_____________________________________________________________________ 

SOURCE WATER MONITORING GUIDANCE 

MANUAL FOR PUBLIC WATER SYSTEMS 

FOR THE FINAL LONG TERM 2 ENHANCED 

SURFACE WATER TREATMENT RULE 

Office of Water (4601M) 

 EPA 815-R06-005 

 February 2006 
U.S. Environmental Protection Agency 

Office of Water (4601M) 

1200 Pennsylvania Avenue NW 

Washington DC 20460 

EPA 815-R-06-005 

http://www.epa.gov/safewater/disinfection/lt2/compliance.html 

February 2006 

Printed on Recycled Paper 
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 

conley.sean@epa.gov 
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 Schedule 4 - 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 

i February 2006 
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. coli Data: 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 

ii February 2006 
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 Raw E. 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 

iii February 2006 
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 E. coli Methods for LT2 Rule .............................................................. 60 

Table 7-2. Examples of Different Combinations of Positive Tubes ...................................... 69 

iv February 2006 
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.................................................................... EnvirochekTM 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. coli 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 v 
ACRONYMS

CFU Colony-forming unit 

CNFG Confluent growth 

DAPI 4, 6-diamidino-2-phenylindole 

DCTS LT2/Stage 2 Data Collection and Tracking System 

DIC Differential interference contrast 

EPA United States Environmental Protection Agency 

FA Fluorescence assay 

FITC Fluorescein isothiocyanate 

GWUDI Ground water under the direct influence [of surface water] 

ICR Information Collection Rule 

IDC Initial demonstration of capability 

IESWTR Interim Enhanced Surface Water Treatment Rule 

IFA Immunofluorescence assay 

IMS Immunomagnetic separation 

IPMC Information Processing and Management Center 

IPR Initial precision and recovery 

IPT Initial proficiency testing 

L Liter 

LT2 Rule Long Term 2 Enhanced Surface Water Treatment Rule 

LT2ESWTR Long Term 2 Enhanced Surface Water Treatment Rule 

mL Milliliter 

MPC Magnetic particle concentrator 

MPN Most probable number 

MS Matrix spike 

MS/MSD Matrix spike/matrix spike duplicate 

Fm Micrometer 

NA-MUG Nutrient agar (NA) with 4-methylumbelliferyl-beta-D-glucuronide (MUG) 

NELAC National Environmental Laboratory Accreditation Conference 

nm Nanometer 

NPDWR National Primary Drinking Water Regulations 

NTU Nephelometric turbidity unit 

OPR Ongoing precision and recovery 

OPT Ongoing proficiency testing 

PBMS Performance-based measurement system 

PT Proficiency testing 

PWS Public water system 

QA Quality assurance 

QAP Quality assurance plan 

QC Quality control 

RSD Relative standard deviation 

SDWA Safe Drinking Water Act 

SOP Standard operating procedure 

TNTC Too numerous to count 

UV Ultraviolet 

vi February 2006 
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 

1 February 2006 
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)(1)]. 

• 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 
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. coli 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. coli 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)(1)]. 

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. 

3 February 2006 
c 

Section 1: General Requirements 

Table 1-1. Summary of LT2 Rule Monitoring Requirements (Initial Round) 

SCHEDULE Monitoring 

begins 

Monitoring 

duration 

Monitoring parameters and sample frequency 

requirements 

Cryptosporidium E. coli Turbidity 

SCHEDULE 1*: 

Large systems 

serving ≥100,000 

October 1, 

2006 2 years a Minimum 

 1 sample/month b Minimum 

1 sample/month c Minimum 1 sample/month c 

SCHEDULE 2*: 

Large systems 

serving >50,000 

and <100,000 

April 1, 2007 2 years a Minimum 

 1 sample/month b Minimum 

1 sample/month c Minimum 1 sample/month c 

SCHEDULE 3*: 

Large systems 

serving >10,000 

and <50,000 

April 1, 2008 2 years a Minimum 

 1 sample/month b Minimum 

1 sample/month c Minimum 1 sample/month c 

SCHEDULE 4*: 

Small filtered 

systems (serving 

fewer than 10,000) 

October 1, 

2008 1 year a,d See next row § Every two weeks N/A 

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 

April 1, 2010 

1 year b

, 2 Cryptosporidium 

samples per month, 

or 

2 years e

, 1 Cryptosporidium 

sample per 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)] 

a 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)] 

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 E. coli/100 mL for systems using lakes/reservoirs or is greater than 50 E. coli/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. 

4 February 2006 
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 Timeline Duration 

Establish contract with a Cryptosporidium 

laboratory approved under EPA’s Lab QA 

Program (see Section 2.3.1) (Reference 8.2) 

As soon as possible, recommended by 

June 2006 

N/A - single event 

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 E. 

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) Required– No later than July 1, 2006 

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 Required (if grandfathering) - No later 

than July 1, 2006 

Submit grandfathered Cryptosporidium data 

package (if applicable) (see Section 5) b,c Required (if grandfathering) - No later 

than December 1, 2006 

Collect monitoring samples c (see Section 4) Required– No later than the month 

beginning October 1, 2006 

At least once per 

month for 2 yearsd 

Submit monitoring results c (see Sections 6.3 and 

7.3) 

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) 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)] 

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 

5 February 2006 

c 
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 Timeline Duration 

Establish contract with a Cryptosporidium 

laboratory Approved under EPA’s Lab QA 

Program (see Section 2.3.1) 

As soon as possible, recommended by 

December 2006 

N/A - single event 

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 E. 

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) Required– No later than January 1, 

2007 

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 Required (if grandfathering) - No later 

than January 1, 2007 

Submit grandfathered Cryptosporidium data 

package (if applicable) (see Section 5) b,c Required (if grandfathering) - No later 

than June 1, 2007 

Collect monitoring samples c (see Section 4) Required– No later than the month 

beginning April 1, 2007 

At least once per 

month for 2 yearsd 

Submit monitoring results c (see Sections 6.3 and 

7.3) 

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) 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)] 

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 

6 February 2006 

c 
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 Timeline Duration 

Establish contract with a Cryptosporidium 

laboratory Approved under EPA’s Lab QA 

Program (see Section 2.3.1) 

Recommended by December 2007 

N/A - single event 

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 E. 

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) Required– No later than January 1, 

2008 

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 Required (if grandfathering) - No later 

than January 1, 2008 

Submit grandfathered Cryptosporidium data 

package (if applicable) (see Section 5) b,c Required (if grandfathering) - No later 

than June 1, 2008 

Collect monitoring samples c (see Section 4) Required– No later than the month 

beginning April 1, 2008 

At least once per 

month for 2 yearsd 

Submit monitoring results c (see Sections 6.3 and 

7.3) 

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) 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)] 

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 

7 February 2006 

c 
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. 

8 February 2006 
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 Timeline Duration 

Verify that the laboratory that will perform your 

E. coli analyses under the LT2 Rule is certified 

under the drinking water laboratory certification Recommended by June, 2008 

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 E. coli 

monitoring by monitoring for Cryptosporidium Required– No later than July 1, 2008 N/A - single event 

Submit sampling location and source water 

monitoring description 

Submit notice of intent to grandfather (if 

applicable) (see Section 5) d,e 

Required (if grandfathering) - No later 

than July 1, 2008 d,e 

Submit grandfathered E. coli data package (if 

applicable) (see Section 5) d,e 

Required (if grandfathering) - No later 

than December 1, 2008 d,e 

Collect E. coli samples e (See Section 4) Required– No later than the month 

beginning October 1, 2008 

One year (one 

sample every two 

weeks) b 

Submit E. coli monitoring results (See Section 

7.3) 

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 

Table 1-5 continued on the following page 

9 February 2006 
c 

Section 1: General Requirements 

Table 1-5. SCHEDULE 4: Timeline for required monitoring (Initial Round) (continued) 

Unfiltered Systems and 

Filtered Systems that Exceed E. coli Trigger Levelsa

 or 

that Elect to Bypass E. coli Monitoring and Proceed Directly to Cryptosporidium Monitoring or 

that fail to conduct E. coli monitoring 

Establish contract with a Cryptosporidium 

laboratory approved under EPA’s Lab QA 

Program 

Recommended by December, 2009 

N/A - single event 

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) Required– No later than January 1, 2010 

Submit sampling location and source water 

monitoring description 

Submit notice of intent to grandfather (if 

applicable) (see Section 5) d,e No later than January 1, 2010 d,e 

Submit grandfathered Cryptosporidium data 

package (if applicable) (see Section 5) d,e No later than June 1, 2010 d,e 

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 E. coli monitoring if 

the E. coli annual mean concentration exceeds 10 E. coli/100 mL for systems using lakes/reservoirs or exceeds 50 E. coli/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)] 

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).d 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 
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)(1) 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. 

11 February 2006 
Section 1: General Requirements 

Table 1-6. Submitting Reports and Notices 

Options for submitting reports and notices 

Report/Notice E mail Hardcopy DCTS 

Sampling Schedule * T 

Sample Location Description T T 

Intent to Provide Maximum Treatment T 

Intent to Grandfather Data T T 

Grandfathered Data T T T 

Grandfathered Data Supporting 

Documentation T T 

Initial Round Monitoring Data * T 

* 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 
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 

13 February 2006 
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14 February 2006 
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 
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? 

L 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 
Section 2: Contracting for Laboratory Services 

L 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. 

L 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)]. 

L 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. coli, 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)(1)] 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 
Section 2: Contracting for Laboratory Services 

For E. coli samples, the sample volume collected should be at least 100 mL.

L 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. 

2.1.2.4 Subsamples 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)(1)]. 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. 

L Clearly specify in your contract any services required in addition to routine sample analysis. 

18 February 2006 
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. 

L	 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 Giardia? 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-only 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 
Section 2: Contracting for Laboratory Services 

L 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. 

L 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 
Section 2: Contracting for Laboratory Services 

L 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 
Section 2: Contracting for Laboratory Services 

PWSs that want to review the raw data for each E. coli analysis should request the following:

• Monitoring sample identification information 

• Monitoring sample result, in E. coli/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. 

L 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. 

22 February 2006 
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). 

L 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 > 20EC 

that results in resampling by your PWS), the laboratory should not be responsible for the additional costs 

that may result. 

L 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 

23 February 2006 
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.

L 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 

24 February 2006 
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 

25 February 2006 
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. 

26 February 2006 
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. 

27 February 2006 
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. 

28 February 2006 
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. coli, 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. 

29 February 2006 
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)(1)]. 

• 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 

30 February 2006 
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 presedimentation 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 Term1Enhanced Surface Water Treatment Rule, as applicable, must take source 

water samples in the surface water prior to bank filtration [40 CFR § 141.703(d)(1)]. 

• 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. 

31 February 2006 
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. coli 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)(1)]. Sample results and explanations should be submitted through the LT2/Stage 2 Data 

Collection and Tracking System. 

32 February 2006 
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 

33 February 2006 
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34 February 2006 
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. 

35 February 2006 
Section 4: Collecting and Shipping Source Water Samples 

Table 4-1. Minimum Data Elements to Record During Sample Collection 

Sampling Information Required Recommended 

PWS name T 

Public Water System Identification 

(PWSID) number a T 

Facility name T 

Facility ID a T 

Sample collection point name T 

Sample collection point ID T 

Source water type b T 

Requested analysis T 

Sample collection date a T 

Sample collection time (start and 

stop time for field-filtered samples) c T 

Meter readings (for field-filtered 

samples only) T 

Source water temperature T 

Turbidity d T 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. c 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 EnvirochekTM or EnvirochekTM 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)]. 

36 February 2006 
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 IDEXX 

(EnvirochekTM and EnvirochekTM HV capsule filters) (Filta-Max® foam filters) 

www.pall.com/gelman www.idexx.com 

600 South Wagner Road Sales: Ann Arbor, MI 48103 Phone: (800) 321-0207 ext.1 

Sales: Fax: (207) 856-0630 

Phone: (800) 521-1520 ext.2 Technical Support: Fax: (734) 913-6114 Phone: (800) 321-0207 ext.2 

Technical Support: Fax: (207) 856-0630 

Phone: (800) 521-1520 ext.3 E-mail: water@idexx.com 

Fax: (734) 913-6114 

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)(1)], PWSs are 

required to analyze, at a minimum, either: 

• 10 L of sample, or 

• 2 mL of packed pellet volume, or 

37 February 2006 
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 EnvirochekTM and EnvirochekTM 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 centrifugation 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 

38 February 2006 
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. coli Sample Collection 

For most large PWSs, E. coli analyses will be conducted onsite and samples will not be shipped. 

However, many small PWSs will collect E. coli 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. coli samples for the LT2 Rule must use an E. coli 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. coli method the laboratory plans to use for LT2 Rule monitoring [40 CFR § 

141.704(b) and 141.705(b)]. Approved E. coli methods and the drinking water certification program are 

provided in the Microbial 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. coli 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. 

coli 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. coli analysis, as well as packing and shipping guidance 

for utilities that transport samples offsite for analysis, is provided as Appendix J. 

39 February 2006 
Section 4: Collecting and Shipping Source Water Samples 

EPA encourages laboratories to analyze samples as soon as possible after collection. E. coli samples must 

be analyzed within 30 hours of sample collection [40 CFR § 141.704(b)(1)]. 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. coli samples should be maintained between 0EC and 10EC 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. coli 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. coli 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 >20EC or frozen, or Cryptosporidium samples that the laboratory has determined exceeded 

20EC or froze during shipment [Section 8.1.3 of EPA Method 1622/1623]. E. coli samples that are 

received at >10EC or frozen, or E. coli samples that the laboratory has determined exceeded 10EC or 

froze during shipment, must also be rejected. 

After receipt, Cryptosporidium samples must be stored at the laboratory between 1EC and 10EC, and not 

frozen, until processed (Section 8.1.3 of EPA Method 1622/1623) [40 CFR § 141.704(a)]. E. coli 

samples must be stored at the laboratory between 0EC and 10EC, and not frozen, until processed [40 CFR 

§ 141.704(b)(3)]. 

The following steps should help maintain acceptable temperatures: 

• If Cryptosporidium and E. coli samples are collected early in the day, chill samples by storing in a 

refrigerator between 1EC and 10EC 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 1EC and 10EC. 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 >20EC. 

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. coli 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: 

40 February 2006 
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 Parmer 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. EPA Manual for 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)(1) and 40 CFR § 141.704(c)]. These 

methods include: 

• Method 2130B, published in Standard Methods for the Examination of Water and Wastewater (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 at NTIS, 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. 

41 February 2006 
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 2130B. 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 reagitate 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 
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)(1)]. 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)(iii)]. 

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. 

43 February 2006 
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)(1)]. 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. 

44 February 2006 
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. coli 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 
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. 

46 February 2006 
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 1⁄4 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 Microbial 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 

47 February 2006 
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)(1)]. 

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 for the PWS [40 CFR § 141.707(g)(1)]. 

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. 

48 February 2006 
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 Bin Classification 

Cryptosporidium < 0.075 oocysts/L Bin 1 

0.075 oocysts/L # Cryptosporidium < 1.0 oocyst/L Bin 2 

1.0 oocyst/L # Cryptosporidium < 3.0 oocysts/L Bin 3 

Cryptosporidium $ 3.0 oocysts/L Bin 4 

PWSs that serve fewer than 10,000 people and NOT 

required to monitor for Cryptosporidium a Bin 1 

a 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 E. coli/100 mL for lake/reservoir sources or 50 E. coli/100 

mL for flowing stream sources or do not exceed an alternative State-approved indicator trigger [40 CFR § 141.701(a)(4) and § 

141.710(c)]. 

49 February 2006 
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 § 

141.710(b)(1)] 

• 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 § 

141.710(b)(5)]. 

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 >0.01oocysts/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)(1)]. 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. 

50 February 2006 
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 

51 February 2006 
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. 

52 February 2006 
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 

EPA does not have access to data 

PWS actions 

• PWS reviews electronic data through the DCTS 

• PWS cannot edit data - only review data and either return to laboratory to resolve errors or submit to 

EPA 

• 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 

53 February 2006 
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 1⁄4 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. 

54 February 2006 
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 1EC to 10EC. 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 

55 February 2006 
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 

#20EC, 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 

56 February 2006 
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 = × 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. 

57 February 2006 
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%) 27 mL 

volume analyzed (L) = 0.74 × 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 × 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. 

58 February 2006 
SECTION 7: E. COLI DATA: 

USE, RECORDING, SUBMITTING AND REVIEW 

When E. coli 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. coli Data 

E. coli data are being collected by large filtered PWSs and applicable wholesalers during LT2 Rule 

monitoring to confirm or refine the E. coli levels that trigger Cryptosporidium monitoring by small, 

filtered PWSs. E. coli data are being collected by small filtered PWSs because only those small filtered 

PWSs with mean E. coli 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. coli trigger levels were set at a mean of 50 E. coli/100 mL for flowing stream–type source waters and 

10 E. coli/100 mL for reservoir/lake source waters. The E. coli 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. coli analysis under the LT2 Rule are listed below in Table 7-1. 

7.2 E. coli Data Recording at the Laboratory 

The laboratories performing E. coli 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. coli 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 
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 Method Format Method Citation 

Standard Methods 

9221B.1/9221F (LTB-EC-MUG) multiple tube fermentation/ 

most probable number 

Standard Methods for the 

Examination of Water and 

Wastewater (Reference 8.6 )2 

Standard Methods for the 

Standard Methods 9223B 

(Colilert®/Colilert-18®) multiple tube/multiple well 

Examination of Water and 

Wastewater (Reference 8.6)2

; 

IDEXX Laboratories, Inc. 

(Reference 8.8) 

Standard Methods 9222B/9222G1 

(mEndo/LES-Endo-NA-MUG) membrane filtration, two step 

Standard Methods for the 

Examination of Water and 

Wastewater (Reference 8.6 )2 

Standard Methods 9222D/9222G 

(mFC-NA-MUG) membrane filtration, two step 

Standard Methods for the 

Examination of Water and 

Wastewater (Reference 8.6)2 

Standard Methods 9213D/ 

EPA Method 1103.1 (mTEC) membrane filtration, one step 

EPA Method 1103.1 (Reference 

8.9); Standard Methods for the 

Examination of Water and 

Wastewater (Reference 8.6)2 

EPA Method 1603 Modified 

mTEC membrane filtration, one step EPA Method 1603 (Reference 8.10) 

EPA Method 1604 MI medium 1 membrane filtration, one step EPA Method 1604 (Reference 

8.11) 

m-ColiBlue24® Broth 1 membrane filtration, one step Hach Company (Reference 8.12) 1 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 9221B.1/9221F, 

and SM 9223B)

2 18th, 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 

60 February 2006 
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 CFU/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 CFU /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. coli/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. 

61 February 2006 
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 

62 February 2006 
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 0EC and 10EC, 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 
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® QuantiTray 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 × = 813 E. coli MPN/100 mL

10 

7.4.3.2 Calculations for Determining the E. coli Concentration Using the Colilert® QuantiTray 51® (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 
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/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 

MPN value × = E. 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 × = 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 
Section 7: E. coli Data: Use, Recording, Submitting and Review 

D. Using the E. coli counts from the appropriate dilution, E. coli CFU/100 mL is calculated based on 

the following equation: 

100

E. coli CFU × 

mL sample filtered 

Example 1: 

Filter 1 volume = 100 mL

Filter 2 volume = 10 mL

Filter 3 volume = 1.0 mL

Filter 4 volume = 0.1 mL 

= E. coli CFU/100 mL

CFU = TNTC

CFU = 40

CFU = 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: 

100 

40 E. coli CFU × = 400 E. coli CFU/100 mL 

 10 mL 

E. If no E. coli colonies are present, the detection limit (i.e., 1 CFU 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 

100 mL 

Detection limit = 

Largest volume filtered 

100 mL 

= <1 E. coli /100 mL 

100 mL 

Example 3: 

Filter 1 volume (mL) = 100 mL

Filter 2 volume (mL) = 10 mL

Filter 3 volume (mL) = 1.0 mL

Calculation of E. coli/100 mL: 

100 mL 

= <10 E. coli CFU /100 mL

10 mL 

CFU = 0

CFU = 0

CFU = 0

= E. coli CFU/100 mL

CFU = Lab accident, no data available

CFU = 0

CFU = 0

F. If there are no filters with E. coli counts in the 20-80 colony range (20-60 for mFC-NAMUG), sum the E. coli counts on all filters, divide by the volume filtered and report as 

number per 100 mL. 

66 February 2006 
Section 7: E. coli Data: Use, Recording, Submitting and Review 

 Example 4: 

Filter 1 volume (mL) = 50 mL CFU = 15

Filter 2 volume (mL) = 25 mL CFU = 6

Filter 3 volume (mL) = 10 mL CFU = 0

The analytical result is calculated as: 

100 

(15 + 6 + 0) × = 25 E. coli CFU/100 mL 

(50+25+10) 

Example 5: 

Filter 1 volume (mL) = 50 mL CFU = 105

Filter 2 volume (mL) = 25 mL CFU = 92

Filter 3 volume (mL) = 10 mL CFU = 85

The analytical result is calculated as: 

100 

(105 + 92 + 85) × = 332 E. coli CFU/100 mL

(50 + 25 + 10) 

 Example 6: 

Filter 1 volume (mL) = 100 mL CFU = 82 

Filter 2 volume (mL) = 10 mL CFU = 18 

Filter 3 volume (mL) = 1.0 mL CFU = 0

The analytical result is calculated as: 

100 

 (82 + 18 + 0) × = 90 E. coli CFU/100 mL 

(100 + 10 + 1) 

Example 7: 

Filter 1 volume (mL) = 50 mL CFU = TNTC

Filter 2 volume (mL) = 25 mL CFU = TNTC

Filter 3 volume (mL) = 10 mL CFU = 83

The analytical result is calculated as: 

100 

83 × = 830 E. coli CFU/100 mL 

10 

67 February 2006 
Section 7: E. coli Data: Use, Recording, Submitting and Review 

7.4.3.4 Calculation of E. 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

68 February 2006 
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 

Least dilute

(Lowest)

 Most dilute 

 (Highest) Combination 

of positives 

MPN Index from 

Standard Methods 

E. coli/100 mL 

(after adjustment) 10 

mL 1 mL 0.1 mL 

0.01 

mL 

0.001 

mL 

A 5 5 1 0 0 5-1-0 33 330 

B 4 5 1 0 0 4-5-1 48 48 

C 0 0 1 0 0 0-0-1 1.8 1.8 

D 5 4 4 1 0 4-4-1 40 400 

E 5 4 4 0 1 4-4-1 40 400 

F 5 5 5 5 2 5-5-2 540 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: 

33 

Analytical result = = 330 E. coli / 100 mL

0.1 

Example B: 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. coli/100 mL. 

Example C: 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. coli/100 mL. 

Examples D and E: 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: 

40 

Analytical result = = 400 E. coli / 100 mL

0.1 

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: 

540 

Analytical result = = 54,000 E. coli / 100 mL

0.01 

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. 

69 February 2006 
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70 February 2006 
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®, QuantiTray®/2000, and ColisureTM 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 Escherichia coli 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 Escherichia 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 
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/cgibin/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: 

 Filtered system providing a total of at least 5.5-log of treatment for Cryptosporidium [40 CFR part 

141.701 (d)(1)]. 

 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: 

Yes No I understand the treatment requirements that my PWS is required to meet and am 

aware of the deadline for providing the treatment. 

Yes No I have discussed these requirements with a State or U.S. EPA representative 

Signature: Date: 

Name (print) 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 
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] 

Laboratory name: 

Laboratory address: 

Laboratory contact: 

Phone/fax/email: 

PWS required field: 

For further information on this bid sheet, refer to Section 2.2 of the Source Water Monitoring Guidance Manual for Public Water Systems 

Submit bid to: 

Bid validity period: 

Bid deadline (day, date, time (including time zone)): 

Estimated award date: 

PWS name: 

PWS address: 

PWS contact: 

Phone/fax/email: 

Extra Services (if applicable): 

Laboratory period of performance: 

Results turnaround time: 

Costs for Cryptosporidium -Only or Cryptosporidium /Giardia Analysis 

(A) (B) (A x B) 

Number of 

plants 

Samples 

required per 

plant 

Total 

samples

 Cost per 

sample 

Total 

cost 

Up to [no.] 

Up to [no.] 

Number of 

plants 

Equipment 

required per 

plant 

Total 

equipment

 Cost per 

unit 

Total 

cost 

Number of 

plants 

Shipments per 

plant 

Total 

shipments

 Cost per 

shipment 

Total 

cost 

Total 

Costs for E. coli analysis 

(A) (B) (A x B) 

Number of 

plants 

Units required 

per plant** 

Total 

units

 Cost per 

unit 

Total 

cost 

Shipment of sample collection bottles to PWS 

Total 

Bulk water samples - full analysis 

Potential replacement samples 

Sampling if conducted by the laboratory or subcontractor (including turbidity) 

Shipment of cubitainer/carboy to PWS 

Sample collection bottles 

Shipment of collected samples to laboratory 

Shipping**** 

Equipment 

Field sample analysis 

Equipment 

Sample 

PWS requests bid for Cryptosporidium analysis using Method 1623* 

[Specify sample volume if other than 10 L] 

Shipping**** 

Sample 

PWS requests bid for E.coli analysis using [Specify method] 

Shipment of collected samples to laboratory 

Cubitainers/Carboys*** 

Matrix spike samples 

Practice samples 

Estimated subsamples** 

*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

***All 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 
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] 

PWS required field: 

Laboratory name: PWS name: 

Laboratory address: PWS address: 

Laboratory contact: PWS contact: 

Phone/fax/email: Phone/fax/email: 

Estimated award date: 

Bid validity period: 

Costs for Cryptosporidium -Only or Cryptosporidium /Giardia Analysis 

(A) (B) (A x B) 

Number of 

plants 

Samples 

required per 

plant 

Total 

samples

 Cost per 

sample 

Total 

cost 

Up to [no.] 

Up to [no.] 

Number of 

plants 

Equipment 

required per 

plant 

Total 

equipment

 Cost per 

unit 

Total 

cost 

Cubitainer/Carboy (for use with each matrix spike) 

Number of 

plants 

Shipments per 

plant 

Total 

shipments

 Cost per 

shipment 

Total 

cost 

Total 

Costs for E. coli analysis 

(A) (B) (A x B) 

Number of 

plants 

Units required 

per plant**  

Total 

units

 Cost per 

unit 

Total 

cost 

Shipment of sample collection bottles to PWS 

Total 

Shipment of cubitainer/carboy (for matrix spike) to laboratory 

Shipment of collected samples to laboratory*** 

Matrix spike samples (bulk sample) 

Practice samples 

Potential replacement samples 

Shipment of filter apparatus to PWS 

Shipment of cubitainer/carboy (for matrix spike) to PWS 

Shipment of filters to PWS*** 

Results turnaround time: 

Extra Services (if applicable): 

Sampling apparatus for rental or purchase (used during monitoring period) 

Sampling if conducted by the laboratory or subcontractor (including turbidity) 

Submit bid to: 

Equipment 

Sample 

PWS requests bid for Cryptosporidium analysis using Method 1623* 

[Specify sample volume if other than 10 L] 

Shipping 

Field-filtered samples - full analysis 

Estimated subsamples** 

Bid deadline (day, date, time (including time zone)): Laboratory period of performance:

Equipment 

Field sample analysis 

Sample collection bottles 

Shipment of collected samples to laboratory 

Shipping  

Sample 

PWS requests bid for E.coli analysis using [Specify method] 

For further information on this bid sheet, refer to Section 2.2 of the Source Water Monitoring Guidance Manual for Public Water Systems 

Filters [EnvirochekTM, EnvirochekTM HV or Filta-Max®]** *(extra if clogging expected) 

*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

***All 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 C-1 
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: _____________________ PWS ID: _____________________

Water Treatment Plant Name: _____________________ 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 
This page intentionally left blank 
1

2

3

4

5

6

7

8

9

10

Figure Description 

Sample tap before chemical treatment and backwash water recycle (if applicable) 

Multiple sources: sample tap after two combined sources 

Multiple sources: two (or more) sources to be composited 

Multiple plants with the same influent 

Bank filtration 

Ground water under the direct influence of surface water 

Presedimentation basin 

Raw water off-stream storage 

Mixed source water: ground water and surface water sources 

Blank schematic for submission to EPA 

E-1
Figure 1. Sample Tap before Chemical Treatment 

and Backwash Water Recycle (if applicable) 

Raw water 

Source water 

LT2 

LT2 

sample 

point 

Chemical 

Backwash water 

recycle 

LT2 Rule: 

E-2

chemical building 

Raw water intake 

Backwash water 

recycle (if applicable) 

sample 

point 

addition 

(if applicable) 

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)]. 
Figure 2. Multiple Sources: Sample Tap after Two Combined 

§ 

Multiple Alternating Sources: 

l

LT2 Rule: 

Source water “B” 

Raw water 

blending station 

Raw water intake 

Backwash water 

recycle (if applicable) 

LT2 

sample 

point 

Source water “A” 

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)] * 

* Systems unable to sample from 

this combined source location 

should refer to Figure 3. 

Chemical 

addition 

Systems that use multiple a ternating 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)]. 

System

samples prior to ch

141.703(b)(1)], unl

collect source wa

treatment [40 CFR § 

recycle filter backwa

water samples prior 

water addition 

Backwa

lag

E-3
Figure 3. Multiple Sources: Two (or More) Sources to be Com

) 

addition 

75% 

of flow 

25% 

off low 

l

Source water “B” 

Raw water 

blending station 

Raw water intake 

Backwash water 

recycle (if applicable

Source water “A” 

Chemical 

Manually 

collect 

sample 

Manually 

collect 

sample 

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)]. 

LT2 Rule: Systems must col ect sour

treatment [40 CFR § 141.703(b)(1)], un

source water samples after chemical t

Systems that recycle filter backwash wat

prior to the point of filter backwash wa

Backwash w

lagoon 

)(2)(i)]. 

Source water 

“A” 

Source water 

“B” 

100% 

l

water 

water 

Analyzed Analyzed 

E-4

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

75% 25% 

To be analyzed 

Collect samples manually at each source near the intaday and analyze each independently, then calcu ate a average of the analysis results. This is done by multipfor each source by the percentage of its contribution to flow at the time the samples were collected, and then values [40 CFR § 141.703(e)(2)(ii)]. 

Source 

“A” 

Source 

“B” 

Multiply result by 

percentage of 

contribution to total 

flow 

Multiply result by 

percentage of 

contribution to total 

flow 

OPTION 2:
Figure 4. Multiple Plants with the Same Influent

LT2 

sample 

point 

Chemical 

addition 

To plant 

)]. 

l

E-5

Source water 

Raw water 

chemical building 

Raw water intake 

Wate

LT2 

sample 

point 

Wate

To plant “B” 

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

LT2 Rule: Systems m

prior to chemica trea

unless approved by t

samples after ch

141.703(b)(2)]. Systems 

must collect source wate

backwash water ad
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: 

stream 

addition 

and 

Raw water 

chemical building 

Backwash water 

recycle (if applicable) 

W

Flowing 

Lateral 

well 

Well Chemical 

Sand 

gravel 

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 § 141.703(d)(1)].* 

Groundwater 

Minimal surface 

water mixing with 

ground water 

Manually 

collect 

sample 

Backwash w

lagoon 

Bank filtration cross section 

i

Cryptosporidium

(

i

Well point 

E-6

Scenario 2: Systems using bank filtered water that is treated by subsequent 

filtrat on 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 

 treatment credit for the bank filtration [40 CFR § 141.703(d)(2)]. 

* Refers to systems using bank filtration to meet Crypto

Interim Enhanced Surface Water Treatment Rule IESW§ 141.173(b) or 40 CFR § 141.522(a). ** Refers to systems where bank filtrat on serves as pr

Raw water 

chemical building 

Backwash water 

recycle (if applicable) 

W

Bank filtration cross section 

Flowing 

stream 

Lateral 

well 

Chemical 

addition 

Sand 

and 

gravel 

Groundwater 

Minimal surface 

water mixing with 

ground water 

LT2 

sample 

Backwash w

lagoon 
Figure 6. Ground Water Under the Direct Influence of Surfac

(GWUDI)

Well 

LT2 

point 

Cryptosporidium 

igure 5). 

LT2 Rule: 

Raw water 

chemical building 

Backwash water 

recycle (if applicable) 

Flowing 

stream 

Chemical 

addition 

Groundwater 

Water table Water table 

sample 

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 

treatment credit for bank 

filtration (see F

System

samples prior to ch

141.703(b)(1)], unl

collect source wa

treatment [40 CFR § 

recycle filter backwa

water samples prior 

water addition 

GWUDI cross section Backwash wa

lagoon 

E-7
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)]. 

LT2 

addition Source water 

Raw water 

chemical building 

Raw water intake 

Backwash water 

recycle (if applicable) 

Wate

sample 

point 

Presed. 

basin 

Chemical 

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. 

LT2 

point 

no 

E-8

Source water 

Raw water intake 

Wate

Presed. 

basin 

sample 

Sampling here will result in 

Cryptosporidium 

treatment credit 
Figure 8. Raw Water Off-Stream Storage

storage 

LT2 

addition 

LT2 Rule: 

E-9

Source water 

Raw water off-stream 

Raw water 

chemical building 

Raw water intake 

Backwash water 

recycle (if applicable) 

sample 

point Chemical 

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. 

System

samples prior to ch

141.703(b)(1)], unl

collect source wa

treatment [40 CFR § 

recycle filter backwa

water samples prior 

water addition 

Ba
Figure 9. Mixed Source Water: Ground Water and Surface Wat

Ground water well 

cross section 

addition 

addition 

Well 

Groundwater 

i

prior to treatment, systems must 

) 

Scenario 2: 

Source water 

Water tre

Raw water 

chemical building 

LT2 

sample 

point Chemical 

Raw water intake 

Ground water well 

cross section 

Well 

Groundwater 

Water table 

Scenario 1: 

Water treatment plant 

Raw water 

chemical building 

LT2 

sample 

point Chemical 

Source water 

Water table 

If a sample tap is ava lable 

where the sources are combined 

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 

recycle (if applicable

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. 

Raw water intake 

Ground water sources are not required 

to be monitored under the LT2 Rule. 

No sample is needed from this source. 

LT2 

tre

unl

so

tre

Sy

wate

prio

Backwash water 

lagoon 

Backwash water 

lagoon 

E-10
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 retype (if applicable): 1. LT2 sampling location 2. Points of chemical treatment prior to the treatm3. Filter backwash water addition 4. Pretreatment processes (e.g., presedimentatio5. Multiple source waters (show by adding additi

Source water 

E-11
Appendix F 

LT2 Sample Collection Form 
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Long Term 2 Enhanced Surface Water Treatment Rule 

Sample Collection Form 

Utility Information Shipping Information For Lab Use Only 

PWS name: 

PWS address: 

Lab name: 

Lab address: 

Date received: 

Time received: 

Received by: 

Sample temperature 

on receipt: 

Sample 

condition 

on receipt: Sampler name: Date shipped: 

Tracking number: 

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 typea

 (circle one): Flowing stream (FS) Reservoir/lake (RL) 

GWUDIb

 - FS GWUDIb - RL 

Requested analysis (circle one): Cryptosporidium field sample Cryptosporidium matrix spike 

E. coli 

Sample Collection Information 

Cryptosporidium E. coli 

Initial meter reading 

(field-filtered samples only): Sample collection time: 

Final meter reading 

(field-filtered samples only): Turbidity (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
Appendix G 

EnvirochekTM Field Filtration for Cryptosporidium 
This page intentionally left blank 
Procedure for Field-Filtering Samples for Cryptosporidium Analysis 

Using the Pall Life Sciences 

EnvirochekTM or EnvirochekTM HV Capsules 

1.0 Materials 

The following materials should be available before collecting sample:

Q Several pairs of new, powder-free latex gloves

Q Sample collection form (Appendix F)

Q Pall Life Sciences EnvirochekTM or EnvirochekTM HV capsule (recommend that two capsules be

kept on hand in case the first one clogs prior to filtering 10 L) 

Q Sanitary $30 L carboy (if sample is not from a pressurized source) 

Q Stopwatch 

Q Sample label 

Q Cooler, approximately 16-quart 

Q Temperature monitoring device (e.g., thermometer vial, ThermochronTM iButton, or equivalent) 

(if measuring temperature during shipment) 

Q Two large plastic trash bags 

Q One 8-lb bag of ice or gel ice packs 

Q Three gallon size ziplock bags 

Q Strapping tape 

Q Two self-adhesive plastic airbill sleeves 

Q 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:

Q 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 

Q Two 0.5-in. X 0.375-in. barbed reducing connectors 

Q Five lengths of clean 12.7-mm (0.5-in.) internal-diameter clear, vinyl, laboratory tubing 

Q Five pairs of hose clamps fit to tubing 

Q One 0.5-in. x 3-in. nipple 

Q One coupling to fit 0.5-in. internal diameter tubing 

Q One roll teflon tape 

Q One 0.5-in. x 0.5-in. x 0.25-in. tee 

Q Six 0.5-in. barbed male adapters 

Q One garden hose barbed adapter 

Q Pressure regulator 

G-1 February 2006 
Q Pressure gauge (capsule’s maximum operating pressure, EnvirochekTM - 30 PSID, EnvirochekTM 

HV - 60 PSID) 

Q Water meter (flow totalizer) 

Q Flow rate meter with valve or flow control valve (capsule’s maximum flow rate, EnvirochekTM ­

2 L/min, EnvirochekTM 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 EnvirochekTM /EnvirochekTM HV capsule. In place of 

the EnvirochekTM /EnvirochekTM 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 EnvirochekTM capsule or 

>60 PSIG for EnvirochekTM HV capsule) sites, the sampling system should be assembled in the 

following order, as shown in Figure 1 below: 

Q Reinforced influent tubing

Q Pressure regulator

Q Pressure gauge

Q Reinforced inlet tubing

Q EnvirochekTM /EnvirochekTM HV capsule

Q Reinforced outlet tubing

Q Flow totalizer (mechanical or graduated collection device)

Q Flow rate meter with valve or flow control valve

Q Effluent tubing to drain

G-2 February 2006 
February 2006

Figure 1.Sample System Setup for Collecting Cryptosporidium Samples from a Pressurized Source

( >30 PSIG for EnvirochekTM capsule or >60 PSIG for EnvirochekTM HV capsule)

For low pressure (1 to 30 PSIG for EnvirochekTM capsule or 1 to 60 PSIG for EnvirochekTM HV capsule)

sites, the sampling system should be assembled at the sample port valve in the following order, as shown

in Figure 2 below :

Q Influent tubing

Q EnvirochekTM /EnvirochekTM HV capsule

Q Outlet tubing

Q Flow rate meter with valve or flow control valve

Q Flow totalizer (mechanical or graduated collection device)

Q Effluent tubing to drain

Figure 2.Sample System Setup for Collecting Cryptosporidium Samples from a Pressurized Source

(1 - 30 PSIG for EnvirochekTM capsule or 1 - 60 PSIG for EnvirochekTM HV capsule)

2.1.5 Connect the sampling system, with the connector in place of the EnvirochekTM /EnvirochekTM

HV capsule, to the pressurized water system, using appropriate fittings and clamps.

G-3
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 

EnvirochekTM capsule or 4 L/min (approximately 1.0 gpm) for the EnvirochekTM 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 EnvirochekTM or 60 PSIG if using the EnvirochekTM 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): 

O Public water system (PWS) name 

O PWS address 

O Sampler name 

O Sample ID (optional) 

O Public Water System Identification (PWS ID) number 

O Public Water System facility ID number 

O Facility name 

O Sample collection point ID 

O Sample collection point name 

O Sample collection date 

O Source water type (optional [but required for E. coli sample forms]) 

O 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: 

O PWS ID 

O Facility name 

O Date of sample collection 

2.1.9 Record the following information on the sample collection form: 

O Initial meter reading 

2.1.10 Remove the connector and in its place, install the EnvirochekTM /EnvirochekTM HV capsule in 

line, securing the inlet and outlet ends with the appropriate fittings/clamps. 

G-4 February 2006 
Note! Retain the vinyl caps provided with the EnvirochekTM /EnvirochekTM 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 

EnvirochekTM capsule or 4 L/min for EnvirochekTM HV capsule, if necessary. Record the 

following information on the capsule label or sample collection form: 

O 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 EnvirochekTM 

/EnvirochekTM 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: 

O Stop time (when the water was shut off) 

O Final meter reading 

O Comments to laboratory, if needed 

2.1.15 With the capsule inlet pointed up, loosen the outlet end of the EnvirochekTM /EnvirochekTM 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 EnvirochekTM /EnvirochekTM 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 

EnvirochekTM /EnvirochekTM 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 
February 2006

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 EnvirochekTM /EnvirochekTM HV capsule. In

place of the EnvirochekTM /EnvirochekTM 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:

G Influent tubing

G EnvirochekTM /EnvirochekTM HV capsule

G Outlet tubing

G Centrifugal or peristaltic pump

G Tubing

G Flow rate meter with valve or flow control valve

G Flow totalizer (mechanical or graduated collection device)

G Effluent tubing to drain

Figure 3. Sample System Setup for Collecting Cryptosporidium Samples from an

Unpressurized Source

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
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 EnvirochekTM 

capsule or 4 L/min (approximately 1.0 gpm) for the EnvirochekTM 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: 

O Public water system (PWS) name 

O PWS address 

O Sampler name 

O Sample ID (optional) 

O Public Water System Identification (PWS ID) number 

O Public Water System facility ID number 

O Facility name 

O Sample collection point ID 

O Sample collection point name 

O Sample collection date 

O Source water type (optional [but required for E. coli sample forms]) 

O 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: 

O PWS ID 

O Facility name 

O Date of sample collection 

2.2.7 Record the following information on the sample collection form: 

O Initial meter reading 

2.2.8 Install the EnvirochekTM /EnvirochekTM HV capsule in line, securing the inlet and outlet ends 

with the appropriate fittings/clamps. 

Note! Retain the vinyl caps provided with the EnvirochekTM /EnvirochekTM 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 
2.2.10 Turn on the pump. Adjust the flow rate to approximately 2 L/min for the EnvirochekTM capsule or 

4 L/min for the EnvirochekTM HV capsule, if necessary. Record the following information on the 

capsule label and/or sample collection form: 

O 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: 

O Stop time (when the pump was shut off) 

O Final meter reading 

O Comments to laboratory, if needed 

2.2.13 With the capsule inlet pointed up, loosen the outlet end of the EnvirochekTM /EnvirochekTM 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 EnvirochekTM /EnvirochekTM 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 

EnvirochekTM /EnvirochekTM 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 ThermochronTM 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. 

O 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 
Appendix H 

Filta-Max® Field Filtration for Cryptosporidium 
This page intentionally left blank 
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:

Q Several pairs of new, powder-free latex gloves

Q Sample collection form (Appendix F)

Q Filta-Max® foam filter module (IDEXX, cat. number, FMC 10603) with housing (IDEXX, cat.

number, FMC 10504) 

Q Sanitary $30 L carboy (if sample is not from a pressurized source) 

Q Stopwatch 

Q Sample number label 

Q Cooler, approximately 16-quart 

Q Temperature monitoring device (e.g., thermometer vial, ThermochronTM iButton, or equivalent) 

(if measuring temperature during shipment) 

Q Two large plastic trash bags 

Q One 8-lb bag of ice or gel ice packs 

Q Three gallon size ziplock bags 

Q Strapping tape 

Q Two self-adhesive plastic airbill sleeves 

Q 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: 

Q Electric peristaltic pump (if sample is not from a pressurized source) 

Q Two 0.5-in. x 0.375-in. barbed reducing connectors 

Q Five lengths of clean 12.7-mm (0.5-in.) internal-diameter clear, vinyl, laboratory tubing 

Q Five pairs of hose clamps fit to tubing 

Q One 0.5-in. x 3-in. nipple (if sample line pressure is >120 PSIG) 

Q One coupling to fit 0.5-in. internal diameter tubing 

Q One roll teflon tape 

Q One 0.5-in. x 0.5-in. x 0.25-in. tee (if sample line pressure is >120 PSIG) 

Q Six 0.5-in. barbed male adapters 

Q One garden hose barbed adapter 

Q Pressure regulator (if sample line pressure is >120 PSIG) 

Q Pressure gauge (filter’s maximum operating pressure 120 PSIG) 

H-1 February 2006 
Q Water meter (flow totalizer)

Q 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: 

Q Reinforced influent tubing 

Q Pressure regulator

Q Pressure gauge

Q Reinforced inlet tubing

Q Filta-Max® filter housing, with the direction of flow depicted with an arrow

Q Reinforce outlet tubing

Q Flow totalizer (mechanical or graduated collection device)

Q Flow rate meter with valve or flow control valve

Q Effluent tubing to drain

H-2 February 2006 
February 2006

®

Figure 1. Sample System Setup for Collecting Cryptosporidium Samples from a Pressurized

Source ( >120 PSIG)

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: 

Q Influent tubing 

Q Filta-Max® filter housing, with the direction of flow depicted with an arrow

Q Outlet tubing

Q Flow rate meter with valve or flow control valve

Q Flow totalizer (mechanical or graduated collection device)

Q Effluent tubing to drain

Figure 2. Sample System Setup for Collecting Cryptosporidium Samples from a Pressurized

Source (7.5 - 120 PSIG)

H-3
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): 

O Public water system (PWS) name 

O PWS address 

O Sampler name 

O Sample ID (optional) 

O Public Water System Identification (PWS ID) number 

O Public Water System facility ID number 

O Facility name 

O Sample collection point ID 

O Sample collection point name 

O Sample collection date 

O Source water type (optional [but required for E. coli sample forms]) 

O 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): 

O 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. 

O 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): 

O Stop time (when the water was shut off) 

O Final meter reading 

O 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: 

O PWS ID 

O Facility name 

O 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 
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:

Q Influent tubing

Q Peristaltic pump

Q Inlet tubing

Q Filta-Max® filter housing, with the direction of flow depicted with an arrow

Q Outlet tubing

Q Flow rate meter with valve or flow control valve

Q Flow totalizer (mechanical or graduated container)

Q Effluent tubing to drain

Figure 3. Sample System Setup for Collecting Cryptosporidium Samples from an Unpressurized

Source

2.2.3 Place the inlet end of the inlet tubing in 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

H-6
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: 

O Public water system (PWS) name 

O PWS address 

O Sampler name 

O Sample ID (optional) 

O Public Water System Identification (PWS ID) number 

O Public Water System facility ID number 

O Facility name 

O Sample collection point ID 

O Sample collection point name 

O Sample collection date 

O Source water type (optional [but required for E. coli sample forms]) 

O 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: 

O 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: 

O Start time 

H-7 February 2006 
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: 

O Stop time (when the pump was shut off) 

O Final meter reading or measured total volume sample 

O 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: 

O PWS ID 

O Facility name 

O 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 ThermochronTM 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. 

O 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 
Appendix I 

Collecting Bulk Water Samples for 

Laboratory Filtration and Cryptosporidium Analysis -

Recommended Procedure 
This page intentionally left blank 
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: 

Q Several pairs of new, powder-free latex gloves 

Q Sample collection form (Appendix F) 

Q Sanitary 10 L cubitainer 

Q Sample number label 

Q Waterproof pen 

Q Cooler, approximately 34-quart 

Q Two large plastic trash bags 

Q Two 8-lb. bags of ice, or the equivalent number of ice packs sufficient to maintain the sample 

between 1°C and 10°C 

Q Four gallon size ziplock bags 

Q Strapping tape 

Q Two self-adhesive plastic airbill sleeves 

Q 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): 

O Public water system (PWS) name 

O PWS address 

O Sampler name 

O Sample ID (optional) 

O Public Water System Identification (PWS ID) number 

O Public Water System facility ID number

O Facility name

I-1
O Sample collection point ID 

O Sample collection point name 

O Sample collection date 

O Source water type (optional [but required for E. coli sample forms]) 

O 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: 

O Sample collection time 

O 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 ThermochronTM 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. 

O 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. coli Analyses - Recommended Procedure 
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Recommended Procedure for Collecting Source Water Samples 

for E. coli Analyses 

1.0 Materials 

The following materials should be available before collecting samples:

Q Several pairs of new, powder-free latex gloves (optional)

Q 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 

Q Sample number label 

The following additional materials may be needed if the sample will be shipped off-site for analysis: 

Q Sample collection form 

Q Gallon size zippered plastic bag 

Q Cooler, approximately 9-quart 

Q Two large plastic trash bags 

Q One 8-lb. bag of ice or gel ice packs 

Q Strapping tape 

Q Bubble wrap 

Q Two self-adhesive plastic airbill sleeves 

Q Airbill for shipment 

Q Temperature monitoring device (thermometer vial, or ThermochronTM 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): 

O Public water system (PWS) name 

O PWS address 

O Sampler name 

O Sample ID (optional) 

O Public Water System Identification (PWSID) number 

O Public Water System facility ID number 

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O Facility name 

O Sample collection point ID 

O Sample collection point name 

O Sample collection date 

O Source water type 

O Requested analysis (circle “E. coli”) 

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. coli 

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. coli 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 ThermochronTM 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. 

O 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. coli 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: / / 

Will you be collecting additional source water monitoring samples 

to meet the requirements of your plant? YES 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 

9 Cryptosporidium laboratory qualifications. 

A list of laboratories approved for LT2 Cryptosporidium analysis is posted at 

http://www.epa.gov/safewater/disinfection/lt2/lab_home.html. 

9 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) 

9 Sampling schedule. Is the sampling schedule designed to monitor for 

Cryptosporidium at least monthly (Section 3.2) on a regular schedule? 

9 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)? 

9 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)? 

9 Method version. Is EPA Method 1622 or EPA Method 1623 used to analyze 

Cryptosporidium samples (Section 5.2.2)? 

9 Sample volume issues. Is a minimum of 10 L collected for each monitoring and 

matrix spike sample (Section 4.2.1)? 

9 E. coli 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. coli methods (Table 7.1)? 

9 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? YES NO 

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 YES NO 

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? 

YES 

YES 

NO 

NO 

Have you included additional documentation regarding dates and reasons for resampling, the 

use of presedimentation, and/or off-stream storage during routine plant operation? YES NO 

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? YES NO 

Are all data elements from field sample results from the monitoring period included? YES NO 

Are all data elements from MS sample results from the monitoring period included? YES NO 

Are the volumes analyzed for all field samples at least 10 L? 

For samples in which less that 10 L was examined, were at least 2 mL of packed pellet volume 

analyzed or did two filters clog? 

YES 

YES 

NO 

NO 

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 YES NO 

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? YES 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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