immediately
before packing the cooler, disperse 6 pounds of ice into 3 to 4 plastic, zippered plastic bags. Gel
packs or blue ice may be used in lieu of wet ice, as long as the sample is maintained in the
appropriate temperature range. Seal the zippered plastic bags, expelling as much air as possible,
and secure top with tape.
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Note! Shipping companies may delay sample shipments if leakage occurs. Double liners and
ziplock bags around ice will prevent leakage and delays.
3.2 Place the bag containing the samples into the shipping container. Cover sample bottles with
bubble wrap, to prevent freezing, and place ice or ice packs around the sample bag.
3.3 If you will be monitoring sample temperature during shipment, place in the cooler the
temperature monitoring device (e.g., extra sample bottle for measuring sample temperature upon
receipt at the laboratory, thermometer vial, or Thermochron™ iButton). Seal each liner bag by
twisting top of bag and tying in a knot.
3.4 Peel the backing off one of the plastic airbill sleeves and attach the sleeve to the inside of the
cooler lid.
%> Sign and date the sample collection form.
Fold the completed sample collection form, and place it inside the plastic sleeve.
3.5 Close the cooler lid, seal the horizontal joints with strapping tape, and secure the lid with tape by
taping the cooler at each end, perpendicular to the seal.
Note! Shipping companies may delay sample shipments if leakage occurs. Be sure to seal the
cooler joints.
3.6 Peel the backing off of the second airbill sleeve and attach the sleeve to the outside of the cooler
lid. Complete the shipping airbill with the laboratory address, billing information, sample weight,
and shipping service. Remove the shipper's copy of the airbill, and place the remainder of the
airbill inside the plastic sleeve.
4.0 Shipping and Tracking
4.1 Ship samples on the day of collection and use a reliable shipping service for next-day delivery.
Note! Under LT2, E. coll samples must be analyzed within 30 hours of sample collection.
Samples that will be shipped off-site for analysis, need to be shipped the same day of
collection. If the sample holding time or temperature requirements are exceeded upon
receipt at the laboratory, the sample will be rejected and will need to be re-collected.
4.2 Contact the laboratory to notify them of the sample shipment. Request that the laboratory contact
you the next day if the sample is not received.
4.3 Using the airbill number on the shipper's copy of the airbill, track the sample shipment using the
shipping company's web page or by contacting the shipping company over the phone.
4.4 If problems are encountered with the shipment, communicate with the shipping company to
resolve, and update the laboratory regarding the status of the shipment.
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Appendix K
Intent to Grandfather
Example Notice
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Long Term 2 Enhanced Surface Water Treatment Rule
Intent to Grandfather - Example Notice
Public Water Systems (PWSs) that will submit grandfathered data for the LT2 Rule must first
submit a notice that they intend to submit previously collected monitoring results. This notice
must specify the number of previously collected results that the system will submit, the dates of
the first and last sample, and whether they will collect additional source water monitoring
samples to meet the requirements of the rule. If a PWS plans to collect additional samples, it
must also submit a monitoring schedule. Submitting this form satisfies the requirement to submit
a notice of intent to grandfather.
Submit this information no later than 3 months prior to the month that the PWS is
required to start monitoring.
PWS Information
PWS Name:
PWS ID:
PWS Address:
Email Address:
Water Treatment Plant Name:
Water System Facility ID:
Grandfathered Data Information
Number of previously collected sample results that will be submitted:
Date of first sample: / /
Date of last sample: / /
Wll you be collecting additional source water monitoring samples
to meet the requirements of your plant? Q YES Q NO
If yes, you must also submit a monitoring schedule that is required for your plant.
Signature:
Date:
Name (print)
Phone:
This notice may be submitted using one of the following options:
• Through the LT2/Stage 2 Data Collection and Tracking system
available at http://www.epa.gov/safewater/disinfection/lt2
• As an email attachment and sent to stage2mdbp@epa.gov
• By mail or fax to the following: LT2ESWTR and Stage 2 DBPR
P.O. Box 98
Dayton, OH 45401
FAX: (937)-586-6557
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Appendix L
Recommended Checklist for Beginning
Grandfathered Cryptosporidium Monitoring
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Recommended Checklist for Beginning
Grandfathered Cryptosporidium Monitoring
a Cryptosporidium laboratory qualifications.
A list of laboratories approved for LT2 Cryptosporidium analysis is posted at
http://www.epa.gov/safewater/disinfection/lt2/labjiome.html.
n Sampling location. Is sample collection from the plant intake prior to chemical
treatment (Section 3.1)? Have you prepared a description of the sampling location
that addresses the position of the sampling location in relation to the system's
water source(s) and treatment processes, including points of chemical addition
and filter backwash water recycle? (Appendices D and E)
n Sampling schedule. Is the sampling schedule designed to monitor for
Cryptosporidium at least monthly (Section 3.2) on a regular schedule?
n Laboratory coordination. Have you verified that your intended sampling
schedule can be accommodated by an approved Cryptosporidium laboratory (to
avoid sample rejection) (Section 2.3.1)?
n Matrix spikes. Does the sampling schedule include collection of an extra bulk 10
L sample, in addition to the required monitoring samples, along with the 1st and
20th samples (Section 4.1.3)?
n Method version. Is EPA Method 1622 or EPA Method 1623 used to analyze
Cryptosporidium samples (Section 5.2.2)?
n Sample volume issues. Is a minimum of 10 L collected for each monitoring and
matrix spike sample (Section 4.2.1)?
n E. co/i laboratory qualifications. Is the laboratory certified under the drinking
water laboratory certification program for total or fecal coliform analysis using the
same technique as one of the LT2 E. coll methods (Table 7.1)?
n Turbidity measurements. Will turbidity measurements be made for each sample
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Appendix M
Grandfathered Cryptosporidium Data Package
Report Checklist
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Long Term 2 Enhanced Surface Water Treatment Rule
Grandfathered Cryptosporidium Data Package - Report Checklist
Public Water Systems (PWSs) that must monitor for Cryptosporidium can use this checklist to prepare
to report required information to EPA, if they wish to grandfather data. PWSs must submit previously
collected monitoring results for grandfathering along with all required documentation no later than 2
months after the month the PWS is required to start monitoring.
PWS Information
PWS Name: PWS ID:
PWS Address:
Email Address:
Water Treatment Plant Name:
Water System Facility ID:
Grandfathered Data Information
Have you submitted the required Intent to Grandfather Report, at least 3 months before you
would be required to conduct monitoring?
Does the data package include a signed cover letter certifying that the data represent the plant's
current source water and that all source water Cryptosporidium monitoring results collected
during LT2 monitoring are included in this package?
Does the data package include a sample collection schedule specifying calendar dates,
including first and last samples?
Have you submitted a sampling schedule for additional new monitoring you will conduct, if any?
Have you included additional documentation regarding dates and reasons for resampling, the
use of presedi mentation, and/or off-stream storage during routine plant operation?
Does the data package include a list of the field and matrix spike (MS) samples submitted in the
data package, identified by sample ID and collection date?
Are all data elements from field sample results from the monitoring period included?
Are all data elements from MS sample results from the monitoring period included?
Are the volumes analyzed for all field samples at least 10 L?
For samples in which less that 1 0 L was examined, were at least 2 ml of packed pellet volume
analyzed or did two filters clog?
Does the data package include a letter from the laboratory certifying that all method QC
requirements were acceptable for every field and MS sample submitted with the package,
including OPR, Method blank, holding times, staining controls, etc.? Alternatively, bench sheets
and report forms documenting this QC information may be included, rather than a laboratory
letter.
Was the temperature of all Cryptosporidium samples <20°C (and not frozen) upon receipt?
DYESQ
DYESQ
DYESQ
DYESD
DYESQ
DYESQ
DYESQ
DYESQ
DYESD
DYESQ
DYESQ
DYESQ
NO
NO
NO
NO
NO
NO
NO
NO
NO
NO
NO
NO
PWSs that must monitor for Cryptosporidium may submit this checklist with the
grandfathered data package using one of the following options:
• As an email attachment sent to stage2mdbp@epa.gov
• By mail or fax to the following: LT2ESWTR and Stage 2 DBPR
P.O. Box 98
Dayton, OH 45401
FAX: (937)-586-6557
Grandfathered data can be submitted separately from supporting grandfathered data
package information through the LT2/Stage 2 Data Collection and Tracking system available
at http://www.epa.gov/safewater/disinfection/lt2.
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