v>EPA
United States
Environmental Protection
Agency
Source Water Monitoring Guidance
Manual for Public Water Systems for the
Long Term 2 Enhanced Surface Water
Treatment Rule (LT2 Rule)
June 2003
Draft
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Office of Water (4607)
EPA815-D-03-005
http://www.epa.gov/safewater/lt2/index.html
June 2003
Printed on Recycled Paper
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Disclaimer
The Standards and Risk Management Division, of the Office of Ground Water and Drinking Water, has
reviewed and approved this guidance for publication. Neither the United States Government nor any of its
employees, contractors, or their employees make any warranty, expressed or implied, or assumes any
legal liability or responsibility for any third party's use of or the results of such use of any information,
apparatus, product, or process discussed in this report, or represents that its use by such party would not
infringe on privately owned rights. Mention of trade names or commercial products does not constitute
endorsement or recommendation for use.
Questions concerning this document or its application should be addressed to:
Mary Ann Feige
U.S. EPA Office of Ground Water and Drinking Water
Technical Support Center
Room 127
26 West Martin Luther King Drive
Cincinnati, OH 45268-1320
(513)569-7944
(513) 569-7191 (facsimile)
feige.maryann@epa.gov
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TABLE OF CONTENTS
Section 1: Introduction 1
1.1 Background 2
1.2 Large System Requirements 2
1.3 Small System Requirements 3
1.4 Use of Cryptosporidium Data 5
1.4.1 Cryptosporidium Monitoring Sample Data 5
1.4.2 Cryptosporidium Matrix Spike Data 6
1.5 Use of E. coli Data 7
Section 2: Grandfathering Cryptosporidium Data 8
2.1 General Guidelines for Generating Cryptosporidium Data for Grandfathering 8
. 1 Sample Collection Location 8
.2 Sample Collection Schedule 9
.3 Cryptosporidium Analytical Methods for Grandfathered Data 9
.4 Cryptosporidium Laboratories for Grandfathered Data 11
.5 E. coli and Turbidity Measurements 11
2.2 Reporting Grandfathered Data 11
2.2.1 Data Package Contents 11
2.2.2 Schedule for Submission of Grandfathered Data 13
2.2.3 Procedures for Submission of Grandfathered Data 13
2.3 Checklists for Grandfathering Cryptosporidium Data 13
Section 3: Understanding Cryptosporidium Analyses 14
3.1 Summary of EPA Methods 1622 and 1623 14
3.2 Cryptosporidium Laboratory Quality Control 15
3.2.1 Initial Precision and Recovery Test 15
3.2.2 Method Blank Test 16
3.2.3 Ongoing Precision and Recovery Test 16
3.2.4 Holding Time Requirements 16
3.2.5 Staining Controls 17
3.2.6 Proficiency Testing Samples 17
3.2.7 Matrix Spike Samples 17
3.3 Archiving Examination Results 17
Section 4: Understanding E. coli Analyses 18
4.1 Summary of LT2 Rule E. coli Methods 18
4.1.1 Most Probable Number (MPN) Methods 18
4.1.2 Membrane Filtration (MF) Methods 19
4.2 E. coli Laboratory Quality Control 21
4.2.1 Dilution/Rinse Water Sterility Check 21
4.2.2 Media Sterility Check 21
4.2.3 Positive/Negative Controls 21
4.2.4 Media Storage 22
4.2.5 Filtration Unit Sterilization 22
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4.2.6 Preparation Blanks 22
4.2.7 Verification 22
Section 5: Contracting for Cryptosporidium Laboratory Services 23
5.1 Defining Your Needs and Developing a Contract 23
5.1.1 Client Information 24
5.1.2 Sample Information 24
5.1.3 Sampling Schedules 26
5.1.4 Analytical Methodology 26
5.1.5 Data Deliverables and Other Contract Issues 28
5.2 Developing a Bid Sheet 31
5.3 Soliciting the Contract 32
5.3.1 Approved Laboratories 32
5.3.2 Primary and Backup Laboratory Contracts 33
5.4 Evaluating Bids 33
5.4.1 Identifying Responsive Bidders 33
5.4.2 References 34
5.5 Communicating with the Laboratory 34
Section 6: Collecting and Shipping Source Water Samples 35
6.1 Sample Volumes 36
6.2 Sample Collection Location 37
6.2.1 Plants That Do Not Have a Sampling Tap Located Prior to Any Treatment . . 37
6.2.2 Plants That Use Different Water Sources at the Same Time 37
6.2.3 Plants That Use Presedimentation 37
6.2.4 Plants That Use Raw Water Off-Stream Storage 38
6.2.5 Plants That Use Bank Filtration 38
6.3 Source Water Monitoring Schedule 38
6.4 Sample Collection Guidance 39
6.4.1 Sample Collection Documentation 40
6.4.2 Cryptosporidium Sample Collection 41
6.4.3 E. coli Sample Collection 44
6.4.4 Measuring Turbidity 44
6.4.5 Monitoring Sample Temperature 46
Section 7: Reviewing Cryptosporidium Data 48
7.1 Cryptosporidium Data Recording at the Laboratory 48
7.1.1 LT2 Sample Collection Form 48
7.1.2 Method 1622/1623 Bench Sheet 48
7.1.3 Method 1622/1623 Cryptosporidium Slide Examination Form 49
7.2 Submitting Cryptosporidium Data through the LT2 Data Collection System 49
7.2.1 Data Entry/Upload 50
7.2.2 PWS DataReview 51
7.2.3 EPA/State Review 51
7.3 What Do the Sample Examination Results Mean? 51
7.3.1 Immunofluorescent Assay (IFA) 51
7.3.2 4',6-diamadino-2-phenylindole (DAPI) Examination 52
7.3.3 Differential Interference Contrast (DIG) Examination 52
7.5 Reviewing and Validating Raw Cryptosporidium Data {Optional} 52
7.5.1 Data Completeness Check 53
7.5.2 Evaluation of Data Against Method Quality Control Requirements 53
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7.5.3 Calculation Verification 54
7.6 Data Archiving Requirements 56
Section 8: Reviewing E. coli Data 57
8.1 E. coli Laboratory Data Recording at the Laboratory 57
8.1.1 Sample Identification Information 57
8.1.2 Primary Data 57
8.1.3 Sample Processing and Quality Control Information 57
8.1.4 Sample Results 58
8.2 Submission of E. coli Data through the LT2 Data Collection System 58
8.2.1 Data Entry/Upload 58
8.2.2 PWS Data Review 59
8.2.3 EPA/State Review 59
8.3 Reviewing and Validating E. coli Data (Optional) 59
8.3.1 Data Completeness Check 59
8.3.2 Evaluation of Data Against Method Quality Control Requirements 60
8.3.3 Calculation Verification 61
8.4 Data Archiving Requirements 67
Section 9: References 68
Section 10: Acronyms 69
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TABLES
Table 1-1. Timeline for Large Systems Regulated under the LT2 Rule 3
Table 1-2. Timeline for Small Systems Regulated under the LT2 Rule 4
Table 1-3. Bin Classifications 5
Table 1-4. Effect of the Number of Oocysts on Bin Classification Based on Mean of 12 Samples . 6
Table 1-5. Effect of the Number of Oocysts on Bin Classification Based on Mean of 48 Samples . 6
Table 6-1. Summary of LT2 Rule Monitoring Requirements 35
Table 6-2. Sample Collection Activities Required for Each Plant Type 40
Table 6-3. Minimum Data Elements to Record During Sample Collection 40
Table 6-4. Contacts for Filters Approved for Use in EPA Method 1622/1623 43
Table 7-1. LT2 Data Collection System Data Entry, Review, and Transfer Process 50
Table 8-1. Incubation Times and Temperatures for Approved E. Coll Methods 61
Table 8-2. Examples of Different Combinations of Positive Tubes 66
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IV
June 2003
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APPENDICES
Appendix A Checklist for Beginning Grandfathered Cryptosporidium Monitoring
Appendix B Checklist for Submitting Grandfathered Cryptosporidium Data
Appendix C Example LT2 Sample Collection Form
Appendix D Example Bulk Sample Collection Protocol for Cryptosporidium
Appendix E Example Envirochek™ Field Filtration Protocol for Cryptosporidium
Appendix F Example Filta-Max™ Field Filtration Protocol for Cryptosporidium
Appendix G Example E. coli Sample Collection Protocol
Appendix H Method 2130B for Turbidity Measurement
Appendix I Great Lakes Instrument Method 2 for Turbidity Measurement
Appendix J Revised EPA Method 180.1 for Turbidity Measurement
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SECTION 1: INTRODUCTION
The Long Term 2 Enhanced Surface Water Treatment Rule (LT2ESWTR or LT2 rule) requires public
water systems (PWSs) that use surface water or ground water under the direct influence of surface water
to monitor their source water (influent water prior to treatment) for Cryptosporidium, E. coli, and
turbidity for a limited period [40 CFRpart 141.701 (a)-(h)]. In support of the monitoring requirements
specified by the rule, three documents have been developed to provide guidance 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 advice.
• Microbial Laboratory Guidance Manual for the Long Term 2 Enhanced Surface Water Treatment
Rule (LT2 Rule). The goal of this manual is to provide 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 Long Term 2 Enhanced Surface Water Treatment Rule (LT2 Rule) Data
Collection System. This manual provides PWSs and laboratories with instructions on the entry,
review, and approval of electronic data using the LT2 Data Collection System, and for generating
reports using the system.
All of these manuals are available at http://www.epa. gov/safewater/lt2/index.html. Responses to
frequently asked questions (FAQs) on sampling, analysis, and data reporting questions for the LT2 rule
also are available on this website.
777/5 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 is intended solely for guidance and does not alter any regulatory or analytical method
requirements not altered by the LT2 rule itself.
This manual provides guidance on the following aspects of the LT2 rule:
• Section 1: Overview of the rule's monitoring requirements and how the Cryptosporidium and E. coli
data collected under the rule will be used
• Section 2: Guidance on submitting historical data ("grandfathering")
• Section 3: Understanding Cryptosporidium analyses
• Section 4: Understanding E. coli analyses
• Section 5: Establishing a Cryptosporidium laboratory contract
Section 6: Guidance on collecting and shipping LT2 monitoring samples
• Section 7: Reviewing Cryptosporidium data
Section 8: Reviewing E. coli data
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Section 1: Introduction
1.1 Background
The LT2 rule is a National Primary Drinking Water Regulation that requires monitoring, reporting, and
public notification requirements for all PWSs that use surface water sources. The rule requires additional
treatment techniques for some systems, based on Cryptosporidium monitoring results (40 CFR part
141.720 - 141.721). The LT2 rule was developed to improve control of microbial pathogens, including
specifically the protozoan Cryptosporidium, in drinking water and to address risk trade-offs with
disinfection byproducts.
The LT2 rule provides for increased protection against microbial pathogens in public water systems that
use surface water sources. The rule focuses on Cryptosporidium, a protozoan pathogen that is widespread
in surface waters. EPA is particularly concerned about Cryptosporidium because it is highly resistant to
inactivation by standard disinfection practices. Ingestion of Cryptosporidium oocysts can cause acute
gastrointestinal illness, and symptoms in sensitive subpopulations may be severe, including
risk of mortality. Cryptosporidium has been identified as the pathogenic agent in a number of waterborne
disease outbreaks.
EPA convened a Federal Advisory Committee to develop recommendations for both the Stage 2
Disinfectants and Disinfection Byproducts Rule and the LT2 rule. As recommended by the Federal
Advisory Committee, the LT2 rule requires public water systems that use surface water or ground water
under the direct influence of surface water to monitor their source water (influent water prior to treatment
plant) for Cryptosporidium, E. coll, and turbidity [40 CFR part 141.701 (a)-(h)]. These data would be
used to determine whether additional treatment is needed at PWSs and to assess whether a relationship
could be established between the Cryptosporidium and E. coli levels in source water.
1.2 Large System Requirements
Large systems affected by the LT2 rule include both filtered and unfiltered systems.
A large, filtered system in the LT2 rule is a system that:
• Uses surface water or ground water under the direct influence of surface water
• 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 systems are required to conduct initial source water monitoring that includes
Cryptosporidium, E. coli, and turbidity sampling [40 CFR part 141.701 (b)].
A large, unfiltered system in the LT2 rule is a system that:
• Uses surface water or ground water under the direct influence of surface water
• Serves at least 10,000 people
Does not currently provide filtration and meets all filtration avoidance criteria
Large unfiltered systems are required to conduct initial source water monitoring that includes
Cryptosporidium sampling only [40 CFR part 141.701 (d)].
All of the Cryptosporidium sampling requirements and guidance discussed in this document apply equally
to both filtered and unfiltered systems and both filtered and unfiltered systems that serve at least 10,000
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Section 1: Introduction
people are referred to as large systems in this document. However, the E. coll and turbidity guidance in
this document does not apply to large unfiltered systems.
The steps required for LT2 rule compliance for large systems, and the schedule for these steps, are
summarized in Table 1-1.
Table 1-1. Timeline for Large Systems Regulated under the LT2 Rule
Event
Establish contract with a
Cryptosporidium laboratory
pending approval under EPA's Lab
QA Program (Section 2.4.1, below)
Verify that your utility laboratory is
certified under the drinking water
laboratory certification program to
perform the technique you plan to
use for performing E. coli analyses
under LT2a
Submit grandfathered
Cryptosporidium data package
Work with your Cryptosporidium
laboratory to establish a mutually
acceptable sampling schedule
Submit sampling schedule through
the LT2 Data Collection System
Collect monitoring samples'1
Submit monitoring results"
Schedule
As soon as possible
As soon as possible
Within 2 months of rule promulgation11
Within 8 months of rule promulgation0
As soon as possible after establishing contract
Within 3 months of rule promulgation
Beginning 6 months after rule promulgation
No later than 10 days after the end of the first month
following the month that the sample was collected
(approximately 40 to 70 days after sample collection,
depending on when during the month the sample is
collected)
Duration
N/A- single event
N/A- single event
N/A - single event
N/A - single event
N/A - single event
At least once per
month for 2 years6
At least once per
month for 2 years6
a Not applicable to large, unfiltered systems because these systems are not required to monitor for E. coli or turbidity
b PWSs with at least 2 years of grandfathered data at the time of LT2 rule promulgation and that intend to use these
data in lieu of monitoring under the LT2 rule
c PWSs with fewer than 2 years of grandfathered data at the time of LT2 rule promulgation, or that have at least 2
years of grandfathered data but intend to conduct monitoring under the LT2 rule
d PWSs may be eligible to use historical (grandfathered) data in lieu of these requirements if certain quality
assurance and quality control criteria are met (see Section 2)
6 PWSs monitoring for Cryptosporidium may collect more than one sample per month if sampling is evenly spaced
over the monitoring period
N/A = Not applicable
1.3 Small System Requirements
A small system in 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
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Section 1: Introduction
• Does not currently provide filtration and meets all filtration avoidance criteria
These systems are required to conduct initial source water monitoring for E. coll as an indicator of
Cryptosporidium and, for those systems exceeding E. coli trigger levels, Cryptosporidium monitoring [40
CFR part 141.701 (c)].
The steps required for LT2 rule compliance for small systems, and the schedule for these steps, are
summarized in Table 1-2.
Table 1-2. Timeline for Small Systems Regulated under the LT2 Rule
Event
Verify that your utility laboratory is
certified under the drinking water
laboratory certification program to
perform the technique you plan to
use for perform £ coli analyses
under LT2
Submit sampling schedule through
the LT2 Data Collection System
Collect £ coli samples
Submit £ coli monitoring results
Schedule
Prior to rule promulgation
Within 27 months of rule promulgation
Beginning 30 months (2.5 years) after
rule promulgation
No later than 10 days after the end of
the first month following the month that
the sample was collected
(approximately 40 to 70 days after
sample collection, depending on when
during the month the sample is
collected)
Duration
N/A - single event
N/A - single event
1 year (2 samples per month)
At least once per month for 1 year
Possible additional monitoring requirement for Cryptosporidium if small systems exceed £. coli trigger levels3
Establish contract with a
Cryptosporidium laboratory
pending approval under EPA's Lab
QA Program (Section 2.4.1, below)
Submit sampling schedule through
the LT2 Data Collection System
Work with your Cryptosporidium
laboratory to establish a mutually
acceptable sampling schedule
Collect Cryptosporidium samples
Submit Cryptosporidium monitoring
results
As soon as possible after you are
notified that your plant has exceeded
the £ coli trigger levels
Within 45 months of rule promulgation
Within 2 months of rule promulgation
48 months (4 years) after
promulgation
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)
N/A - single event
N/A - single event
N/A - single event
1 year (2 samples per month)"
At least once per month for 1 year
a Small systems may be required to monitor for Cryptosporidium for 1 year, beginning 6 months after completion of
£ coli monitoring; Cryptosporidium monitoring required if the £ coli annual mean concentrations exceed 10 £
CO///100 ml_ for systems using lakes/reservoirs or exceed 50 £ co///100 ml_ for systems using flowing streams
b PWSs monitoring for Cryptosporidium may collect more than two samples per month if sampling is evenly spaced
over the monitoring period
N/A = Not applicable
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Section 1: Introduction
Details on the use of the Cryptosporidium and E. coll data collected under the LT2 rule are provided in
Sections 1.4 and 1.5.
1.4 Use of Cryptosporidium Data
Two types of Cryptosporidium data are collected under the LT2 rule: Cryptosporidium occurrence data
from the analysis of monitoring samples, and method performance data from the analysis of matrix spike
(MS) samples. The use of occurrence data from monitoring samples is discussed in Section 1.4.1; the use
of method performance data from MS samples is discussed in Section 1.4.2.
1.4.1 Cryptosporidium Monitoring Sample Data
The concentration of Cryptosporidium oocysts in source water samples analyzed during the LT2 rule will
be used to calculate a mean Cryptosporidium concentration for a PWS and classify the PWSs into a
treatment requirements "bin" (40 CFRpart 141.709). These bin classifications are provided in Table 1-3.
The treatment bin classification established for each PWSs will be 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 and source water protection to address their
greater risk for high Cryptosporidium source water concentrations.
Table 1-3. Bin Classifications
Cryptosporidium Bin Concentration
Cryptosporidium < 0.075 oocysts/L
0
075 oocysts/L < Cryptosporidium < 1.0 oocyst/L
1.0 oocyst/L < Cryptosporidium < 3.0 oocysts/L
Cryptosporidium > 3.0 oocysts/L
Bin Classification
Bin 1
Bin 2
Bin 3
Bin 4
1.4.1.1 Calculating Bin Classifications
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 a PWS serving at least 10,000 people, bin classification would be based on the following:
For PWSs that collect at least 48 samples during the required monitoring period, the Cryptosporidium
bin calculation is equal to the arithmetic mean of all sample concentrations
• For PWSs that collect at least 24 samples, but not more than 47 samples, during the required
monitoring period, the Cryptosporidium bin calculation is equal to the highest arithmetic mean of all
sample concentrations in any 12 consecutive months in the monitoring period
For PWS serving fewer than 10,000 people, and that monitor for Cryptosporidium for 1 year, bin
classification would be based on the simple arithmetic mean of all sample concentrations.
In all cases, the bin concentration is calculated using individual sample concentrations. These
concentrations are calculated as "number of oocysts detected / volume (in L) analyzed." Individual
sample concentrations are not calculated as "oocysts detected / 10 L," nor are bin concentrations
calculated as the "sum of the oocysts detected / the sum of the volumes analyzed." As a result, each
sample has an equal weight on the final bin concentration. In cases where no oocysts are detected, the
number of oocysts used to calculate the sample concentration is "0."
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Section 1: Introduction
1.4.1.2 Number of Oocysts Detected Versus Bin Classification
To better understand the relationship between the absolute number of oocysts detected in your samples
and the resulting bin classification, several crosswalks are provided below. Table 1-4 applies to large
plants conducting monthly monitoring over 2 years. This table provides a crosswalk between the sum of
the oocysts detected in 10- and 50-L samples during the highest 12-month period and the corresponding
bin classification.
Table 1-4. Effect of the Number of Oocysts on Bin Classification Based on Mean of 12 Samples
Sum of oocysts
found in 12, 10-L
samples3
0-8 oocysts
9-125 oocysts
126-365 oocysts
366 or more oocysts
Sum of oocysts
found in 12, 50-L
samples'1
0-44 oocysts
45 - 629 oocysts
630 - 1829 oocysts
1830 or more oocysts
Corresponding range of mean
Cryptosporidium concentrations
From
To
< 0.075 oocysts/L
0.075 oocysts/L
1.0 oocyst/L
< 1.0 oocyst/L
< 3.0 oocysts/L
> 3.0 oocysts/L
Corresponding bin
classification
1
2
3
4
a Representing the highest 12-month mean; assumes that 10-L samples are analyzed for each event
b Representing the highest 12-month mean; assumes that 50-L samples are analyzed for each event
Table 1-5 applies to large plants conducting semimonthly monitoring over 2 years. This table provides a
crosswalk between the sum of the number of oocysts detected in samples during the entire 2-year
monitoring period and the corresponding bin classification. Again, because this crosswalk is based on
analysis of exactly 10 L or 50 L for all samples, it may not apply to all plants that monitor for
Cryptosporidium on a semimonthly basis, but it helps put into perspective the impact that one high
sample result may have on bin classification.
Table 1 -5. Effect of the Number of Oocysts on Bin Classification Based on Mean of 48 Samples
Sum of oocysts
found in 48, 10-L
samples3
0-35
36 - 503
504 - 1463
1464 or more
Sum of oocysts
found in 48, 50-L
samples'1
0-179 oocysts
180-2519 oocyst
2520 -73 19 oocysts
7320 or more oocysts
Corresponding range of mean
Cryptosporidium concentrations
From
To
< 0.075 oocysts/L
0.075 oocysts/L
1.0 oocyst/L
< 1.0 oocyst/L
< 3.0 oocysts/L
>3.0 oocysts/L
Corresponding bin
classification
1
2
3
4
a Assumes that 10-L samples are analyzed for each event
b Assumes that 50-L samples are analyzed for each event
Systems may analyze larger volumes than 10 L, and larger volumes analyzed should increase analytical
sensitivity (detection limit), provided method performance is acceptable. Because these tables are based
on analysis of exactly 10 L or exactly SOL for all samples, it may not apply to all plants that monitor
monthly for Cryptosporidium, but it helps put into perspective the impact that one high sample result may
have on bin classification. In addition, filtering higher volumes may not result in the same high volume
analyzed if the source is turbid and the PWS analyzes only a portion of the concentrated sample. The
calculations used to determine the volume analyzed if less than the entire sample volume is analyzed are
discussed in Section 7.5.3.
1.4.2 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, per the requirements in EPA Methods
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Section 1: Introduction
1622/1623 (Section 9.1.8). A description of MS samples is provided in Section 3.2.7 of this document.
For large systems that perform monthly monitoring for 2 years and collect 24 monitoring samples and for
small systems that are triggered into monitoring for 1 year and collect 24 monitoring samples, two MS
samples will be analyzed. For large systems that perform semimonthly or more frequent monitoring for 2
years and collect 48 or more samples, a minimum of three MS samples will be analyzed.
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. The descriptive statistics of the MS sample results will be compared
to the performance of the methods during the Information Collection Rule Supplemental Surveys to
verify the assumptions on method performance upon which the LT2 rule is based.
When considering the method performance that could be achieved for analysis of Cryptosporidium under
the LT2 rule, the Federal Advisory Committee (FACA) evaluated the results of EPA Methods 1622/1623
in the ICRSS, which involved 87 PWSs sampling twice per month over 1 year for Cryptosporidium and
other parameters. During the ICRSS, the mean Cryptosporidium recovery and mean relative standard
deviation of the MS samples were 43% and 49%, respectively (Reference 9.1).
1.5 Use of E. coll Data
E. coll data are being collected by large systems during LT2 rule monitoring to assess whether a
relationship can be established between the Cryptosporidium and E. coli levels in source water and a
microbial index developed to establish trigger levels for E. coli that would indicate high Cryptosporidium
concentrations in a source water. If a relationship can be established, small systems initially will be
permitted to monitor for E. coli, rather than conducting more expensive Cryptosporidium analyses. Only
those systems with E. coli levels above the trigger level established in the microbial index would then be
required to monitor for Cryptosporidium to determine bin placement (40 CFR part 141.702).
A preliminary index was developed during development of the FACA agreement using data from the
Information Collection Rule (ICR) and ICRSS (Reference 9.2). These data were evaluated for parameters
that could indicate the likelihood that a source water mean Cryptosporidium level would be above the Bin
2 threshold concentration of 0.075 oocysts/L. Fecal coliforms, total coliforms, E. coli, viruses (ICR only),
and turbidity were assessed for development of the microbial index. Data analyses placed greater
emphasis on E. coli and fecal coliforms because of the direct relationship between these parameters and
fecal contamination.
E. coli was determined to provide the best performance as a Cryptosporidium indicator with the available
data. Based on the data from the ICR and ICRSS, the preliminary E. coli trigger levels were set at a mean
of 10 E. co///100 mL for reservoir/lake-type source waters and 50 E. co///100 mL for flowing stream-type
source waters.
These levels may potentially be revised based on the much larger, more reliable Cryptosporidium and E.
coli data set collected through LT2 rule monitoring.
Draft 1 June 2003
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SECTION 2: GRANDFATHERING CRYPTOSPORIDIUM DATA
"Grandfathered" Cryptosporidium data are results generated before monitoring under the Long Term 2
Enhanced Surface Water Treatment Rule (LT2ESWTR or LT2 rule) starts and that a public water system
(PWS) intends to use in determining its bin classification (Section 1.4.1) under the rule. Grandfathered
data may be used in lieu of, or in addition to, results generated during LT2 rule implementation (40 CFR
part 141.708). This section of the manual is designed to assist PWSs in producing grandfathered data that
should be equivalent to the data collected during LT2 rule implementation and, therefore, eligible for use
in bin classification. The final LT2 rule will establish requirements for reporting and acceptance of
grandfathered monitoring results.
2.1 General Guidelines for Generating Cryptosporidium Data for
Grandfathering
A PWS's grandfathered Cryptosporidium data package should meet the following general conditions (40
CFR part 141.708):
• Samples were collected from the appropriate location(s)
Samples were representative of a plant's source water(s) and the source water(s) have not changed
Samples were 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)
Samples were collected in equal intervals of time over the entire collection period (e.g., weekly,
twice-per-month, monthly)
The data set includes all source water Cryptosporidium monitoring results generated during the
grandfathered data monitoring period (see details below—data from monitoring not directed towards
LT2 rule binning will not be a component of the binning data set)
Sample volumes of at least 10 L were analyzed or, in cases where 10 L were not analyzed, at least 2
mL of packed pellet volume were analyzed (additional details below)
The data were generated using the validated versions of EPA Methods 1622 or 1623
The data are fully compliant with the QA/QC criteria specified in the version of Method 1622 or
Method 1623 used to generate the data, including analysis of matrix spike (MS) samples at a
frequency of at least 5% (1 MS sample for every 20 monitoring samples)
The following sections discuss these recommendations in more detail.
2.1.1 Sample Collection Location
The sample collection location requirements are the same for LT2 rule monitoring and for grandfathered
data and are discussed in Section 6.2. If the PWS does not collect samples as recommended in Section
6.2, the data may not be acceptable for grandfathering.
Draft 8 June 2003
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Section 2: Grandfathering Cryptosporidium Data
2.1.2 Sample Collection Schedule
During LT2 rule monitoring, PWSs will be required to collect samples at least monthly and in accordance
with a schedule established by the PWS prior to initiation of monitoring (40 CFRpart 141.703). PWSs
may collect samples more frequently (e.g., twice-per-month, weekly), provided the same frequency is
maintained throughout the monitoring period [40 CFRpart 141.701 (e)]. Sampling for grandfathered data
should follow these same criteria.
EPA recommends that, prior to initiation of grandfathered monitoring, PWSs 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 to EPA. PWSs that have begun grandfathered
monitoring without establishing a sampling schedule should develop a schedule for the collection of
remaining samples. PWSs should collect samples within 2 days before or after the dates indicated in their
sampling schedules. Exceptions to 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 should sample as close to the scheduled date as feasible and submit an explanation for the
alternative sampling date with the analytical results.
PWSs that are unable to report a valid Cryptosporidium analytical result for a scheduled sampling
date due to failure to comply with the analytical method quality control standards (e.g., sample is lost
or contaminated; laboratory exceeds an analytical method holding time) should collect a replacement
sample within 14 days of being notified by the laboratory that a result cannot be reported for that
date. PWSs should submit an explanation for the replacement sample with the analytical results.
Alternative sample collection dates should be timed so as not to coincide with another scheduled
Cryptosporidium sample collection date. Documentation of alternate sample collection, including the
reason, should be provided with the grandfathered data package.
Water treatment plants that use surface water or ground water under the direct influence (GWUDI), but
are operated only seasonally (e.g., during times of high-water demand) should monitor at least monthly
during the period when the plant is in operation.
The Federal Advisory Committee Agreement in Principle (Agreement) for the LT2 rule recommends that
if PWSs collect atotal of at least 48 samples (regardless of whether all of the samples were collected
before LT2 rule promulgation or some were collected before and some after rule promulgation), the
Cryptosporidium bin concentration will be equal to the arithmetic mean of all sample concentrations [40
CFR part 141.709 (b)(l)]. For PWSs that collect a total of at least 24 samples, but not more than 47
samples, the Cryptosporidium bin concentration will be equal to the highest arithmetic mean of all sample
concentrations in any 12 consecutive months during which Cryptosporidium samples were collected [40
CFR part 141.709 (b)(2)].
2.1.3 Cryptosporidium Analytical Methods for Grandfathered Data
Methods 1622 or 1623 should be used for Cryptosporidium analyses for the LT2 rule [40 CFRpart
141.708 (b)(l)]. The following are EPA-validated versions of Methods 1622 and 1623 acceptable for
monitoring for Cryptosporidium before LT2 rule implementation:
• Method 1623: Cryptosporidium and Giardia in Water by Filtration/IMS/FA. U.S. Environmental
Protection Agency, Office of Water. 2001. EPA-821-R-01-025
• Method 1622: Cryptosporidium in Water by Filtration/IMS/FA. U.S. Environmental Protection
Agency, Office of Water. 2001. EPA-821-R-01-026
Draft 9 June 2003
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Section 2: Grandfathering Cryptosporidium Data
• Method 1623: Cryptosporidium and Giardia in Water by Filtration/IMS/FA. U.S. Environmental
Protection Agency, Office of Water. 1999. EPA-821-R-99-006 (Note: The 2001 version of the
method should be used to generate data after January 1, 2002.)
• Method 1622: Cryptosporidium in Water by Filtration/IMS/FA. U.S. Environmental Protection
Agency, Office of Water. 1999. EPA-821-R-99-001 (Note: The 2001 version of the method should be
used to generate data after January 1, 2002.)
The procedures in EPA Method 1622/1623 are performance-based, and allow for modifications. The 2001
versions of EPA Method 1622/1623 also approve for nationwide use modified versions of the methods
using the following components:
Whatman Nuclepore CrypTest® filter
• IDEXX Filta-Max™ filter
• Waterborne Aqua-Glo™ G/C Direct FL antibody stain
• Waterborne Crypt-a-Glo™ and Giardi-a-Glo™ antibody stains
Since release of the 2001 versions of Methods 1622/1623, EPA also has approved a modified version of
the methods using the Pall Gelman Envirochek™ F£V filter and has approved the use of irradiated, flow
cytometer-sorted spiking suspensions for routine QC sample spiking.
Laboratories that analyze Cryptosporidium samples using other modified procedures, as allowed under
the performance criteria of Methods 1622/1623, should be approved to use the modified procedures under
the Lab QA Program discussed in Section 2.1.4, below, and in detail in the Microbial Laboratory
Guidance Manual for the Long Term 2 Enhanced Surface Water Treatment Rule (LT2 Rule).
Other notable differences between the 1999 and 2001 versions of EPA Method 1622/1623 include the
following:
Clarified sample acceptance criteria
Modified capsule filter elution procedure
• Modified concentrate aspiration procedure
• Modified IMS acid dissociation procedure
Updated QC acceptance criteria for initial precision and recovery (IPR) and ongoing precision and
recovery (OPR) tests
• Addition of a troubleshooting section for QC failures
Modified holding times
Inclusion of flow cytometry-sorted spiking suspensions (required for spiked samples analyzed during
LT2 monitoring)
2.1.3.1 Minimum Sample Volume and Subsampling Analysis
The requirements for sample volume analyses are the same for LT2 rule monitoring and for grandfathered
data [40 CFRpart 141.708 (b)(5)]. These requirements are discussed in Section 6.1 of this manual.
2.1.3.2 Analysis of Matrix Spike Samples
The requirements for analysis of matrix spike (MS) samples are the same for LT2 rule monitoring and for
grandfathered data [40 CFRpart 141.708 (e)]. These requirements, and guidance on MS sample
collection, are discussed in Section 6.4.2 of this manual.
Draft 10 June 2003
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Section 2: Grandfathering Cryptosporidium Data
2.1.4 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 (see http://www.epa.gov/safewater/lt2/index.html). 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 will be evaluated under the Lab QA Program before the data are submitted
to EPA. Note that PWSs will not submit grandfathered data packages until after the LT2 rule is final,
currently scheduled for mid- or late 2004. Samples analyzed by laboratories that do not meet the criteria
for approval under the LT2 rule may not be accepted for grandfathering.
Laboratories should also participate in the EPA Protozoa PT Program. EPA does not expect there to be
restrictions on the number of laboratories involved in the generation of a PWS's grandfathered data.
2.1.5 E. co/i and Turbidity Measurements
The Agreement would not exclude the use of previously collected Cryptosporidium data if E. coll and
turbidity samples are not collected. However, the Agreement recommends that PWSs serving at least
10,000 people should collect E. coli and turbidity samples along with Cryptosporidium samples when
monitoring under the LT2 rule. EPA recommends that PWSs conducting early (i.e., grandfathered)
monitoring collect and analyze E. coli samples with each Cryptosporidium sample and measure turbidity
during each sampling event.
2.2 Reporting Grandfathered Data
The final LT2 rule will establish reporting requirements for grandfathered data. The following
recommendations are intended to give PWSs an indication of potential reporting requirements for
consideration when establishing their grandfathered data monitoring programs.
For consideration of grandfathered data, PWSs should submit to EPA a complete data package as
described below.
2.2.1 Data Package Contents
The grandfathered data package should include the following:
1. 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 collected during the LT2 rule
monitoring period (defined below) are included in the package
2. Sample collection schedule established before beginning monitoring
3. 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
4. A list of the field and MS samples submitted in the data package (see Section 2.2.1.1, below, for
details), identified by sample ID and collection date
5. Sample results for all field and MS samples (see Section 2.2.1.2, below, for details) and
6. Documentation that all method-required quality control requirements were acceptable for every field
and MS sample submitted with the package (see Section 2.2.1.3, below, for details).
Draft 11 June 2003
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Section 2: Grandfathering Cryptosporidium Data
2.2.1.1 Sample Results to be Reported
PWSs that conduct monitoring for grandfathering should submit results for all source water
Cryptosporidium samples analyzed during the LT2 rule monitoring period, as defined below (40 CFR part
141.707). This will include all samples that were:
• Collected from the sampling location used for LT2 rule monitoring,
• Not spiked, and
Analyzed using the laboratory's routine process for Method 1622/1623 analyses, including analytical
technique and QA/QC.
EPA plans that the LT2 rule monitoring period for a specific PWS will begin with the collection of the
first sample submitted for LT2 rule binning and end with the collection of the final sample submitted for
LT2 rule binning (as long as a minimum of 2 years of acceptable data have been submitted). With the use
of grandfathered data, the final sample may be collected before the end of the LT2 rule implementation
schedule. Sample results generated after the last sample result in the PWS's data package would be
considered outside the PWS's LT2 rule monitoring period and would not need to be submitted to EPA for
LT2 rule binning purposes. However, these results may be subject to reporting requirements under other
federal or State regulations.
2.2.1.2 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 part 141.708 (d)]:
• PWS ID
• Facility ID
• Sample collection point
Sample collection date
Sample type (field or MS)
• Sample volume filtered (L), to nearest % L
• Number of oocysts counted
• For samples in which less than 10 L is filtered or less than 100% of the sample volume is examined,
PWSs should 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 should also report the
volume of resuspended concentrate and volume of this resuspension processed through
immunomagnetic separation.
For matrix spike samples, PWSs should also report the sample volume spiked and estimated number
of oocysts spiked. These data are not applicable to monitoring samples.
EPA recommends that these data elements be reported by submitting a completed sample collection form,
laboratory bench sheet, and Cryptosporidium report form for each sample. Example bench sheets and
report 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/lt2/index.html. Sample documentation forms that are different from these
examples, but that contain the minimum required data elements listed above, may be acceptable.
Draft 12 June 2003
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Section 2: Grandfathering Cryptosporidium Data
2.2.1.3 Supporting Quality Control Information
The data package should include a signed letter from the laboratory certifying that all method-required
quality control elements (including sample temperature upon receipt, ongoing precision and recovery 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
(however, the actual MS sample results are not required to meet the methods' MS QC acceptance
criteria). The letter should include a list of the applicable monitoring and MS samples, and the
corresponding OPR and method blank sample ID for each.
Alternately, the PWS may include the bench sheet and Cryptosporidium report form (or comparable
detailed data reporting forms) for each OPR and method blank sample associated with the field and MS
samples in the grandfathered data package. If this option is selected, the letter from the laboratory still
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 and MS sample results.)
2.2.2 Schedule for Submission of Grandfathered Data
EPA's current intent is that PWSs with at least 2 years of grandfathered data at the time of LT2 rule
promulgation and that intend to use these data in lieu of monitoring under the LT2 rule (i.e., do NOT
intend to conduct additional monitoring) should submit these data to EPA within 2 months following LT2
rule promulgation (currently planned for mid- or late 2004). EPA plans to notify these PWSs within 4
months following LT2 rule promulgation as to whether their data are sufficient for bin classification [40
CFR part 141.708 (f)].
PWSs with fewer than 2 years of grandfathered data at the time of LT2 rule promulgation, or that have at
least 2 years of grandfathered data but intend to conduct monitoring under the LT2 rule, should submit
these data to EPA within 8 months of LT2 rule promulgation (which provides the systems with 2 months
to review data from the last potential historical sampling event). Data collected when LT2 rule monitoring
begins (6 months after promulgation) will be submitted through the LT2 Data Collection System [40 CFR
part 141.708 (g)].
Under the Agreement, PWSs should conduct monitoring under the LT2 rule unless notified in writing by
EPA that they have 2 years of acceptable data.
2.2.3 Procedures for Submission of Grandfathered Data
EPA does not intend to formally accept grandfathered Cryptosporidium data until the LT2 rule is
finalized. The final rule will include procedures for submission of grandfathered data.
2.3 Checklists for Grandfathering Cryptosporidium Data
To help PWSs interested in monitoring for Cryptosporidium before LT2ESWTR apply the information
provided in this guidance, two checklists have been developed. The "Checklist for Beginning
Grandfathered Cryptosporidium Monitoring"(Appendix A) is designed to be used by PWSs to check
their monitoring plans against this guidance document before proceeding with monitoring. The "Checklist
for Submitting Grandfathered Cryptosporidium Data" (Appendix B) is designed to be used by PWSs to
check their data package against the information in this guidance document before submitting the data
package to EPA for review.
Draft 13 June 2003
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SECTION 3: UNDERSTANDING CRYPTOSPORIDIUM ANALYSES
The LT2 rule requires the use of EPA Method 1622 or EPA Method 1623 for Cryptosporidium
monitoring [40 CFR part 141.705 (a)]. This section provides utility personnel unfamiliar with
Cryptosporidium sample analyses with information on how the analyses are performed and on the quality
control (QC) measures the laboratory uses to verify data quality.
3.1 Summary of EPA Methods 1622 and 1623
EPA Methods 1622 and 1623 resulted from an EPA effort initiated in 1996 to identify new and
innovative technologies for analysis of source water samples for Cryptosporidium and Giardia. The
methods are identical in most respects, generally differing only in the addition of Giardia antibodies in
EPA Method 1623's purification and staining procedures. Both EPA Methods 1622 and 1623 were
subjected to interlaboratory validation studies using various source waters, and used in a national survey
of 87 surface water plants (the Information Collection Rule Supplemental Surveys) to provide EPA with a
realistic indication of how the methods would perform when they were used in the monitoring study
(Reference 9.1).
Both EPA Methods 1622 and 1623 also were developed as "performance-based" methods. The methods
include quantitative criteria requirements (minimum recovery and maximum variability) for initial and
ongoing QC samples. These criteria are used to verify acceptable laboratory performance using the
version of the method originally validated or to determine whether a modified version of the method
performs acceptably.
In EPA Methods 1622 and 1623, the following steps are performed:
Filtration. The sample is filtered in the field or in the laboratory using one of the filters approved for
use with EPA Methods 1622 and 1623:
• Pall Gelman Envirochek™ capsule filter
• Pall Gelman Envirochek™ HV capsule filter
• IDEXX Filta-Max™ foam filter
The oocysts, cysts, and extraneous materials are retained on the filter.
Elution. Materials on the filter are removed by elution with an aqueous buffered salt detergent
solution. This elution process is performed differently for each filter:
• For the Pall Gelman Envirochek™ and Envirochek™ HV filters, elution is performed by filling
the capsule with elution buffer, attaching the filter to a "wrist shaker" type lab shaker, and
allowing the filter to shake for 5 minutes at a time in three different orientations.
• For the IDEXX Filta-Max™ filter, the elution technique differs by laboratory. Some laboratories
may add the foam filter and elution buffer to a manual plunger chamber to expand the foam filter
and flush any oocysts out of the pores in the foam. Other laboratories may add the foam filter
rings and elution buffer to a stomacher bag and use a stomacher to elute the filter.
Draft 14 June 2003
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Section 3: Understanding Cryptosporidium Analyses
• For the Whatman CrypTest® filter, elution is performed by adding elution buffer to the filter
housing and using sonication and pressurized backwashing to separate oocysts from the filter
fabric.
Concentration. After the filter is eluted, the eluate is centrifuged to concentrate the eluted particles
into a "packed pellet" at the bottom of the centrifuge tube. This packed pellet is measured by the
laboratory analyst. If the pellet volume is < 2 mL (and 10 L was filtered) the entire sample must be
analyzed. If the pellet volume is > 2 mL, only 2 mL is required to be analyzed under the LT2 rule
(although the utility may request that more be analyzed).
Aspiration and resuspension. The analyst aspirates the supernatant from the top of the packed pellet
to minimize the total sample volume, and resuspends the pellet material by vortexing the sample. The
analyst measures the total resuspended concentrate volume. If the packed pellet volume was > 2 mL,
and the entire sample volume will not be analyzed, only a portion of the concentrate volume will be
processed through the remainder of the method. By dividing the concentrate volume processed
through the remainder of the method by the total concentrate volume, the laboratory can determine
what percent of the sample volume filtered was actually analyzed. By multiplying this percentage by
the sample volume filtered, the laboratory can determine the volume analyzed.
Purification. Magnetic beads conjugated to ax&i-Cryptosporidium antibodies are added to the sample
concentrate and allowed to mix with the sample, where they attach themselves to any oocysts present.
The magnetized oocysts are separated from the extraneous materials using a magnet, and the
extraneous materials are discarded. The magnetic bead complex is then detached from the oocysts.
Application of the purified sample to a slide. After immunomagnetic separation, the purified
sample is applied to a microscope slide.
Drying the sample. The sample is dried to the slide for several hours to several days to allow the
sample to be stained and rinsed without loss of organisms.
Staining the sample. Two stains are added to the sample before it is examined to help the analyst
identify any Cryptosporidium that may be present. The oocysts and cysts are stained on the slide with
fluorescently labeled monoclonal antibodies and 4',6-diamidino-2-phenylindole (DAPI).
Examining the sample. During microscopic examination of the slide, three evaluation techniques are
required by EPA Methods 1622 and 1623 to determine whether an object is a Cryptosporidium
oocyst. (Guidance on interpreting examination results is provided in Section 7.3.)
3.2 Cryptosporidium Laboratory Quality Control
As required by both EPA Method 1622/1623 and the Laboratory QA Program, laboratories approved to
perform Cryptosporidium analyses for the LT2 rule must perform specific quality control (QC) steps
during sample analyses to demonstrate that data are reliable [40 CFRpart 141.705 (a)(3)]. These QC steps
are described below, in Sections 3.2.1 - 3.2.7.
3.2.1 Initial Precision and Recovery Test
Before performing field sample analyses using EPA Methods 1622 or 1623, the laboratory must
demonstrate acceptable performance. This is demonstrated by the initial precision and recovery (IPR) test,
which consists of four reagent water samples spiked with 100 to 500 oocysts. The results of the four
analyses are used to calculate the average percent recovery and the relative standard deviation (RSD) of
Draft 15 June 2003
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Section 3: Understanding Cryptosporidium Analyses
the recoveries for Cryptosporidium. For EPA Methods 1622/1623, the mean Cryptosporidium recovery
must be in the range of 24% to 100% and the RSD of the four recoveries must be less than 55%. If more
than one process will be used for filtration and/or separation of samples, a separate set of IPR samples
must be analyzed for each process.
3.2.2 Method Blank Test
The method blank test in EPA Method 1622/1623 consists of analysis of an unspiked reagent water
sample to demonstrate freedom from contamination. One method blank sample must be analyzed each
week or every 20 samples, whichever is more frequent. If more than one process will be used for filtration
and/or separation of samples, a separate method blank must be analyzed for each process. If one or more
Cryptosporidium oocysts are found in a blank, analysis of additional samples is halted until the source of
contamination is eliminated and a blank shows no evidence of contamination.
3.2.3 Ongoing Precision and Recovery Test
The ongoing precision and recovery (OPR) in EPA Method 1622/1623 entails analysis of a reagent water
sample spiked with 100 to 500 oocysts to demonstrate ongoing acceptable performance. One OPR sample
must be analyzed each week or every 20 samples, whichever is more frequent. If more than one process
will be used for filtration and/or separation of samples, a separate OPR sample must be analyzed for each
process. OPR samples must be analyzed before any monitoring samples are processed for each batch to
verify acceptable performance. OPR Cryptosporidium recovery must be in the range of 11% to 100% to
be considered acceptable.
3.2.4 Holding Time Requirements
During Cryptosporidium analyses for the LT2 rule, sample processing should be completed as soon as
possible by the laboratory. The laboratory should complete sample filtration, elution, concentration,
purification, and staining the day the sample is received wherever possible. However, the laboratory is
permitted to split up the sample processing steps if processing a sample completely in one day is not
possible. If this is necessary, sample processing can be halted after filtration, application of the purified
sample onto the slide, or staining.
The following holding times must be met for samples analyzed by EPA Methods 1622/1623 during the
LT2 rule:
• Sample collection and filtration. Sample elution must be initiated within 96 hours of sample
collection (if shipped to the laboratory as a bulk sample) or filtration (if filtered in the field).
• Sample elution, concentration, and purification. The laboratory must complete the elution,
concentration, and purification in one work day. It is critical that these steps be completed in one
work day to minimize the time that any target organisms present in the sample sit in eluate or
concentrated matrix. This process ends with the application of the purified sample on the slide for
drying.
• Staining. The sample must be stained within 72 hours of application of the purified sample to the
slide.
Examination. Although fluorescence assay (FA) and 4',6-diamidino-2-phenylindole (DAPI) and
differential interference contrast (DIC) microscopy examination and confirmation should be
performed immediately after staining is complete, laboratories have up to 7 days from completion of
sample staining to complete the examination and confirmation of samples. However, if
fading/diffusion of fluorescien isothiocyanate (FITC) or DAPI staining is noticed, the laboratory must
reduce this holding time. In addition, the laboratory may adjust the concentration of the DAPI
staining solution so that fading/diffusion does not occur.
Draft 16 June 2003
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Section 3: Understanding Cryptosporidium Analyses
3.2.5 Staining Controls
Positive staining controls entail staining and examination of a slide with positive antigen or 200 to 400
intact oocysts to verify that the stain is fluorescing appropriately. These controls are prepared with each
batch of slides that are stained. Negative staining controls entail staining and examining a slide with
phosphate buffered saline solution to verify that no oocysts or interfering particulates are present.
3.2.6 Proficiency Testing Samples
As part of the Lab QA Program, laboratories must successfully analyze initial proficiency testing (IPX)
samples initially, and an ongoing proficiency testing (OPT) samples three times per year. These samples
and the Lab QA Program are discussed in more detail in the Microbial Laboratory Guidance Manual for
the Long Term 2 Enhanced Surface Water Treatment Rule (LT2 Rule).
3.2.7 Matrix Spike Samples
The matrix spike (MS) test in EPA Method 1622/1623 entails analysis of a separate sample aliquot spiked
with 100 to 500 oocysts to determine the effect of the matrix on the method's oocyst recovery.
One MS sample must be analyzed for every 20 samples from your PWS. The first MS sample should be
collected and analyzed during the first sampling event under the monitoring program and at least 12
months must elapse between the first and last MS sample. You should evaluate the MS recoveries, as well
as other attributes of sample processing and examination, and work with the laboratory to determine
whether sample filtration and processing procedures are working acceptably, or need to be re-evaluated.
If it is not possible to analyze an MS sample for the first sampling event due to laboratory sample
processing burden or other reasons, the first MS sample should be analyzed as soon as possible to identify
potential method performance issues with the matrix. The requirement that at least 12 months must elapse
between the first and last MS sample still applies. For example, if a PWS that is monitoring monthly for
24 months is unable to process an MS sample until the 8th sampling event, due to laboratory sample
processing load, the second MS sample can be processed no earlier than the 20th sampling event.
EPA Method 1622/1623 specifies the following additional requirements for MS sample analyses:
• The MS sample volume analyzed must be within 10% of the volume analyzed for the associated field
sample.
• The MS sample must be analyzed in the same QC batch as the field sample, using the same method.
• The MS sample must be collected as a split sample or immediately before or after the associated field
sample.
Under the LT2 rule, 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 CFRpart 141.705 (a)(2)(ii)].
3.3 Archiving Examination Results
Although not required, laboratories also can archive slides and/or take photographs of slides to maintain
for clients. Slides should be stored in a humid chamber in the dark at 0°C to 10°C. An alternative
mounting medium also may be used, which may potentially preserve slides longer. Details are provided in
the Microbial Laboratory Guidance Manual for the Long Term 2 Enhanced Surface Water Treatment
Rule (LT2 Rule).
Draft 17 June 2003
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SECTION 4: UNDERSTANDING E. COL/ANALYSES
As noted in Section 1, E. coll and turbidity data generated under the LT2 rule are used differently for
large systems than small systems. E. coll and turbidity are reported with Cryptosporidium data by large
systems to enable EPA to determine whether an E. coll trigger level can be established through the
microbial index. If a defensible trigger level can be established between E. coll concentrations and
Cryptosporidium levels, small systems will be able to perform less-expensive E. coll analyses initially to
determine whether more expensive Cryptosporidium monitoring is even necessary.
Although E. coll data will not be used to determine whether additional treatment is needed for large
systems, as Cryptosporidium data will, it is nonetheless critical that the large systems generate reliable E.
coll data to establish relevant trigger levels for use by the small systems. The E. coll data generated by
small systems will be used to determine whether Cryptosporidium monitoring is required, so it is critical
that these data be reliable, as well.
This section provides utility personnel unfamiliar with E. coll sample analyses with an overview of the
methods used under the LT2 rule and the quality control (QC) measures the laboratory uses to verify data
quality.
4.1 Summary of LT2 Rule E. coli Methods
E. coli sample analyses performed under the LT2 rule must be quantitative; presence/absence E. coli
results are unacceptable under LT2. The methods described below are approved for the analysis ofE. coli
samples under the LT2 rule [40 CFRpart 141.705 (b)].
4.1.1 Most Probable Number (MPN) Methods
4.1.1.1 Standard Methods 9223B: Colilert® and Colilert-18®
Colilert® and Colilert-18® tests are chromogenic/fluorogenic enzyme substrate tests for the simultaneous
determination of total coliforms and E. coli in water. These tests use commercially available media
containing the chromogenic substrate ortho-nitrophenyl-p-D-galactopyranoside (ONPG), to detect total
coliforms and the fluorogenic substrate 4-methylumbelliferyl-p-D-glucuronide (MUG), to detect E. coli.
Media formulations are available in disposable tubes for the multiple-tube procedure or packets for the
multiple-well procedure. Appropriate preweighed portions of media for mixing and dispensing into
multiple-tubes and wells are also available. The use of commercially prepared media is required for
quality assurance and uniformity. All tests must be conducted in a format that provides quantitative
results [40 CFRpart 141.705 (b)].
• Multiple-Tube. For the multiple-tube procedure, a well-mixed sample and/or sample dilution/volume
is added to tubes containing predispensed media. Tubes are then capped and mixed vigorously to
dissolve the media. Alternatively, this procedure can be performed by adding appropriate amounts of
substrate media to a bulk diluted sample (with appropriate dilutions for enumeration), then mixing
and dispensing into multiple-tubes. A 15-tube MPN should be used to obtain quantitative results. The
Draft 18 June 2003
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Section 4: Understanding E. coli Analyses
number of dilutions/volumes are determined based on the type, quality, and character of the water
sample.
Multiple-Well. A multiple-well procedure may be performed with sterilized disposable packets. The
commercially available Quanti-Tray® or Quanti-Tray®/2000 multiple-well tests use Colilert® or
Colilert-18® media to determine E. coli (IDEXX, 1999b,c). In these tests, the packet containing
media is added to a 100-mL sample (or appropriate dilutions for enumeration). The sample is then
mixed and poured into the tray. A tray sealer separates the sample into 51 wells (Quanti-Tray) or 97
wells (Quanti-Tray/2000) and seals the package.
After the appropriate sample dilutions/volumes are added, the tubes or trays are incubated at 35°C ±
0.5°C for 18 h when using Colilert-18® or 24 h when using Colilert®. If the response is questionable
after the specified incubation period, the sample is incubated for up to an additional 4 h at 35°C ±
0.5°C for both Colilert® tests. Each tube or well is then compared to the reference color "comparator"
provided with the media. A yellow color greater or equal to the comparator indicates the presence of
total coliforms in the sample, and the tube or well is then checked for fluorescence under long-
wavelength UV light (365-nm). The presence of fluorescence greater than or equal to the comparator
is a positive test for E. coli. If water samples contain humic acid or colored substances, inoculated
tubes or wells should also be compared to a sample water blank without Colilert® reagent added. The
concentration in MPN/100 mL is then calculated from the number of positive tubes or wells using
MPN tables provided by the manufacturer.
4.1.1.2 Standard Methods 9221B/9221F: LTB -EC-MUG
The multiple-tube fermentation method for enumerating E. coli in water uses multiple-tubes and
dilutions/volumes in a two-step procedure to determine E. coli concentrations. In the first step, or
"presumptive phase," a series of tubes containing lauryl tryptose broth (LTB) are inoculated with
undiluted samples and/or dilutions/volumes of the samples and mixed. Inoculated tubes are incubated for
24 ± 2 h at 35°C ± 0.5°C. Each tube then is swirled gently and examined for growth (i.e., turbidity) and
production of gas in the inner Durham tube. If there is no growth, acid, or gas, tubes are re-incubated for
24 ± 2 h at 35°C ± 0.5°C and re-examined. Production of growth and gas within 48 ± 3 h constitutes a
positive presumptive test for coliforms, which include E. coli.
After enrichment in the presumptive medium, positive tubes are subjected to a second step for
enumeration of E. coli. Presumptive tubes are agitated, and growth is transferred using a sterile loop or
applicator stick to tubes containing EC broth supplemented with 4-methylumbelliferyl-p-D-glucuronide
(MUG). Inoculated tubes are incubated at 44.5°C ± 0.2°C for 24 ± 2 h in a water bath. All tubes
exhibiting growth and gas production are examined for bright blue fluorescence under long-wavelength
UV light (366-nm) indicating a positive test for E. coli. The density of E. coli in MPN/100 mL is then
calculated from the number of positive EC-MUG tubes, using MPN tables or formulas. A 15-tube MPN is
required under the LT2 Rule.
4.1.2 Membrane Filtration (MF) Methods
4.1.2.1 Standard Methods 9222B/9222G: mEndo/LES-Endo-NA-MUG and Standard Methods
9222D/9222G: mFC-NA-MUG
These membrane filter methods for enumerating E. coli are two-step incubation procedures. First, a
sample is filtered through a 0.45 |im filter, then the filter is placed on a pad saturated with mEndo broth or
a plate containing mEndo or LES-Endo agar and incubated for 24 ± 2 h at 35°C ± 0.5°C. Pink to red
colonies with a metallic (golden-green) sheen on the filter are considered to be total coliforms. If initial
determination of fecal coliforms is desired, mFC media can be substituted for mEndo/LES-Endo.
Following initial isolation of total coliforms (or fecal coliforms), the filter is transferred to nutrient agar
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Section 4: Understanding E. coli Analyses
containing 4-methylumbelliferyl-p-D-glucuronide (NA-MUG) and incubated for 4 h at 35°C ± 0.5°C.
Sheen colonies on mEndo or blue colonies on mFC that fluoresce under a long-wavelength UV light
(366-nm) are positive for E. coli. If high levels of non-E1. coli total coliforms interfere with the ability to
accurately enumerate E. coli despite additional dilutions, transfer from mFC or an alternate method (e.g.,
SM 9213D, EPA Method 1603) should be used.
4.1.2.2 Standard Methods 9213D: mTEC
The mTEC agar method is a two-step procedure that provides a direct count of E. coli in water, based on
the development of colonies on the surface of a membrane filter when placed on a selective nutrient and
substrate media. This method originally was developed by EPA to monitor the quality of recreational
water. This method was also used in health studies to develop the bacteriological ambient water quality
criteria for E. coli. In this method, a water sample is filtered through a 0.45|im membrane filter, the filter
is placed on mTEC agar (a selective primary isolation medium), and the plate is incubated first at 35°C ±
0.5°C for 2 h to resuscitate injured or stressed bacteria and then at 44.5°C ± 0.2°C for 22-24 h in a water
bath. Following incubation, the filter is transferred to a filter pad saturated with urea substrate medium.
After 15 minutes, all yellow or yellow-brown colonies (occasionally yellow-green) are counted as
positive for E. coli using a fluorescent lamp and either a magnifying lens or a stereoscopic microscope.
4.1.2.3 EPA Method 1603: Modified mTEC
The modified mTEC agar method is a single-step MF procedure that provides a direct count of E. coli in
water based on the development of colonies on the surface of a filter when placed on selective modified
mTEC media. This is a modification of the standard mTEC media that eliminates bromcresol purple and
bromphenol red from the medium, adds the chromogen 5-bromo-6-chloro-3-indolyl-p-D-glucuronide
(Magenta Glue), and eliminates the transfer of the filter to a second substrate medium. In this method, a
water sample is filtered through a 0.45|im membrane filter, the filter is placed on modified mTEC agar,
incubated at 35°C ± 0.5°C for 2 h to resuscitate injured or stressed bacteria, and then incubated for 22-24
h in a 44.5°C ± 0.2°C water bath. Following incubation, all red or magenta colonies are counted as E.
coli.
4.1.2.4 EPA Method 1604: MI Medium
The MI medium method is a single-step membrane filtration procedure used to simultaneously enumerate
total coliforms and E. coli. In this EPA-developed method, a water sample is filtered through a 0.45-|im
membrane filter, the filter is placed on an MI agar or broth plate, and the medium is incubated at 35°C ±
0.5°C for 24 h. If high levels of non-E. coli total coliforms interfere with the ability to accurately
enumerate E. coli despite additional dilutions, an alternate method (e.g., SM 9213D, EPA Method 1603)
should be used.
E. coli colonies exhibit a blue color and also may fluoresce under a long-wave length UV light (366-nm).
If desired, the plates can also be observed under long-wavelength UV light (366-nm) for the presence of
total coliform species that fluoresce. Because the blue color from the breakdown of indoxyl-p-D-
glucuronide (IBDG) can mask fluorescence, non-fluorescent blue colonies are included in the total
coliform count. Water samples with high turbidity can clog the membrane filter, interfering with filtration
and potentially interfering with the identification of target colonies.
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Section 4: Understanding E. coli Analyses
4.1.2.5 m-ColiBlue24® Broth
This broth method is a single-step MF test for enumerating total coliforms and E. coli. As with NA-MUG,
modified mTEC, and MI media, the selective identification of E. coli is based on the detection of the P-
glucuronidase enzyme. The test medium includes the chromogen 5-bromo-4-chloro-3-indoxyl-p-D-
glucuronide (BCIG or X-Gluc). The chromogen BCIG is hydrolyzed by p-glucuronidase, releasing an
insoluble indoxyl salt that causes the colonies to exhibit a blue color. M-ColiBlue24® broth is a
commercially available format of this method and contains a nutritive lactose-based medium containing
inhibitors to eliminate the growth of non-coliforms. With m-ColiBlue24® broth, a water sample is filtered
through a 0.45|im membrane filter, and the filter is transferred to a plate containing an absorbent pad
saturated with m-ColiBlue24® broth. The filter is incubated at 35°C ± 0.5°C for 24 h and examined for
colony growth. The presence of E. coli is indicated by blue colonies. The presence of total coliforms
(non-E1. coif) is indicated by red colonies. If enumeration of total coliforms is desired, blue and red
colonies should be included in the total coliform count. If high levels of non-E1. coli total coliforms
interfere with the ability to accurately enumerate E. coli despite additional dilutions, an alternate method
(e.g., SM 9213D, EPA Method 1603) should be used.
4.2 E. coll Laboratory Quality Control
E. coli sample results reported under the LT2 rule should meet the quality control (QC) specifications set
forth in the approved versions of the methods described above. Sections 4.2.1 - 4.2.7 describe quality
control specifications for E. coli analyses performed under the LT2 rule. This guidance is provided to
help summarize the QC specifications in the methods and does not substitute for or alter the method
specifications. Sample results that do not meet these specifications are not considered valid, and cannot be
reported under the LT2 rule. Additional information on the QC specifications is available in Section 4.2
of the Microbial Laboratory Guidance Manual for the Long-Term 2 Enhanced Surface Water Treatment
Rule (LT2 Rule).
4.2.1 Dilution/Rinse Water Sterility Check
Each batch (or lot, if commercially prepared) of dilution/rinse water should be checked for sterility by
adding 50 mL of water to 50 mL of a double-strength non-selective broth (e.g., tryptic soy, trypticase soy,
or tryptose broth). Incubate at 35°C ± 0.5°C, check for growth after 24 hours and 48 hours (or for the
longest incubation time specified in the method), and record results. The dilution/rinse water batch should
be discarded if growth is detected.
4.2.2 Media Sterility Check
To test sterility of newly prepared media prior to the analysis of field samples, incubate one plate per each
media batch at the appropriate temperature for 24 and 48 hours (or for the longest incubation time
specified in the method) and observe for growth. If any contamination is observed, determine the cause,
correct, and reject any data from samples tested with the media.
4.2.3 Positive/Negative Controls
For each new lot or batch of medium, check the analytical procedures and integrity of the medium before
use by testing with known positive and negative control cultures. Laboratories using commercially-
prepared media with manufacturer shelf-lives of greater than 90 days should run positive and negative
controls each quarter in addition to running the batch/lot-specific controls and sterility checks.
Laboratories are encouraged to perform positive and negative control tests each day that field samples are
analyzed. Positive and negative controls should be chosen based on the method-specific requirements. For
example if a 44.5°C water bath is not required by the method, it is not necessary to include Enterobacter
aerogenes as a negative control.
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Section 4: Understanding E. coli Analyses
4.2.4 Media Storage
The following media storage specifications should be met for E. coli analyses:
• Agar plates may be held for up to 2 weeks at 1°C to 5°C in plastic bags or containers. Protect media
containing dyes from exposure to light.
• Broth in loose fitting caps (e.g., snap caps) should be stored at 1°C to <30°C for no more than 2
weeks
Broth in tight fitting caps (e.g., screw caps) should be stored at 1°C to <30°C for no longer than 3
months
All media should be at room temperature prior to use
Media exhibiting growth or gas should be discarded
4.2.5 Filtration Unit Sterilization
Membrane filter equipment should be autoclaved before the beginning of a filtration series. A filtration
series ends when 30 minutes or longer elapses after a sample is filtered. Ultraviolet (UV) light (254 nm)
may be used to sanitize equipment (after initial autoclaving for sterilization), if all supplies are pre-
sterilized. UV light can also be used to reduce bacterial carry-over between samples during a filtration
series. The UV lamp should be tested quarterly with a UV light meter or an agar plate. Appropriate
corrective actions should be taken, if necessary.
4.2.6 Preparation Blanks
Preparation blanks should be analyzed to detect potential contamination of dilution/rinse water during the
course of analyses.
4.2.6.1 Membrane Filter Preparation Blank
If membrane filtration is used, an MF preparation blank is performed at the beginning and the end of each
filtration series by filtering 20-30 mL of dilution water through the membrane filter and testing for
growth. If the control indicates contamination with the target organism, all data from affected samples
should be rejected. A filtration series ends when 30 minutes or more elapse between sample filtrations.
4.2.6.2 Most Probable Number Preparation Blank
EPA recommends that a volume of sterilized, buffered water be analyzed exactly like a field sample each
day samples are analyzed. The preparation blank should be incubated with the sample batch and observed
for growth of the target organism. If the control indicates contamination with the target organism, all data
from affected samples should be rejected.
4.2.7 Verification
Verification specifications are detailed in the Certification Manual (Reference 9.3), Standard Methods
(Reference 9.4), and Appendices J through L of the Microbial Laboratory Guidance Manual for the
Long-Term 2 Enhanced Surface Water Treatment Rule (LT2 Rule).
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SECTION 5: CONTRACTING FOR CRYPTOSPORIDIUM
LABORATORY SERVICES
Although many public water systems (PWSs) have established procedures and policies governing the
purchase of services and supplies, these procedures seldom lend themselves to the purchase of analytical
services. This section provides a basic framework for defining the technical and contractual requirements
associated with purchasing laboratory services for Cryptosporidium analyses for the LT2 rule, awarding
contracts, and working with a contract laboratory.
Successfully contracting for Cryptosporidium 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 and
standardized bid sheet
Step 3: Solicit qualified laboratories
Step 4: Award contracts to a primary laboratory and a backup laboratory
Step 5: Work closely with your laboratory before monitoring begins and maintain communications
throughout monitoring
Each of these general steps, and details on the activities associated with each, are discussed in Sections
5.1 through 5.5.
5.1 Defining Your Needs and Developing a Contract
The first step in developing an analytical services contract for Cryptosporidium 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 Cryptosporidium 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.
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Section 5: Contracting for Cryptosporidium Laboratory Services
5.1.1 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 Cryptosporidium
analyses to fulfill monitoring requirements for multiple plants in a system?
Clearly identify in your contract the name and identification number 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 L T2 Data
Collection System, and the laboratory you use for Cryptosporidium sample analyses will need
to know this information. (Alternately, you can provide this information after award to the
awarded laboratory only.)
5.1.2 Sample Information
"What" describes the samples to be analyzed. As noted in Sections 5.1.2.1 through 5.1.2.5, this
encompasses a variety of factors, each of which needs to be evaluated and defined before you develop
your contract.
5.1.2.1 Number of Samples
What is the total number of samples the laboratory will need to analyze? This total includes not only
routine monitoring samples (field samples), but also the matrix spike (MS) samples (Section 3.2.7) that
are required at a frequency of 1 per 20 field samples. Field 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 amortize the costs of these samples across billable samples.
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 "make-up" sample (see Section 6.3 for details). You should
add, as an option to be exercised at your direction in such an event, two additional sample analyses to the
total.
Clearly indicate in your contract the total number of: (1) field samples and (2) MS samples
that the laboratory will be required to analyze. Add two additional, optional, sample analyses
to be exercised if "make-up "samples are required due to problems unrelated to laboratory
performance.
5.1.2.2 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 on-site and ship the filter to the laboratory? Shipping and analytical costs are
likely to be lower if you filter your samples on-site, but you will need to purchase or rent sample filtration
equipment (see Section 6.4 for details) and have staff trained to use the required procedures or pay for the
laboratory or another firm to perform these tasks.
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Section 5: Contracting for Cryptosporidium Laboratory Services
Clearly specify in the SOW 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 5.1.4.2).
If you will be filtering on-site, and will be using your own equipment to filter the samples, you should
consider purchasing filters directly from the vendor, rather than from the laboratory, to reduce costs.
(Additional information on filtering samples on-site and purchasing filters is provided in Section 6.4.2).
If your PWS will be purchasing filters directly, specify this in the contract, so the
laboratory knows not to include this in their per-sample price.
5.1.2.3 Anticipated Sample Volume
The LT2 rule will require that at least 10 L be analyzed for each sample (with some exceptions - see
Section 6.1) [40 CFRpart 141.705 (a)(l)]. 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 5.1.2.4).
as= Clearly indicate in your contract the volume you anticipate collecting for each sample.
5.1.2.4 Subsamples and Filter Clogs
As noted in Section 3.1, additional steps are required at the laboratory for samples that generate a larger
packed pellet volume than can be processed as one sample through the method's purification step.
Specifically, the laboratory will need to process the packed pellet from the sample as two or more
"subsamples" through the remainder of the method (purification, staining, and examination) to meet LT2
rule sample volume analysis requirements. If a sample clogs before 10 L have been filtered, at least two
filters must be used to meet LT2 rule sample volume analysis requirements [40 CFRpart 141.705 (a)(l)].
If the source water(s) to be monitored by your PWS are characterized by high turbidity, some of your
samples may need to be processed as multiple subsamples or may require two filters to enable you to
meet LT2 rule monitoring requirements. Even if your source water(s) typically is characterized by low
turbidity, you should allow for the possibility that some samples may result in larger packed pellet
volumes on occasion. By including this in the original contract, you will avoid changes to the contract on
short notice if subsamples are required during monitoring.
Clearly indicate in your contract 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.
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Section 5: Contracting for Cryptosporidium Laboratory Services
5.1.2.5 Extra Services
Will any additional services be required of the laboratory outside of actual sample analyses? Possible
services include:
Sampling kit rental for on-site filtration
Sample shipping containers
• Sample archiving (laboratories can archive slides and some can take photographs of slides to maintain
for clients)
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.
I ^ Clearly specify in your contract any services required in addition to routine sample analysis. I
5.1.3 Sampling Schedules
"When " refers to your anticipated schedule for shipping samples to the laboratory. Will your PWS begin
monitoring before implementation of the LT2 rule with the intent to grandfather some or all of the data or
will your PWS monitor according to the rule schedule?
The minimum monitoring frequency for the LT2 rule is once per month [40 CFRpart 141.701 (e)].
During LT2 monitoring, will your PWS collect and ship samples once per month, or will you monitor
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 is will comply with LT2 rule
requirements and is mutually acceptable to your PWS and the laboratory.
Indicate in your contract the month that you plan to begin monitoring and whether you will be
monitoring on a monthly or more frequent basis. 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.
5.1.4 Analytical Methodology
"How " describes the analytical method that the laboratory will use. This involves two sets of options:
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.
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Section 5: Contracting for Cryptosporidium Laboratory Services
5.1.4.1 EPA Method 1622 Versus EPA Method 1623
Will your PWS monitor for Cryptosporidium only or Cryptosporidium and Giardial Most laboratories
analyze samples for both Cryptosporidium and Giardia using EPA Method 1623. If EPA Method 1623 is
used by the laboratory to analyze your LT2 rule samples, only Cryptosporidium data need to be
submitted. If Giardia data are collected, they do not need to be submitted to EPA.
Your contract should specify that EPA Method 1622 be used only if you are interested in monitoring for
Cryptosporidium only (this method only detects Cryptosporidium). Although reagent costs for this
method are slightly less than for EPA Method 1623, actual sample analysis costs may not be lower
because laboratories may not be able to allocate the QC sample costs for this method across as many
clients.
5.1.4.2 Filter Options
Although EPA validated EPA Method 1622 and EPA Method 1623 using one filter type, modified
versions of the methods using alternate filter options have been approved by EPA since validation. The
following available filters are considered acceptable by EPA for use with EPA Methods 1622 and 1623:
• Original Pall Gelman Envirochek™ capsule filter
• IDEXX Filta-Max™ foam filter
• Pall Gelman Envirochek™ F£V capsule filter
Unless your PWS has experience with Cryptosporidium sampling, and a basis for requesting a specific
filter type, you should indicate in the contract that all are acceptable.
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.
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.
5.1.4.3 Quality Control Requirements
Although EPA Methods 1622 and 1623 specify the QC requirements that must be met during
performance of the method, your contract should reiterate that the following QC tests must be performed
at the required frequency during processing and analysis of your samples:
• Method blank test (Section 3.2.2)
Ongoing precision and recovery (OPR) test (Section 3.2.3)
• Holding time requirements (Section 3.2.4)
• Staining controls (Section 3.2.5)
None of these QC measures should be billable, however. As noted above, in Section 5.1.2.1, the costs for
the method blank, OPR, and staining control tests should be amortized by the laboratory across the cost of
monitoring samples for all of their clients.
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Section 5: Contracting for Cryptosporidium Laboratory Services
Reiterate in the contract that method blanks, OPRs, and staining controls must be performed
at the frequency required in the method, and that all holding times must be met.
5.1.5 Data Deliverables and Other Contract Issues
In addition to the "who, " "what, " "when, " and "how " questions that need to be addressed by the
contract, you also will need to provide details on data delivery, adjustments for lateness, and sample
reanalysis cost issues. These issues are discussed in Sections 5.1.5.1 through 5.1.5.5.
5.1.5.1 Data Submission
EPA has developed the web-based LT2 Data Collection System to allow laboratories to report data to
PWSs electronically and allow PWSs to verify the data electronically before submitting the monitoring
results to EPA. This reporting process is summarized in Section 7.2 for Cryptosporidium data, and
discussed in detail in the Users' Manual for the Long Term 2 Enhanced Surface Water Treatment Rule
(LT2 Rule) Data Collection System. The laboratory, at a minimum, will need to submit the results for each
Cryptosporidium monitoring sample to you electronically. (Although your PWS also could enter these
data, 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
LT2 Data Collection System.)
Clearly indicate in your contract that the laboratory is required to enter Cryptosporidium
monitoring results for your samples into the L T2 data Collection System.
5.1.5.2 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.-lfyour PWS does intend to review all of the raw data associated with your
LT2 samples (discussed in Section 7), 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. (If your PWS will store and
maintain all sample results, rather than the laboratory, then the original forms should be requested.)
Suggestions for the materials that should be requested include the following:
• Sample result summary sheet, which should include 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
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Section 5: Contracting for Cryptosporidium Laboratory Services
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
If you need the laboratory to submit hardcopy results (this is not necessary, unless you
intend to review all of the raw data), clearly indicate in your contract the materials that are
required.
5.1.5.3 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 (approximately 40 to 70 days after sample
collection, depending on when during the month the sample is collected ) [40 CFRpart 141.707 (d)]. For
example, if a sample is collected on March 17, data must be submitted by May 10.
The required data turnaround must 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. As a general rule, the data turnaround time should not be shorter than the sum of the maximum
holding times in the method—15 days. 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 LT2
Data Collection System.
Using the 15 days allowed for sample analysis by the methods (plus additional time to compile the data
package and mail the results) as the shortest realistic turnaround time, determine when you will actually
need the results. The same turnaround time can be specified for both submission of electronic data and
receipt of hardcopy materials.
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Section 5: Contracting for Cryptosporidium Laboratory Services
Do not specify a data turnaround time shorter than you really need, as it may increase the per-sample
price quoted by the laboratories. This turnaround time should be short enough to provide time to carefully
evaluate the results before they must be submitted to EPA, but long enough that it does not unreasonably
burden the laboratory and potentially increase the per-sample quotes you receive when you solicit the
project.
Specify in the contract the data turnaround requirement for electronic and hardcopy
submission of data. This turnaround time should be calculated as the time between sample
collection and receipt of the hardcopy data by your PWS.
5.1.5.4 Liquidated Damages and Penalties
You should consider including penalty or damage clauses in your contracts as incentives to preclude
laboratories from submitting data late or performing analyses improperly. Due to the nature of the
services provided, it is often difficult to assess actual damages caused by improperly performed analyses.
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% of the analysis price per calendar day of delay, to a
maximum reduction of 50% of the analysis price.
If liquidated damages or penalties are involved, they should (1) be based on actual damage caused (in
terms of cost) by each day of lateness, (2) be strong enough to discourage late delivery, and (3) be
reasonable enough that they will not discourage laboratories from bidding. If liquidated damages or
penalties will be applied to meet the required data turnaround time, this information should be included.
The contract should specify that the laboratory will not be charged with liquidated damages when the
delay in delivery or performance arises out of causes beyond the control and without the fault or
negligence of the laboratory. It also may be necessary to limit damages to a certain dollar value or scope.
Other types of damages that should be considered, and may be included in the contract, include costs for
resampling and administrative costs associated with the evaluation and processing of unacceptable data
(data that do not meet the requirements specified in the contract or the QC requirements specified in the
analytical method).
Clearly indicate in your contract whether liquidated damages will be applied to late data or
other problems, how these liquidated damages are calculated, and the limits and conditions
associated with the damages.
5.1.5.5 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 sample received at > 10°C that results in
resampling by your PWS), the laboratory should not be responsible for the additional costs that may
result.
Draft 30 June 2003
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Section 5: Contracting for Cryptosporidium Laboratory Services
Clearly indicate in your contract when the laboratory would be required to bear the costs of
sample re-analysis costs 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
payment. Rejection of data should be based on sound technical review of the results. It also obligates you
to make no use of those results without making some payment to the laboratory.
Clearly indicate in your contract that your PWS has the right to inspect results and reject
the results if they do not meet contract requirements.
5.2 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 chart, with analytical requirements along
one axis and number of samples and prices along the other.
The bid sheet should include the following information:
• Project identifier (e.g. "LT2 Cryptosporidium Monitoring Sample Analyses for [PWS name and/or
facility name]")
Space for laboratory identification information
Day, date, and time (including time zone) of the bid deadline
Bid submission information (contact and mailing address, fax number, and/or email address)
Estimated award date
Laboratory period of performance (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)
• Required delivery date (data turnaround time and the basis of its calculation, such as from collection
of each sample)
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)
Draft 31 June 2003
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Section 5: Contracting for Cryptosporidium Laboratory Services
A summary of the analytical requirements:
• Method (e.g., Cryptosporidium and Giardia by EPA Method 1623)
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, it is important that you
specify this)
• Whether samples will be shipped as filtered samples or bulk water samples
• Sample volume (e.g., 10 L, 50 L)
Total number of field samples to be analyzed, plus two extra, in case of "make-up" samples
Total number of MS samples to be analyzed
Total number of potential subsamples to be analyzed (expressed as "Up to [no.] subsamples")
The number generally should not exceed three per sample
• If you have high-turbidity water, you may need to specify up to three subsamples for all of your
field 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 need to be analyzed)
Total number of potential extra filters (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 needs to be used)
Columns for laboratories to enter per-analysis and total costs
5.3 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.
5.3.1 Approved Laboratories
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 will need to 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)
(67 FR 9731, March 4, 2002). Information on the Laboratory QA Program is posted on
Draft 32 June 2003
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Section 5: Contracting for Cryptosporidium Laboratory Services
http://www.epa.gov/safewater/lt2/index.html and this program is described in detail in the Microbial
Laboratory Guidance Manual for the Long Term 2 Enhanced Surface Water Treatment Rule (LT2 Rule).
Briefly, the objectives of the program are to evaluate laboratories' capacity and competency to reliably
measure 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:
• Acceptably perform initial proficiency testing (IPX) on blind samples
Participate in an on-site evaluation of their technical, data management, and quality assurance
procedures
• Acceptably perform ongoing proficiency testing (OPT) on blind samples every four months
To improve Cryptosporidium data quality and consistency during LT2 rule monitoring, EPA requires that
only those laboratories approved for Cryptosporidium analysis under the Lab QA Program be used for
LT2 rule monitoring analyses [40 CFRpart 141.706 (a)]. A list of laboratories approved through the Lab
QA Program is available from http://www.epa. gov/safewater/lt2/index.html.
5.3.2 Primary and Backup Laboratory Contracts
Because a laboratory's approval status may 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.
The award of primary and backup contracts should be discussed in the contract solicitation. All other
things considered equal, the award for the primary contract could be made to the lowest responsive,
responsible bidder and the award for the backup contract could be made to the second lowest responsive,
responsible bidder.
5.4 Evaluating Bids
After the laboratories have received the solicitation and submitted their bids, you must 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.
5.4.1 Identifying Responsive Bidders
You should consult your legal department or purchasing department to identify any applicable
requirements for evaluating competitive bids within their organization. At a minimum, however, you
should review all bids and recalculate subtotals and totals to ensure that the bidding laboratories did not
make any mathematical errors. In addition, you should verify that there are no unacceptable contingencies
associated with any of the bids, such as the use of a filter other than the filter that was specified in the
contract solicitation. 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.
Draft 33 June 2003
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Section 5: Contracting for Cryptosporidium Laboratory Services
Of the remaining (responsive) bids, identify the lowest bidder to award the primary contract and the
second lowest bidder to award the backup contract. If additional assessments of a laboratory's
performance or responsibility are needed, you may want to contact references.
5.4.2 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 which the laboratory has performed
Cryptosporidium 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?
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?
5.5 Communicating with the Laboratory
After the analytical services contract is awarded, you should request from the 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, you should always notify the
laboratory of sample shipments and confirm that the laboratory received the sample on time and in
acceptable condition.
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.
Draft 34 June 2003
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SECTION 6: COLLECTING AND
SHIPPING SOURCE WATER SAMPLES
Large systems (PWSs serving a population of at least 10,000 people) monitoring under the LT2 rule are
required to collect and analyze source water samples for Cryptosporidium, E. coll, and turbidity for a
minimum of 2 years. Small systems (PWSs that serve fewer than 10,000 people) are required to monitor
their source water for E. coli for a minimum of 1 year. A subset of small systems would then be required
to conduct Cryptosporidium analyses over a 1-year period if they exceed E. coli trigger levels (40 CFR
part 141.701).
Monitoring requirements for each system size and the schedule for each stage of monitoring is described
in Table 6-1.
Table 6-1. Summary of LT2 Rule Monitoring Requirements
Public water
system size
Large systems
(serving 10,000 or
more people)
Small systems
(serving fewer than
10,000 people)
Monitoring begins
6 months after
promulgation of LT2
rule
30 months (2 1/2
years) after
promulgation of LT2
rule
Monitoring
duration
2 years3
1 year3'"
Monitoring parameters and sample
frequency requirements
Cryptosporidium
minimum
1 sample/month0
see below §
£. co//
minimum
1 sample/month11
1 every 2 weeks
§ Possible additional monitoring requirement for Cryptosporidium
If small systems exceed £ coli trigger levels, then. . .
Small systems
(serving fewer than
10,000 people)
48 months (4 years)
after promulgation
of LT2 rule
1 year
2 sample/month
N/A
a PWSs may be eligible to use historical (grandfathered) data in lieu of these requirements if certain quality
assurance and quality control criteria are met (see Section 2)
b Small systems may be required to monitor for Cryptosporidium for 1 year, beginning 6 months after completion of
£ coli monitoring; Cryptosporidium monitoring would be required if the £. coli annual mean concentrations exceed
10 £ CO///100 ml_ for systems using lakes/reservoirs or exceed 50 £ co///100 ml_ for systems using flowing streams
c PWSs monitoring for Cryptosporidium may collect more than 1 sample per month if sampling is evenly spaced over
the monitoring period
d Large unfiltered systems are required to conduct source water monitoring that includes Cryptosporidium sampling
only
N/A = Not applicable. No monitoring required.
Draft
35
June 2003
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Section 6: Collecting and Shipping Source Water Samples
6.1 Sample Volumes
Sample volume guidance is provided in Section 6.1.1 for Cryptosporidium samples and Section 6.1.2 for
E. coll samples.
6.1.1 Cryptosporidium Samples
Under LT2 rule Cryptosporidium sample volume requirements [40 CFRpart 141.705 (a) (1)], PWSs are
required to analyze, at a minimum, either:
• 10 L of sample, or
• 2 mL of packed pellet volume, or
As much volume as two filters can accommodate before clogging (this condition applies only to
filters that have been approved by EPA for nationwide use with EPA Method 1622/1623—the Pall
Gelman Envirochek™ and Envirochek™ HV filters, or the IDEXX FiltaMax™ foam filter).
The LT2 rule sample volume analysis requirement of 10 L (rather than 10.0 or 10.00 L) accommodates
the potential for imprecisely filled sample containers or filters. Sample volumes > ##.5 L would be
rounded up and sample volumes < ##.4 L would be rounded down. For example, 9.8 L would be rounded
to 10 L, and would meet rule requirements.
Systems may analyze larger volumes than 10 L, and larger volumes analyzed should increase analytical
sensitivity (detection limit), provided method performance is acceptable. EPA encourages systems to
analyze similar sample volumes throughout the monitoring period. However, data sets including different
samples volumes will be accepted, provided the system analyzes the minimum sample volume
requirements noted above.
PWSs with highly turbid water may be able to collect the required minimum packed pellet volume by
avoiding filtration altogether, and shipping a bulk water sample to the laboratory for centrifugation. The
laboratory can mix the sample thoroughly and centrifuge 250-mL or greater aliquot volumes sequentially
according to Section 13.2 of Method 1622/1623, until 2 mL of packed pellet volume is generated.
If the PWS encounters variable water quality that clogs the filter unpredictably, the PWSs should
routinely bring two filters plus a cubitainer to the sampling point for each sampling event:
• If the water quality allows a full 10 L to be filtered without clogging, the 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, and the volume is not anticipated to yield 2
mL of packed pellet volume, the PWS should be able to filter the remaining volume through the
second filter and ship both filters to the laboratory for processing.
6.1.2 E. co/i Samples
PWSs should analyze up to 100-mL of sample for LT2 monitoring. EPA recommends that the PWS
collect and ship more than 100-mL of sample to ensure sufficient volume for sample analysis is available
in the event of spillage at the laboratory. If spillage or leakage occurs during shipment, there is an
opportunity for sample contamination to occur and the sample should not be analyzed (see Section 8.3.1).
Additional details on sample collection procedures are provided in Section 6.4.3. The capacity of sample
containers should be 120-mL (6 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.
Draft 36 June 2003
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Section 6: Collecting and Shipping Source Water Samples
6.2 Sample Collection Location
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. 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), the same set of monitoring results may be applied to each plant. E. coll
samples should be collected at the same location as Cryptosporidium samples.
PWSs are required to collect source water samples for the LT2 rule from the plant intake prior to any
treatment [40 CFR part 141.704 (a)]. Guidance on sampling at plants where this may not be feasible, or
where other factors, such as the use of multiple sources, need to be addressed, is provided below, in
Sections 6.2.1 through 6.2.5.
6.2.1 Plants That Do Not Have a Sampling Tap Located Prior to Any Treatment
Plants in this situation should 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.
Establish a sampling location prior to treatment
•
6.2.2 Plants That Use Different Water Sources at the Same Time
This includes multiple surface water sources and blended surface water and ground water sources. Plants
in this situation should pursue one of the following options:
• If there is a sampling tap where the sources are combined prior to treatment, the sample should be
collected from the tap.
• Samples can be manually collected 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% of the
drinking water is from Source A and 25% 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.
Separate samples can be manually collected at each source near the intake on the same day and
analyzed independently. The results would then be used to calculate a weighted average of the
analysis results. The weighted average would 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% of the
drinking water is from Source A and 25% 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 the concentration of the sample from Source B was 2 oocysts/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]).
6.2.3 Plants That Use Presedimentation
For these plants, source water samples must be collected after the presedimentation basin but before any
other treatment [40 CFR 141.704 (b)]. Use of presedimentation basins during monitoring should be
consistent with routine operational practice and should be documented. For systems taken samples after
Draft 37 June 2003
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Section 6: Collecting and Shipping Source Water Samples
presedimentation basin, no "Microbial Toolbox" credits will be allowed for presedimentation, if the plant
is classified into a bin that requires additional treatment [40 CFR 141.726 (a)].
6.2.4 Plants That Use Raw Water Off-Stream Storage
For these plants, source water samples must be collected after the off-stream storage reservoir [CFR
141.704 (c)]. Use of off-stream storage during monitoring should be consistent with routine operational
practice and should be documented.
6.2.5 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 Surface Water
Treatment Rule (SWTR) [40 CFR 141.704 (c)].
PWSs using bank filtered water that is treated by subsequent filtration for compliance with the SWTR
must collect source water samples from the well (i.e., after bank filtration) but before any other
treatment. Use of bank filtration during monitoring should be consistent with routine operational
practice and should be documented. Systems collecting samples after a bank filtration process may
not receive microbial toolbox credit for the bank filtration [40 CFR 141.726 (c)].
PWSs using bank filtered water without additional filtration must take source water samples in the
surface water source (e.g., the river). Use of bank filtration during monitoring should be consistent
with routine operational practice and should be documented.
Before monitoring begins, all plants must establish a source water monitoring schedule, as discussed in
Section 6.3.
6.3 Source Water Monitoring Schedule
PWSs are required to collect samples at least monthly and in accordance with a schedule established by
the PWS prior to initiation of monitoring. PWSs may collect samples more frequently (e.g., twice-per-
month, weekly), provided the same frequency is maintained throughout the monitoring period [40 CFR
part 141.701 (e)].
Water treatment plants that use surface water or ground water under the direct influence (GWUDI), but
are operated only seasonally (e.g., during times of high-water demand) should monitor at least monthly
during the period when the plant is in operation.
Systems regulated under the LT2 rule are required to submit source water monitoring schedule to EPA
within 3 months of rule promulgation [40 CFR part 141.703 (a)]. The schedule is entered using the
scheduler function within the LT2 Data Collection System. Details on the use of the scheduler are
provided in the Users' Manual for the Long Term 2 Enhanced Surface Water Treatment Rule (LT2 Rule)
Data Collection System. Systems are required to collect samples within 2 days before or after the dates
indicated in their sampling schedules [40 CFR part 141.703 (b)].
The scheduler function will be available for PWSs to establish their LT2 monitoring schedule for a 3-
month period, beginning on the date of final rule publication. The use of a predetermined monthly or
semimonthly sampling schedule at each PWS during LT2 is designed to capture storm events and other
factors that affect water quality on a periodic basis. Because a PWS can potentially bias the results of the
monitoring by avoiding sample collection during periods of low water quality, the submission of pre-
scheduled sampling dates will be used to assess compliance.
Draft 38 June 2003
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Section 6: Collecting and Shipping Source Water Samples
6.4 Sample Scheduling Compliance Issues
Permissible exceptions to 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 system should sample as close to the scheduled date as feasible and submit an explanation for the
alternative sampling date to EPA concurrent with shipment of the sample to the laboratory.
EPA will evaluate the explanation and update the schedule in the LT2 Data Collection System, if
acceptable, to permit the analytical result to be submitted through the system (results with sample
collection dates that do not comply with the schedule entered by the PWS before monitoring began
will be rejected from the system).
Systems that are unable to report a valid Cryptosporidium analytical result for a scheduled sampling
date due to failure to comply with the analytical method quality control requirements (e.g., sample is
lost or contaminated; laboratory exceeds analytical method holding time) must collect a replacement
sample within 14 days of being notified by the laboratory that a result cannot be reported for that
date. Systems must submit an explanation for the replacement sample with the analytical results.
Systems should collect an E. coll sample at the same time as the Cryptosporidium replacement
sample.
Alternative sample collection dates should be timed so as not to coincide with another scheduled
Cryptosporidium sample collection date. Documentation of alternate sample collection, including the
reason, should be provided with the grandfathered data package.
6.4 Sample Collection Guidance
Large plants must begin collecting source water samples 6 months after rule promulgation and small
plants must begin 30 months after rule promulgation. Because the LT2 monitoring program is designed to
assess source water Cryptosporidium andE. coll concentrations, not the concentrations of these
organisms at points after any treatment, samples must be collected prior to any treatment and where the
water is no longer subject to surface runoff during LT2 monitoring (40 CFR part 141.704).
During each of the scheduled sampling events, several actions must be performed in addition to collecting
the sample. These actions, and an indication of which plant types each applies to, are summarized in
Table 6-2.
Draft 39 June 2003
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Section 6: Collecting and Shipping Source Water Samples
Table 6-2. Sample Collection Activities Required for Each Plant Type
Action
Document sample collection
information
Collect Cryptosporidium
sample
Collect £. co// sample
Measure turbidity
Monitor sample temperature
during sample transport
Large filtered
plants
/
/
/
/
/a
Large unfiltered
plants
/
/
/a
All small plants
/
/
/b
Small plants that
exceed the £.
co// trigger level
/
/
/
a Those utilities with on-site Cryptosporidium analytical capabilities will not need to transport samples unless the
laboratory is not located in close proximity to the sample collection location
b Those small plants with on-site £. co// analytical capabilities will not need to transport samples unless the laboratory
is not located in close proximity to the sample collection location
Guidance and procedures for each of these sample collection activities is provided in Sections 6.4.1 -
6.4.5, below.
6.4.1 Sample Collection Documentation
The information in Table 6-3 should be recorded during sample collection to link the monitoring result to
the plant, and to provide information required for development of the microbial index.
Table 6-3. Minimum Data Elements to Record During Sample Collection
Sampling Information
PWS name
Public Water System Identification
(PWSID) number3
Facility name
Facility IDa
Sample collection point name
Sample collection point IDa
Sample collection date3
Source water typeb
Requested analysis
Sample collection time (start time
for field-filtered samples)
Meter readings (for field-filtered
samples only)
Required
/
/
/
/
/
Recommended
/
/
/
/
/
/
Draft
40
June 2003
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Section 6: Collecting and Shipping Source Water Samples
Sample collection stop time (for
field-filtered samples only)
Turbidity"
/
/
a The combination of these elements constitute the unique sample identifier for LT2 monitoring samples
b This information should be recorded with the E. coll sample collection information, as it will be entered into the LT2
data collection system with the E. coll sample results, for use in reassessing the microbial index. It does not need to
be reported with the Cryptosporidium sample collection information
For samples that are shipped off-site, this information should be documented on an LT2 sample collection
form (Appendix C), or similar form provided by your contract laboratory. For samples analyzed on-site
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.
The source water type for the sample will be used to reassess the relationship between Cryptosporidium
and E. coll concentrations (the microbial index discussed in Section 1.5). 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 flowing stream surface water
GWUDI of reservoir/lake surface water
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% reservoir/lake water and 45% flowing/stream water, the "reservoir/lake"
option should be circled on the LT2 sample collection form.
The majority of source water for plants that use GWUDI is ground water. However, as noted above, the
selection of source water type under the LT2 rule is based on the majority of surface water used as source
water. As a result, the selection of source water type is based on the type of surface water that accounts
for the majority of the influence of the ground water source.
The turbidity of the source water also needs to be measured. Cryptosporidium sample collection
procedures are discussed in Section 6.4.2; E. coli sample collection procedures and turbidity measurement
procedures are discussed in Section 6.4.3 and 6.4.4, respectively.
6.4.2 Cryptosporidium Sample Collection
Several options are available to the PWS in collecting untreated surface water samples for
Cryptosporidium analysis, including the following:
• 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 D.
• On-site filtration of water samples using the Pall Gelman Envirochek™ or Envirochek™ F£V capsule
filter. A detailed protocol for filtering samples on-site from pressurized or unpressurized sources is
provided as Appendix E.
• On-site filtration of water samples using the IDEXX™ Filta-Max foam filter. A detailed protocol for
filtering samples on-site from pressurized or unpressurized sources is provided as Appendix F.
Draft 41 June 2003
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Section 6: Collecting and Shipping Source Water Samples
Regardless of the procedure used to collect Cryptosporidium samples, the sample must be eluted from the
filter within 96 hours of sample collection, per EPA Method 1622/1623 (Section 8.2). If this holding time
is violated, the laboratory will reject the sample, and your PWS will be required to recollect and reship
the sample.
X
L T2 rule requirement: Each sample must meet the QC criteria for the methods [40 CFR
part 141.705 (a) (3)]. Per EPA Method 1622/1623, samples must be
processed or examined within each of the holding times specif led by
the method (Section 8.2).
6.4.2.1 Matrix Spike Samples
Method 1622/1623 requires matrix spike (MS) samples to be analyzed at a frequency of 1 MS sample for
every 20 monitoring samples from each plant. This frequency translates to the following, for each plant
category:
• For large PWSs that perform monthly monitoring for 2 years (resulting in 24 monitoring samples), 2
MS samples must be collected and analyzed
• For large PWSs that perform semi-monthly or more frequent monitoring for 2 years (resulting in 48
or more samples), a minimum of 3 MS samples will be collected and analyzed
• For small PWSs that are triggered into Cryptosporidium monitoring and collect semi-monthly
samples for 1 year (resulting in 24 samples), 2 MS samples must be collected and analyzed
The MS sample and the associated unspiked sample must be analyzed by the same procedure and the MS
sample must be the same volume as 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.
Utilities collecting and shipping bulk water samples for filtration and analysis at the laboratory 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. A mixing chamber (filter housing without filter) can be
added immediately upstream from the Y to aid in equalizing the distribution of sample particulates to
either side.
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 would not be used to adjust Cryptosporidium recoveries at any individual source
water; but MS results would be used collectively to assess overall recovery and variability for EPA
Method 1622/1623 in source water. No resampling would be necessary for MS samples that do not meet
Method 1622/1623 recovery guidelines.
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Section 6: Collecting and Shipping Source Water Samples
L T2 rule requirements: (1) The MS and field sample must be collected from the same
sampling location by splitting the sample stream or collecting the
samples sequentially. (2) The volume of the MS sample analyzed
must be within 10% of the volume of the field sample analyzed. (3)
The MS and field sample must be analyzed by the same procedure
[40 CFKpart 141.705 (a) (2) (i)].
6.4.2.2 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 it may be cheaper than purchasing the filters
from your Cryptosporidium contract laboratory as part of the sampling kit. This approach also provides
your PWS with a ready supply of extra filters on-site, if a filter clogs during a sampling event. Plants
wishing to explore this option should call one of the contacts in Table 6-4.
Table 6-4. Contacts for Filters Approved for Use in EPA Method 1622/1623
Pall Life Sciences
(Envirochek™ and Envirochek™ HV capsule filters)
IDEXX
(Filta-Max™ foam filters)
vwwv.pall.com/qelman
600 South Wagner Road
Ann Arbor, Ml 48103
Sales:
Phone: (800) 521-1520 ext.2
Fax: (734)913-6495
Technical Support:
Phone: (800) 521-1520 ext.3
Fax: (734)913-6495
www.idexx.com
Sales:
Phone: (800) 321-0207 ext.1
Fax: (207) 856-0630
Technical Support:
Phone: (800) 321-0207 ext.2
Fax: (207) 856-0630
E-mail: water@idexx.com
The PWS also can 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 on-site by the utility, the utility should use disposable materials
wherever possible to mitigate the risk of cross-contamination between samples or sampling events, and
must disinfect the non-disposable sampling equipment between uses (if the laboratory provides the
sampling kit, this disinfection step is performed at the laboratory.)
Sampling kit cleaning should consist of the following:
Cleaning equipment by scrubbing with warm detergent solution and exposing to hypochlorite
solution (minimum of a 5% solution of 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
6.4.2.3 Filter Clogs and Highly Turbid Water Samples
PWSs with highly turbid source waters are likely to generate larger packed pellet volumes after
centrifugation and to clog filters than PWSs with low-turbidity waters. As noted in Section 6.1, at least 2
mL of packed pellet volume must be analyzed (for samples in which 10 L is filtered), or as much volume
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Section 6: Collecting and Shipping Source Water Samples
as two filters can accommodate before clogging. (If more than 10 L is filtered, then less of the packed
pellet volume needs to be analyzed.)
PWSs with highly turbid water may be able to collect the required minimum packed pellet volume by
avoiding filtration altogether, and shipping a bulk water sample to the laboratory for centrifuging. The
laboratory can centrifuge 250-mL or greater aliquot volumes sequentially, until a packed pellet volume of
2 mL is generated.
6.4.3 E. co/i Sample Collection
For most large systems, E. coll analyses will be conducted on-site, so samples will not be shipped in most
cases, unlike Cryptosporidium samples. However, many small systems will collect E. coli samples and
ship them off-site 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 under the drinking water
certification program for the general coliform analysis technique corresponding to the method the
laboratory plans to use for LT2 rule monitoring [40 CFR part 141.705 (b) and 141.706 (b)]. Approved E.
coli methods and their corresponding drinking water certification program coliform techniques are
discussed in the Microbial Laboratory Guidance Manual for the Long Term 2 Enhanced Surface Water
Treatment Rule (LT2 Rule). Summary information on these methods is also provided in Section 4 of this
document.
Collect E. coli samples in sterile, non-toxic, plastic, or glass containers with aleak-proof lid. The capacity
of sample containers should be 120-mL (6 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 off-site for analysis, is provided as Appendix G.
EPA strongly encourages laboratories to analyze samples as soon as possible after collection. E. coli
samples must be analyzed within 24 hours of sample collection [40 CFR part 141.705 (b)(l)]. Note: This
is a longer time period than currently permitted in Standard Methods and the Manual for the Certification
of Laboratories Analyzing Drinking Water, and is based on data demonstrating that surface water samples
could be held, chilled, for up to 24 hours and still yield valid results (Reference 9.5).
Samples should be maintained above freezing and below 10°C in a refrigerator or in a cooler with wet ice,
blue ice, or gel packs, etc. Additional guidance on monitoring sample temperature is available in Section
6.4.5 of this manual.
6.4.4 Measuring Turbidity
PWSs must measure the turbidity of the source at the time of Cryptosporidium and E. coli sample
collection during LT2 rule monitoring. Turbidity must be measured by a party approved by the State [40
CFR part 141.706 (c)] using methods for turbidity measurement approved at 40 CFR part 141.74 [40CFR
part 141.705 (c)]. These methods include:
• Method 2130B, published in Standard Methods for the Examination of Water and Wastewater (19th
or 20th Edition). The full text of the 19th Edition is provided as Appendix H.
Great Lakes Instrument (GLI) Method 2. The full text of this method is provided as Appendix I.
• Revised EPA Method 180.1, approved in August 1993 in Methods for the Determination of Inorganic
Substances in Environmental Samples (EPA-600/R-93-100). The full text of this method is provided
as Appendix J.
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Section 6: Collecting and Shipping Source Water Samples
Systems must use turbidimeters that conform to one of the approved methods for measuring turbidity,
such as Hach Turbidimeter 1720D with EPA Method 180.1, GLI Turbidimeter Accu 4 with GLI Method
2, or equivalents (Note: These examples do not constitute an endorsement of specific instrumentation.
Approved methods provide specifications that turbidimeters must meet, and conformance of instruments
with these particular specifications must be determined prior to analysis.). For regulatory reporting
purposes, either an on-line or a benchtop turbidimeter may be used, and systems must comply with all
quality control requirements specified in methods and regulations. If a system chooses to utilize on-line
units for monitoring, the system must validate the continuous measurements for accuracy on a regular
basis using a protocol approved by the State [40 CFRpart 141.74 (c) (1)].
6.4.4.1 Measuring Sample Turbidity During LT2 Monitoring
When measuring turbidity, cuvettes must be clear, colorless glass or plastic. The tube must 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 by warm water bath for a short time, then re-
agitate the sample before placing it in the turbidimeter. Read turbidity directly from instrument
display. Note: Measurements should be within the calibration range.
• Measuring Sample Turbidity Using GLI Method 2 or Revised EPA Method 180.1. Different
procedures should be followed, depending on the turbidity of the sample:
For turbidities estimated to be less than 40 NTU. Shake the sample thoroughly to disperse the
solids. After waiting for the air bubbles to disappear, pour the sample into the turbidimeter tube
and read directly from the instrument scale.
For turbidities estimated to be greater than 40 NTU. Dilute the sample with turbidity-free water
and compute the turbidity with the dilution factor included.
6.4.4.2 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
part 141.706 (c)]. Each utility laboratory is required to operate a formal quality control (QC) program and
to maintain performance records that define the quality of the data generated. Two types of calibration are
required 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 (a recipe for preparation can be found at EPA
Method 180.1 and Standard Method 2130B), AMCO-AEPA-1 (available from Advanced Polymer
Systems), 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 1 year (at 4000 NTU). Consult an approved method for more
information.
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Section 6: Collecting and Shipping Source Water Samples
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
corrected 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.
6.4.5 Monitoring Sample Temperature
Source water samples are dynamic environments and, depending on sample constituents and
environmental conditions, Cryptosporidium oocysts present in a sample can degrade and E. coll present in
a sample can grow or die off, biasing analytical results. Cryptosporidium and E. coli samples for LT2 rule
monitoring that are not analyzed the same day they are collected must be maintained below 10°C to
reduce biological activity. This is specified in Section 8.0 of the June 2003 versions of EPA Method
1622/1623 for Cryptosporidium samples and at 40 CFR part 705 (b) (1) and Chapter V, Section 6.3, of
the Laboratory Certification Manual (Reference 5.2) for E. coli samples.
Samples for all analyses should remain above freezing at all times. This is a requirement in Section 8.0 of
the June 2003 versions of EPA Method 1622/1623. Although not a significant concern for 10-L water
samples, this is a real concern for Cryptosporidium filters and 120- or 250-mL E. coli samples that are
shipped off-site with coolant materials, such as wet ice, blue ice, or gel packs. E. coli holding time studies
performed in support of the LT2 rule (Reference 9.5) demonstrated that E. coli samples can freeze under
these conditions if samples are not packed properly.
The sample collection protocols procedures in Appendices D, E, F, and G provide sample packing
procedures for E. coli and Cryptosporidium samples. Utility personnel should follow these procedures to
ensure that samples remain at acceptable temperatures during shipment.
Because Cryptosporidium samples collected for the LT2 rule must meet the QC criteria in the methods
[40 CFR part 705 (a) (3)], and because these QC criteria include receipt of samples at <10°C and not
frozen, laboratories must reject LT2 Cryptosporidium samples received at >10°C or frozen (this is
discussed further in Section 3.3.12 in this manual). In these cases, the PWS must re-collect and re-ship the
sample.
L T2 rule requirement: Each sample must meet the QC criteria for the methods [40 CFR
part 141.705 (a) (3)]. Per EPA Method 1622/1623, samples not
processed on the day of collection must be received at the
laboratory at < 10°C and not frozen (Section 8.1)
The sample collection protocols discussed in Section 6.4.2 for Cryptosporidium samples and Section
6.4.3 for E. coli samples provide guidance on packing samples to maintain appropriate temperatures.
Utility personnel should follow these procedures to ensure that samples remain at acceptable temperatures
during shipment.
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Section 6: Collecting and Shipping Source Water Samples
Several options are available to measure sample temperature upon receipt at the laboratory and, in some
cases, during shipment:
• 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 sample. The temperature of this extra
sample volume is measured upon receipt to estimate the temperature of the field sample. Temperature
sample bottles are not appropriate for use with bulk samples because of the potential effect that the
difference in sample volume may have in temperature equilibration in the sample cooler. Example
product: Cole Farmer cat. no. U-06252-20.
Thermometer vial. A similar option is to use a thermometer that is securely housed in a liquid-filled
vial. 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.
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. Information
on Thermocron® iButtons is available from http://www.ibutton.com/. Distributors include
http://www.pointsix.com/, http://www.rdsdistributing.com. and http://www.scigiene.com/.
• Stick-on temperature strips. Another option is for the laboratory to apply a stick-on temperature
strip to the outside of the sample container upon receipt at the laboratory. This option does not
measure temperature as precisely as the other options, but still mitigates the risk of sample
contamination while providing an indication of sample temperature to verify that the sample
temperature is acceptable. Example product: Cole Farmer cat. no. U-90316-00.
All temperature measurement devices should be calibrated routinely to ensure accurate measurements.
See the U.S. EPA Manual for the Certification of Laboratories Analyzing Drinking Water (Reference 9.3)
for more information.
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SECTION 7: REVIEWING CRYPTOSPORIDIUM DATA
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 and
reported to the PWS through the LT2 Data Collection System and via hardcopy forms. This section
provides an overview of the data recording and reporting processes and discusses the significance of the
examination results reported by the laboratory. This section also provides guidance to those PWSs
interested in reviewing laboratory data.
7.1 Cryptosporidium Data Recording at the Laboratory
The Cryptosporidium laboratory records LT2 rule monitoring data using the following standardized
forms:
7.1.1 LT2 Sample Collection Form
This form (an example of which is provided as Appendix C) 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 data collection system, it provides
documentation for the utility, the laboratory, and EPA or State officials on sample receipt information
re levant to LT2 rule requirements regarding sample temperatures and sample holding times.
7.1.2 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:
• Sample ID
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.)
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Section 7: Reviewing Cryptosporidium Data
• Filter clog and packed pellet information, which may need to 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
7.1.3 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:
• Sample ID
Date and time the examination was completed
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 revealed the presence of nuclei, and, if so, how
many were observed by the analyst
Whether the analyst observed internal structures during DIG examination
7.2 Submitting Cryptosporidium Data through the LT2 Data
Collection System
During the LT2 rule, laboratories will report Cryptosporidium data to their PWS clients electronically
through EPA's LT2 Data Collection System. The LT2 Data Collection System 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 LT2 Data Collection System 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 LT2 Data
Collection System is provided in Table 7-1, below.
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Section 7: Reviewing Cryptosporidium Data
Table 7-1. LT2 Data Collection System Data Entry, Review, and Transfer Process
Laboratory actions
Laboratory posts analytical results to the LT2 Data Collection System
LT2 Data Collection System reduces data and checks data for completeness and compliance with LT2
rule requirements
Laboratory Principal Analyst confirms that data meet quality control requirements
Laboratory "releases" results electronically to the PWS for review
Laboratory user cannot edit data after it is released to the PWS
o
o
ro
ro
"o
D.
LU
PWS actions
PWS cannot edit data - only review data and either return to laboratory to resolve errors or submit to
EPA
PWS reviews electronic data through LT2 Data Collection System
PWS "releases" data back to the laboratory if 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 are not valid for use in LT2 binning)
EPA and State actions
EPA and State users cannot edit data - only review data
EPA and State review data through LT2 Data Collection System
The data reporting process is discussed in more detail below, in Sections 7.2.1 through 7.2.3, and
discussed in detail in the Users' Manual for the Long Term 2 Enhanced Surface Water Treatment Rule
(LT2 Rule) Data Collection System. The LT2 data system users' guide also provides detailed information
on the PWS user registration process. Information on the LT2 Data Collection System, as well as a
downloadable users' manual, is available at http://www.epa.gov/safewater/lt2/index.html.
7.2.1 Data Entry/Upload
The analyst or another laboratory staff member enters a subset of the data recorded at the bench (Section
7.1) into the LT2 Data Collection System, 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 point
• Sample collection date
Sample type (field or MS)
• Sample volume filtered (L), to nearest % L
• Was 100% of filtered volume examined?
Number of oocysts counted
• For samples in which less than 10 L is filtered or less than 100% of the sample volume is examined,
the laboratory also must enter or upload the number of filters used and the packed pellet volume.
• For samples in which less than 100% of sample volume is examined, the laboratory also must report
the volume of resuspended concentrate and volume of this resuspension processed through
immunomagnetic separation.
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Section 7: Reviewing Cryptosporidium Data
• 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 samples.
The laboratory must verify that all holding times and other QC requirements were met.
After the information has been entered or uploaded into the system, the system 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 6.1), and calculate
MS recoveries.
The laboratory's Primary Analyst under the Lab QA Program is responsible for verifying the quality and
accuracy of all sample results in the laboratory, and is required to review and approve the results before
they are submitted to the PWS for review. If inaccuracies or other problems are identified, the primary
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 Primary Analyst 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 system to the PWS, and the data can
no longer be changed by the laboratory.
7.2.2 PWS Data Review
After the laboratory has released Cryptosporidium data electronically to the PWS using the LT2 Data
Collection System, 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 sample collection point ID or
collection date is incorrect, the PWS can release the results back to the laboratory for issue resolution. In
addition to noting the reason in the LT2 Data Collection System for the return of the data to the
laboratory, you also should contact the laboratory verbally to discuss the issue.
If the PWS determines that the data are accurate, the PWS releases the results to EPA (and the State, if
applicable) as "approved" results. If the PWS determines that the data are accurate, but 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 you contend are not
valid for use in LT2 binning, and have submitted to EPA for evaluation.
7.2.3 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 LT2 Data Collection System. EPA and State users cannot edit the data.
7.3 What Do the Sample Examination Results Mean?
As noted in Section 3.1, the laboratory applies two stains to a sample slide, and then examines the sample
using three different techniques to determine whether objects that cannot be ruled out as Cryptosporidium
oocysts are on a sample slide. A description of these stains and techniques-and how each is used to
evaluate objects examined by the analyst, is provided below.
7.3.1 Immunofluorescent Assay (IFA)
One of the two stains added to the sample before examination is a fluorescent antibody stain that reacts
with Cryptosporidium. The antibodies in this stain, which exhibit an intense apple-green fluorescence
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Section 7: Reviewing Cryptosporidium Data
when the slide is examined using ultraviolet light, will attach to Cryptosporidium oocysts that may be
present in the sample. During IFA, the analyst scans the entire well at relatively low magnification (200X)
for apple-green fluorescing objects the size and shape of oocysts. If such an object is located, the analyst
proceeds to the next step in the examination process. The analyst cannot conclude at this stage that an
apple-green fluorescing organism the size and shape of a Cryptosporidium oocyst is indeed an oocyst
because the object may be another organism that has cross-reacted with the antibody stain. Additional
examination procedures are used to determine whether this is the case.
7.3.2 4',6-diamadino-2-phenylindole (DAPI) Examination
The second stain added to the sample before examination is DAPI, a dye that interacts with nucleic acids
and stains nuclei that may be present within the oocyst. The DAPI stain fluoresces when the slide is
examined using ultraviolet light. During the DAPI examination, the analyst observes the object at medium
magnification (400X) to determine whether it contains stained nuclei. Cryptosporidium oocysts contain
four nuclei.
Although looking for four nuclei during DAPI examination, if the object has less than four nuclei, the
analyst cannot rule out the possibility that the organism is a Cryptosporidium oocyst. For example, if less
than four stained nuclei are observed, the object may actually have four nuclei, but some may not be
visible in the plane of focus. Similarly, objects in which no stained nuclei are observed may be organisms
other than Cryptosporidium, may be dead Cryptosporidium oocysts, or may even be live oocysts that are
resistant to DAPI staining.
The DAPI examination is one of several tools for the analyst to use to determine whether an object is an
oocyst. The analyst cannot conclude whether the object is an oocyst based on this examination alone, nor
can the analyst conclude, based on negative results, that the organism is non-infectious. As a result, the
analyst must proceed to the next step in the examination process, even if less than four nuclei are
observed.
7.3.3 Differential Interference Contrast (DIG) Examination
The third evaluation performed by the analyst is an examination of the object at high magnification
(1000X). Using DIC, the analyst looks at the object's external or internal morphological characteristics
(this does not require the use of a stain). The analyst looks for characteristics atypical of Cryptosporidium
oocysts (e.g., spikes, stalks, appendages, pores, one or two large nuclei filling the cell, crystals, spores,
etc.). If atypical structures are not observed, and the object cannot be ruled out as an oocyst based on the
results of the IFA and the DAPI examination, the analyst reports this object as a Cryptosporidium oocyst.
Based on the DIC examination, the size of the object is determined and compared to the acceptable range
for the target organism. If the size and shape of the object is within the acceptable range, the analyst
records the size and shape and characterizes the Cryptosporidium oocyst in one of three ways: (1) an
oocyst with internal structures, i.e., those having recognizable structures consistent with Cryptosporidium,
(2) an oocyst with amorphous structures, or (3) an empty oocyst. Assignment of these characterizations is
dependent on analyst judgement and none of these characterizations is a direct indicator of whether
oocysts are viable and infectious.
7.5 Reviewing and Validating Raw Cryptosporidium Data (Optional)
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 5.1.5). However, this step
is not required. However, for those PWSs interested in taking this extra step, Sections 7.5.1 through 7.5.3
provide guidance on how to review and validate hardcopy data and verify accuracy.
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7.5.1 Data Completeness Check
Upon receipt of the hardcopy sample results for a monitoring sample, verify that the laboratory has
submitted the following materials:
• Sample result summary sheet, which should include the following:
• 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 met
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
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.
7.5.2 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
sample was received in acceptable condition (not leaking, etc.), and at a temperature between 0°C and
10°C, and 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.
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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 report 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 1 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
(generally, the date next to the slide staining step should be no more than 3 days later than the
date next to the slide preparation step)
The laboratory examined the slide within 7 days of staining (the examination date should be no
more than 7 days from the slide staining date)
• Positive and negative staining controls. Based on the information at the top of the Method
1622/1623 Cryptosporidium reporting form, verify that the laboratory performed positive and
negative staining controls, and that the results of these controls were acceptable.
7.5.3 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 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 still may wish to
verify them. Guidance on recalculating and verifying final results using primary measurements is
provided below.
7.5.3.1 Field Sample Calculations
To calculate the concentration of Cryptosporidium in your field 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
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Section 7: Reviewing Cryptosporidium Data
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 < 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 <100% of the sample filtered is processed through the remainder of the method and examined
(such as when the volume filtered yields > 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 7.1.2)
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 > 5 mL
Packed pellets with a volume >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 sample was filtered and processed, producing a packed pellet volume of 2.7 mL.
The laboratory transferred 20 mL of the 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.
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
7.5.3.2 Matrix Spike Sample Calculations
For matrix spike (MS) samples, the laboratory records all of the same information that is recorded for
field samples, in addition to information specific to matrix spike samples. The sample volume spiked and
estimated number of oocysts spiked into the sample are used to calculate the concentration of spiked
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organisms in the sample. To correct for background concentration, the number of organisms detected in
the unspiked field 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 sample (to correct for background
concentration)
oocysts counted in MS sample - oocysts counted in unspiked field sample
percent recovery = * 100%
oocysts spiked into MS sample
7.6 Data Archiving Requirements
LT2 rule monitoring data must keep monitoring results until 36 months after source water monitoring has
been completed. Although it is the PWS's responsibility to meet LT2 rule data storage requirements for
compliance monitoring samples, the PWS may designate this responsibility to the laboratory.
Although not required, laboratories also can archive slides and/or take photographs of slides to maintain
for clients. As noted in Section 5.1.2.5, this may be considered an extra service and result in extra costs,
as these steps may not be routinely performed by the laboratory. Slides should be stored in a humid
chamber in the dark at 0°C to 10°C. An alternative mounting medium also may be used, which may
potentially preserve slides longer. Details are provided in the Microbial Laboratory Guidance Manual for
the Long Term 2 Enhanced Surface Water Treatment Rule (LT2 Rule).
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SECTION 8: REVIEWING E. COLI DATA
When E. coli samples are processed and analyzed by the laboratory, data on sample measurements,
sample processing times, and slide examination results are recorded at the laboratory and reported to the
PWS through the LT2 Data Collection System. 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.
8.1 E. coll Laboratory Data Recording at the Laboratory
The laboratories performing E. coli analyses during the LT2 rule record the following general types of
information:
• 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.)
8.1.1 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).
8.1.2 Primary Data
The laboratory records 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.
8.1.3 Sample Processing and Quality Control Information
The laboratory records information on the bench sheet on how they processed and analyzed the sample,
including incubation start/end date and times and temperature, 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
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.
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8.1.4 Sample Results
The final E. coli concentration for field 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, a low censored value
based on the volume of sample analyzed must be reported (e.g. <1CFU /100 mL or <1.8 MPN/100 mL).
E. coli concentration will never be reported as a zero.
8.2 Submission of E. coll Data through the LT2 Data Collection
System
During the LT2 rule, laboratories will report E. coli data electronically through EPA's LT2 Data
Collection System to the PWS staff responsible for approving and submitting monitoring results to EPA.
The LT2 Data Collection System 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 LT2 Data Collection System 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 LT2 Data Collection System for
both Cryptosporidium and E. coli samples is provided in Table 7-1, in Section 7.2, above.
The data reporting process is summarized below, in Sections 8.2.1 through 8.2.3, and discussed in detail
in the Users' Manual for the Long Term 2 Enhanced Surface Water Treatment Rule (LT2 Rule) Data
Collection System. The LT2 data system users' guide also provides detailed information on the laboratory
registration process. Information on the LT2 Data Collection System, as well as a downloadable users'
manual, is available at http://www.epa.gov/safewater/lt2/index.html.
8.2.1 Data Entry/Upload
The analyst or another laboratory staff member enters a subset of the data recorded at the bench (Section
8.1) into the LT2 Data Collection System 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 point
• 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)
• E. CO///100 mL (see note below)
Note: The laboratory may then enter the final result for the sample that was calculated at the laboratory or
may enter the primary measurements recorded at the bench, and have the LT2 Data Collection System
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
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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.
The laboratory's official contact is responsible for verifying the quality and accuracy of all sample results
in the laboratory, and is required to 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 system to the PWS, and the data can
no longer be changed by the laboratory.
8.2.2 PWS Data Review
After the laboratory has released E. coli data electronically to the PWS using the LT2 Data Collection
System, 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 sample collection point ID or collection date
is incorrect, the PWS can release the results back to the laboratory for issue resolution. In addition to
noting the reason in the LT2 Data Collection System for the return of the data to the laboratory, you also
should contact the laboratory verbally to discuss the issue.
If the PWS determines that the data are accurate, the PWS releases the results to EPA (and the State, if
applicable) as "approved" results. If the PWS determines that the data are accurate, but believes that the
data are not valid for other reasons, the PWS can release the results as "contested."
8.2.3 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 LT2 Data Collection System. EPA and State users cannot edit the data.
8.3 Reviewing and Validating E. coli Data (Optional)
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 8.3.1
through 8.3.3 provides guidance on how to review and validate the hardcopy data and verify accuracy.
8.3.1 Data Completeness Check
Upon receipt of hardcopy sample results for a monitoring sample, verify that the following information is
included:
• Sample result summary sheet, which should include the following:
• 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)
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Section 8: Reviewing E. coli Data
LT2 sample collection form initiated at the time of sample collection and completed with sample
receipt information by the laboratory
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 (i.e., leaking)
Sample holding time exceeded
• Sample holding temperature not within acceptable range
• Unacceptable blank sample result
Unacceptable positive or negative control result
Media sterility checks were not acceptable
• Method incubation times or temperatures were not within acceptable range
• Membrane filtration: Too much sediment on the filter
• Membrane filtration: Confluent growth of non-target organism
• 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 organism)
Quanti-Tray® was damaged or leaked
• Sample was not distributed to all wells in Quanti-Tray®
• All rows of tubes were not inoculated
Positive presumptive 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 data collection system. If the laboratory enters
the results into the LT2 data collection system, 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.
8.3.2 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 below 10°C, but not frozen.
• QC samples associated field samples. The frequency of analysis of quality control samples
including method blanks, positive and negative controls, etc. varies according to method requirements
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Section 8: Reviewing E. coli Data
and specifications in the Certification Manual. Verify that the required QC samples were run with the
field sample. A summary of these QC specifications is provided in Section 4.2 of this document.
• 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 24 hours of sample collection.
• Incubation times and temperatures. Using the dates and times of the method steps recorded by the
laboratory on the E. coli method bench sheet, verify that the method-specified incubation times and
temperatures, specified in Table 8-1 were met.
Table 8-1. Incubation Times and Temperatures for Approved E. Coli Methods
Method
Standard Methods 9223B
Standard Methods 9221 B/F
Standard Methods 9222B/9222G
Standard Methods 9222D/9222G
Standard Methods 921 3D
EPA 1603
EPA 1604
Other Membrane Filter Method
Media
Colilert®
Colilert-18®
LIB
EC-MUG
mENDO -» NA-MUG
LES-ENDO -» NA-MUG
mFC -» NA-MUG
mTEC agar
Modified mTEC
Ml medium
m-ColiBlue24®® Broth
Incubation Time/Temperature
24 to 28 hours at 35°C ± 0.5°C
18to22hoursat35°C±0.5°C
24 ± 2 and 48 ± 3 hours at 35°C ± 0.5°C
24 ± 2 hours at 44.5°C ± 0.2°C
24 ± 2 hours at 35°C ± 0.5°C -» 4 hours at
35°C ± 0.5°C
24 ± 2 hours at 35°C ± 0.5°C -» 4 hours at
35°C ± 0.5°C
24 ± 2 hours at 44.5°C ± 0.2°C -» 4 hours at
35°C ± 0.5°C
2 hours at 35°C ± 0.5°C -» 22 to 24 hours at
44.5°C ± 0.2°C
2 hours at 35°C ± 0.5°C -» 22 to 24 hours at
44.5°C ± 0.2°C
24hoursat35°C±0.5°C
24 hours at 35°C ± 0.5°C
8.3.3 Calculation Verification
Method-specific data to record for each of the individual method types as well as standardized
calculations for each method type are discussed in Sections 8.3.3.1 through 8.3.3.4.
8.3.3.1 Calculations for Determining the K coli Concentration Using the Colilert® Quanti-Tray
2000® (97-well)
A. Select appropriate dilution to yield countable results. If multiple dilutions are used, the tray
exhibiting positive wells in the 40% to 80% range (39 to 78 total positive large and small wells)
should be used to determine MPN value.
B. Determine MPN. Using the number of large positive wells and small positive wells from the
appropriate dilution, 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 5 small
positive wells, the corresponding MPN would be 49.6 MPN/100 mL.
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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:
MPN value
Analytical result =
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 10 and 2 in the table.
MPN = 81.3
100
Analytical result = 81.3 x = 813 £. coli MPN/100 mL
10
8.3.3.2 Calculations for Determining the K coli Concentration Using the Colilert® Quanti-Tray
51® (51-well)
A. Select appropriate dilution. If multiple dilutions are used, the tray exhibiting positive wells around
the 80% range (41 positive wells) should be used to determine MPN value.
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 26 positive wells, the corresponding MPN would be 36.4 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 x = £. coli MPN/100 mL
mL sample analyzed
Example:
Volume analyzed (mL) = 10 mL (in 90 mL of dilution water)
Number of positive wells = 41
MPN = 83.1
The analytical result is calculated as follows:
100
83.1 x = 831 E. coli MPN/100 mL
10
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Section 8: Reviewing E. coli Data
8.3.3.3 Calculations for determining the E. coli concentration using membrane filter data (adapted
from Reference 9.4)
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 Data Collection System.
See Section 8.2.1 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 plates results
in CNFG, then potentially 0.01 mL should be analyzed during the next sampling event. 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 simultaneously with E. coli determination.
Note: Results that are TNTC or CNFG are not appropriate for L T2 tnicrobial data analysis,
and cannot be entered into the L T2 data Collection System.
J
D. Using the E. coli counts from the appropriate dilution, E. coli CPU/100 mL is calculated based on the
following equation:
100
£ coli CPU x = £ coli CFU/100 mL
mL sample filtered
Example 1:
Filter 1 volume = 100 mL CPU = TNTC
Filter 2 volume = 10 mL CPU = 40
Filter 3 volume = 1.0 mL CPU = 9
Filter 4 volume = 0.1 mL CPU = 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 £ coli CPU x = 400 £ coli CFU/100 mL
10 mL
E. If no E. coli colonies are present, the detection limit is calculated as
< largest volume filtered per 100 mL.
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Example 2:
Filter 1 volume (mL) = 100 mL CPU = 0
Filter 2 volume (mL) = 10 mL CPU = 0
Filter 3 volume (mL) = 1.0 mL CPU = 0
100mL
Detection limit =
Largest volume filtered
= £. co//CFU/100ml_
100 mL
100mL
= <1 £. co///100mL
Example 3:
Filter 1 volume (mL) =100 mL
Filter 2 volume (mL) =10 mL
Filter 3 volume (mL) =1.0 mL
Calculation of E. co//'/100 mL:
CPU = Lab accident, no data available
CFU = 0
CFU = 0
100 mL
10 mL
= <10E. coli CPU 7100 mL
F. If there are no filters with E. coli counts in the 20-80 colony range (20-60 for mFC-NA-MUG), sum
the E. coli counts on all filters, divide by the volume filtered and report as number per 100 mL.
Example 4:
Filter 1 volume (mL) = 50 mL
Filter 2 volume (mL) = 25 mL
Filter 3 volume (mL) =10 mL
The analytical result is calculated as:
CPU = 15
CPU = 6
CFU = 0
100
0)
(50+25+10)
= 25£. co//CFU/100mL
Example 5:
Filter 1 volume (mL) = 50 mL CPU =105
Filter 2 volume (mL) = 25 mL CPU = 92
Filter 3 volume (mL) = 10 mL CPU = 85
The analytical result is calculated as:
100
(105+ 92 +85)
(50 + 25 + 10)
= 332£. co//CFU/100mL
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Example 6:
Filter 1 volume (mL) =100 mL
Filter 2 volume (mL) =10 mL
Filter 3 volume (mL) =1.0 mL
The analytical result is calculated as:
CPU = 82
CPU =18
CFU = 0
100
(82+ 18 + 0)x
(100+ 10+ 1)
= 90£. co//CFU/100ml_
Example 7:
Filter 1 volume (mL) = 50 mL
Filter 2 volume (mL) = 25 mL
Filter 3 volume (mL) =10 mL
The analytical result is calculated as:
CPU = TNTC
CPU = TNTC
CPU = 83
100
83
= 830£. co//CFU/100ml_
10
8.3.3.4 Calculation of E. coli Concentrations Using Multiple-Tube Methods (adapted from
Reference 9.6):
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, approved by the Standard Methods Committee in 1999.
Note: The analytical result can be automatically calculated using the LT2 Data Collection System.
See Section 8.2.1 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 8-2, 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
8-2, Example A).
If the lowest dilution (least dilute) tested has less than five tubes with positive results, select it and
the two next succeeding higher dilutions (Table 8-2, 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 8-2,
Example D).
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• 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 8-2, Example E).
If there were not enough higher dilutions tested to select three dilutions, then select the next lower
dilution (Table 8-2, Example F).
C. MPN values need to 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 dilution series, per
method requirements, the MPN value must be adjusted if these are not the significant dilution series
selected. Use the following calculation to adjust the MPN when the 10 mL, 1 mL, and 0.1 mL
dilution series are not the significant dilution series selected:
MPN value
Analytical result =
# of mL in middle dilution
= £. co//MPN/100 mL
Table 8-2. Examples of Different Combinations of Positive Tubes (Significant Dilution Results Are
in Bo/dand Underlined)
Example
A
B
C
D
E
F
Least dilute ^ Most dilute
(Lowest) v' (Highest)
10
mL
5
4
0
5
5
5
1
mL
5
5
0
4
4
5
0.1
mL
1_
1_
1_
4
4
5
0.01
mL
0
0
0
1
0
5
0.001
mL
0
0
0
0
1
2
Combination
of positives
5-1-0
4-5-1
0-0-1
4-4-1
4-4-1
5-5-2
MPN Index from
Standard Methods
33
48
1.8
40
40
540
£. co/;7100 mL
(after adjustment)
330
48
1.8
400
400
54,000
Example A: The significant dilution series for the 5-1-0 combination of positives includes the 1
mL, 0.1 mL, and 0.01 mL dilutions. Since 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£. coli /100mL
0.1
Example B:
Example C:
Examples D and E:
Since 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.
Since 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/WO mL.
The significant dilution series for the 4-4-1 combination of positives includes the
1 mL, 0.1 mL, and 0.01 mL dilutions. Since the 10 mL, 1 mL, and 0.1 mL
dilutions were not selected, an adjustment is necessary to account for the
dilutions selected:
Draft
66
June 2003
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Section 8: Reviewing E. coli Data
40
Analytical result = = 400 E. coli 1100 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. Since 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 £. coli 1100 mL
0.01
8.4 Data Archiving Requirements
Under the LT2 rule, monitoring data must keep until 36 months after source water monitoring has been
completed [40 CFRpart 141.731 (a)]. Although it is the PWS's responsibility to meet LT2 rule data
storage requirements for compliance monitoring samples, the PWS may designate this responsibility to
the laboratory.
Draft 67 June 2003
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SECTION 9: REFERENCES
9.1 Cornell, Kevin, et al. 2000. ICRSS - Building a Better Protozoa Data Set, J. AWWA. 91(10): 30
-43.
9.2 Pope, Misty, et al. 2003. "Using E. coll To Indicate Source Water Susceptibility to High
Concentrations of Cryptosporidium" in Information Collection Rule Data Analysis. AWWARF,
Denver, CO.
9.3 USEPA. 1997. Manual for the Certification of Laboratories Analyzing Drinking Water; Criteria
and Procedures; Quality Assurance: Fourth Edition. EPA 815-B-97-001.
9.4 APHA. 1998. Standard Methods for the Examination of Water and Wastewater; 20th Edition.
American Public Health Association, American Waterworks Association, Washington, D.C.
9.5 Pope, M., et al. 2002. Assessment of the effects of holding time and temperature on E. coll
concentrations in surface water samples. Appl. Environ. Micro, (submitted).
9.6 2001 Supplement to the 20th Edition of Standard Methods 9221 C: Explanation of Bacterial
Density. This supplement is available for download at http://www.techstreet.com/cgi-
bin/detail?product id=923645.
Draft 68 June 2003
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SECTION 10: ACRONYMS
CPU
CNFG
DAPI
DIG
EPA
FA
FITC
GWUDI
ICR
IFA
IMS
IPR
IPX
L
LT2 rule
LT2ESWTR
MPN
MS
MS/MSD
(im
NA-MUG
nm
NPDWR
NTU
OPR
OPT
PBMS
PT
PWS
QA
Colony-forming unit
Confluent growth
4, 6-diamidino-2-phenylindole
Differential interference contrast
U.S. Environmental Protection Agency
Immunofluorescense assay
Fluorescien isothiocyanate
Ground water under the direct influence of surface water
Information Collection Rule
Immunofluorescence assay
Immunomagnetic separation
Initial precision and recovery
Initial proficiency testing
Liter
Long Term 2 Enhanced Surface Water Treatment Rule
Long Term 2 Enhanced Surface Water Treatment Rule
Most probable number
Matrix spike
Matrix spike/matrix spike duplicate
Micrometer
Nutrient agar (NA) with 4-methylumbelliferyl-beta-D-glucuronide (MUG)
Nanometer
National Primary Drinking Water Regulations
Nephelometric turbidity unit
Ongoing precision and recovery
Ongoing proficiency testing
Performance-based measurement system
Proficiency testing
Public water system
Quality assurance
Draft
69
June 2003
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Section 10: Acronyms
QAP Quality assurance plan
QC Quality control
RSD Relative standard deviation
SDWA Safe Drinking Water Act
TNTC Too numerous to count
UV Ultraviolet
Draft 70 June 2003
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