OMB Control Number: 2040 0246 Expiration Date: 07/ 31/ 02 United States Environmental Protection Agency Office of Ground Water and Drinking Water Technical Support Center April 29, 2002 Dear Laboratory Manager:

Thank you for your interest in the U. S. EPA  s Laboratory Quality Assurance Evaluation Program for Analysis of Cryptosporidium under the Safe Drinking Water Act ( Lab QA Program). This is a voluntary program open to laboratories analyzing Cryptosporidium in water using EPA Method 1622 and EPA Method 1623. To increase the likelihood that laboratories analyzing water samples for Cryptosporidium generate reliable data, EPA has established the following process for evaluating laboratory performance and quality assurance practices:

Step 1. Application. Laboratories must first submit the Laboratory QA Program application. The application forms are enclosed with this letter, and the application requirements are described in detail below. EPA will evaluate laboratory applications to confirm the following: ( 1) the laboratory has the equipment required in EPA Method 1622 and/ or EPA Method 1623 ( April 2001), ( 2) laboratory personnel have the recommended experience to analyze samples, and ( 3) the laboratory has successfully completed the initial precision and recovery ( IPR) and matrix spike/ matrix spike duplicate ( MS/ MSD) tests specified in the method. Laboratories that do not meet these requirements will be requested to correct any deficiencies before proceeding to the next step in the evaluation process.

Step 2. Performance evaluation. After an application has been accepted, the laboratory will be sent a set of eight initial proficiency testing ( IPT) samples consisting of a suspension of oocysts in a concentrated matrix. Laboratories will resuspend these spikes in reagent water to produce simulated source water samples, and analyze the samples using the version of Method 1622/ 1623 ( April 2001) that the laboratory plans to use for routine Cryptosporidium analyses. If a laboratory wishes to be evaluated for more than one version of the method, the laboratory will receive a set of eight PT samples for each version.

Laboratory IPT data will be evaluated against the mean recovery and precision ( as relative standard deviation) criteria for the IPT samples. Laboratories will receive two opportunities to pass the IPT test. If a laboratory fails two times, it will not be eligible for another set until after the laboratory staff has received additional training in performing the method.

Laboratories already participating in the EPA Protozoa Performance Evaluation ( PE) Program, may use the initial round of samples from the PE program to meet the IPT sample requirement.

Step 3. On site evaluation. After a laboratory passes the IPT, an on site evaluation of the laboratory will be scheduled. The on site evaluation will include two separate, but concurrent assessments: ( 1) assessment of the laboratory  s sample processing and analysis procedures, including microscopic examination, and ( 2) evaluation of the laboratory  s personnel qualifications, quality control program, equipment, and record keeping procedures. Each laboratory will receive an audit report, which will document deficiencies, if any, that should be corrected by the laboratory. After a laboratory has corrected any deficiencies noted in the audit report, EPA will confirm that the laboratory meets the performance criteria of the Laboratory Quality Assurance Program for Analysis of Cryptosporidium under the Safe Drinking Water Act.

Laboratories that meet the program performance criteria will also receive a set of three ongoing proficiency testing ( OPT) samples approximately every four months that must be analyzed in the same manner as the IPT samples. EPA will evaluate the precision and recovery data for OPT samples to determine if the laboratory continues to meet the performance criteria of the Laboratory QA Program.

Application Requirements The first step in the laboratory evaluation process is submission of a laboratory application package. The following materials should be submitted for each laboratory application package:

1. Signed, completed application form ( attached).

2. Completed self audit checklist ( attached). This checklist is similar to the checklist that will be used to audit your laboratory during the on site evaluation.

3. Resumes detailing qualifications of your laboratory  s proposed principal analyst/ supervisor and each analyst and technician listed on the application form and documentation of the training, including the number of samples analyzed by each ( the list for each analyst and technician should include at a minimum the number of samples specified below for personnel prerequisites).

The recommended personnel prerequisites for the laboratory evaluation program are as follows:

Principal Analyst/ Supervisor ( one required per laboratory)   BS/ BA in microbiology or closely related field   A minimum of 1 year of continuous bench experience with Cryptosporidium and IFA microscopy   A minimum of 6 months experience using EPA Method 1622 and/ or EPA Method 1623   A minimum of 100 samples analyzed using EPA Method 1622 and/ or EPA Method 1623 ( minimum 50 samples if the person was an approved analyst for Cryptosporidium under the Information Collection Rule( ICR))

Other Analysts ( no minimum requirement per laboratory)   Two years of college in microbiology or equivalent or closely related field   A minimum of 6 months of continuous bench experience with Cryptosporidium and IFA microscopy   A minimum of 3 months experience using EPA Method 1622 and/ or EPA Method 1623   A minimum of 50 samples analyzed using EPA Method 1622 and/ or EPA Method 1623 ( minimum 25 samples if the person was an ICR approved analyst)

Technician ( no minimum requirement per laboratory)   Three months experience with the specific parts of the procedure he/ she will be performing   A minimum of 50 samples analyzed using EPA Method 1622 and/ or EPA Method 1623 ( minimum 25 samples if the person was an ICR approved technician) for the specific analytical procedures they will be using. 4. Detailed laboratory standard operating procedures for each version of the method your laboratory plans on using for routine Cryptosporidium analyses. SOP  s for the following should be included:

  Performance of each method step including, sample spiking, filtration, elution, concentration, purification, slide preparation, sample staining and examination   Dividing pellets greater than 0.5mL   Preparation of reagents   Dishwashing   Staff training   Corrective action procedures for failing to meet OPR, method blank, staining controls, sample acceptance, and performance verification criteria   Sampling procedures to be followed by field or utility personnel   Procedures for data recording, checking manual calculations, and checking accuracy of all data transcriptions 5. EPA Method 1622 or EPA Method 1623 initial demonstration of capability ( IDC) data which include initial precision and recovery ( IPR) test results and matrix spike and matrix spike duplicate ( MS/ MSD) test results for Cryptosporidium. The IPR test consists of four reagent water samples spiked with between 100  500 oocysts and one method blank. The MS/ MSD test consists of one unspiked and two spiked source water samples. These tests are described in Section 9 of EPA Method 1622 and EPA Method 1623 and the results should meet the criteria in the method ( April 2001 version). The following data should be submitted:

  Completed EPA Method 1622/ 1623 bench sheets and report forms for each of the eight samples ( attached)   Initial demonstration of capability summary form ( attached)   Spiking suspension preparation data. This should include completed spiking suspension sheets ( attached) if spikes were prepared by your laboratory or flow cytometer calibration forms if spikes were obtained from an external source.

Laboratories wishing to be evaluated for more than one version of the method ( different volumes, filters, elution and concentration procedures, and immunomagnetic separation kits) should submit a complete set of IPR and MS data for each version.

If your laboratory currently participates in the EPA PT sample program and the required IDC data have already been submitted, the data do not need to be resubmitted. Please indicate this is the case on the initial demonstration of capability summary form.

6. Table of contents from your laboratory  s quality assurance plan. The quality assurance plan should specifically address the requirements of protozoa analysis under the programs for which the laboratory intends to analyze samples.

7. An example of the data reporting form used to submit Cryptosporidium results to your clients.

8. A statistical summary of percent recoveries for all OPR and MS samples analyzed at your laboratory for the past six months.

Application materials should be submitted to the following address: Jennifer Scheller Cryptosporidium Laboratory QA Program Coordinator DynCorp Biology Studies Group 6101 Stevenson Avenue Alexandria, VA 22304 Send comments on the Agency's need for this information, the accuracy of the provided burden estimates, and any suggested methods for minimizing respondent burden, including through the use of automated collection techniques to the Director, Collection Strategies Division, U. S. Environmental Protection Agency ( 2822T), 1200 Pennsylvania Ave., NW, Washington, D. C. 20460. Include the OMB control number in any correspondence. Do not send the completed form to this address.

When your application package has been received and reviewed, you will be notified whether it is complete or has any deficiencies. After your application has been accepted, you will be notified of when you should expect your initial set of PT samples. If you have any questions about the laboratory application materials or evaluation process, please feel free to contact either me at 513 569 7944 or Jennifer Scheller at 703 461 2118.

Sincerely,

Mary Ann Feige Cryptosporidium Laboratory Quality Assurance Evaluation Program Manager 26 West Martin Luther King Drive Cincinnati, OH 45268 Attachments Burden Statement: The public reporting and recordkeeping burden for this collection of information is estimated to average 18 hours per response or 72 hours per respondent annually. Burden means the total time, effort, or financial resources expended by persons to generate, maintain, retain, or disclose or provide information to or for a Federal agency. This includes the time needed to review instructions; develop, acquire, install, and utilize technology and systems for the purposes of collecting, validating, and verifying information, processing and maintaining information, and disclosing and providing information; adjust the existing ways to comply with any previously applicable instructions and requirements; train personnel to be able to respond to a collection of information; search data sources; complete and review the collection of information; and transmit or otherwise disclose the information. An agency may not conduct or sponsor, and a person is not required to respond to, a collection of information unless it displays a currently valid OMB control number. OMB Control Number: 2040 0246 Expiration Date: 07/ 31/ 02 Application for the Laboratory Quality Assurance Evaluation Program for Analysis of Cryptosporidium under the Safe Drinking Water Act Part 1. Laboratory Information Laboratory Name:

Address:

City: State: Zip:

Contact Person:

Title:

Telephone: Fax:

Email address:

Type of laboratory ( circle one): Commercial Utility State Academic Other Was your laboratory ICR approved for protozoa? 9 Yes 9 No Is your laboratory currently participating in the EPA PE Program? 9 Yes 9 No Number of field samples analyzed by your laboratory using Method 1622/ 1623:

Number of spiked samples analyzed by your laboratory using Method 1622/ 23:

Number of fields samples your laboratory can currently analyze per month using Method 1622/ 1623: Number of field samples your laboratory could potentially analyze per month using Method 1622/ 1623 during LT2:

1 April 30, 2002 Part 2. Method Information: Versions of Method 1622/ 1623 for which the lab is seeking evaluation Method step Method 1622 ( Cryptosporidium only) Method 1623 ( Cryptosporidium & Giardia)

Filtration ( check all that apply and indicate the volume filtered for each)

Gelman Envirochek Gelman HV Envirochek IDEXX FiltaMax Whatman CrypTest Other ( describe)

Elution ( check all that apply)

Wrist action shaker ( Envirochek)

Stomaching of FiltaMax filter FiltaMax wash station plunger Back Wash/ Sonication ( CrypTest)

Other ( describe)

Concentration ( check all that apply)

Centrifugation Filtration through membrane Other ( describe)

Purification ( check all that apply)

Dynal anti Crypto, Dynal GC combo Other ( describe)

Staining ( check all that apply)

Waterborne AquaGlo Waterborne Crypt a Glo Waterborne Giardi a Glo Meridian Merifluor Other ( describe)

Descriptions of   other  method steps and other comments:

2 April 30, 2002 Part 3. Personnel Information ( attach additional sheets if necessary) 1. Principal Analyst/ Supervisor : one required per approved laboratory Name Current position Academic training/ degree( s) Time in current position No. of samples processed for protozoa analyses No. of samples processed using Methods 1622/ 1623 Was this person approved as an analyst under the ICR? 9 Yes 9 No Portions of method currently performed ( circle all that apply): Filtration Elution Concentration IMS Staining Examination 2. Analyst or Technician ( circle one)

Name Current position Academic training/ degree( s) Time in current position No. of samples processed for protozoa analyses No. of samples processed using Methods 1622/ 1623 Was this person approved under the ICR? 9 Yes 9 No If yes then check one: 9 Analyst 9 Technician Portions of method currently performed ( circle all that apply): Filtration Elution Concentration IMS Staining Examination 3. Analyst or Technician ( circle one)

Name Current position Academic training/ degree( s) Time in current position No. of samples processed for protozoa analyses No. of samples processed using Methods 1622/ 1623 Was this person approved under the ICR? 9 Yes 9 No If yes then check one: 9 Analyst 9 Technician Portions of method currently performed ( circle all that apply): Filtration Elution Concentration IMS Staining Examination 4. Analyst or Technician ( circle one)

Name Current position Academic training/ degree( s) Time in current position No. of samples processed for protozoa analyses No. of samples processed using Methods 1622/ 1623 Was this person approved under the ICR? 9 Yes 9 No If yes then check one: 9 Analyst 9 Technician Portions of method currently performed ( circle all that apply): Filtration Elution Concentration IMS Staining Examination 5. Analyst or Technician ( circle one)

Name Current position Academic training/ degree( s) Time in current position No. of samples processed for protozoa analyses No. of samples processed using Methods 1622/ 1623 Was this person approved under the ICR? 9 Yes 9 No If yes then check one: 9 Analyst 9 Technician Portions of method currently performed ( circle all that apply): Filtration Elution Concentration IMS Staining Examination 3 April 30, 2002 Part 4. Laboratory Equipment Confirmation Checklist for Methods 1622 and 1623 Key Equipment and Reagents Manufacturer/ Model If not Present, Proof of Purchase Attached ( Y/ N)

Filtration and elution Flow control valve  0.5 gpm Centrifugal or other pump Low flow meter or graduated container Laboratory shaker for agitating capsule filters ( Envirochek only)

Laboratory shaker side arms ( Envirochek only)

Filter housing ( CrypTest or Filta Max)

Wash station ( Filta Max only)

Stomacher ( Filta Max only)

Compressed air source ( CrypTest only)

Sonicator ( CrypTest only)

Concentration Concentrator apparatus ( Filta Max only)

1500 X G, swinging bucket centrifuge for 15 mL  250 mL tubes Immunomagnetic separation Sample mixer/ rotator for 10 mL tubes Magnetic particle concentrator for 10 mL tubes Magnetic particle concentrator for 1.5 mL tubes Flat sided sample tubes Examination Epifluorescence/ differential interference contrast microscope with stage and ocular micrometers and 20X to 100X objectives Excitation/ band pass microscope filters for fluorescein isothiocyanate ( FITC) assay Excitation/ band pass filters for 4', 6 diamidino 2 phenylindole ( DAPI) assay The above application information is complete and accurate to the best of my knowledge.

Name and Signature Laboratory Manager or Designee Date Submit application package to: Jennifer Scheller, Cryptosporidium Laboratory Quality Assurance Evaluation Program Coordinator, DynCorp I& ET, 6101 Stevenson Avenue, Suite 500, Alexandria, VA 22304 4 April 30, 2002 Audit Checklist ( To Be Used for Self Audit)

Part A: Facilities, Equipment, and Quality Assurance Item to be Evaluated Classification Yes, No, Unknown*, or NA 1 Laboratory Equipment and Supplies 1.1 Reagent grade water testing 1.1.1 Is reagent water tested monthly for these minimum parameters: conductivity, total chlorine residual; and annually for metals Pb, Cd, Cr, Cu, Ni, Zn? Requirement 1.1.2 Were the results for the above parameters acceptable, total chlorine residual not greater than 0.1 mg/ L, conductivity not greater than 2 µ mhos/ cm, and each metal not greater than 0.05 mg/ L and collectively not greater than 0.1 mg/ L? Requirement 1.1.3 Is reagent water tested monthly for heterotrophic plate count? Requirement 1.1.4 Are the results for the heterotrophic plate count acceptable, < 500/ mL? Requirement 1.2 Laboratory pH meter:

1.2.1 Accuracy ± 0.1 units, scale graduations, 0.1 units? Requirement 1.2.2 Is a record maintained for pH measurements and calibrations used? Requirement 1.2.3 Is pH meter standardized each use period with pH 7, 4 or 10 standard buffers ( selection dependant upon desired pH)? Requirement 1.2.4 All pH buffers are dated when received and opened and are discarded before expiration date? Requirement 1.3 Balances ( top loader or pan balance):

1.3.1 Are balances calibrated monthly using Class S/ S 1 weights, or weights traceable to Class S/ S 1 weights? Requirement 1.3.2 Is correction data available with S/ S 1 weights? Requirement 1.3.3 Is preventative maintenance conducted yearly at a minimum? Recommendation 1.4 Autoclave:

1.4.1 Is unit equipped with a temperature gauge/ operational safety valve? Requirement 1.4.2 Are date, contents, sterilization time and temperature recorded for each cycle? Requirement 1.4.3 Is a maximum registering thermometer or continuous monitoring device used during each autoclave cycle? Requirement 1.4.4 Is automatic timing mechanism checked with stopwatch quarterly? Requirement 1.4.5 Are spore strips or ampules used monthly to confirm sterilization? Requirement 1.5 Refrigerator/ Freezer:

1.5.1 Is refrigerator able to maintain temperature of 1 ° C to 5 ° C? Requirement 1.5.2 Is temperature recorded once daily for days in use? Requirement 1.6 Temperature recording device:

1.6.1 Are calibration of glass/ mercury thermometers checked annually ( dial thermometers quarterly) at the temperature used against a reference NIST thermometer or equivalent? Requirement 1.7 Micropipetters:

1.7.1 Have micropipetters been calibrated within the past year? [ Section 9.2.1] Requirement 1.8 Centrifuge 1.8.1 Is a maintenance contract in place, or internal maintenance protocol available? Requirement 1.8.2 Is RPM and RCF calibrated yearly? Requirement 1.9 General 1.9.1 Are calibration and maintenance records complete and well organized? Recommendation 5 April 29, 2002 Item to be Evaluated Classification Yes, No, Unknown*, or NA 2 Quality Assurance 2.1 Does the laboratory have a formal QA laboratory plan prepared and ready for examination? Requirement 2.2 Are employee resumes present and complete? Requirement 2.3 Is a training protocol for new employees present? Recommendation 2.4 Is the laboratory performing analyst verification of examination monthly and does the lab have corrective action procedures in place if criteria are not met? ( Section 10.5) Requirement 2.5 Are employee training records available and up to date? Requirement 2.5.1 Have technicians/ analysts analyzed the required number of samples using Method 1622/ 1623? Requirement 2.6 Are all relevant SOPs present and current? Requirement 2.7 Are sampling instructions present for clients collecting and/ or filtering samples in the field? Requirement 2.8 Does the laboratory have criteria for sample acceptance and corrective action procedures? Requirement 2.9 Are data recording procedures present? Requirement 2.9.1 Does the laboratory have an SOP for checking all manual calculations? Requirement 2.10 Are corrective action contingencies present? Requirement 2.10.1 For OPR failures? [ Section 9.7.4] Requirement 2.10.2 For method blank contamination? Requirement 2.10.3 For positive/ negative staining control failures? Requirement 2.11 Does the quality assurance plan specifically address requirements for protozoa analysis under the programs for which the laboratory intends to analyze samples? Requirement 2.12 Is a laboratory organization chart or other information available listing staff organization and responsibilities? Does it identify the QA manager? Requirement 2.12.1 Is the QA manager separate from the lab manager? Recommendation 2.13 Does the laboratory have a list of preventative maintenance procedures and schedules? Requirement 2.14 Date range covered for quality control ( QC) sample audit?

2.15 When did the laboratory begin processing samples with the Envirochek filter? / /

2.16 When did the laboratory begin processing samples with the Filta Max filter ( if applicable)? / /

2.17 When did the laboratory begin processing samples with the CrypTest filter ( if applicable)? / /

2.18 Approximately how many field samples were analyzed using methods 1622/ 1623 since the lab started using Method 1622/ 1623? Field samples ___ MS_____

2.19 Have acceptable initial precision and recovery analyses been performed for each version of the method the laboratory is using? Requirement 2.20 Were method blanks run once per week or per 20 samples during this period? [ Section 9.6.1] Requirement 2.20.1 If the answer to 2.20 is no, then at what frequency where method blanks performed?

2.20.2 What percentage of method blanks evaluated were without contamination?

2.20.3 Was an acceptable method blank associated with each field sample examined? Requirement 2.20.4 How many method blanks were evaluated?

2.21 Were ongoing precision and recovery ( OPR) samples run once per week or per 20 samples during this period? [ Section 9.7] Requirement 2.21.1 If the answer to 2.21 is no, then at what frequency where OPR samples performed?

2.21.2 What percentage of OPR samples evaluated met the recovery criteria? [ Table 3; Section 9.7.3]

6 April 29, 2002 Item to be Evaluated Classification Yes, No, Unknown*, or NA 2.21.3 Does the laboratory maintain control charts of OPR results? [ Section 9.7.5] Requirement 2.21.4 Was an acceptable OPR associated with each field sample examined? Requirement 2.21.5 How many OPR samples were evaluated?

2.21.6 How many OPR samples were analyzed during the past six months?

2.21.7 What is the mean and relative standard deviation of the recoveries of the OPR samples analyzed during the past six months? Mean_______ RSD________

2.22 Were matrix spike ( MS) samples analyzed at the method  specified frequency? [ Section 9.1.8] Requirement 2.22.1 If the answer to 2.22 is no, then at what frequency were MS samples analyzed?

2.22.2 How many MS samples were evaluated?

2.22.3 How many MS samples were analyzed during the past six months?

2.22.4 What is the mean and relative standard deviation of the MS samples analyzed during the past six months? Mean_______ RSD________

2.23 Were OPR and MS samples spiked with 100  500 organisms? [ Section 9.7] Requirement 2.23.1 If the answer to 2.23 is no, then at what level were samples spiked?

2.24 Are the laboratory personnel performing the QC analyses representative of the personnel seeking approval under this program? Requirement 2.25 Does the laboratory have records of all QC checks available for inspection? Requirement 2.26 Does the laboratory have an adequate record system for tracking samples from collection through log in, analysis, and data reporting? Requirement 2.27 Are results from each sample maintained electronically?

2.28 If data are stored electronically, are files backed up on more than one disk to ensure data are not lost in the eventuality of some hardware failure? Requirement 2.29 If data is stored electronically, does the laboratory have an SOP for checking the accuracy of data entry into an electronic system? Requirement 2.30 Is the laboratory using the April 2001 version of Method 1622/ 1623? Requirement 3 Data Recording Procedures 3.1 Is shipping information complete, including the time and date of sample receipt, sample condition, and noting any discrepancies between samples on the traffic report and samples received? Requirement 3.2 Do sample numbers on the shipping forms match the sample numbers on the report forms? Requirement 3.3 Are current Method 1622/ 1623 bench sheets used to record sample processing data? Recommendation 3.4 Are all primary measurements during each step recorded, including all raw data used in calculations? Requirement 3.5 Name of analyst or technician performing the elution is recorded? Requirement 3.6 Date and time of elution is recorded? Requirement 3.7 Name of analyst or technician performing the concentration is recorded? Requirement 3.8 Date and time of concentration is recorded? Requirement 3.9 Are batch and lot numbers of reagents used in the analysis of the sample recorded? Requirement 3.10 Lot number for the IMS kit is recorded? Requirement 3.11 Are Method 1622/ 1623 Cryptosporidium report forms used to record sample examination results? Requirement 3.12 Name of examining analyst is recorded? Requirement 3.13 Date and time of sample examination is recorded? Requirement 3.14 Are calculations of final concentrations and recoveries complete and correct? Requirement 7 April 29, 2002 Item to be Evaluated Classification Yes, No, Unknown*, or NA 3.15 Do values recorded on the data sheets match the reported values? Requirement 3.16 Are mistakes on all forms crossed out with a single line, initialed, and dated? Requirement 3.17 Are data always recorded in pen? Requirement 3.18 Are hardcopy records well organized, complete, and easily accessible? Requirement 3.19 Does the laboratory include a disclaimer on the report to the client if method QC requirements were not met? Recommendation 3.20 Is the manually recorded data legible? Requirement 3.21 Do records demonstrate each analyst's characterization of 3 oocysts and 3 cysts from positive control for each microscopy session? [ Section <IP_ADDRESS>] Requirement 3.22 Data shows that no more than 0.5 mL of pellet was used per IMS? [ Section 13.2.4] Requirement 4 Holding Times 4.1 Samples analyzed according to December 1999 version of Method 1622/ 1623 4.1.1 Is time from initiation of sample collection to completion of concentration 72 hours or less? [ Section 8.1] Requirement 4.1.2 Concentrate is held no longer than 24 hours between IMS and staining? [ Section 8.2] Requirement 4.1.3 Are stained slides read and confirmed within 72 hours of staining? [ Section 8.4] Requirement 4.2 Samples analyzed according to April 2001 version of Method 1622/ 1623 4.2.1 Is sample elution initiated within 96 hours of sample collection or field filtration? [ Section 8.2.1] Requirement 4.2.2 Are sample elution, concentration, and purification steps completed in one work day? [ Section 8.2.2] Requirement 4.2.3 Are slides stained within 72 hours of application of the purified sample to the slide? [ Section 8.2.3] Requirement 4.2.4 Are stained slides read and confirmed within 7 days of staining? [ Section 8.2.4] Requirement 5 Spike enumeration procedures 5.1 What method does the laboratory currently use to estimate spike doses:( A) flow sorted spikes, ( B) well slide counted spikes, ( C) hemacytometer counted spikes, or ( D) membrane filter counted spikes Circle one: A B C D 5.1.1 If flow sorted spikes are used, on what date did the laboratory begin using flow sorted spikes? / /

5.1.2 If counted manually, does the laboratory follow Method 1622/ 1623 procedures for establishing spike level? [ Section 11.3] Requirement 5.1.3 What were the relative standard deviations of the last four spike enumerations? 1.

2.

3.

4.

5.2 Source of oocysts for spikes 5.3 If 50 L samples are analyzed, what positive control procedure does the laboratory follow for OPR and MS samples: ( A) spike entire 50 L, ( B) spike and filter 10 L before filtering 40 L, or ( C) filter 40 L before spiking and filtering 10 L.

* Unknown response requires an explanation Note: All section references in [ ] refer to Method 1623 April 2001 8 April 29, 2002 Part B: Sample Processing and Examination Item to be evaluated Classification Yes, No, NA or Unknown 6 Laboratory Facilities and Laboratory Safety 6.1 Are laboratory coats and gloves worn in the laboratory? Requirement 6.2 No other safety or facility issues were observed?

7 Sample Spiking Technician:

7.1 What method does laboratory currently use to estimate spike doses:( A) flow sorted spikes, ( B) well slide counted spikes, ( C) hemacytometer counted spikes, or ( D) membrane filter counted spikes Circle one: A B C D 7.2 With what filter type did the laboratory demonstrate their spiking procedure?

7.3 Is the carboy used for negative control randomly selected from carboy stock to check efficacy of cleaning system? Requirement 7.4 If flow sorted spikes are used, was suspension vial vortexed for two minutes or per manufacturers instructions? [ Section 11.4.3] Method Procedure 7.5 Was the suspension vial adequately rinsed? [ Section <IP_ADDRESS>] Method Procedure 7.6 Does the laboratory have an acceptable SOP for sample spiking? Requirement 7.7 Other than the issues noted for items 7.2 through 7.6 ( if any) was sample spiking demonstrated successfully?

8 Envirochek ( Complete Sections that apply)

8.1 Envirochek Filtration Technician:

8.1.1 Are all components required for sample filtration present and in good condition? [ Section 6.2] Requirement 8.1.2 Is the filter assembly set up correctly? [ Figure 3, pg 48] Method Procedure 8.1.3 Is the pump adequate for needs? [ Section 6.3.3] Requirement 8.1.4 Is the appropriate flow rate maintained ( approximately 2L/ min)? [ Section <IP_ADDRESS>] Method Procedure 8.1.5 Is the volume filtered measured using a flow meter or calibrated carboy? [ Section <IP_ADDRESS>] Requirement 8.1.6 Is the system well maintained and cleaned appropriately following use? Requirement 8.1.7 Is the system able to maintain seal during use with no leaks? Requirement 8.1.8 Does the laboratory have an acceptable SOP for Envirochek filtration? Requirement 8.1.9 Other than the issues noted in items 8.1.1 through 8.1.8, was Envirochek filtration demonstrated successfully?

8.2 Envirochek capsule filter elution Technician:

8.2.1 Is the elution buffer prepared as per Method 1622/ 1623? [ Section 7.4] Method Procedure 8.2.2 Is the wrist shaker assembly set up correctly? [ Section <IP_ADDRESS>.1] Method Procedure 8.2.3 Does the eluting solution cover the membrane? [ Section <IP_ADDRESS>.2] Method Procedure 8.2.4 Are the samples shaken at an appropriate speed? [ Section <IP_ADDRESS>.3] Method Procedure 8.2.5 Are the samples shaken three times for 5 minutes each time, and each in a different orientation? [ Section <IP_ADDRESS>] Method Procedure 8.2.6 Does the laboratory have an acceptable SOP for Envirochek capsule filter elution? Requirement 8.2.7 Other than the issues noted for items 8.2.1 through 8.2.7 ( if any) was Envirochek filter elution demonstrated successfully?

9 CrypTest 9.1 CrypTest Filtration Technician:

9.1.1 Are all components required for sample filtration present and in good condition? [ Section 6.2.3] Requirement 9 April 29, 2002 Item to be evaluated Classification Yes, No, NA or Unknown 9.1.2 Is the filter assembly set up correctly? Method Procedure 9.1.3 Is the pump adequate for needs? [ Section 6.3.3] Requirement 9.1.4 Is the appropriate flow rate maintained ( approximately 2L/ min)? Method Procedure 9.1.5 Is the volume filtered measured using a flow meter or a calibrated carboy? Requirement 9.1.6 Is the system well maintained and cleaned appropriately following use? Requirement 9.1.7 Is the system able to maintain seal during use with no leaks? Requirement 9.1.8 Does the laboratory have an acceptable SOP for CrypTest Filtration? Requirement 9.1.9 Other than the issues noted in items 9.1.3 through 9.1.10 ( if any) was CrypTest filtration demonstrated successfully?

9.2 CrypTest cartridge filter elution Technician:

9.2.1 Does the filter seat properly in the filter housing, so there are no leaks? Requirement 9.2.2 Is the elution buffer prepared according to manufacturer  s instructions? [ Section 7.4.2] Method Procedure 9.2.3 Is an appropriate amount of elution solution backwashed into the filter housing? ( approx. 150 mL) Method Procedure 9.2.4 Is the assembly well sealed ( no leaks)? Requirement 9.2.5 Is sonication performed for 2 minutes? Method Procedure 9.2.6 Is the filter elution repeated, according to the manufacturer  s instructions? Method Procedure 9.2.7 Following the last elution, is the remaining elution buffer driven from the outlet side to the inlet side and into the sample bottle? Requirement <IP_ADDRESS> Is the regulated compressed air source used, sufficient to drive the eluting buffer from the filter? Requirement 9.2.8 After elution is complete, is the filter removed from the housing and the base, lid, and lip of the filter housing rinsed using eluting solution and added to the sample bottle? Requirement 9.2.9 Does the laboratory have an acceptable SOP for CrypTest elution? Requirement 9.2.10 Other than the issues noted in items 9.2.1 through 9.2.9 ( if any) was CrypTest filter elution demonstrated successfully?

10 Filta Max 10.1 Filta Max filtration Technician:

10.1.1 Are all components required for sample filtration present and in good condition? [ Section 6.2.4] Requirement 10.1.2 Is the filter assembly set up correctly? Method Procedure 10.1.3 Is appropriate flow rate maintained of < 4 L per minute? Method Procedure 10.1.4 Is the volume filtered measured correctly using a flow meter or calibrated carboy? Requirement 10.1.5 Is system well maintained and cleaned appropriately following use? Requirement 10.1.6 Is system able to maintain seal during use with no leaks? Requirement 10.1.7 Does the laboratory have an acceptable SOP for Filta Max filtration? Requirement 10.1.8 Does the laboratory indicate on the filter housing the correct direction of flow? Requirement 10.1.9 Other than the issues noted in items 10.1.1 through 10.1.8 ( if any) was Filta Max filtration demonstrated successfully?

10.2 Filta Max filter wash station elution Technician:

10.2.1 Is an automatic or manual wash station used?

10.2.2 Is the filter wash station set up correctly? Requirement 10.2.3 Is PBST used to elute the filter? [ Section 7.4.2] Method Procedure 10 April 29, 2002 Item to be evaluated Classification Yes, No, NA or Unknown 10.2.4 Is an appropriate amount of PBST used for each wash? ( approx. 600 mL) Method Procedure 10.2.5 During the first wash, is the plunger moved up and down 20 times? Method Procedure 10.2.6 Is the plunger moved up and down gently to avoid generating excess foam? Method Procedure 10.2.7 During the second wash, is the plunger moved up and down 10 times? Method Procedure 10.2.8 If the automatic washer is used, is the machine operating properly? Requirement 10.2.9 Is the wash station cleaned adequately between samples? Requirement 10.2.10 Does the laboratory have an acceptable SOP for Filta Max elution with the wash station? Requirement 10.2.11 Other than the issues noted for items 10.2.2 through 10.2.10 ( if any) was elution of the Filta max filter using the wash station demonstrated successfully?

10.3 Filta Max filter stomacher elution Technician 10.3.1 Is PBST used to elute the filter? [ Section <IP_ADDRESS>] Method Procedure 10.3.2 Is an appropriate amount of PBST used for each wash? ( approx. 600 mL) Method Procedure 10.3.3 Are two washes performed for 5 minutes each? Method Procedure 10.3.4 Is the stomacher in good condition and operating properly? Requirement 10.3.5 Does the laboratory have an acceptable SOP for Filta Max elution using a stomacher? Requirement 10.3.6 Other than the issues noted for items 10.3.1 through 10.3.5 ( if any) was elution of the Filta Max filter using the stomacher demonstrated successfully?

10.4 Filta Max filter sample concentration ( as an alternative to Section 11) Technician:

10.4.1 Is concentrator set up correctly? Requirement 10.4.2 Is the force of the vacuum maintained below 30 cm Hg? Method Procedure 10.4.3 Is concentration performed after each of the washes? Method Procedure 10.4.4 Is the concentrate from the first wash added to the 600mL of eluate from the second wash? Method Procedure 10.4.5 Is the sample concentrated so that some liquid remains above the filter ( enough to cover the stirbar about half way)? Method Procedure 10.4.6 Is the stir bar and concentration tube rinsed after each concentration and the liquid added to the concentrate? Requirement 10.4.7 Was the filter membrane washed twice? Method Procedure 10.4.8 Was 5 mL of PBST used each time? Method Procedure 10.4.9 Is the membrane adequately washed to remove oocysts from filter? Method Procedure 10.4.10 Is the pellet volume determined? Requirement 10.4.11 Is there a set of standards for comparison of pellet size? Recommendation 10.4.12 Does the laboratory have an acceptable SOP for concentration using the Filta Max concentrator? Requirement 10.4.13 Other than the issues noted in items 10.4.1 through 10.4.12 ( if any) was sample concentration using the Filta Max concentrator demonstrated successfully?

11 Concentration 11.1 Envirochek, CrypTest, and Filta Max filter sample centrifugation Technician:

11.1.1 Is the sample centrifuged at 1500 x G using a swinging bucket rotor? [ Section 13.2.1] Method Procedure 11.1.2 Are the centrifuge tubes properly balanced prior to centrifugation? Requirement 11.1.3 Is the sample centrifuged for 15 minutes? [ Section 13.2.1] Method Procedure 11.1.4 Is the centrifuge slowly decelerated at the end without the brake? [ Section 13.2.1] Method Procedure 11.1.5 Is the pellet volume determined? Requirement 11 April 29, 2002 Item to be evaluated Classification Yes, No, NA or Unknown 11.1.6 Is there a set of standards for comparison of pellet size? Recommendation 11.1.7 Does the laboratory have an acceptable SOP for sample concentration? Requirement 11.1.8 Is residual suspension rinsed from all containers and gloves? Requirement 11.1.9 Other than the issues noted in items 11.1.1 through 11.1.8 ( if any) was sample concentration demonstrated successfully?

12 Reagents, equipment and clean up 12.1 Source for reagent grade water:

12.1.1 Is still or DI unit maintained according to manufacturer's instructions? Requirement 12.1.2 Is reagent grade water used to prepare all media and reagents? [ Section 7.3] Requirement 12.2 Centrifuge:

12.2.1 Does centrifuge have a swinging bucket rotor? [ Section 6.8.1] Requirement 12.2.2 Is the centrifugation nomograph for determining relative centrifugal force located close to the centrifuge( s)? Requirement 12.3 SOP  s for Reagents 12.3.1 Are SOP  s available for the preparation of all essential chemicals and reagents? Requirement 12.3.2 Are SOP  s posted or easily accessible at the bench? Recommendation 12.3.3 Are all reagents clearly labeled with date of preparation, technician initials, and expiration date? Requirement 12.4 Clean up 12.4.1 Is all glassware and plasticware washed well and stored appropriately between uses? Requirement 12.4.2 Is distilled or deionized water used for final rinse? Requirement 12.4.3 Is an SOP available for glassware washing? Requirement 13 Purification and Slide Preparation Technician:

13.1 What IMS kit/ manufacturer is used?

13.2 Is the supernatant from the centrifuged sample aspirated no lower than 5 mL above the pellet? [ Section 13.2.2] Requirement 13.3 Is the pellet vortexed a sufficient time for resuspension? [ Section 13.2.3] Method Procedure 13.4 Does the lab have an appropriate SOP for dividing pellets greater than 0.5mL into subsamples and analyzing? Requirement 13.5 Is no more than 0.5 mL of pellet used per IMS? [ Section 13.2.4] Method Procedure 13.6 Is the leighton tube rotated at 18 rpm for 1 hour at room temperature? Method Procedure 13.7 Is the resuspended pellet volume quantitatively transferred to the Leighton tube ( 2 rinses)? [ Section <IP_ADDRESS>] Method Procedure 13.8 Are the IMS beads thoroughly resuspended prior to addition to the Leighton tube? [ Section <IP_ADDRESS>] Method Procedure 13.9 Is the sample quantitatively transferred from the Leighton tube to the microcentrifuge tube ( 2 rinses)? [ Section <IP_ADDRESS>] Method Procedure 13.10 Is standard NaOH ( 5 µ L, 1N) and standard HCl ( 50 µ L, 0.1N) used? [ See note on pg 37] Requirement 13.11 Is sample vortexed vigorously for 50 seconds immediately after the addition of acid and 30 seconds after the sample has set for 10 minutes at room temperature? [ Section 13.3.3] Method Procedure 13.12 Is a second dissociation performed? [ Section <IP_ADDRESS>] Method Procedure 13.13 When the second dissociation is performed, does the laboratory: ( A) use a second slide, or ( B) add the additional volume to the original slide? Circle one: A B 12 April 29, 2002 Item to be evaluated Classification Yes, No, NA or Unknown 13.14 Are the slides clearly labeled so they can be associated with the correct sample? Requirement 13.15 What type of slides are used?

13.16 Is slide dried at a) room temperature or b) 35 to 42 C? [ Section <IP_ADDRESS>] Circle one: A B 13.17 If the slide is warmed, is incubator or slide tray calibrated and labeled? Requirement 13.18 Does the laboratory have an acceptable SOP for sample purification? Requirement 13.19 Other than the issues noted in items 13.1 through 13.18 ( if any) were sample purification and slide preparation performed successfully?

14 Sample staining Technician:

14.1 What staining kit/ manufacturer is used?

14.2 Is FITC stain applied according to manufacturer  s directions? Method Procedure 14.3 Are positive and negative staining controls performed? Requirement 14.4 Are the direct labeling reagents applied properly? [ Section 15.2.1] Method Procedure 14.5 Are the slides incubated in a humid chamber in the dark at room temperature for approximately 30 minutes or per manufacturer  s directions? [ Section 14.4] Method Procedure 14.6 Are the labeling reagents rinsed away properly after incubation, without disturbing the sample? [ Section 14.5] Method Procedure 14.7 Was the working DAPI stain prepared the day it was used? [ Section 7.7.2] Method Procedure 14.8 Is stock DAPI stored at 0 to 8oC in the dark? [ Section 7.7.2] Method Procedure 14.9 Is the DAPI stain applied properly and allowed to stand for a minimum of 1 minute? [ Section 14.6] Method Procedure 14.10 Is the DAPI stain rinsed away properly without disturbing the sample? [ Section 14.7] Method Procedure 14.11 Is the mounting media applied properly? Method Procedure 14.11.1 What type of mounting media is used?

14.11.2 Are all the edges of the cover slip sealed well with clear fingernail polish, unless Elvenol is used? [ Section 14.9] Method Procedure 14.12 Are the finished slides stored in a humid chamber in the dark at 0 to 8oC ( humid chamber not required for Evenol)? [ Section 14.10] Method Procedure 14.13 Does the laboratory have an acceptable SOP for sample staining? Requirement 14.14 Other than the issues noted in items 14.2 through 14.13 ( if any) was sample staining demonstrated successfully?

15 Microscope and Examination 15.1 Is microscope equipped with appropriate excitation and band pass filters for examining FITC labeled specimens? ( Exciter filter  450 490 nm, dichroic beam splitting mirror  510 nm, barrier or suppression filter: 515 520 nm)? [ Section 6.9.2] Requirement 15.2 Is microscope is equipped with appropriate excitation and band pass filters for examining DAPI labeled specimens? ( Exciter filter  340 380 nm, dichroic beam splitting mirror  400 nm, barrier or suppression filter  420 nm) [ Section 6.9.3] Requirement 15.3 Does the microscope have HMO or DIC, objectives? [ Section 6.9.1] Requirement 15.4 Is microscope operation easily changed from epifluorescence to DIC/ HMO? Recommendation 15.5 Does the microscope have a 20 X scanning objective? [ Section 6.9.1] Requirement 15.6 Does the microscope have a 100 X oil immersion objective? [ Section 6.9.1] Requirement 15.7 Is the microscope equipped with an ocular micrometer? [ Section 6.9.1] Requirement 15.8 Is a stage micrometer available to laboratory? [ Section 10.3.5] Requirement 15.9 Is a calibration table for each objective located close to the microscope( s)? [ Section 10.3.5] Requirement 13 April 29, 2002 Item to be evaluated Classification Yes, No, NA or Unknown 15.10 Does the wattage of the mercury lamp meet the microscope specifications? Requirement 15.11 Has the mercury bulb been used less than the maximum hours recommended by the manufacturer? [ Section <IP_ADDRESS>] Recommendation 15.12 Does the positive control contain Cryptosporidium oocysts at the appropriate fluorescence intensity for both FITC and DIC? Requirement 15.13 Does the laboratory have an acceptable SOP for sample examination? Requirement 15.14 Other than the issues noted for items 15.1 through 15.13 ( if any) were other microscope or examination issues acceptable?

Note: All section references in [ ] refer to Method 1623 April 2001 14 April 29, 2002 Initial Demonstration of Capability Data Summary Form Laboratory Name Laboratory ID Date Method Information Which method was used? 9 Method 1622 9 Method 1623 Filter used: Elution method: Concentration method:

IMS kit used: Staining kit used:

Volume of water spiked ( L): Volume of water filtered ( L):

Initial Demonstration of Capability Summary Data Sample Giardia ( not required) Cryptosporidium Equivalent Sample Volume Analyzed ( to nearest 1/ 4 L) Turbidity ( NTU) Estimated No. of Cysts Spiked No. of Cysts Detected Estimated No. of Oocysts Spiked No. of Oocysts Detected Method blank Spiked reagent water 1 Spiked reagent water 2 Spiked reagent water 3 Spiked reagent water 4 Mean recovery Precision ( RSD)

Matrix unspiked Matrix spike 1 Matrix spike 2 Mean recovery Precision ( RPD)

15 April 29, 2002 Burden Statement: The public reporting and recordkeeping burden for this collection of information is estimated to average 18 hours per response or 72 hours per respondent annually. Burden means the total time, effort, or financial resources expended by persons to generate, maintain, retain, or disclose or provide information to or for a Federal agency. This includes the time needed to review instructions; develop, acquire, install, and utilize technology and systems for the purposes of collecting, validating, and verifying information, processing and maintaining information, and disclosing and providing information; adjust the existing ways to comply with any previously applicable instructions and requirements; train personnel to be able to respond to a collection of information; search data sources; complete and review the collection of information; and transmit or otherwise disclose the information. An agency may not conduct or sponsor, and a person is not required to respond to, a collection of information unless it displays a currently valid OMB control number.

16 April 29, 2002


[Federal Register Volume 79, Number 163 (Friday, August 22, 2014)]
[Rules and Regulations]
[Pages 49688-49690]
[FR Doc No: 2014-19988]

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DEPARTMENT OF HOMELAND SECURITY

Coast Guard

33 CFR Part 165

[Docket Number USCG-2014-0296]
RIN 1625-AA00

Security Zone, Change of Enforcement Period, Chesapeake Bay;
Between Sandy Point and Kent Island, MD

AGENCY: Coast Guard, DHS.

ACTION: Final rule.

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SUMMARY: The Coast Guard is changing the enforcement period of a
security zone regulation within the Baltimore COTP Zone. This
regulation applies to a recurring event that takes place on the William
P. Lane Jr. Memorial Bridges, across the Chesapeake Bay, between Sandy
Point and Kent Island, MD. This action is necessary to protect persons
and property, and prevent terrorist acts or incidents on navigable
waters during the event. This rule prohibits vessels and people from
entering the security zone and requires vessels and persons in the
security zone to depart the security zone, unless specifically exempt
under the provisions in this rule or granted specific permission from
the Coast Guard Captain of the Port Baltimore.

DATES: This rule is effective September 22, 2014 and will be enforced
annually on the second Sunday in November from 7 a.m. to 11 a.m., and
if necessary due to inclement weather, on the third Sunday in November
from 7 a.m. to 11 a.m.

ADDRESSES: Documents mentioned in this preamble are part of docket
[USCG-2014-0296]. To view documents mentioned in this preamble as being
available in the docket, go to http://www.regulations.gov, type the
docket number in the ``SEARCH'' box and click ``SEARCH.'' Click on Open
Docket Folder on the line associated with this rulemaking. You may also
visit the Docket Management Facility in Room W12-140 on the ground
floor of the Department of Transportation West Building, 1200 New
Jersey Avenue SE., Washington, DC 20590, between 9 a.m. and 5 p.m.,
Monday through Friday, except Federal holidays.

FOR FURTHER INFORMATION CONTACT: If you have questions on this rule,
call or email Mr. Ronald Houck, U.S. Coast Guard Sector Baltimore, MD;
telephone<PHONE_NUMBER>, email<EMAIL_ADDRESS>If you have
questions on viewing or submitting material to the

docket, call Cheryl Collins, Program Manager, Docket Operations,
telephone<PHONE_NUMBER>.

SUPPLEMENTARY INFORMATION:

Table of Acronyms

DHS Department of Homeland Security
FR Federal Register
NPRM Notice of Proposed Rulemaking

A. Regulatory History and Information

    On May 16, 2014, we published a notice of proposed rulemaking
(NPRM) entitled ``Security Zone, Change of Enforcement Period,
Chesapeake Bay; Between Sandy Point and Kent Island, MD'' in the
Federal Register (79 FR 28468). We received no comments on the proposed
rule. No public meeting was requested, and none was held.

B. Basis and Purpose

    The legal bases and authorities for this rule are found in 33
U.S.C. 1231; 46 U.S.C. Chapter 701, 3306, 3703; 50 U.S.C. 191, 195; 33
CFR 1.05-1, 6.04-1, 6.04-6, and 160.5; Public Law 107-295, 116 Stat.
2064; Department of Homeland Security Delegation No. 0170.1, which
collectively authorize the Coast Guard to propose, establish, and
define regulatory security zones. The purpose of this security zone is
to protect persons and property, mitigate potential terrorist acts or
incidents, and enhance public and maritime safety and security in order
to safeguard life, property, and the environment on or near the
navigable waters.

C. Discussion of Comments, Changes and the Final Rule

    The Coast Guard received no comments in response to the NPRM. No
public meeting was requested and none was held.

D. Regulatory Analyses

    We developed this rule after considering numerous statutes and
executive orders related to rulemaking. Below we summarize our analyses
based on these statutes and executive orders.

1. Regulatory Planning and Review

    This rule is not a significant regulatory action under section 3(f)
of Executive Order 12866, Regulatory Planning and Review, as
supplemented by Executive Order 13563, Improving Regulation and
Regulatory Review, and does not require an assessment of potential
costs and benefits under section 6(a)(3) of Executive Order 12866 or
under section 1 of Executive Order 13563. The Office of Management and
Budget has not reviewed it under those Orders.
    Although this regulation would restrict access to this area, the
effect of this rule will not be significant because: (i) The security
zone will only be in effect annually on the second Sunday in November
from 7 a.m. through 11 a.m., and if necessary due to inclement weather,
on the third Sunday in November from 7 a.m. through 11 a.m., and (ii)
the Coast Guard will give advance notification via maritime advisories
so mariners can adjust their plans accordingly, and will continue such
advisories on the status of the security zone until the completion of
the event.

2. Impact on Small Entities

    The Regulatory Flexibility Act of 1980 (RFA), 5 U.S.C. 601-612, as
amended, requires federal agencies to consider the potential impact of
regulations on small entities during rulemaking. The term ``small
entities'' comprises small businesses, not-for-profit organizations
that are independently owned and operated and are not dominant in their
fields, and governmental jurisdictions with populations of less than
50,000. The Coast Guard received no comments from the Small Business
Administration on this rule. The Coast Guard certifies under 5 U.S.C.
605(b) that this rule will not have a significant economic impact on a
substantial number of small entities.
    This rule may affect the following entities, some of which may be
small entities: the owners or operators of vessels intending to operate
or transit through or within, or anchor in, the security zone during
the enforcement period. This security zone will not have a significant
economic impact on a substantial number of small entities for the
reasons provided under Regulatory Planning and Review.

3. Assistance for Small Entities

    Under section 213(a) of the Small Business Regulatory Enforcement
Fairness Act of 1996 (Pub. L. 104-121), we want to assist small
entities in understanding this rule. If the rule affects your small
business, organization, or governmental jurisdiction and you have
questions concerning its provisions or options for compliance, please
contact the person listed in the FOR FURTHER INFORMATION CONTACT,
above.
    Small businesses may send comments on the actions of Federal
employees who enforce, or otherwise determine compliance with, Federal
regulations to the Small Business and Agriculture Regulatory
Enforcement Ombudsman and the Regional Small Business Regulatory
Fairness Boards. The Ombudsman evaluates these actions annually and
rates each agency's responsiveness to small business. If you wish to
comment on actions by employees of the Coast Guard, call 1-888-REG-FAIR
(1-888-734-3247). The Coast Guard will not retaliate against small
entities that question or complain about this rule or any policy or
action of the Coast Guard.

4. Collection of Information

    This rule will not call for a new collection of information under
the Paperwork Reduction Act of 1995 (44 U.S.C. 3501-3520).

5. Federalism

    A rule has implications for federalism under Executive Order 13132,
Federalism, if it has a substantial direct effect on the States, on the
relationship between the national government and the States, or on the
distribution of power and responsibilities among the various levels of
government. We have analyzed this rule under that Order and determined
that this rule does not have implications for federalism.

6. Protest Activities

    The Coast Guard respects the First Amendment rights of protesters.
Protesters are asked to contact the person listed in the FOR FURTHER
INFORMATION CONTACT section to coordinate protest activities so that
your message can be received without jeopardizing the safety or
security of people, places or vessels.

7. Unfunded Mandates Reform Act

    The Unfunded Mandates Reform Act of 1995 (2 U.S.C. 1531-1538)
requires Federal agencies to assess the effects of their discretionary
regulatory actions. In particular, the Act addresses actions that may
result in the expenditure by a State, local, or tribal government, in
the aggregate, or by the private sector of $100,000,000 (adjusted for
inflation) or more in any one year. Though this rule will not result in
such an expenditure, we do discuss the effects of this rule elsewhere
in this preamble.

8. Taking of Private Property

    This rule will not cause a taking of private property or otherwise
have taking implications under Executive Order 12630, Governmental
Actions and Interference with Constitutionally Protected Property
Rights.

9. Civil Justice Reform

    This rule meets applicable standards in sections 3(a) and 3(b)(2)
of Executive Order 12988, Civil Justice Reform, to

minimize litigation, eliminate ambiguity, and reduce burden.

10. Protection of Children

    We have analyzed this rule under Executive Order 13045, Protection
of Children from Environmental Health Risks and Safety Risks. This rule
is not an economically significant rule and does not create an
environmental risk to health or risk to safety that may
disproportionately affect children.

11. Indian Tribal Governments

    This rule does not have tribal implications under Executive Order
13175, Consultation and Coordination With Indian Tribal Governments,
because it does not have a substantial direct effect on one or more
Indian tribes, on the relationship between the Federal Government and
Indian tribes, or on the distribution of power and responsibilities
between the Federal Government and Indian tribes.

12. Energy Effects

    This action is not a ``significant energy action'' under Executive
Order 13211, Actions Concerning Regulations That Significantly Affect
Energy Supply, Distribution, or Use.

13. Technical Standards

    This rule does not use technical standards. Therefore, we did not
consider the use of voluntary consensus standards.

14. Environment

    We have analyzed this rule under Department of Homeland Security
Management Directive 023-01 and Commandant Instruction M16475.lD, which
guide the Coast Guard in complying with the National Environmental
Policy Act of 1969 (NEPA) (42 U.S.C. 4321-4370f), and have determined
that this action is one of a category of actions that do not
individually or cumulatively have a significant effect on the human
environment. This rule involves establishing a security zone. This rule
is categorically excluded from further review under paragraph 34(g) of
Figure 2-1 of the Commandant Instruction. An environmental analysis
checklist supporting this determination and a Categorical Exclusion
Determination are available in the docket where indicated under
ADDRESSES.

List of Subjects in 33 CFR Part 165

    Harbors, Marine safety, Navigation (water), Reporting and
recordkeeping requirements, Security measures, Waterways.

    For the reasons discussed in the preamble, the Coast Guard amends
33 CFR part 165 as follows:

PART 165--REGULATED NAVIGATION AREAS AND LIMITED ACCESS AREAS

0
1. The authority citation for part 165 continues to read as follows:

    Authority:  33 U.S.C. 1231; 46 U.S.C. Chapter 701, 3306, 3703;
50 U.S.C. 191, 195; 33 CFR 1.05-1, 6.04-1, 6.04-6, and 160.5; Pub.
L. 107-295, 116 Stat. 2064; Department of Homeland Security
Delegation No. 0170.1.

0
2. Revise Sec.  165.507 to read as follows:

Sec.  165.507  Security Zone; Chesapeake Bay, between Sandy Point and
Kent Island, MD.

    (a) Definitions. The ``Captain of the Port, Baltimore, Maryland''
means the Commander, Coast Guard Sector Baltimore, Maryland or any
Coast Guard commissioned, warrant, or petty officer who has been
authorized by the Captain of the Port, Baltimore, Maryland to act on
his or her behalf.
    (b) Location. The following area is a security zone: All waters of
the Chesapeake Bay, from the surface to the bottom, within 250 yards
north of the north (westbound) span of the William P. Lane Jr. Memorial
Bridge, and 250 yards south of the south (eastbound) span of the
William P. Lane Jr. Memorial Bridge, from the western shore at Sandy
Point to the eastern shore at Kent Island, Maryland.
    (c) Regulations. (1) All persons are required to comply with the
general regulations governing security zones found in Sec.  165.33 of
this part.
    (2) Entry into or remaining in this zone is prohibited unless
authorized by the Coast Guard Captain of the Port, Baltimore, Maryland.
    (3) Persons or vessels requiring entry into or passage through the
security zone must first request authorization from the Captain of the
Port, Baltimore to seek permission to transit the area. The Captain of
the Port, Baltimore, Maryland can be contacted at telephone number
(410) 576-2693. The Coast Guard vessels enforcing this section can be
contacted on VHF Marine Band Radio, VHF channel 16 (156.8 MHz). Upon
being hailed by a U.S. Coast Guard vessel by siren, radio, flashing
light, or other means, the operator of a vessel shall proceed as
directed. If permission is granted, all persons and vessels must comply
with the instructions of the Captain of the Port, Baltimore, Maryland
and proceed at the minimum speed necessary to maintain a safe course
while within the zone.
    (d) Enforcement. The U.S. Coast Guard may be assisted in the patrol
and enforcement of the zone by Federal, State, and local agencies.
    (e) Enforcement period. This section will be enforced annually on
the second Sunday in November from 7 a.m. to 11 a.m., and if necessary
due to inclement weather, on the third Sunday in November from 7 a.m.
to 11 a.m.

    Dated: August 7, 2014.
K. C. Kiefer,
Captain, U.S. Coast Guard, Captain of the Port Baltimore.
[FR Doc. 2014-19988 Filed 8-21-14; 8:45 am]
BILLING CODE 9110-04-P


[Federal Register Volume 77, Number 160 (Friday, August 17, 2012)]
[Notices]
[Pages 49793-49794]
[FR Doc No: 2012-19953]

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ENVIRONMENTAL PROTECTION AGENCY

[EPA-HQ-OPP-2012-0570; FRL-9358-4]

Ortho-Phthalaldehyde; Receipt of Application for Emergency
Exemption, Solicitation of Public Comment

AGENCY: Environmental Protection Agency (EPA).

ACTION: Notice.

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SUMMARY: EPA has received a specific exemption request from the
National Aeronautics and Space Administration (NASA) to use the
pesticide ortho-phthalaldehyde (OPA) (CAS No. 643-79-8) to treat the
International Space Station internal active thermal control system
(IATCS) coolant to control Cupriavidus metallidurans, Variovorax
paradoxus, Acidovorax sp., Sphingomonas parapaucimobilis,
Stenotrophomonas maltophilia, Methylobacterium extorquens, and
unidentified gram negative rods. This emergency exemption involves the
use of a chemical which has not been registered by the EPA. EPA is
soliciting public comment on the exemption.

DATES: Comments must be received on or before September 4, 2012.

ADDRESSES: Submit your comments, identified by docket identification
(ID) number EPA-HQ-OPP-2012-0570, by one of the following methods:
     Federal eRulemaking Portal: http://www.regulations.gov. Follow the online instructions for submitting
comments. Do not submit electronically any information you consider to
be Confidential Business Information (CBI) or other information whose
disclosure is restricted by statute.
     Mail: OPP Docket, Environmental Protection
Agency Docket Center (EPA/DC), Mail Code: 28221T, 1200 Pennsylvania
Ave. NW., Washington, DC 20460-0001.
     Hand Delivery: To make special arrangements for
hand delivery or delivery of boxed information, please follow the
instructions at http://www.epa.gov/dockets/contacts.htm.

Additional instructions on commenting or visiting the docket, along
with more

information about dockets generally, is available at http://www.epa.gov/dockets.

FOR FURTHER INFORMATION CONTACT: Debra Rate, Registration Division
(7505P), Office of Pesticide Programs, Environmental Protection Agency,
1200 Pennsylvania Ave. NW., Washington, DC 20460-0001; telephone
number:<PHONE_NUMBER>; fax number:<PHONE_NUMBER>; email address:
<EMAIL_ADDRESS>
SUPPLEMENTARY INFORMATION:

I. General Information

A. Does this action apply to me?

    You may be potentially affected by this action if you are a
pesticide manufacturer (NAICS code 32532) or involved with the
International Space Station. This listing is not intended to be
exhaustive, but rather provides a guide for readers regarding entities
likely to be affected by this action. Other types of entities not
listed in this unit could also be affected. The North American
Industrial Classification System (NAICS) codes have been provided to
assist you and others in determining whether this action might apply to
certain entities. If you have any questions regarding the applicability
of this action to a particular entity, consult the person listed under
FOR FURTHER INFORMATION CONTACT.

B. What should I consider as I prepare my comments for EPA?

    1. Submitting CBI. Do not submit this information to EPA through
www.regulations.gov or email. Clearly mark the part or all of the
information that you claim to be CBI. For CBI information in a disk or
CD-ROM that you mail to EPA, mark the outside of the disk or CD-ROM as
CBI and then identify electronically within the disk or CD-ROM the
specific information that is claimed as CBI. In addition to one
complete version of the comment that includes information claimed as
CBI, a copy of the comment that does not contain the information
claimed as CBI must be submitted for inclusion in the public docket.
Information so marked will not be disclosed except in accordance with
procedures set forth in 40 CFR part 2.
    2. Tips for preparing your comments. When submitting comments,
remember to:
    i. Identify the document by docket ID number and other identifying
information (subject heading, Federal Register date and page number).
    ii. Follow directions. The Agency may ask you to respond to
specific questions or organize comments by referencing a Code of
Federal Regulations (CFR) part or section number.
    iii. Explain why you agree or disagree; suggest alternatives and
substitute language for your requested changes.
    iv. Describe any assumptions and provide any technical information
and/or data that you used.
    v. If you estimate potential costs or burdens, explain how you
arrived at your estimate in sufficient detail to allow for it to be
reproduced.
    vi. Provide specific examples to illustrate your concerns and
suggest alternatives.
    vii. Explain your views as clearly as possible, avoiding the use of
profanity or personal threats.
    viii. Make sure to submit your comments by the comment period
deadline identified.
    3. Environmental justice. EPA seeks to achieve environmental
justice, the fair treatment and meaningful involvement of any group,
including minority and/or low income populations, in the development,
implementation, and enforcement of environmental laws, regulations, and
policies. To help address potential environmental justice issues, the
Agency seeks information on any groups or segments of the population
who, as a result of their location, cultural practices, or other
factors, may have atypical or disproportionately high and adverse human
health impacts or environmental effects from exposure to the
pesticide(s) discussed in this document, compared to the general
population.

II. What action is the agency taking?

    Under section 18 of the Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA) (7 U.S.C. 136p), at the discretion of the
Administrator, a Federal or State agency may be exempted from any
provision of FIFRA if the Administrator determines that emergency
conditions exist which require the exemption. NASA has requested the
Administrator to issue a specific exemption for the use of OPA in the
International Space Station IATCS coolant to control Cupriavidus
metallidurans, Variovorax paradoxus, Acidovorax sp., Sphingomonas
parapaucimobilis, Stenotrophomonas maltophilia, Methylobacterium
extorquens, and unidentified gram negative rods. Information in
accordance with 40 CFR part 166 was submitted as part of this request.
    As part of this request, the applicant asserts that OPA is the most
effective biocide which meets the requisite IATCS criteria, including:
the need for safe, non-intrusive implementation and operation in a
functioning system; the ability to control existing planktonic and
biofilm residing micro-organisms; a negligible impact on system-wetted
materials of construction; and a negligible reactivity with existing
coolant additives. Non-use of OPA in the requested manner would leave
NASA's International Space Station without an adequate long-term
solution for controlling the micro-organisms in the coolant systems.
    The Applicant proposes to make no more than one application of OPA
per module in the International Space Station IATCS coolant including
the U.S. Laboratory, the Japanese Experiment Module, the Columbus and
Node with a total treated coolant volume not to exceed 829 Liters with
a maximum of 1,974 cubic centimeters (cm3) OPA resin.
    The regulations governing section 18 of FIFRA require publication
of a notice of receipt of an application for a specific exemption
proposing use of a new chemical (i.e., an active ingredient) which has
not been registered by EPA. The notice provides an opportunity for
public comment on the application. Owing to the long lead time
necessary for sending products to the International Space Station, EPA
has evaluated NASA's application in advance of seeking public comment,
and has concluded that the emergency exemption should be approved.
Although EPA has approved this emergency exemption, EPA still welcomes
public comment. EPA's regulations provide that an emergency exemption
may be modified or revoked if, among other things, additional
information indicates that the product may cause unreasonable adverse
effects or may not be effective at controlling the target pests.
Accordingly, the Agency will review and consider all comments received
during the comment period in determining whether the specific exemption
should be modified or revoked.

List of Subjects

    Environmental protection, Pesticides and pests.

    Dated: August 3, 2012.
 Lois Rossi,
Director, Registration Division, Office of Pesticide Programs.
[FR Doc. 2012-19953 Filed 8-16-12; 8:45 am]
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[Federal Register Volume 80, Number 217 (Tuesday, November 10, 2015)]
[Notices]
[Pages 69652-69653]
[FR Doc No: 2015-28663]

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ENVIRONMENTAL PROTECTION AGENCY

[FRL-9936-92-OA]

Notification of a Public Teleconference of the Science Advisory
Board Hydraulic Fracturing Research Advisory Panel

AGENCY: Environmental Protection Agency (EPA).

ACTION: Notice.

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SUMMARY: The EPA Science Advisory Board (SAB) Staff Office announces a
public teleconference of the SAB Hydraulic Fracturing Research Advisory
Panel as part of the peer review of the EPA draft report, Assessment of
the Potential Impacts of Hydraulic Fracturing for Oil and Gas on
Drinking Water Resources, (May, 2015 External Review Draft, EPA/600/R-
15/047). The teleconference will serve to complete agenda items from
the October 28-30, 2015 Panel meeting, namely to develop preliminary
key points in response to charge questions on the agency's draft
assessment.

DATES: The public teleconference will be held on Thursday, December 3,
2015, from 2:00 p.m. to 6:00 p.m. (Eastern Time).

ADDRESSES: The teleconference will be conducted by telephone only.

FOR FURTHER INFORMATION CONTACT: Members of the public who wish to
obtain further information regarding this public teleconference may
contact Edward Hanlon, Designated Federal Officer, by telephone: (202)
564-2134 or email at<EMAIL_ADDRESS>The SAB mailing address is:
U.S. EPA Science Advisory Board (1400R), U.S. Environmental Protection
Agency, 1200 Pennsylvania Avenue NW., Washington, DC 20460. General
information about the SAB, including information concerning the SAB
meeting and teleconferences announced in this notice, may be found on
the SAB Web site at http://www.epa.gov/sab.
    Technical Contact for EPA's Draft Report: Any technical questions
concerning EPA's draft report should be directed to Dr. Jeffrey
Frithsen, National Center for Environmental Assessment, Office of
Research and Development, U.S. EPA, 1200 Pennsylvania Avenue NW., Mail
Code 8601P, Washington, DC 20460, telephone<PHONE_NUMBER> or via email
at<EMAIL_ADDRESS>
SUPPLEMENTARY INFORMATION:
    Background: The SAB was established pursuant to the Environmental
Research, Development, and Demonstration Authorization Act (ERDDAA),
codified at 42 U.S.C. 4365, to provide independent scientific and
technical advice to the Administrator on the technical basis for Agency
positions and regulations. The SAB is a Federal Advisory Committee
chartered under the Federal Advisory Committee Act (FACA), 5 U.S.C.,
App. 2. The SAB will comply with the provisions of FACA and all
appropriate SAB Staff Office procedural policies. Pursuant to FACA and
EPA policy, notice is hereby given that the SAB Hydraulic Fracturing
Research Advisory Panel will hold a public teleconference as part of
the peer review of the EPA draft report, Assessment of the Potential
Impacts of Hydraulic Fracturing for Oil and Gas on Drinking Water
Resources, (May, 2015 External Review Draft, EPA/600/R-15/047).
    The EPA's Office of Research and Development (ORD) has developed a
draft assessment report concerning the relationship between hydraulic
fracturing and drinking water in the United States. The purpose of the
report, Assessment of the Potential Impacts of Hydraulic Fracturing for
Oil and Gas on Drinking Water Resources, (May, 2015 External Review
Draft, EPA/600/R-15/047), is to synthesize available scientific
literature and data to assess the potential for hydraulic fracturing
for oil and gas to impact the quality or quantity of drinking water
resources, and identify factors affecting the frequency or severity of
any potential impacts. As noticed in 80 FR 32111-32113, the SAB
Hydraulic Fracturing Research Advisory Panel held a face-to-face
meeting on October 28-30, 2015, to conduct a peer review of the
agency's draft report.
    The purpose of the December 3, 2015, public teleconference is for
the SAB Hydraulic Fracturing Research Advisory Panel to complete agenda
items from the October 28-30, 2015 Panel meeting, namely to develop
preliminary key points in response to charge questions on the agency's
draft assessment.
    Availability of Meeting Materials: Additional background on this
SAB activity, the teleconference agenda, and other materials for the
teleconference will be posted on the SAB Web site at http://yosemite.epa.gov/sab/sabproduct.nsf/02ad90b136fc21ef85256eba00436459/f7a9db9abbac015785257e540052dd54!OpenDocument&Highlight=0,hydraulic,fracturing in advance of the teleconference.
    Procedures for Providing Public Input: Federal advisory committees
and panels, including scientific advisory committees, provide
independent advice to the EPA. Interested members of the public may
submit relevant information on the topic of this advisory activity,
and/or the group conducting the activity, for the SAB to consider
during the advisory process. Input from the public to the SAB will have
the most impact if it provides specific scientific or technical
information or analysis for SAB committees and panels to consider or if
it relates to the clarity or accuracy of the technical information.
Throughout the Panel review process, there will be opportunities for
the public to provide comments. For example, the public was invited to
provide comments to the Docket on the draft EPA report and will have an
opportunity to provide comments to the Docket on the SAB Panel's draft
report, provide oral statements to the Panel during the Panel
teleconferences and meeting, and provide comments in preparation for
quality review of the SAB Panel's draft report by the Chartered SAB.
Members of the public wishing to provide written comments may submit
them to the EPA Docket electronically via www.regulations.gov, by
email, by mail, by facsimile, or by hand delivery/courier. Please
follow the detailed instructions provided in the written statements
section of this notice. Members of the public wishing to

provide oral statements to the SAB Panel should contact the DFO
directly.
    Oral Statements: In general, individuals or groups requesting to
present an oral statement at a public teleconference will be limited to
three minutes per speaker. To be placed on the public speaker list for
the December 3, 2015 teleconference, interested parties should notify
Mr. Edward Hanlon, DFO, by email no later than November 25, 2015.
    Written Statements: Written statements for the December 3, 2015,
teleconference should be received in the EPA Docket by November 25,
2015, so that the information may be made available to the SAB Panel
sufficiently in advance of the teleconference for the Panel's
consideration.
    Written statements should be identified by Docket ID No. EPA-HQ-OA-
2015-0245 and submitted to the Docket at www.regulations.gov by one of
the following methods:
     www.regulations.gov: Follow the on-line instructions for
submitting comments.
     Email<EMAIL_ADDRESS>Include the docket number in
the subject line of the message.
     Mail: Office of Environmental Information (OEI) Docket
(Mail Code: 28221T), Docket ID No. EPA-HQ-OA-2015-0245, U.S.
Environmental Protection Agency, 1200 Pennsylvania Ave. NW.,
Washington, DC 20460. The phone number is<PHONE_NUMBER>.
     Hand Delivery: The OEI Docket is located in the EPA
headquarters Docket Center, Room 3334, EPA West Building, 1301
Constitution Ave. NW., Washington, DC. The EPA Docket Center Public
Reading Room is open from 8:30 a.m. to 4:30 p.m., Monday through
Friday, excluding legal holidays. The telephone number for the Public
Reading Room is<PHONE_NUMBER>. Deliveries are only accepted during the
docket's normal hours of operation, and special arrangements should be
made for deliveries of boxed information.
     Fax:<PHONE_NUMBER>.
    Public comments submitted after November 25, 2015 will be marked
late, and should be submitted to the Docket by email, mail, hand
delivery or fax (see detailed instructions above). Consistent with SAB
Staff Office general practice, comments received after November 25,
2015 will be made available to the SAB Panel as soon as practicable. It
is EPA's policy to include all comments received in the public docket
without change and to make the comments available on-line at
www.regulations.gov, including any personal information provided,
unless a comment includes information claimed to be Confidential
Business Information (CBI) or other information whose disclosure is
restricted by statute. Do not submit information that you consider to
be CBI or otherwise protected through www.regulations.gov or email. The
www.regulations.gov Web site is an ``anonymous access'' system, which
means EPA will not know your identity or contact information unless you
provide it in the body of your comment. If you send an email directly
to EPA without going through www.regulations.gov, your email address
will be automatically captured and included as part of the comment that
is placed in the public docket and made available on the Internet. If
you submit an electronic comment, EPA recommends that you include your
name and other contact information in the body of your comment and with
any disk or CD-ROM you submit. If EPA cannot read your comments due to
technical difficulties and cannot contact you for clarification, the
SAB Panel may not be able to consider your comments. Electronic files
should avoid the use of special characters and any form of encryption
and be free of any defects or viruses. For additional information about
EPA's public docket, visit the EPA Docket Center homepage at
www.epa.gov/epahome/dockets.htm.
    Documents in the docket are listed in the www.regulations.gov
index. Although listed in the index, some information is not publicly
available, e.g., CBI or other information whose disclosure is
restricted by statute. Certain other material, such as copyrighted
material will be publicly available only in hard copy. Publicly
available docket materials are available either electronically at
www.regulations.gov or in hard copy at the OEI Docket in the EPA
Headquarters Docket Center.
    Accessibility: For information on access or services for
individuals with disabilities, please contact Mr. Edward Hanlon at the
phone number or email address noted above, preferably at least ten days
prior to the meeting to give EPA as much time as possible to process
your request.

    Dated: November 3, 2015.
Thomas H. Brennan,
Deputy Director, EPA Science Advisory Board Staff Office.
[FR Doc. 2015-28663 Filed 11-9-15; 8:45 am]
BILLING CODE 6560-50-P


EPA BIOPESTICIDES AND POLLUTION PREVENTION DIVISION COMPANY NOTICE OF FILING FOR PESTICIDE PETITIONS PUBLISHED IN THE FEDERAL REGISTER

EPA Biopesticides and Pollution Prevention Division contact: [Daniel Schoeff,<PHONE_NUMBER>]

AgBiTech Pty Ltd 8 Rocla Court Glenvale, Queensland, 4350 Australia

[8F8697]

	EPA has received a pesticide petition ([8F8697]) from AgBiTech Pty Ltd, 8 Rocla Court; Glenvale, Queensland, 4350  Australia proposing, pursuant to section 408(d) of the Federal Food, Drug, and Cosmetic Act (FFDCA), 21 U.S.C. 346a(d), to amend 40 CFR part 180.

(Options (pick one)

	2. to establish an exemption from the requirement of a tolerance for

(Options (pick one)        	1. microbial pesticide  Autographa californica Multiple Nucleopolyhedrovirus, isolate R3 [SD-7095]

Pursuant to section 408(d)(2)(A)(i) of  FFDCA, as amended, AgBiTech Pty Ltd has submitted the following summary of information, data, and arguments in support of their pesticide petition. This summary was prepared by AgBiTech Pty Ltd and EPA has not fully evaluated the merits of the pesticide petition. The summary may have been edited by EPA if the terminology used was unclear, the summary contained extraneous material, or the summary unintentionally made the reader conclude that the findings reflected EPA's position and not the position of the petitioner.

I. AgBiTech Pty Ltd  Petition Summary        	[8F8697]

A. Product Name and Proposed Use Practices

	Autographa californica Multiple Nucleopolyhedrovirus (AcMNPV) is a baculovirus characterized by rod-like nucleocapsids contained within a viral envelope.  Baculoviridae genomes are supercoiled circular double stranded DNA ranging from 80-180 kbp in size. Virons consist of one or more nucleocapsids enclosed within a single envelope. Virons are embedded in protein bodies called occlusion bodies (OBs). These OBs are approximately 1 to 10 um in diameter and visible through a light microscope, appearing as translucent irregular crystals.

      Baculoviruses are ancient organisms, likely discovered as early as 5000 years ago as a disease impacting the silk industry in Asia. They have been exclusively isolated from arthropods, specifically from Lepidoptera, Hymenoptera and Diptera, and usually identified by the species that they infect along with the type of occlusion body that they form.        Most nucleopolyhedrovirus' infect one or a few host species, with the exception of AcMNPV, Anagrapha falcifera NPV and MbMNPV, which have a broader host range infecting up to 10 different Lepidopteran families. Autograpaha californica Multiple Nucleopolyhedrovirus (AcMNPV; formerly known as Angrapha falcifera MNPV (AfMNPV) and Rachiplusia ou MNPV (RoMNPV)), the subject of this regulatory application has an narrow host range specific for Noctuidae Lepidoptera. The target pests are Helicoverpa zea  (corn earworm, cotton bollworm, tomato fruitworm), Heliothis virescens (tobacco budworm), Chrysodeixis includens (soybean looper) and Trichoplusia ni (cabbage looper).

      AcMNPV and NPVs in general are ubiquitous in nature, found in nearly every location where investigated. This natural product will be an excellent addition to growers' options for H. zea, H virescens, C. includens and T. ni control that reduces or eliminates the need for chemical inputs and fits well within an integrated pest management program for organic or conventional production.

B. Product Identity/Chemistry

	1. Identity of the pesticide and corresponding residues. The insect viral strain, Autographa californica Multiple Nucleopolyhedrovirus  -  R3 [SD-7095] was isolated from Autographa biloba larvae collected in Rolla, Missouri in 2001. The strain is a natural insect viral strain that has not been modified in any way and is identical to AcMNPV that are ubiquitous in nature. NPVs are known for their ability to control their hosts and have been shown to reduce larval damage below economic levels leading to improved crop production.

	2. Magnitude of residues at the time of harvest and method used to determine the residue. [NA remove  -  see 1. above]

	3. A statement of why an analytical method of detecting and measuring the levels of the pesticide residue are not needed. No analytical method is included since this petition requests an exemption from the requirement of a tolerance.

C. Mammalian Toxicological Profile

	Baculoviruses in general have a long history of safe use as microbial pesticides in the USA. Baculoviruses have narrow host ranges and virtually no adverse effects in people, wildlife, or non-target insects. The active ingredient for all baculoviruses, including AcMNPV, is the occlusion bodies (OBs) that are formed in the nuclei of infected cells. Target larvae ingest OBs and until about day 4 post-infection, no symptoms are observed. From 4-6 days post-infection, the larvae begin to appear diseased and typically stop eating at day 6-7 with death occurring between 7-10 days post-infection. This mode-of-action occurs across all baculoviruses demonstrating the similarity of this group of insect control viruses.

The body of toxicology data is quite extensive for NPVs, addressing different routes of exposure  -  oral, pulmonary, dermal and injection  -  with many different species, rodents, dogs, monkeys and even humans for Helicoverpa zea NPV (EPA, 1990). Since the reregistration actions completed in 1990, other NPV and GV strains have been registered, beet armyworm, Indian Meal Moth, etc. In addition, a full set of toxicology results are available from other less related NPVs (e.g., OpNPV, LdNPV) addressing the same routes of exposure but limited to rodent and a dog study.

The last re-registration documents prepared by EPA that provide detailed historical regulatory data and extensive review of health and safety data on NPVs were published in 1990s (EPA, 1990; EPA, 1996). The full body of toxicology data regardless of the baculovirus species is important to cite given the close relatedness of all baculoviruses and their common mode of action (see Figure 2, Harrison et al., 2008). What makes each strain unique is the pest complex that they control, which is typically very narrow.

Perhaps one of the best description of the lack of human toxicity is provided in by OECD (2002):        `Baculoviruses are naturally occurring pathogens of arthropods. Their host range is exclusively restricted to arthropods. No member of this virus family is infective to plants or vertebrates. Baculoviruses are ubiquitously resent in the environment and have been used for biological insect control for more than 100 years. Circumstantial evidence for the safety of baculoviruses emerges from the history of contact between baculoviruses and humans without any detrimental effect.'

As part of the scientific rationale, a bridging study was conducted using mammalian cell lines to characterize any mammalian cell effects from AcMNPV. Non-occluded AcMNPV isolates, control insect medium (NPV control), spent medium, inactivated virus as appropriate and a commercial NPV product were included in the tests.

In this study, no cytopathic effects or replication of AcMNPV exposed to any of the mammalian cell lines were observed. If NPV nucleic acid had been detected in the cells, then viral antigen would have been assayed; and, the presence of infectious virus in the supernatant at the same days of incubation would also have been assayed in a sensitive insect bioassay. However, since no AcMNPV nucleic acid was detected, no further testing was necessary.

The results of this cell culture study, demonstrating a total lack of infectivity or toxicity of AcMNPV to mammalian cells, allows for a bridge to the many different types of mammalian toxicology studies conducted with commercial NPV strains. The common mode of action shared by all NPV adds to the weight of evidence that NPVs as a group, and specifically AcMNPV, will not cause mammalian toxicity. Therefore, AcMNPV should be considered as safe as any baculovirus used in past and current commercial end products.

D. Aggregate Exposure

	1. Dietary exposure. Since AcMNPV is applied to the crop, there will be negligible to non-existent dietary, dermal or inhalation exposure. Use of this baculovirus in pesticide products will not increase the exposure of humans beyond normal background levels.

	i. Food. There will be no accumulation of the insect virus or this AcMNPV product in any plant tissues or food.

	ii. Drinking water.  Drinking water is unlikely to be contaminated with AcMNPV, because AcMNPV only grows and replicates within the C. includens host, which are limited to plant and soil habitats and do not live in water environments.

	2. Non-dietary exposure. Non-dietary exposure of infants, children or the US population in general, to AcMNPV are not expected due to the uses of this product within agricultural settings.

E. Cumulative Effects

	The unique mode-of-action and narrow host range AcMNPV, and of NPV occlusion bodies in general, coupled with the lack of mammalian toxicity provides no basis for the expectation of cumulative exposure with other compounds.

F. Safety Determination

	1. U.S. population. The lack of mammalian toxicity to the insect virus AcMNPV provides support for our request of an exemption from the requirement of a tolerance set forth in this petition, including infants and children.

	2. Infants and children. (See F.1. above)

G. Effects on the Immune and Endocrine Systems

	The unique mode-of-action and narrow host range AcMNPV, and of NPV occlusion bodies in general, coupled with the lack of mammalian toxicity provides no basis for the expectation of effects on the immune or endocrine systems.

H. Existing Tolerances

	No tolerances or tolerance exemptions have been granted for AcMNPV. However, these naturally occurring insect viruses have all been granted exemptions from the requirement of a tolerance; Spodoptera frugiperda NPV (§180.1339), Heliocoverpa zea NPV (§180.1027), Spodoptera exigua NPV (§180.1118), Anagrapha falcifera MNPV (§180.1149) and Cydia pomonella GV (§180.1148) and Indian Meal Moth GV (§180.1218). The first registered product was based on H. zea NPV under the brand name Elcar in 1975 for use on cotton and tobacco. And as of 2002, the EPA had registered10 products from five NPVs and from two granuloviruses (EPA, 1990).

I. International Tolerances

	No international tolerances or tolerance exemptions have been granted for AcMNPV.