[Federal Register Volume 85, Number 172 (Thursday, September 3, 2020)]
[Notices]
[Pages 55065-55067]
[FR Doc No: 2020-19366]

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DEPARTMENT OF TRANSPORTATION

Office of the Secretary

[Docket No. DOT-OST-2020-0049]

Information Collection Activities; Requests for Comments

AGENCY:  Office of the Secretary (OST), DOT.

ACTION: Notice and request for public comment and submission to OMB for
clearance of renewed approval of information collection.

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SUMMARY: In compliance with the Paperwork Reduction Act of 1995, this
notice announces that the Information Collection Request (ICR)
abstracted below will be forwarded to the Office of Management and
Budget (OMB) for review and comments. The ICR describes the nature of
the information collection and its expected burden. A Federal Register
Notice with a 60-day comment period soliciting comments on the
following information collection was published on June 11, 2020. The
purpose of this Notice is to allow 30 days for public comment.

DATES: Comments to this notice must be received by October 5, 2020.

ADDRESSES: Written comments and recommendations for the proposed
information collection should be sent within 30 days of publication of
this notice to www.reginfo.gov/public/do/PRAMain. Find this information
collection by selecting ``Currently under Review--Open for Public
Comments'' or by using the search function. Please note that comments
submitted in response to this Notice are public record. Before
including any detailed personal information, you should be aware that
your comments as submitted, including your personal identification

information, will be available for public view.

FOR FURTHER INFORMATION CONTACT: Bohdan Baczara, Office of Drug and
Alcohol Policy and Compliance, Office of the Secretary, U.S. Department
of Transportation, 1200 New Jersey Avenue SE, Washington, DC 20590;
202-366-3784 (voice),<PHONE_NUMBER> (fax), or<EMAIL_ADDRESS>
When submitting comments or requesting information, please include the
docket number and information collection title for reference.

SUPPLEMENTARY INFORMATION:
    OMB Control Number: 2105-0529.
    Title: Procedures for Transportation Workplace Drug and Alcohol
Testing Programs.
    Type of Review: Clearance of a renewal of an information
collection.
    Form Numbers: DOT F 1385; DOT F 1380.
    Respondents: The information will be used by transportation
employers, Department representatives, and a variety of service agents.
    Abstract: Under the Omnibus Transportation Employee Testing Act of
1991, DOT is required to implement a drug and alcohol testing program
in various transportation-related industries. This specific requirement
is elaborated in 49 CFR part 40, Procedures for Transportation
Workplace Drug and Alcohol Testing Programs. This request for a renewal
of the information collection for the program includes 43 burden items
including the U.S. Department of Transportation Alcohol Testing Form
(ATF) [DOT F 1380] and the DOT Drug and Alcohol Testing Management
Information System (MIS) Data Collection Form [DOT F 1385].
    The ATF includes the employee's name, the type of test taken, the
date of the test, and the name of the employer. Data on each test
conducted, including test results, is necessary to document that the
tests were conducted and is used to take action, when required, to
ensure safety in the workplace. The MIS form includes employer specific
drug and alcohol testing information such as the reason for the test
and the cumulative number of test results for the negative, positive,
and refusal tests. No employee specific data is collected. The MIS data
is used by each of the affected DOT Agencies (i.e., Federal Aviation
Administration, Federal Transit Administration, Federal Railroad
Administration, Federal Motor Carrier Safety Administration, and the
Pipeline and Hazardous Materials Safety Administration) and the United
States Coast Guard when calculating their industry's annual random drug
and/or alcohol testing rate.
    Estimated Total Number of Respondents: 3,593,202.
    Estimated Number of Responses: 11,841,478.
    Frequency of Response: The information will be collected annually.
    Estimated Total Number Burden Hours: 1,287,811.

----------------------------------------------------------------------------------------------------------------
                                                     Number of       Number of                     Salary costs
                    PRA item                        respondents      responses     Burden  hours        ($)
----------------------------------------------------------------------------------------------------------------
Exemptions from Regulation Provisions Requests                 1               1               3            $104
 [40.7(a)]......................................
Employer Stand-down Waiver Requests [40.21(b)]..               0               0               0               0
Employee Testing Records from Previous Employers         584,628       3,538,179         471,757      16,379,410
 [40.25(a)].....................................
Employee Release of Information [40.25(f)]......       3,538,179       3,538,179         235,878       8,189,704
MIS Form Submission [40.26].....................          17,840          17,840          26,760         929,107
Collector (Qualification and Refresher) Training           5,000           5,000             333          11,561
 Documentation [(40.33(b) & (e)]................
Collector Error Correction Training                       12,000          19,625           1,308          45,425
 Documentation [40.33(f)].......................
Laboratory Reports to DOT Regarding Unlisted                   0               0               0               0
 Adulterant [40.91(e)]..........................
Semi-Annual Laboratory Reports to Employers                   23         385,854          25,723         893,123
 [40.111(a)]....................................
Semi-Annual Laboratory Reports to DOT                         23              46               3             106
 [40.111(d)]....................................
Medical Review Officer (MRO) (Qualifications and           1,000           1,000              66           2,291
 Continuing Education) Training Documentation
 [40.121(c) & (d)]..............................
MRO Review of Negative Results Documentation               5,000         381,055          25,403         873,000
 [40.127(b)(2)(ii)].............................
MRO Failure to Contact Donor Documentation                 5,000          63,827           4,255         147,738
 [40.131(c)(1)].................................
MRO Effort to Contact DER Documentation                    5,000          63,827           4,255         147,738
 [40.131(c)(2)(iii)]............................
DER Successful Contact Employee Documentation             51,061          51,061           3,404         118,190
 [40.131(d)]....................................
DER Failure to Contact Employee Documentation             12,765          12,765             851          29,547
 [40.131(d)(2)(i)]..............................
MRO Verification of Positive Result Without                5,000          12,765             851          29,547
 Interview Documentation [40.133]...............
Adulterant/Substitution Evaluation Physician                   0               0               0               0
 Statements [40.145(g)(2)(ii)(d)]...............
MRO Cancellation of Adulterant/Substitution for                0               0               0               0
 Legitimate Reason Reports [40.145(g)(5)].......
Employee Admission of Adulterating/Substituting               40              40               3             104
 Specimen MRO Determination [40.159(c)].........
Split Specimen Requests by MRO [40.171(c)]......           5,000           7,206             480          16,680
Split Failure to Reconfirm for Drugs Reports by               35              34               2              69
 MRO [40.187(b)]................................
Split Failure to Reconfirm for Adulterant/                     5               5               1              34
 Substitution Reports by MRO [40.187(c)]........
Shy Bladder Physician Statements [40.193(f)]....             773             773              64           2,238
MRO Statements Regarding Physical Evidence of                  0               0               0               0
 Drug Use [40.195(b) & (c)].....................
Drug Test Correction Statements [40.205 (b)(1) &          25,000         154,732          20,630         716,308
 (2)]...........................................
Breath Alcohol Technician (BAT)/Screening Test             2,000           2,000             133           4,617
 Technician (STT) (Qualification and Refresher)
 Training Documentation [40.213(b)(c)&(e)]......
BAT/STT Error Correction Training Documentation              168             168              11             390
 [40.213(f)]....................................
Complete DOT Alcohol Testing Forms [40.225(a)]..          10,000       3,378,454         450,460      15,639,989
Evidential Breath Testing Device Quality                  10,000          10,000             666          23,123
 Assurance/Calibration Records [40.233(c)(4)]...
Shy Lung Physician Statements [40.265(c)(2)]....             168             168              11             390
Alcohol Test Correction Statements                           337             337              22             781
 [40.271(b)(1)&(2)].............................

Substance Abuse Professional (SAP)                         3,334           3,334             222           7,707
 (Qualification and Continuing Education)
 Training Documentation [40.281(c)&(d)].........
Employer SAP Lists to Employees [40.287]........          10,000         115,713           7,714         267,837
SAP Reports to Employers [40.311(c),(d) & (e)]..          10,000          94,456           6,297         218,634
Correction Notices to Service Agents [40.373(a)]              25              25              25             868
Notice of Proposed Exclusion (NOPE) to Service                 5               5              50           1,736
 Agents [40.375(a)].............................
Service Agent Requests to Contest Public                       2               2               2              69
 Interest Exclusions (PIE) [40.379(b)]..........
Service Agent Information to Argue PIE                         2               2               8             277
 [40.379(b)(2)].................................
Service Agent Information to Contest PIE                       2               2               8             277
 [40.381(a) & (b)]..............................
Notices of PIE to Service Agents [40.399].......               1               1               1              34
Notices of PIE to Employer and Public [40.401                  1               1               1              34
 (b) & (d)].....................................
Service Agent PIE Notices to Employers [40.403                 1             300             150           5,208
 (a)]...........................................
                                                 ---------------------------------------------------------------
    Total New...................................       3,593,202      11,841,478       1,287,811      44,703,995
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    Public Comments Invited: (1) Whether the proposed collection of
information is necessary for the proper performance of the functions of
the Department, including whether the information will have practical
utility; (2) the accuracy of the Department's estimate of the burden of
the proposed information collection; (3) ways to enhance the quality,
utility and clarity of the information to be collected; and (4) ways to
minimize the burden of the collection of information on respondents,
including the use of automated collection techniques or other forms of
information technology.

    Authority: The Paperwork Reduction Act of 1995; 44 U.S.C.
Chapter 35, as amended.

    Issued in Washington, DC, on August 28, 2020.
Bohdan Baczara,
Deputy Director, DOT, Office of Drug and Alcohol Policy and Compliance.
[FR Doc. 2020-19366 Filed 9-2-20; 8:45 am]
BILLING CODE 4910-9X-P


[Federal Register Volume 80, Number 211 (Monday, November 2, 2015)]
[Notices]
[Page 67392]
[FR Doc No: 2015-27842]

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DEPARTMENT OF ENERGY

Federal Energy Regulatory Commission

Combined Notice of Filings #1

    Take notice that the Commission received the following electric
rate filings:

    Docket Numbers: ER15-1451-001.
    Applicants: California Independent System Operator Corporation.
    Description: Compliance filing: 2015-10-26 Filing in Compliance
with Sept 25 2015 Order in Docket ER15-1451 to be effective 9/25/2015.
    Filed Date: 10/26/15.
    Accession Number: 20151026-5417.
    Comments Due: 5 p.m. ET 11/16/15.

    Docket Numbers: ER15-2208-002.
    Applicants: ISO New England Inc.
    Description: Compliance filing: Winter Reliability Compliance
Filing to be effective 9/14/2015.
    Filed Date: 10/26/15.
    Accession Number: 20151026-5241.
    Comments Due: 5 p.m. ET 11/16/15.

    Docket Numbers: ER16-142-000.
    Applicants: Pacific Gas and Electric Company.
    Description: Tariff Cancellation: Notice of Termination of
McAllister Ranch ID IA and WDT SA to be effective 12/31/2015.
    Filed Date: 10/26/15.
    Accession Number: 20151026-5007.
    Comments Due: 5 p.m. ET 11/16/15.

    Take notice that the Commission received the following land
acquisition reports:

    Docket Numbers: LA15-3-000.
    Applicants: Alabama Electric Marketing, LLC, Astoria Generating
Company, L.P., Big Sandy Peaker Plant, LLC, California Electric
Marketing, LLC, Crete Energy Venture, LLC, CSOLAR IV South, LLC, CSOLAR
IV West, LLC, High Desert Power Project, LLC, Kiowa Power Partners,
LLC, Lincoln Generating Facility, LLC, New Covert Generating Company,
LLC, New Mexico Electric Marketing, LLC, Rolling Hills Generating,
L.L.C., Tenaska Alabama Partners, L.P., Tenaska Alabama II Partners,
L.P., Tenaska Frontier Partners, Ltd., Tenaska Gateway Partners, Ltd.,
Tenaska Georgia Partners, L.P., Tenaska Power Management, LLC, Tenaska
Power Services Co., Tenaska Virginia Partners, L.P., Texas Electric
Marketing, LLC, TPF Generation Holdings, LLC, Wolf Hills Energy, LLC.
    Description: Quarterly Land Acquisition Report of the Tenaska MBR
Sellers.
    Filed Date: 10/26/15.
    Accession Number: 20151026-5430.
    Comments Due: 5 p.m. ET 11/16/15.

    Take notice that the Commission received the following PURPA
210(m)(3) filings:

    Docket Numbers: QM14-3-000.
    Applicants: Entergy Arkansas, Inc., Entergy Gulf States Louisiana,
L.L.C., Entergy Louisiana, LLC, Entergy Mississippi, Inc., Entergy New
Orleans, Inc., Entergy Services, Inc., Entergy Texas, Inc., Entergy
Services, Inc.
    Description: Response to Third Deficiency Letter of Entergy
Services, Inc., et al.
    Filed Date: 10/23/15.
    Accession Number: 20151023-5371.
    Comments Due: 5 p.m. ET 11/20/15.

    Take notice that the Commission received the following electric
reliability filings:

    Docket Numbers: RD15-5-000; RD15-5-001; RD15-6-000.
    Applicants: North American Electric Reliability Corporation.
    Description: Clarifying Supplemental Information of the North
American Electric Reliability Corporation Updating Implementation Plan.
    Filed Date: 10/23/15.
    Accession Number: 20151023-5403.
    Comments Due: 5 p.m. ET 11/3/15.

    The filings are accessible in the Commission's eLibrary system by
clicking on the links or querying the docket number.
    Any person desiring to intervene or protest in any of the above
proceedings must file in accordance with Rules 211 and 214 of the
Commission's Regulations (18 CFR 385.211 and Sec.  385.214) on or
before 5:00 p.m. Eastern time on the specified comment date. Protests
may be considered, but intervention is necessary to become a party to
the proceeding.
    eFiling is encouraged. More detailed information relating to filing
requirements, interventions, protests, service, and qualifying
facilities filings can be found at: http://www.ferc.gov/docs-filing/efiling/filing-req.pdf. For other information, call<PHONE_NUMBER>
(toll free). For TTY, call<PHONE_NUMBER>.

    Dated: October 27, 2015.
Nathaniel J. Davis, Sr.,
Deputy Secretary.
[FR Doc. 2015-27842 Filed 10-30-15; 8:45 am]
BILLING CODE 6717-01-P


IDEXX 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 Pall Gelman Sciences www. pall. com/ gelman 600 South Wagner Road Ann Arbor, MI 48103 Sales: Phone: ( 800) 521 1520 ext. 2 Fax: ( 734) 913 6495 Technical Support: Phone: ( 800) 521 1520 ext. 3 Fax: ( 734) 913 6495 PPG Industries www. ppg. com One PPG Place Pittsburgh, PA 15272 Sales and Technical Support: Phone: ( 412) 434 3131 Tygon www. tygon. com Saint Gobain Performance Plastics Corp. Distributor: Ryan Herco. Products 1501 Waco St. Richmond, VA 23244 Sales: Phone: ( 804) 672 1158 Fax: ( 804) 672 0878 Technical Support: Saint Gobain Phone: ( 800) 798 1539 Fax: ( 800) 488 9466 Email: linda. d. anderson@ saint gobain. com 3.11.2 Sample Concentration and Purification Supplies Dynal, Inc. www. dynal. net 5 Delaware Drive Lake Success, NY 11042 Sales: Phone: ( 800) 638 9416 ext. 1 Fax: ( 516) 326 3298 E mail: uscustserv@ dynalbiotech. com Technical Support: Phone: ( 800) 638 9416 ext. 2 Fax: ( 516) 326 3298 E mail: ustechserv@ dynalbiotech. com International Equipment Company www. labcentrifuge. com 300 Second Avenue Needham Heights, MA 02492 Sales and Technical Support: Phone: ( 800) 841 1113 ext. 9723 Fax: ( 781) 444 6743 E mail: pat. webb@ thermoiec. com Section 3: Guidance for Cryptosporidium Laboratories 41 Draft June 2003 3.11.3 Slides and Related Supplies Dynal, Inc. www. dynal. net 5 Delaware Drive Lake Success, NY 11042 Sales: Phone: ( 800) 638 9416 ext. 1 Fax: ( 516) 326 3298 E mail: uscustserv@ dynalbiotech. com Technical Support: Phone: ( 800) 638 9416 ext. 2 Fax: ( 516) 326 3298 E mail: ustechserv@ dynalbiotech. com Hauser Scientific www. deltasci. com Distributed by Delta Scientific 1979 Stout Drive, Bldg. 5 Ivyland, PA 38974 Sales and Technical Support: Phone: ( 800) 428 9400 Fax: ( 800) 428 3271 E mail: dsirep@ aol. com Meridian Diagnostics, Inc. 3471 River Hills Drive Cincinnati, OH 45244 Sales: Phone: ( 800) 543 1980 Fax: ( 513) 271 0124 Technical Support: Phone: ( 800) 343 3858 Fax: ( 513) 271 0124 PGC Scientifics www. pgcscientifics. com P. O. Box 7277 Gaithersburg, MD 20898 7277 Sales and Technical Support: Phone: ( 800) 424 3300 Fax: ( 800) 662 1112 E mail: cs@ pgcscientifics. com Waterborne, Inc. www. waterborneinc. com 6047 Hurst Street New Orleans, LA 70118 6129 Sales and Technical Support: Phone: ( 504) 895 3338 Fax: ( 504) 895 3338 E mail: custserv@ waterborneinc. com 3.11.4 Antibody Stains BioTechnology Frontiers ( BTF) www. biotechnologyfrontiers. com Unit 1 35 41 Waterloo Road North Ryde NSW Australia Fax: + 61 2 9889 1805 Email: contact@ biotechnologyfrontiers. com Meridian Diagnostics, Inc. 3471 River Hills Drive Cincinnati, OH 45244 Sales: Phone: ( 800) 543 1980 Fax: ( 513) 271 0124 Technical Support: Phone: ( 800) 343 3858 Fax: ( 513) 271 0124 Waterborne, Inc. www. waterborneinc. com 6047 Hurst Street New Orleans, LA 70118 6129 Sales and Technical Support: Phone: ( 504) 895 3338 Fax: ( 504) 895 3338 E mail: custserv@ waterborneinc. com Section 3: Guidance for Cryptosporidium Laboratories 42 Draft June 2003 3.11.5 Microscope Equipment Chroma Technology Corp. www. chroma. com 74 Cotton Mill Hill Brattleboro, VT 05301 Sales: Phone: ( 800) 824 7662 Fax: ( 802) 257 9400 Technical Support: Phone: ( 800) 824 7662 Fax: ( 802) 257 9400 Nikon www. nikonusa. com NIKON 1300 Walt Whitman Road Melville, NY 11747 Customer Information: Phone: ( 800) 52 NIKON Email: Available through website Olympus www. olympusamerica. com Olympus America INC. 2 Corporate Center Drive, Melville, NY 11747 3157, U. S. A. Product Support: Phone: ( 800) 446 5967 Email: Available through website Zeiss www. zeiss. com Carl Zeiss Microimaging, Inc. One Zeiss Drive Thornwood, N. Y. 10594 Sales and Technical Support: Phone: ( 800) 233 2343 Fax: ( 914) 684 7446 E mail: micro@ zeiss. com 3.11.6 Spiking Suspensions and Positive Staining Control Materials BioTechnology Frontiers ( BTF) www. biotechnologyfrontiers. com Unit 1 35 41 Waterloo Road North Ryde NSW Australia Fax: + 61 2 9889 1805 Email: contact@ biotechnologyfrontiers. com Hyperion Research, Ltd. 1008 Allowance Avenue, SE Medicine Hat, Alberta TIA 3G8 Canada Sales and Technical Support: Peter Wallis Phone: ( 403) 529 0847 Fax: ( 403) 529 0852 E mail: hyperion@ telusplanet. net Waterborne, Inc. www. waterborneinc. com 6047 Hurst Street New Orleans, LA 70118 6129 Sales and Technical Support: Phone: ( 504) 895 3338 Fax: ( 504) 895 3338 E mail: custserv@ waterborneinc. com Sterling Parasitology Laboratory, University of Arizona, Tucson Building 90, Rm. 202 Tucson, AZ 85721 Sales and Technical Support: Marilyn Marshall Phone: ( 520) 621 4433 Fax: ( 520) 621 3588 E mail: marshalm@ u. arizona. edu Wisconsin State Laboratory of Hygiene, Flow Cytometry Unit 2601 Agriculture Drive Madison, WI 53718 Sales and Technical Support: Phone: ( 608) 224 6260 Fax: ( 608) 224 6213 Section 3: Guidance for Cryptosporidium Laboratories 43 Draft June 2003 3.11.7 Other Laboratory Supplies for EPA Method 1622/ 1623 Fisher Scientific www. fishersci. com 9999 Veterans Memorial Drive Houston, TX 77038 Sales: Phone: ( 800) 766 7000 Fax: ( 800) 926 1166 Technical Support: Phone: ( 800) 766 7000 Fax: ( 800) 926 1166 Sigma www. sigma aldrich. com P. O. Box 14508 St. Louis, MO 63178 Sales: Phone: ( 800) 325 3010 Fax: ( 800) 325 8070 E mail: custserv@ sial. com Technical Support: Phone: ( 800) 325 5832 Fax: ( 314) 286 7828 E mail: techserv@ sial. com VWR www. vwr. com 05 Heron Drive Bridgeport, NJ 08014 Sales: Phone: ( 800) 932 5000 Fax: ( 856) 467 3336 Technical Support: Phone: ( 800) 932 5000 Fax: ( 856) 467 3336 44 Draft June 2003 SECTION 4: GUIDANCE FOR E. COLI LABORATORIES Although public water system ( PWS) treatment requirements will be determined from Cryptosporidium monitoring results, the Long Term 2 Enhanced Surface Water Treatment ( LT2) rule also requires that large PWSs ( those serving > 10,000) perform E. coli and turbidity analyses on source water samples [ 40 CFR part 141.701 ( b)]. The Cryptosporidium, E. coli, and turbidity data will be analyzed to evaluate whether E. coli and/ or turbidity levels can be used as reliable indicators of Cryptosporidium occurrence in surface waters used as source waters for drinking water facilities. Depending upon the results of these analyses, EPA may permit small PWSs to use E. coli monitoring, or a combination of E. coli and turbidity monitoring, to determine the need for implementing more expensive Cryptosporidium monitoring or improved treatment.

Cryptosporidium analysis requirements and recommendations are discussed in Section 3 of this guidance manual. Turbidity measurement requirements and recommendations are addressed in the Source Water Monitoring Guidance Manual for Public Water Systems for the Long Term 2 Enhanced Surface Water Treatment Rule ( LT2 Rule) ( available for download from http:// www. epa. gov/ safewater/ lt2/ index. html). E. coli analysis requirements and recommendations for the LT2 rule, including laboratory certification, quality assurance/ quality control ( QA/ QC), and the procedures for each of the methods approved for use during LT2 E. coli monitoring, are presented below.

All E. coli sample analyses performed under the LT2 rule must be quantitative [ 40 CFR 141.705 ( b)]. Presence/ absence data cannot be used to assess whether E. coli concentration can be used to indicate Cryptosporidium occurrence levels.

4.1 Laboratory Certification Program PWS laboratories and other laboratories performing LT2 rule E. coli analyses must be certified for coliform analyses under the drinking water certification program for a similar method type ( e. g., membrane filtration, multiple tube, multiple well) [ 40 CFR part 141.706 ( b)]. The drinking water certification program was established to fulfill requirements stipulated by the Safe Drinking Water Act ( SDWA) and the requirements of the National Primary Drinking Water Regulations ( NPDWR) ( 40 CFR 141.28). The certification program requires that all laboratories analyzing drinking water must be certified by EPA, including EPA's Regional labs, laboratories on Federal Indian Lands, principal state laboratories, and laboratories that perform analyses under the SDWA in states without primacy. States that have primary enforcement responsibility ( primacy) are required to have laboratory facilities available which have been certified by EPA ( see 40 CFR 142.10) or National Environmental Laboratory Accreditation Conference ( NELAC). All labs that perform testing for compliance purposes must be certified by the state or EPA. Public water systems serving at least 25 persons or having at least 15 service connections must comply with the SDWA and the requirements of the NPDWR.

The certification program consists of annual performance evaluation samples and on site lab audits every three years by EPA or the state. During laboratory audits EPA or the state will evaluate the laboratory's Quality Assurance Plan ( QAP), performance on routine water samples, and chain of custody procedures. The certification process and detailed specifications for certification are described in the EPA Manual for the Certification of Laboratories Analyzing Drinking Water Certification Manual) ( Reference 5.2) ( http:// www. epa. gov/ safewater/ certlab/ labindex. html) Section 4: E. coli Laboratory Guidance 45 Draft June 2003 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 a similar method type ( e. g., membrane filtration) 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 provided in Table 4 1.

Table 4 1. Approved E. coli Methods for LT2 Rule and Corresponding Drinking Water Certification Program Techniques E. coli Methods Approved for LT2 Rule Appendix References for Methods Included in this Manual Corresponding Drinking Water Certification Program Coliform Analysis Technique Standard Methods 9223B ( Colilert) ® Appendix I Most probable number ( MPN): Multiple tube and multiple well techniques Standard Methods 9223B ( Colilert 18) ® Appendix J Standard Methods 9222B/ 9222G1 ( mEndo/ LES Endo6NA MUG) Appendix K Membrane filtration technique Standard Methods 9222D/ 9222G ( mFC6NA MUG)

Standard Methods 9213D/ EPA Method 1103.1 ( mTEC) Appendix L EPA Method 1603 Modified mTEC Appendix M EPA Method 1604 MI medium 1 Appendix N m ColiBlue24 ® Broth 1 Appendix O Standard Methods 9221B/ 9221F ( LTB6EC MUG) Appendix P MPN: Multiple tube fermentation technique 1 If high levels of non E. coli total coliforms interfere with the ability to accurately enumerate E. coli despite additional dilutions, an alternate method should be used ( i. e, SM 9222D/ 9222G, SM 9213D/ EPA Method 1103.1, EPA Method 1603, SM 9221B/ 9221F)

4.2 Quality Assurance/ Quality Control for E. coli Analyses Laboratory QA for E. coli analyses during LT2 monitoring should meet the specifications in the Certification Manual ( Reference 5.2) and Standard Methods for the Examination of Water and Wastewater, 20th Edition ( 1998) ( Standard Methods) ( Reference 5.3) for facilities, personnel, laboratory equipment, instrumentation, and supplies used for LT2 E. coli analyses. In addition, each laboratory should have implemented a written QA plan describing the QA program and QC activities necessary to meet the laboratory's specific needs. The program should address the following issues: personnel policies, equipment and instrument specifications, specifications for supplies, analytical methods and QC measures, standard operating procedures ( SOPs), documentation specifications, performance evaluation samples, internal and external lab audits, and corrective actions.

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 listed in Table 4.1 above. Section 4.2.1 describes overall quality control specifications for E. coli analyses performed under the LT2 rule. E. coli QC specifications specific to membrane filtration methods and MPN methods are provided in Sections 4.2.2 to 4.2.3. This guidance is provided to help implement 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. A checklist for these specifications is provided as Appendices Q and R. Section 4: E. coli Laboratory Guidance 46 Draft June 2003 Note: E. coli samples that do not meet the QC specifications set forth in these sections are considered to be invalid, and may not be reported under the LT2 rule.

Table 4 2. Quality Control Procedures for E. coli Methods Quality Control Procedure Reference All E. coli Methods Holding time and temperature requirements ( Section <IP_ADDRESS>) LT2 Rule: Section IV. K. 2 Dilution/ rinse water sterility check ( Section <IP_ADDRESS>) Certification Manual: Section 4.4.3 Media sterility check ( Section <IP_ADDRESS>) Certification Manual: Section <IP_ADDRESS> Positive/ negative controls ( Section <IP_ADDRESS>) Certification Manual: Section <IP_ADDRESS> Media storage ( Section <IP_ADDRESS>) Certification Manual: Section <IP_ADDRESS> MPN Methods ( includes multiple tube and multiple well techniques)

Incubation time and temperature specifications ( Section <IP_ADDRESS>) Specified in Method MPN preparation blank ( Section <IP_ADDRESS>) Recommended Verification ( Section <IP_ADDRESS>) Specified in Method Membrane Filtration Methods Incubation time and temperature specifications ( Section <IP_ADDRESS>) Specified in Method Filtration unit sterilization ( Section <IP_ADDRESS>) Certification Manual: Section 4.1 Membrane filter preparation blank ( Section <IP_ADDRESS>) Certification Manual: Section <IP_ADDRESS> Verification ( Section <IP_ADDRESS>) Specified in Method 4.2.1 Quality Control Specifications Applicable to All E. coli Methods Independent of QC specifications for the type of method used for analysis, there are QC measures that should be taken by all laboratories performing E. coli analyses under the LT2 rule to ensure the laboratory is in control during the analysis of LT2 monitoring samples. Quality assurance/ quality control procedures for all E. coli methods are summarized in Sections <IP_ADDRESS> through <IP_ADDRESS>.

<IP_ADDRESS> Holding Time and Temperature Requirements for Field Samples EPA strongly encourages laboratories to analyze samples as soon as possible after collection. However, due to the need by some utilities to ship samples overnight to an off site laboratory for analysis, the holding time for E. coli sample analyses has been extended to 24 hours. Based on studies to assess the Section 4: E. coli Laboratory Guidance 47 Draft June 2003 Note: These QC specifications are meant to be performed routinely for all samples, at the frequency specified in the Certification Manual. The QC specifications are not additional analyses specifically for LT2 E. coli samples.

Note: These QC specifications are meant to be performed routinely for all samples, at the frequency specified in the Certification Manual. The QC specifications are not additional analyses specifically for LT2 E. coli samples. If any contamination is observed, determine the cause, correct, and reject any data from samples associated with: ( 1) dilution or rinse water, or ( 2) media. effect of increased sample holding time in E. coli analysis results, EPA has concluded that E. coli samples can be held for up to 24 hours prior to analysis without compromising the data quality objectives of the LT2 rule ( Reference 5.4).

Samples must be maintained between 0 ° C and 10 ° C during transit [ CFR part 141.705 ( b) ( 1)] and should not be allowed to freeze. For samples shipped off site, utilities should consider including a temperature blank along with the sample so that laboratories can measure the temperature of the sample upon receipt. Alternately, utilities could use Thermochron iButtons ® or other measuring devices to record the temperature throughout transport ( discussed in Section 2.2 in this manual). Additional information pertaining to temperature monitoring is provided in the Source Water Monitoring Guidance Manual for Public Water Systems for the Long Term 2 Enhanced Surface Water Treatment Rule ( LT2 Rule).

<IP_ADDRESS> Dilution/ Rinse Water Sterility Check QC specifications for dilution/ rinse water checks are as follows:

° 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. Discard batch if growth is detected.

<IP_ADDRESS> 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. Section 4: E. coli Laboratory Guidance 48 Draft June 2003 Note: Additional QC analyses are not required for the analysis of E. coli samples under LT2, provided that laboratories perform required QC analyses at the frequency specified in the Certification Manual. <IP_ADDRESS> 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. See Table 4 3 for examples of test 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.

Table 4 3. Positive and Negative Control Cultures Positive Control Culture Negative Control Culture E. coli Enterobacter aerogenes ( ATCC # 13048) 1 Klebsiella pneumoniae ( ATCC # 31488) 2 1 E. aerogenes normally does not grow at 44.5 ° C; if growth occurs, the waterbath or incubator is not holding the 44.5 ° C temperature. The recommended strain is MUG negative and is not thermotolerant. 2 K. pneumoniae is MUG negative and should not fluoresce when transferred to NA MUG. In addition, this strain is thermotolerant.

Positive and negative controls should be chosen based on the method specific requirements. For example if a 44.5 ° C waterbath is not required by the method, it is not necessary to include Enterobacter aerogenes as a negative control.

<IP_ADDRESS> 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 4.2.2 Quality Control Specifications for Most Probable Number Methods In addition to the overall QC specifications set forth in Section 4.2.1, laboratories using MPN methods ( e. g., multiple tube, multiple well) for LT2 rule E. coli analyses should meet method specific incubation time and temperature specifications and method specific E. coli verification specifications. Section 4: E. coli Laboratory Guidance 49 Draft June 2003 Note: Additional QC analyses are not required for the analysis of E. coli samples under LT2, provided that laboratories perform the verifications at the frequency specified in the Certification Manual. <IP_ADDRESS> Incubation Time/ Temperature Specifications for MPN Methods The required incubation times and temperatures for MPN methods are provided in Table 4 4.

Table 4 4. Incubation Time and Temperature Specifications for MPN Methods Method Media Incubation Time/ Temperature Standard Methods 9223B Colilert ® 24  28 hours at 35 ° C ± 0.5 ° C Coliert 18 ® 18  22 hours at 35 ° C ± 0.5 ° C Standard Methods 9221B/ 9221F LTB 22  51 hours at 35 ± 0.5 ° C EC MUG 22  26 hours at 44.5 ° C ± 0.2 ° C <IP_ADDRESS> 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.

<IP_ADDRESS> Verification Verification specifications are detailed in the Certification Manual ( Reference 5.2), Standard Methods ( Reference 5.3), and Appendices I and J of this document. Verifications should be performed in accordance with method specific requirements.

4.2.3 Quality Control Specifications for Membrane Filtration Methods In addition to the overall QC specification in Section 4.2.1, laboratories using membrane filtration methods should meet method specific incubation times and temperatures, and should perform additional QC steps to demonstrate that membrane filtration equipment is free from contamination and cross over.

Note: Additional QC analyses are not required for the analysis of E. coli samples under LT2, provided that laboratories perform required QC analyses at the frequency specified in the Certification Manual.

<IP_ADDRESS> Incubation Time and Temperature The required incubation times and temperatures for membrane filtration methods are provided in Table 4 5. Section 4: E. coli Laboratory Guidance 50 Draft June 2003 Table 4 5. Incubation Time and Temperature Specifications for Membrane Filter Methods Method Media Incubation Time/ Temperature Standard Methods 9222B/ 9222G mENDO6NA MUG 22  26 hours at 35 ° C ± 0.5 ° C6 4 hours at 35 ° C ± 0.5 ° C Les ENDO6NA MUG 22  26 hours at 35 ° C ± 0.5 ° C6 4 hours at 35 ° C ± 0.5 ° C mFC6NA MUG 22  26 hours at 44.5 ° C ± 0.2 ° C6 4 hours at 35 ° C ± 0.5 ° C Standard Methods 9213D mTEC agar 2 hours at 35 ° C ± 0.5 ° C 6 22  24 hours at 44.5 ° C ± 0.2 ° C EPA 1603 Modified mTEC 2 hours at 35 ° C ± 0.5 ° C 6 22  24 hours at 44.5 ° C ± 0.2 ° C EPA 1604 MI agar 24 hours at 35 ° C ± 0.5 ° C Other Membrane Filter Methods m ColiBlue24 Broth 24 hours at 35 ° C ± 0.5 ° C <IP_ADDRESS> 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 presterilized 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.

<IP_ADDRESS> 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.

<IP_ADDRESS> Verification Verification specifications are detailed in the Certification Manual ( Reference 5.2), Standard Methods ( Reference 5.3), and in Appendices K through O of this document. Verifications should be done in accordance with method specific requirements.

Note: Additional verifications are not required for the analysis of E. coli samples under LT2, provided that laboratories perform verifications at the frequency specified in the Certification Manual.

4.3 Sample Collection Procedures During the LT2 rule, PWSs must take source water samples at a location prior to any treatment and where the water is no longer subject to surface runoff [ 40 CFR part 141.704 ( a)]. Guidance on sample collection locations and detailed procedures for collecting E. coli samples for on site analysis and for shipment to an off site laboratory for analysis are provided in the Source Water Monitoring Guidance Manual for Public Water Systems for the Long Term 2 Enhanced Surface Water Treatment Rule ( LT2 Rule). This manual is available for download from http:// www. epa. gov/ safewater/ lt2/ index. html. Section 4: E. coli Laboratory Guidance 51 Draft June 2003 4.3.1 Acceptance Criteria for Receipt of Field Samples Source water samples are dynamic environments and, depending on sample constituents and environmental conditions, E. coli present in a sample can grow or die off, biasing analytical results. Samples that are not analyzed immediately after sample collection during LT2 rule monitoring must be chilled to reduce biological activity, and preserve the state of source water samples between collection and analysis. Samples for E. coli analyses must be maintained between 0 ° C and 10 ° C if they are shipped [ 40 CFR 141.705 ( b)( 1)]. Samples should not be allowed to freeze.

Given the importance of maintaining sample temperatures for E. coli, laboratories approved for performing E. coli analyses during the rule should establish acceptance criteria for receipt of E. coli samples transported to their laboratory. Several options are available to measure sample temperature upon receipt at the laboratory and, in some cases, during shipment are provided in Section 2.2.

4.4 Sample Volume and Dilution Guidance Because E. coli analyses will be performed on source waters, rather than finished drinking waters, multiple sample/ volumes dilutions may necessary to assess high E. coli levels. Initially all PWSs will be required to analyze four sample volumes ( 100, 10, 1.0, and 0.1 mL) for all methods except MTF. For MTF methods the PWS will be initially required to analyze five sample volumes of 10, 1.0, 0.1, 0.01, and 0.001 mL. Sample volumes may need to be adjusted based on confounding conditions ( e. g., high turbidity, heavy rainfall, etc.).

If the PWS has historical data demonstrating that E. coli levels are consistently low, they may drop the smaller sample volumes. However, if the PWS decides to eliminate any of the required volumes due to historical data, it should be noted that changes in conditions ( e. g., increased precipitation, seasonality, etc.) may impact water quality. As a result, sample volumes may need to be adjusted on a per sampling event basis. For example, if there has been a substantial rainfall in the 24 hours prior to sample collection causing runoff, sample volumes would need to be adjusted in order to obtain valid results and avoid data that are above the analytical range of the method. This could also be a concern if the PWS eliminates the 100 mL sample volume due to historical data indicating that values tend to be very high and the E. coli levels are lower than anticipated, forcing the PWS to report data that are below the analytical range of the method. EPA recommends bracketing the target sample volume ( i. e., analyzing a sample volume above and below the sample volume expected to yield useable data) to account for potential variability.

4.4.1 Sample Volume and Dilution Guidance for Multiple Well Methods The analysis of water samples under LT2 using multiple well methods generally requires the use of four aliquots of samples ( 100, 10, 1.0, and 0.1 mL). Sample volumes may need to be adjusted based on confounding conditions ( e. g., high turbidity, heavy rainfall, etc.). Because a 100 mL volume is necessary to fill all of the wells, it will be necessary to add the aliquots of sample to sterile reagent water blanks. The total volume after the sample aliquot is added to the reagent water blank should be approximately 100 mL ( i. e., a 90 mL blank should be used for the 10 mL sample volume, a 99 mL reagent water blank should be used for the 1.0 mL aliquot).

4.4.2 Sample Volume and Dilution Guidance for Membrane Filtration The analysis of water samples under LT2 requires four different aliquots of sample ( 100, 10, 1.0, and 0.1 mL) for membrane filtration methods. lternate sample volumes may be used if necessary. To ensure adequate distribution of bacteria during filtration, dispense aliquots of the sample, with the exception of the 100 mL volume, into sterile buffered water blanks ( at least 30 mL, depending on the sample volume Section 4: E. coli Laboratory Guidance 52 Draft June 2003 analzyed). Alternately, 10 mL of sterile buffer may be added to the filter apparatus prior to adding the sample. The buffered water minimizes clumping of the bacteria on the filter surface.

4.4.3 Sample Volume and Dilution Guidance for Multiple Tube Methods The analysis of water samples by a multiple tube technique ( e. g., Colilert ® in a multiple tube format, SM 9222B LTB/ EC MUG) under LT2 requires the use of a 15 tube most probable number format ( i. e., 5 tubes at each of three dilutions, 10.0 mL, 1.0 mL, and 0.1 mL). Five tubes must be inoculated for each sample volume. Initially, the PWS will be required to analyze four sample volumes ( 10.0, 1.0, 0.1, and 0.01 mL). Additional guidance on selection of dilutions for multiple tube methods is available in Standard Methods 9221 ( Reference 5.3).

4.5 E. coli Data Recording and Calculations Laboratories performing E. coli analyses during the LT2 rule must be certified for the analytical technique used for LT2 E. coli analyses under the drinking water certification program, and therefore the data recording practices required under the drinking water certification program should be followed [ 40 CFR part 141.706 ( b)]. These practices include the following:

° Maintaining sample identification information ( e. g., including sample collection and receipt dates, times, and conditions)

° Recording all raw data ( i. e., primary measurements) used to calculate final sample concentration for each sample ° Recording the incubation/ read times for each method to verify that method specifications were met ° Recording the name of the analyst performing the sample analysis At a minimum, the data elements discussed in detail below should be recorded for E. coli samples during LT2 monitoring. These data elements are critical to ensure that final sample results can be verified using primary data, if necessary, and to demonstrate that sample analyses were performed within methodspecified holding times and incubation times. Standardized bench sheets are available for download on the LT2 website ( http:// www. epa. gov/ safewater/ lt2/ index. html). Please note that, while E. coli laboratories should record the data elements discussed in this section to comply with certification manual specifications, not all of the recorded data will be reported in the LT2 data system. E. coli data reporting under the LT2 rule is discussed in Section 4.6.

In addition to data elements, this section also provides standardized procedures for determining E. coli concentration for LT2 samples for the various analytical techniques that are approved for use under the rule. Because these analyses will be performed on source waters rather than finished drinking waters, and multiple dilutions may be necessary to assess higher E. coli levels, it is critical that laboratories follow the same procedures for determining E. coli sample concentrations to reduce variability in reporting.

Table 4 6 specifies the minimum sample ID information that must be recorded for each E. coli sample, either on a sample collection form used in the field and maintained in the same file as the E. coli laboratory data, or on the laboratory data form ( examples of all forms are provided Appendices S through AB) [ 40 CFR part 141.707 ( 2)]. Section 4: E. coli Laboratory Guidance 53 Draft June 2003 Table 4 6. Minimum Sample ID Information and General Sample Data to Record PWS ID Facility ID Sample collection point Sample collection date Analytical method number Method type Source type E. coli/ 100 mL Turbidity 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 4.5.1 to 4.5.3.

4.5.1 Multiple Well Data In addition to the data elements listed in Table 4 6, laboratories using Colilert ® and Colilert 18 ® methods for E. coli sample analyses in support of the LT2 rule should record the data elements specific to multiple well techniques. Data elements to be recorded for the 97 well format ( Quanti Tray 2000 ® ) are noted in Table 4 7; data to be recorded for the 51 well format ( Quanti Tray ® ) are noted in Table 4 8. These elements include the primary measurements needed to calculate the E. coli concentration in the sample as well as all method required incubation and read times needed to verify that the sample analyses were conducted under analytical control.

Calculations for determining the E. coli concentration using the Quanti Tray 2000 ® ( 97 well) and Quanti Tray ® ( 51 well) formats are provided after each table.

<IP_ADDRESS> Data Elements for Quanti Tray 2000 ® ( 97 well) Analyses Table 4 7. Minimum Data to Record for Quanti tray 2000 ® Colilert ® and Colilert 18 ® Analyses Primary Measurements mL of sample added to tray ( does not include reagent water volume)

Large wells positive: Total coliform positive and UV fluorescence Small wells positive: Total coliform positive and UV fluorescence Holding Time / Incubation Time Information Incubation start/ read date Incubation start/ read time Incubation start/ read temperature Additional incubation start/ read date Additional incubation start/ read time Section 4: E. coli Laboratory Guidance 54 Draft June 2003 <IP_ADDRESS> Determining E. coli Concentration Using Colilert ® and Colilert 18 ® Quanti Tray 2000 ® Data A. Select appropriate dilution to yield countable results. If multiple dilutions are used, the tray exhibiting positive wells in the 40% to 80% range ( 39 to 78 total positive large and small wells) should be used to determine MPN value.

Note: The analytical result can be automatically calculated using the LT2 Data Collection System. See Section 4.6 for additional information.

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.

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:

Analytical result = MPN value × 100 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 Analytical result = 81.3 × 100 = 813 E. coli MPN/ 100 mL 10 <IP_ADDRESS> Data Elements for Quanti Tray ® ( 51 well) Analyses The recommended data elements to record for Quanti Tray ® ( 51 well) analyses are provided in Table 4 8. Section 4: E. coli Laboratory Guidance 55 Draft June 2003 Table 4 8. Minimum Data to Record for Quanti Tray ® Colilert ® and Colilert 18 ® Analyses Primary Measurements mL of sample added to tray ( does not include reagent water volume)

Number of wells positive: Total coliform positive and UV fluorescence Holding Time / Incubation Time Information Incubation start/ read date Incubation start/ read time Incubation start/ read temperature Additional incubation start/ read date Additional incubation start/ read time Additional incubation start/ read temperature <IP_ADDRESS> Determining E. coli Concentration Using Colilert ® and Colilert 18 ® ( 51 well) Data A. Select appropriate dilution. If multiple dilutions are used, the tray exhibiting 80% positive wells ( 41 positive wells) should be used to determine MPN value.

Note: The analytical result can be automatically calculated using the LT2 Data Collection System. See Section 4.6 for additional information.

B. Determine MPN. Using the number of positive wells from the appropriate dilution, identify the corresponding MPN/ 100 mL in the table provided by the vendor. For example, if a 100 mL sample was analyzed, and there were 41 positive wells, the corresponding MPN would be 83.1 MPN/ 100 mL C. Adjust for dilution factor. Because the MPN/ 100 mL values in the table are based on 100 mL samples, the MPN value should be adjusted if less than 100 mL of sample volume was analyzed. Use the following calculation to adjust the MPN to account for the dilution:

MPN value × 100 = E. coli MPN/ 100 mL mL sample analyzed Example:

Volume analyzed ( mL) = 10 mL ( in 90 mL of dilution water) Number of positive wells = 41 MPN = 83.1 The analytical result is calculated as follows:

83.1 × 100 = 831 E. coli MPN/ 100 mL 10 4.5.2 Membrane Filtration Data In addition to the general sample data listed in Table 4 6, laboratories using membrane filtration methods for E. coli sample analyses in support of the LT2 rule should record the data elements specific to this technique. These are noted in Table 4 9, and include the primary measurements needed to calculate the E. Section 4: E. coli Laboratory Guidance 1 Adapted from Standard Methods for the Examination of Water and Wastewater, APHA, AWWA, WEF ( 20th Edition)

56 Draft June 2003 coli concentration in the sample, as well as all method required incubation and read times needed to verify that the sample analyses were conducted under analytical control.

<IP_ADDRESS> Data Elements for Membrane Filtration Analyses Table 4 9. Minimum Data Elements for Record for Membrane Filtration Analyses Primary Measurements Filter 1 volume ( mL) ( e. g., 100 mL) CFU on Filter 1 Filter 2 volume ( mL) ( e. g., 10 mL) CFU on Filter 2 Filter 3 volume ( mL) ( e. g., 1.0 mL) CFU on Filter 3 Filter 4 volume ( mL) ( e. g., 0.1 mL) CFU on Filter 4 Holding Time / Incubation Time Information Primary isolation medium ( e. g., mENDO, mFC) incubation start/ read date Primary isolation medium ( e. g., mENDO, mFC) incubation start/ read time Primary isolation medium ( e. g., mENDO, mFC) incubation start/ read temperature Secondary isolation medium ( e. g, NA MUG) incubation start/ read date Secondary isolation medium ( e. g, NA MUG) incubation start/ read time Secondary isolation medium ( e. g, NA MUG) incubation start/ read temperature <IP_ADDRESS> Determining E. coli Concentrations Using Membrane Filter Data1:

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 4.6 for additional information.

B. If there are greater than 200 colonies per membrane, even for the lowest dilution, the result is recorded as " too numerous to count" ( TNTC). These results cannot be reported for LT2 monitoring, as they cannot be used for the required data analyses. During the next sampling event, analyze an additional, lower dilution volume ( the highest dilution volume may be omitted) unless conditions were unusual ( e. g., heavy rains, flooding, etc.) during the sampling event yielding TNTC for all dilutions.

C. If colonies are not sufficiently distinct for accurate counting, the result is recorded as " confluent growth" ( CNFG). To prevent CNFG from occurring, smaller sample aliquots should be filtered. For example, if sample volumes of 100, 10, 1 and 0.1 mL are analyzed and even the 0.1 mL plate results in CNFG, then potentially 0.01 mL should be analyzed during the next sampling event. For sample volumes less than 1 mL, serial dilutions should be used, and 1 mL volumes of the dilutions should be filtered. The 100 mL volume can be eliminated. Note: If growth is due to high levels of total coliforms but low E. coli then another method should be chosen for analyses that does not rely on total coliform determination prior to or simultaneously with E. coli determination. Section 4: E. coli Laboratory Guidance 57 Draft June 2003 Note: Results that are TNTC or CNFG are not appropriate for LT2 microbial data analysis, and cannot be entered into the LT2 Data Collection System. These results should not be reported.

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

E. coli CFU × 100 = E. coli CFU/ 100 mL mL sample filtered Example 1:

Filter 1 volume = 100 mL CFU = TNTC Filter 2 volume = 10 mL CFU = 40 Filter 3 volume = 1.0 mL CFU = 9 Filter 4 volume = 0.1 mL CFU = 0 Using the guidance on countable colonies in Step A, the counts from the 10 mL plate will be used to calculate the E. coli concentration for the sample:

40 E. coli CFU × 100 = 400 E. 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.

Example 2:

Filter 1 volume ( mL) = 100 mL CFU = 0 Filter 2 volume ( mL) = 10 mL CFU = 0 Filter 3 volume ( mL) = 1.0 mL CFU = 0 Detection limit = 100 mL = E. coli CFU/ 100 mL Largest volume filtered 100 mL = < 1 E. coli / 100 mL 100 mL Section 4: E. coli Laboratory Guidance 58 Draft June 2003 Example 3:

Filter 1 volume ( mL) = 100 mL CFU = Lab accident, no data available Filter 2 volume ( mL) = 10 mL CFU = 0 Filter 3 volume ( mL) = 1.0 mL CFU = 0 Calculation of E. coli/ 100 mL:

100 mL = < 10 E. coli CFU / 100 mL 10 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 CFU = 15 Filter 2 volume ( mL) = 25 mL CFU = 6 Filter 3 volume ( mL) = 10 mL CFU = 0 The analytical result is calculated as:

( 15 + 6 + 0) × 100 = 25 E. coli CFU/ 100 mL ( 50+ 25+ 10)

Example 5 Filter 1 volume ( mL) = 50 mL CFU = 105 Filter 2 volume ( mL) = 25 mL CFU = 92 Filter 3 volume ( mL) = 10 mL CFU = 85 The analytical result is calculated as:

( 105 + 92 + 85) × 100 = 332 E. coli CFU/ 100 mL ( 50 + 25 + 10)

Example 6:

Filter 1 volume ( mL) = 100 mL CFU = 82 Filter 2 volume ( mL) = 10 mL CFU = 18 Filter 3 volume ( mL) = 1.0 mL CFU = 0 The analytical result is calculated as:

( 82 + 18 + 0) × 100 = 90 E. coli CFU/ 100 mL ( 100 + 10 + 1) Section 4: E. coli Laboratory Guidance 59 Draft June 2003 Example 7 Filter 1 volume ( mL) = 50 mL CFU = TNTC Filter 2 volume ( mL) = 25 mL CFU = TNTC Filter 3 volume ( mL) = 10 mL CFU = 83 The analytical result is calculated as:

83 × 100 = 830 E. coli CFU/ 100 mL 10 4.5.3 Multiple Tube Data In addition to the general sample data listed in Table 4 6, laboratories using multiple tube methods for E. coli sample analyses in support of the LT2 rule should record the data elements specific to these techniques. These data elements are noted in Table 4 10 for 15 tube most probable number methods and Table 4 11 for 15 tube multiple tube fermentation methods. The data elements include the primary measurements needed to calculate the E. coli concentration in the sample, as well as all method required incubation and read times needed to verify that the sample analyses were conducted under analytical control.

Calculations for determining the E. coli concentration using multiple tube formats are provided in Section <IP_ADDRESS>.

<IP_ADDRESS> Data Elements for Multiple Tube Analyses Table 4 10. Minimum Data Elements to Record for 15 Tube MPN Methods Primary Measurements Number of positive 10.0 mL tubes: Total coliform positive and UV fluorescence Number of positive 1.0 mL tubes: Total coliform positive and UV fluorescence Number of positive 0.1 mL tubes: Total coliform positive and UV fluorescence Number of positive 0.01 mL tubes: Total coliform positive and UV fluorescence Number of positive 0.001 mL tubes: Total coliform positive and UV fluorescence Holding Time / Incubation Time Information Incubation start/ read date Incubation start/ read time Incubation start/ read temperature Additional incubation start/ read date Additional incubation start/ read time Additional incubation start/ read temperature Section 4: E. coli Laboratory Guidance 2Adapted from 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 ( Accessed April 7, 2003).

60 Draft June 2003 Table 4 11. Minimum Data Elements to Record for 15 Tube Fermentation Methods Primary Measurements Number of positive tubes 10.0 mL Number of positive tubes 1.0 mL Number of positive tubes 0.1 mL Number of positive tubes 0.01 mL Number of positive tubes 0.001 mL Number of positive tubes 0.0001 mL Holding Time / Incubation Time Information LTB incubation start date LTB incubation start time LTB incubation start temperature LTB 24 hour incubation read date LTB 24 hour incubation read time LTB 24 hour incubation read temperature LTB 48 hour incubation read date LTB 48 hour incubation read time LTB 48 hour incubation read temperature EC MUG incubation 24 hour read date ( from 24 hour LTB)

EC MUG incubation 24 hour read time ( from 24 hour LTB)

EC MUG incubation 24 hour read temperature ( from 24 hour LTB)

EC MUG incubation 24 hour read date ( from 48 hour LTB)

EC MUG incubation 24 hour read time ( from 48 hour LTB)

EC MUG incubation 24 hour read temperature ( from 48 hour LTB)

<IP_ADDRESS> Determination of E. coli Concentrations Using Multiple Tube Methods2:

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 4.6 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 Section 4: E. coli Laboratory Guidance 61 Draft June 2003 significant dilutions and are selected according to the following criteria. Examples of significant dilution selections are provided in Table 4 12, 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 4 12, 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 4 12, 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 4 12, Example D).

° When the selection rules above have left unselected any higher dilutions ( more dilute) with positive results, add those higher dilution positive results to the results for the highest selected dilution ( Table 4 12, Example E).

° If there were not enough higher dilutions tested to select three dilutions, then select the next lower dilution ( Table 4 12, 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:

Analytical result = MPN value = E. coli MPN/ 100 mL # of mL in middle dilution Table 4 12. Examples of Different Combinations of Positive Tubes ( Significant Dilution Results Are in Bold and Underlined)

Example Least dilute Most dilute ( Lowest) ( Highest) Combination of positives MPN Index from Standard Methods E. coli/ 100 mL ( after adjustment) 10 mL 1 mL 0.1 mL 0.01 mL 0.001 mL A 5 5 1 0 0 5 1 0 33 330 B 4 5 1 0 0 4 5 1 48 48 C 0 0 1 0 0 0 0 1 1.8 1.8 D 5 4 4 1 0 4 4 1 40 400 E 5 4 4 0 1 4 4 1 40 400 F 5 5 5 5 2 5 5 2 540 54,000 Section 4: E. coli Laboratory Guidance 62 Draft June 2003 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:

Analytical result = 33 = 330 E. coli / 100 mL 0.1 Example B: 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.

Example C: 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/ 100 mL.

Examples D and E: The significant dilution series for the 4 4 1 combination of positives includes the 1 mL, 0.1 mL, and 0.01 mL dilutions. Since the 10 mL, 1 mL, and 0.1 mL dilutions were not selected, an adjustment is necessary to account for the dilutions selected:

Analytical result = 40 = 400 E. coli / 100 mL 0.1 Example F: The significant dilution series for the 5 5 2 combination of positives includes the 0.1 mL, 0.01 mL and .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:

Analytical result = 540 = 54,000 E. coli / 100 mL 0.01 4.6 Electronic Data Reporting 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 3 6, in Section 3.8, above.

The data reporting process is summarized below, in Sections 4.6.1 through 4.6.3, 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. Section 4: E. coli Laboratory Guidance 63 Draft June 2003 4.6.1 Data Entry/ Upload The analyst or another laboratory staff member enters a subset of the data recorded at the bench ( Section 4.5) into the LT2 Data Collection System either by entering the data using web forms or by uploading data in XML format [ 40 CFR part 141.707 ( 2)]. This information includes the following:

° Sample ID ( optional)

° PWS ID ° Facility ID ° Sample collection point ID ° Sample collection date ° Analytical method number ° Method type ° Source water type ( provided by PWS on sample collection form)

° E. coli/ 100 mL ( see note below)

° Turbidity result ( provided by PWS on sample collection form)

Note: The laboratory may enter the final result for the E. coli sample or may enter the primary measurements recorded at the bench, and have the LT2 Data Collection System automatically calculate the final E. coli concentration. Because this information is specific to method type ( membrane filtration, multiple tube, 51 well, and 97 well), the system provides different entry screens for each method type. The laboratory 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 released to the PWS for 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 by the laboratory, 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.

4.6.2 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." Section 4: E. coli Laboratory Guidance 64 Draft June 2003 4.6.3 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.

4.7 Vendor Contact List for E. coli Methods Contact information for the vendors for materials used in the methods described in Section 4 is provided below. Footnotes indicate which vendors are associated with each method.

American Type Culture Collection1 www. atcc. org P. O. Box 1549 Manassas, VA 20108 Sales: Phone: ( 800) 638 6597 Fax: ( 703) 365 2750 Technical Support: Phone: ( 800) 638 6597 E mail: tech@ atcc. org Becton, Dickinson and Company2 www. bd. com 1 Becton Drive Franklin Lakes, NJ 07417 Sales: Phone: ( 800) 675 0908 Fax: ( 410) 316 4723 Technical Support: Phone: ( 800) 638 8663 Fax: ( 410) 527 0244 E mail: rep. technical_ services@ bd. com bioMérieux 6 www. biomerieux. com 595 Anglum Drive Hazelwood, MO 63042 Sales: Phone: ( 800) 638 4835 Fax: ( 800) 657 3053 Technical Support: Phone: ( 800) 638 4835 Fax: ( 800) 657 3053 Cole Parmer1 www. coleparmer. com 625 East Bunker Court Vernon Hills, IL 60061 Sales: Phone: ( 800) 323 4340 Fax: ( 847) 247 2929 E mail: sales@ coleparmer. com Technical Support: Phone: ( 800) 323 4340 ext. 122 Fax: ( 847) 327 2987 E mail: techinfo@ coleparmer. com Fisher Scientific1 www. fishersci. com 9999 Veterans Memorial Drive Houston, TX 77038 Sales: Phone: ( 800) 766 7000 Fax: ( 800) 926 1166 Technical Support: Phone: ( 800) 766 7000 Fax: ( 800) 926 1166 Hach 5 www. hach. com P. O. Box 389 Loveland, CO 80539 Sales: Phone: ( 800) 227 4224 Fax: ( 970) 669 2932 Technical Support: Phone: ( 800) 227 4224 Fax: ( 970) 669 2932 E mail: techhelp@ hach. com Biosynth International Inc. 2 www. biosynth. com 1665 West Quincy Avenue Naperville, IL 60540 Sales: Phone: ( 800) 270 2436 Fax: ( 800) 276 2436 Technical Support: Phone: ( 800) 270 2436 Fax: ( 800) 276 2436 E mail: craig@ biosynth. com Section 4: E. coli Laboratory Guidance 65 Draft June 2003 Hardy Diagnostics 2 www. hardydiagnostics. com 1430 West McCoy Lane Santa Maria, CA 93455 Sales: Phone: ( 800) 266 2222 Fax: ( 805) 346 2760 E mail: sales@ hardydiagnostics. com Technical Support: Phone: ( 800) 266 2222 ext 5658 Fax: ( 805) 346 2760 IDEXX 4 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 Pall Gelman Sciences 3 www. pall. com/ gelman 600 South Wagner Road Ann Arbor, MI 48103 Sales: Phone: ( 800) 521 1520 ext. 2 Fax: ( 734) 913 6495 Technical Support: Phone: ( 800) 521 1520 ext. 3 Fax: ( 734) 913 6495 Oxoid Inc. 2 www. oxoid. com Suite 100, 1926 Merivale Road Nepean, Ontario K2G1E8, Canada Sales: Phone: ( 613) 226 1318 Fax: ( 613) 226 3728 E mail: oxoid@ oxoid. com Technical Support: Phone: ( 800) 521 1520 ext. 3 Fax: ( 734) 913 6495 E mail: oxoid@ oxoid. com Millipore 3 www. millipore. com 80 Ashby Road Bedford, MA 01730 Sales: Phone: ( 800) 645 5476 Fax: ( 781) 533 3110 Technical Support: Phone: ( 800) 645 5476 Fax: ( 781) 533 3110 Sigma 2 www. sigma aldrich. com P. O. Box 14508 St. Louis, MO 63178 Sales: Phone: ( 800) 325 3010 Fax: ( 800) 325 8070 E mail: custserv@ sial. com Technical Support: Phone: ( 800) 325 5832 Fax: ( 314) 286 7828 E mail: techserv@ sial. com VWR1 www. vwr. com 405 Heron Drive Bridgeport, NJ 08014 Sales: Phone: ( 800) 932 5000 Fax: ( 856) 467 3336 Technical Support: Phone: ( 800) 932 5000 Fax: ( 856) 467 3336 1 Possible vendors for all E. coli methods 2 Vendors for methods: 9221B/ 9221F, 9222B/ 9222G, 9222D/ 9222G, 9213D, EPA Method 1603, EPA Method 1604 3 Vendors for methods: 9222B/ 9222G, 9222D/ 9222G, 9213D, EPA Method 1603, EPA Method 1604 4 Vendor for methods: 9221F, 9223B 5 Vendor for method: m ColiBlue24 broth 6 Vendor for API 20E for EPA Method 1604 66 Draft June 2003 SECTION 5: REFERENCES 5.1 Connell, Kevin, et al. 2000. ICRSS  Building a Better Protozoa Data Set, J. AWWA. 91( 10): 30  43.

5.2 USEPA. 1997. Manual for the Certification of Laboratories Analyzing Drinking Water; Criteria and Procedures; Quality Assurance: Fourth Edition. EPA 815 B 97 001.

5.3 APHA. 1998. Standard Methods for the Examination of Water and Wastewater; 20th Edition. American Public Health Association, American Water Works Association, Washington, D. C.

5.4 Pope, M., et al. 2002. Assessment of the effects of holding time and temperature on E. coli concentrations in surface water samples. Appl. Environ. Micro. ( submitted). 67 Draft June 2003 SECTION 6: ACRONYMS CFU Colony forming unit CNFG Confluent growth DAPI 4, 6 diamidino 2 phenylindole DIC Differential interference contrast microscopy EPA U. S. Environmental Protection Agency FA Immunofluorescense assay FITC Fluorescien isothiocyanate ICR Information Collection Rule IDC Initial demonstration of capability IFA Immunofluorescence assay IMS Immunomagnetic separation IPR Initial precision and recovery IPT Initial proficiency test L Liter LT2 rule Long Term 2 Enhanced Surface Water Treatment Rule MPC Magnetic particle concentrator MPN Most probable number MS Matrix spike MS/ MSD Matrix spike/ matrix spike duplicate NA MUG Nutrient Agar ( NA) with 4 methylumbelliferyl beta D glucuronide ( MUG)

NELAC National Environmental Laboratory Accreditation Conference NPDWR National Primary Drinking Water Regulations OPR Ongoing precision and recovery OPT Ongoing proficiency testing PBMS Performance based measurement system PT Proficiency testing PWS Public water system QA Quality assurance QAP Quality Assurance Plan QC Quality control Section 4: E. coli Laboratory Guidance 68 Draft June 2003 RSD Relative standard deviation SDWA Safe Drinking Water Act SOP Standard operating procedure TNTC Too numerous to count UV Ultraviolet