Quantitative Method for Evaluating the Efficacy of Antimicrobial Test Substances on Soft Surface Textiles Against Bacteria
                                 (08/11/2023)

Scope
The Environmental Protection Agency (EPA) Office of Pesticide Programs (OPP) recommends that applicants utilize this method to support efficacy criteria for the registration of products bearing claims for use on soft surface textiles. The method provides a quantitative assessment of the performance of antimicrobial substances against Pseudomonas aeruginosa and Staphylococcus aureus on soft surface textiles.
This method provides log reduction (LR) as the quantitative measure of efficacy for disinfectants against the test microbes on a soft surface textile.

Method Overview
In brief, the method uses 1 cm diameter discs or 1 cm[2] squares (carriers) of a set of representative soft surface textiles.  Each carrier receives 10 uL of microbial inoculum (with a three-part soil load) deposited in the center of each carrier. The inoculum is allowed to dry and is then exposed to 50 uL of the antimicrobial treatment; control carriers receive an equivalent volume of an innocuous fluid (e.g., phosphate buffered saline). The exposure time is allowed to elapse; a liquid neutralizer is then added to the vial to halt the antimicrobial action. Each vial with the carrier is vortexed, serially diluted, and the contents are filtered to recover viable microorganisms. Based on the difference between the mean log10 density values of the untreated control and treated carriers, a mean log10 reduction (LR) in viable bacteria is calculated. The LR value is used as the measure of product effectiveness.
Appropriate safety procedures should always be used when working with laboratory test systems which include human pathogenic microorganisms. Laboratory safety is discussed in the current edition of "Biosafety in Microbiological and Biomedical Laboratories (BMBL)" 6[th] edition, from the subject matter experts within the U.S. Department of Health and Human Services (HHS), including experts from the Centers for Disease Control and Prevention (CDC) and National Institutes of Health (NIH).
                               TABLE OF CONTENTS

1) Special Apparatus and Materials...............................................................
3
2) Carriers.............................................................................................
5
3) Preparation of Test Cultures and Carrier Inoculation ......................................
6
4) Performance Assessment  -  Efficacy..........................................................
9
5) Data Requirements...............................................................................
12
Appendix 1: Preparation of Frozen Stock Culture..............................................
14
Appendix 2: Neutralization Assay and Flowcharts...............................................
16
Appendix 3: Carrier Examples......................................................................
19

 Special Apparatus and Materials
 Test microbes: Pseudomonas aeruginosa (ATCC #15442) and Staphylococcus aureus (ATCC #6538).
 Additional bacteria may be tested (for an additional label claim) per the Agency's guidance.
 Culture media
 Tryptic Soy Broth (TSB). Use to rehydrate lyophilized cultures. Purchase broth from a reputable source or prepare according to manufacturer's instructions.
 Synthetic broth (SB). Growth medium for test cultures. Commercial media (HIMEDIA, Synthetic Broth, AOAC, #M334-500G). Suspend 16.9 g in 1000 mL DI water. Heat if necessary, to dissolve the medium completely. Final pH at 25°C should be 7.1+-02. Medium may be dispensed in 10 mL amounts in 20x150 mm culture tubes or alternatively in 500 mL volumes in a 1 L bottle; steam sterilize at 121°C for 15 minutes. Store prepared SB at 2-8°C.
 Alternatively, SB made in-house per the recipe provided in AOAC Methods 955.15, 964.02, and 955.14 may be substituted.
 10% dextrose solution. Add 5.0 g dextrose to 50 mL de-ionized water and mix by stirring. Filter sterilize the solution using a 0.2 um filter. Store the sterile solution at 2-5°C for up to 30 days.
 TSB with 15% (v/v) glycerol. Use as a cryoprotectant. Suspend 7.5 g tryptic soy broth in 212.5 mL de-ionized water. Add 37.5 mL glycerol and stir, warm slightly to dissolve. Dispense into bottles and steam sterilize for 15 min at 121°C.
 Tryptic soy agar (TSA) and TSA with 5% sheep blood. Use for culturing, isolation, and characterization of the test microbes. Purchase plates from a reputable source or prepare according to manufacturer's instructions.
 Selective media (optional). Mannitol salt agar and Cetrimide agar. Use for quality control of test microbes listed in this procedure. Purchase plates or prepare according to manufacturer's instructions.
 Reagents
 Neutralizer. A liquid reagent used to inactivate and/or dilute the antimicrobial treatment to end the contact time.
 Phosphate buffered saline stock solution (e.g., 10X). To prepare 1X phosphate buffered saline. The stock solution has a pH of approximately 7.2+-0.2.
 Phosphate buffered saline (PBS), 1X. Dilution blanks and filtration. PBS with a pH of approximately 7.0+-0.5 is desirable.
 Soil load, 3-part. Use as the soiling agent.
 BSA: Add 0.5 g bovine serum albumin (BSA, radio immunoassay (RIA) grade or equivalent, CAS# 9048-46-8) to 10 mL of PBS, mix and pass through a 0.2 um pore diameter (polyethersulfone) membrane filter, aliquot (e.g., a minimum of 50 uL), and store at -20+-2°C.
 Yeast Extract: Add 0.5 g yeast extract to 10 mL of PBS, mix, and pass through a 0.2 um pore diameter (polyethersulfone) membrane filter, aliquot (e.g., a minimum of 70 uL), and store at -20+-2°C.
 Mucin: Add 0.04 g mucin (from bovine submaxillary gland, CAS # 84195-52-8) to 10 mL of PBS, stir or vortex-mix until thoroughly dissolved, and pass through a 0.2 um pore diameter (polyethersulfone) membrane filter, aliquot, and store at -20+-2°C.
 The three stock solutions of the soil load are single use only. Do not refreeze; store up to one year at -20+-2°C.
 Antimicrobial Test substance. Ready-to-use, activated, or concentrated antimicrobial. If the antimicrobial test substance is prepared by diluting a concentrate in hard water, adequately mix antimicrobial test substance with the appropriate diluent (e.g., hard water), then use prepared test substance within 3 hours of preparation or as otherwise instructed by the manufacturer. Measuring error increases as delivery volume decreases. To minimize variability due to measuring error, a minimum of 1.0 mL or 1.0 g of concentrated antimicrobial test substance should be used when preparing use-dilutions for testing. Use v/v dilutions for liquids antimicrobial test substances and w/v dilutions for solid antimicrobial test substances. The use of a positive displacement pipette is recommended for viscous liquids.
 1 N NaOH and 1 N HCl. Used for pH adjustment of media/reagents.
 Water. De-ionized (DI), distilled water or water with equivalent quality for making reagent solutions and culture media.
 Gram stain. Used for diagnostic staining.
 Apparatus
 Carriers: Discs (1 cm in diameter) or 1 cm[2] squares cut from soft surface textile. Carriers are single use only. See Section 2 for carrier specifications.
 Hole punch: If necessary, for use in the preparation of 1 cm disc from material.  Model number: SKU# HP-MEI448R or equivalent
 Calibrated 10 uL positive displacement pipette with corresponding 10 uL tips, for carrier inoculation.
 Filter paper. Whatman No. 2, to line glass Petri plates.
 Calibrated micropipettes (e.g., 200 uL, 1 mL) with appropriate corresponding tips, for deposition of test substance on carriers and preparing dilutions.
 Bottle-top dispensers, squirt bottles, pre-measured volumes in tubes, or pipettes, bottles, etc. For rinsing vials and filters.
 Forceps, straight or curved, non-magnetic, disposable with smooth flat tips to handle membrane filters, appropriate to pick up the carriers for placement in vials.
 Polyethersulfone (PES) membranes. Use for recovery of test microbe, 47 mm diameter and 0.2 um pore size.
 Use filter membranes in either a reusable or disposable filtration unit.
 Filter Sterilization Unit (with PES, 0.2 um pore size). Use to filter sterilize soil components.
 20 x 150 m glass culture tubes with Morton closures for test culture preparation.
 Spectrophotometer. For culture standardization (if deemed necessary)
 Vials with lids (plastic or comparable). Sterile, flat-bottomed, wide-mouthed (at least 25 mm diameter), approximately 20 mL capacity, for holding inoculated carriers to be exposed to the test chemical and for accommodating neutralizer (e.g., Thermofisher #2116-0015).
 Transparent vials are more desirable to facilitate application of 50 uL test substance or control substance to inoculated carrier.
 Certified timer. Readable in minutes and seconds, for tracking of timed events and intervals.
 Desiccation unit (with gauge to measure vacuum level) with fresh desiccant (e.g., anhydrous CaCO3). For drying inoculated carriers.
 Vacuum source. In-house line or suitable vacuum pump capable of achieving 0.068 to 0.085 MPa, for drying inoculated carriers in desiccation unit and to perform membrane filtration.
 Titration kit. (i.e., Hach digital titrator) Used for measuring water hardness.
 Vortex-style mixer. Used for vortex-mixing of various solutions.
 Conical centrifuge tubes (e.g., 15 mL). Used for centrifugation of test cultures.
 Centrifuge (with rotor capable of achieving 5,000g). Used for test culture preparation.

 Carriers
 Carrier Materials (see Figure 1)
 Privacy Curtain Fabric (PCF-03): 56% Polyester, 44% Fire Resistant (FR) Polyester, 12.93 oz./lin. yd. +- 1.0 oz. CF Stinson, LLC. Mambo MAM34.
 Non-PVC Fabric (NVF-01): Polyurethane Face made with Polycarbonate and Polyether Resins, Polyester Backing, 20.3 oz./lin. yd. +- 1.0 oz. CF Stinson, LLC. KID17.
 Vinyl Seating Fabric (VF-01): Non-Phthalate Vinyl Surface with Polyester Backing, 30.0 oz./lin. yd. +- 1.0 oz. CF Stinson, LLC. Hopsack HOP24.
            Figure 1: Examples of carrier materials cut into 1 cm discs; materials 2.a.i, 2.a.ii, and 2.a.iii (from left to right)

                                           Vinyl
                                    (VF-01)
                                     Vinyl
                                    (VF-01)
                                    Non-PVC
                                   (NVF-01)
                                    Non-PVC
                                   (NVF-01)
                                Privacy Curtain
                                   (PCF-03)
                                Privacy Curtain
                                   (PCF-03)

 Carrier Preparation
 Punch or obtain approximately 1 cm round carriers or use comparable cutting procedure from fabric.  Alternatively, 1 cm[2] square carriers can be utilized if punching is unavailable or too difficult.
 Note: punch can be sharpened by punching aluminium foil.
 Visually screen carriers to ensure consistent surface characteristics; trim any jagged edges or loose fabric.
 Carriers that demonstrate excessive fraying, ripping, backing separation, discoloration, etc., should not be used.
 No pre-cleaning of carriers is necessary. Sterilize carriers using a gravity cycle at 121°C for 20 minutes; ensure carriers are dry following sterilization. Test sterility of carriers prior to or concurrently with efficacy testing.
 Carriers may not be entirely flat after autoclaving; however, minor distortion, such as cupping or doming, of carriers is acceptable for testing if the test substance and inoculum are not affected when applied to the surface of the carrier.
 Prior to use in testing, document the condition of the screened and sterile carriers (e.g., digital photographs or documentation on the paperwork).
 To assess carrier sterility, place a carrier in tryptic soy broth (TSB) and incubate at 36+-1°C for 3-10 days.
 Use sterilized carriers for up to 6 months. After 6 months, re-sterilize any remaining carriers.

 Preparation of Test Culture and Carrier Inoculation
 Refer to Appendix 1 for preparation of the frozen stock cultures.
 Defrost a cryovial; defrost rapidly to avoid loss in the viability of the preserved cells. Each cryovial is single use only.
 Prior to inoculation, on the day inoculated, use a calibrated pipette to aseptically add 0.1 mL of 10% sterile dextrose (w/v) solution to each 10 mL tube of SB.
 Using a calibrated micropipette, add 100 uL of defrosted stock culture to 10 mL SB with dextrose, briefly vortex-mix and incubate for 24+-2 h at 36+-1°C.
 Incubate without disrupting the culture.
 To assess culture purity, inoculate an agar plate (e.g., TSA or TSA with 5% sheep blood) from the inoculated tube and streak for isolation. Incubate plate with the test culture.
 Following incubation, use the SB cultures to prepare a test suspension for each organism.
 The 24+-2 h culture should exhibit a titer of at least 10[8] CFU/mL.
 For P. aeruginosa, inspect culture prior to harvest; visible pellicle on the surface of the culture is expected to form during incubation (record its presence). Discard the culture if pellicle has been disrupted (fragments in culture).
 Remove visible pellicle on surface of medium and around associated interior edges of the tube with vacuum suction.
 Using a serological pipette, withdraw the remaining broth culture (at least 5 mL) avoiding any sediment on the bottom of the tube and transfer it into a conical centrifuge tube.
 If necessary, culture may be harvested from two 10 mL 24+-2 h broth cultures to centrifuge a maximum of 10 mL P. aeruginosa; record the associated information.
 For S. aureus, briefly vortex-mix the 24+-2 h culture and transfer to a conical centrifuge tube.
 Within 15 min, centrifuge the 24+-2 h harvested broth cultures at 5,000g for 20 min.
 Remove the supernatant without disrupting the pellet. Re-suspend the pellet in 5-10 mL PBS. Record resuspension volume.
 Prepare the final test suspension within 30 min of resuspending the culture.
 If necessary, disrupt the pellet using vortex-mixing or repetitive tapping/striking against a hard surface to disaggregate the pellet completely prior to re-suspending it in 10 mL. If necessary, add 1 mL of PBS to the pellet to aid in the disaggregation.
 If needed, dilute the 5-10 mL of resuspended culture in PBS to achieve a mean control carrier count level of 4.0-5.5 logs CFU/carrier for S. aureus and P. aeruginosa.
 Optical density/absorbance (e.g., 650 nm) may be used as a tool to monitor/adjust the diluted test suspension.
 Use the resuspended or diluted culture to prepare the final test suspension with the addition of the soil load.
 To obtain 500 uL of the final test suspension with the 3-part soil load, vortex-mix each component and combine in the following order using a calibrated micropipette:
 25 uL BSA stock
 35 uL yeast extract stock
 100 uL mucin stock
 Vortex soil suspension for 10 s prior to adding microbial test suspension.
 340 uL microbial test suspension
 Briefly vortex the final test suspension with 3-part soil load (at room temperature, 21+-3°C) and use to inoculate carriers within 30 min of preparation.
 Streak inoculate an agar plate with a loopful of the final test suspension. Incubate plate with the treated and control carrier plates and examine for purity after incubation at 36+-1°C for 72+-4 h.
 It is advisable to briefly rescreen each sterilized carrier for abnormalities prior to inoculation. Place carriers screened side up inside an empty, sterile Petri dish (no more than 20 carriers/dish).
 Privacy curtain carriers have no backing material and may be inoculated on either side.
 Non-PVC and vinyl carriers are layered materials comprised of a smooth, colored top surface and a white fabric bottom; only inoculate the top surface.
 Vortex-mix the final test suspension for 10 s following the addition of the soil load and immediately prior to use.
 Inoculate the number of carriers required for the evaluation of the test substance (3 controls and 5 treated) along with a few extra carriers.
 Using a calibrated positive displacement pipette with a 10 uL tip, withdraw 10 uL of the final test suspension and deposit it at the center of each carrier (screened and sterile), keeping the pipette perpendicular to the carrier during deposition of the final test suspension.  Avoid contact of pipette tip with carrier and do not spread the final test suspension with the pipette tip.
 For consistency, vortex-mix the inoculum frequently during inoculation of the carrier set.
 The same pipette tip may be used to inoculate all carriers (unless the tip is compromised).
 Discard any inoculated carrier where the final test suspension has run over the edge.
 Discard any inoculated carriers that flip over.
 Note: If inoculum soaks/absorbs into the carrier and is no longer visible, carriers are still acceptable to use. Refer to Appendix 3 for examples of inoculated carriers and carriers with 50 uL of test substance added.
 Transfer the Petri dish(es) with the inoculated carriers into a desiccation unit (with desiccant) and completely remove the lid of the Petri dish(es). Close the desiccation unit door (or lid) and seal the unit. Apply vacuum to evacuate the desiccation unit.
 Pressurize the desiccator slowly to lessen the potential for carries to move or flip.
 Note: do not exceed 40 inoculated carriers per desiccator to ensure carriers dry within the prescribed time.
 Maintain and monitor the vacuum level using a gauge. Achieve and maintain consistent level of vacuum (at 20-25 in of mercury, 508-635 torr, 677-847 mbar, or 68000-85000 Pascal) by leaving the vacuum on during the drying period with the desiccator stopcock opened or turning the vacuum off with the stopcock closed.
 Hold the inoculated carriers in the evacuated desiccation unit at 21+-3°C for 45 to 60 min. Visually inspect inoculated carriers to verify that they have completely dried and remove from desiccation unit. Do not use carriers that are visibly wet for testing.
 Record the time for all timed events.
 Depressurize the desiccator slowly to lessen the potential for carriers to move or flip.
 Carriers that become stuck or flip during desiccation should be discarded.
 Use dried inoculated carriers for testing within 30 min following removal from desiccation unit; hold carriers in closed Petri dish(es) at room temperature (21+-3°C) until use.

 Performance Assessment  -  Efficacy
 Evaluate 3 control carriers and 5 treated carriers for each test substance tested (one test organism and contact time/carrier type combination) unless specified otherwise.
 One set of control carriers per carrier type may be used for evaluating multiple test substances against one organism on one test day (assuming the carrier material, neutralizer, and soil load are the same).
 Using sterile forceps, transfer each dried carrier with the inoculated side up to a flat-bottom vial and cap the vial. Repeat until all carriers are transferred.
 Note: An anti-static gun may be aseptically used to reduce the carriers clinging to the side of the vial.
 Prepare the antimicrobial test substance. Use antimicrobial test substance within 3 hours of preparation or as specified by the manufacturer.
 In a timed fashion with appropriate intervals, sequentially deposit 50 uL of the test substance (equilibrated to 21+-3°C) with a calibrated micropipette over the dried inoculum on each test carrier, ensuring complete coverage.
 Note: Gently apply the antimicrobial test substance at a perpendicular angle to the inoculated carrier; do not forcefully deposit the disinfectant.
 Use a new tip for each carrier; do not touch the carrier surface with a pipette tip during the application of the test substance or the control substance; replace with new carrier(s) and vial(s) if this occurs. Do not cap the vials.
 For non-foaming aerosols and pump/trigger spray products, obtain the test substance by dispensing the product into a sterile vessel for collection. Cap the vessel and use dispensed product within 30 min.
 For foaming spray formulations, allow the foam to break down for at least 5-10 minutes for the generation of a 1-2 mL liquid sample. Cap the vessel and use dispensed product within 30 min.
 For NFV-01 and VF-01 carriers, do not process carriers where the test substance runs off the carrier or does not completely cover the inoculum spot; replace with new carrier(s) and vial(s) if this occurs. When test substance is applied to the PCF-03 carriers it is anticipated to soak into and through the carrier (e.g., the test substance may be observed to pool around the carrier).
 If the inoculum spot is no longer observable, place the test substance in the area where the inoculum spot was placed (e.g., the center of the carrier).
 Conduct the test at room temperature (21+-3°C) for the selected contact time. Use a certified timer to ensure that each carrier receives the required contact time.
 Process control carriers last. Each control carrier receives 50 uL PBS, equilibrated to 21+-3°C, instead of the test substance. Hold the control carriers for the same contact time as used for the test substance.
 Within +-5 s of the end of the contact period, add 10 mL of neutralizer equilibrated to 21+-3°C to each vial in the specified order according to the predetermined schedule.  Briefly vortex-mix (2-3 s) each vial following the addition of the neutralizer.
 For calculation purposes, the solution in the neutralized vial with carrier is considered to be 10[0] dilution.
 The neutralizer for the control carriers is the same as that for the treated carriers.
 Ensure that carrier's treated surface comes into contact with the neutralizer by swirling the vial to submerge the carrier prior to briefly vortex-mixing.
 Note: The carrier may float back to the surface after vortex-mixing.
 Following the neutralization of the entire set of carriers, vortex-mix vials at high speed for 30+-5 s then proceed as follows:
 Allow carriers to sit undisturbed in the vials for approximately 5 minutes.
 Vortex-mix vials at high speed for 30+-5 s (vortex-mix #2).
 Ensure that the carriers are submerged in the neutralizer during this step.
 Ensure that the liquid and carrier are both spinning in the vial during the vortex-mix.
 Allow carriers to sit undisturbed in the vials for approximately 5 minutes.
 Vortex-mix vials at high speed for 30+-5 s (vortex-mix #3).
 Ensure that the carriers are submerged in the neutralizer during this step.
 Ensure that the liquid and carrier are both spinning in the vial during the vortex-mix.
 Initiate dilutions within 30 min after completion of the vortex-mixing. Initiate filtration within 30 min of preparing the dilutions.
 Dilute and filter samples from the treated and control carriers; process treated carriers first.
 Serially dilute the eluate from the 10[0] dilution prior to filtration by transferring 1 mL into 9 mL PBS in a dilution tube.
 Turn on vacuum and leave on for the duration of the filtration process.
 Prior to filtration, pre-wet each membrane filter with ~10 mL PBS.
 Use separate membrane filters for each eluate (neutralized solution); however, the same filtration unit may be used for processing eluates from a given carrier set starting with the most dilute sample first.
 Filter each sample through a separate 0.2 um PES membrane filter.
 For eluates from treated carriers remaining in the vial (10[0] dilution), vortex-mix the vial for ~5 s, carefully pour the eluate into the filter unit.
 If a carrier falls onto the filter membrane, aseptically remove it using sterile forceps.
 Rinse the treated vial with ~20 mL PBS, vortex-mix for ~5 s, pour the wash into the same filter unit. For dilution tubes, rinse tube once with ~10 mL PBS, briefly vortex-mix, and pour into filter unit.
 Swirl the contents of the filter unit and quickly filter with limited pooling of liquid in the filter apparatus.
 Rinse the inside surface of the funnel unit with at least 20 mL PBS and filter the contents.
 Aseptically remove the membrane filter and place on the appropriate recovery medium.  Avoid trapping any air bubbles between the filter and the agar surface.
 Sterility controls.
 On the day of the test, filter ~20 mL of neutralizer and ~20 mL of the PBS used in the test using two separate membrane filters and place on individual TSA plates.
 Incubate these filters along with a plate of recovery medium (e.g., TSA) for 72+-4 h at 36+-1°C, record sterility results.
 Incubate plates at 36+-1°C for 48+-4 h for control carriers and 72+-4 h for treated carriers.
 It is recommended to monitor filters daily to optimize counting of colonies. CFUs may be counted daily. Record controls after 48+-4 h and treated carriers after 72+-4 h.
 Count colonies and record results.
 Any level of contamination which interferes with the recording and interpretation of results will result in invalid data.
 For example, contamination occuring on multiple filters within one set of serial dilutions and/or across multiple carriers is considered systemic and the test is deemed invalid.
 For colony counts on filters in excess of 200 record as Too Numerous to Count (TNTC).
 If no colonies are present, record as zero.
 Report non-conforming data (e.g., systemic contamination and atypical serial dilution results) and repeat tests as necessary.
 Systemic contamination
 Atypical serial dilution results (e.g., higher CFUs at more dilute levels).
 Inspect the growth on the filters for purity and typical characteristics of the test microbe, see Appendix 1, Table 1.
 If isolated colonies are present, assess one representative colony per 5-carrier set (treated) or 3-carrier set (controls) using a Gram stain.
 If confluent growth is present, perform a streak isolation on TSA or TSA with 5% sheep blood on growth taken from at least 1 carrier incubate at 36+-1°C for 24-48 h.
 If additional verification of the test organism is required due to observation of atypical morphology, perform further confirmatory analyses (e.g., Vitek, biochemical analyses) and isolation streaks on selective media.

 Data Requirements
 Per test, use colony counts to determine log reduction.
 For an acceptable test, each of the three control carriers must exhibit counts between 4.0-5.5 logs CFU/carrier.
 Use values with at least three significant figures when performing calculations (e.g., log density, mean log density).  Report the final mean log reduction value with two significant figures (e.g., round up to the nearest tenth).
 Calculate the Colony Forming Units (CFU)/carrier using the following equation:  Log10i=1nYii=1nCixDixV,
where:
Y
                                       =
CFU per filter,
C
                                       =
volume filtered,
V
                                       =
total volume of neutralizer,
D
                                       =
10[-k],
k
                                       =
dilution,
n
                                       =
number of dilutions, and
i
                                      =
lower limit of summation (the fewest number of dilutions).
 When TNTC (Too Numerous To Count) values are observed for each dilution filtered, substitute 200 for the TNTC at the highest (most dilute) dilution and account for the dilution factor in the calculations.
 Calculate the log density of each carrier by taking the log10 of the density per carrier.
 Calculate the mean log10 density across treated carriers.
 Calculate the mean log10 density across control carriers.
 Calculate the mean log10 reduction (LR) for treated carriers:
 Mean log10 reduction = the mean log10 density for control carriers minus the mean log10 density for treated carriers.
 For a set of treated carriers: when the 10[0] dilution (the contents of the vial with the carrier) is filtered either by itself or in addition to other dilutions and the data for each carrier result in zeros for each dilution filtered, report the mean LR as greater than or equal to the mean log10 density for the control carriers.
 Log reduction data based on estimates due to the occurrence of TNTC outcomes at each dilution in a dilution series for control and treated carriers is deemed unacceptable.

Appendix 1
                      Preparation of Frozen Stock Culture

 Initiate new stock cultures from lyophilized cultures of Pseudomonas aeruginosa and Staphylococcus aureus from a reputable vendor at least every 18 months.
          New frozen stock culture may be initiated one time using an existing, unexpired frozen stock culture as the source. Begin process at step 3 below, by streaking a loopful of the frozen stock culture onto 2 TSA plates.
 Rehydrate lyophilized culture.
 For lyophilized cultures received as ampules, open ampule of freeze-dried organism per manufacturer's instructions. Using a tube containing 5-6 mL of TSB, aseptically withdraw 0.5 to 1.0 mL and rehydrate the lyophilized culture. Aseptically transfer the entire rehydrated pellet back into the original tube of broth. Mix thoroughly. Incubate broth culture at 36+-1°C for 24+-2 h.
 For lyophilized cultures received on loops/swabs, as pellets, etc., open and rehydrate the lyophilized organism as indicated by the manufacturer. Transfer the rehydrated culture to a tube of 5-6 mL TSB (e.g., swish inoculated swab/loop in the TSB tube or transfer the rehydrated pellet to the TSB tube). Mix thoroughly. Incubate broth culture at 36+-1°C for 24+-2 h.
 At the end of the incubation timeframe, streak a loopful of the broth culture onto 2 TSA plates to obtain isolated colonies. Perform a streak isolation of the broth culture onto BAP as a purity check and streak the broth culture onto the appropriate selective media. Refer to appropriate selective media in Table 1.  Incubate all plates for 24+-2 h at 36+-1°C.
       Record results at the end of the incubation timeframe. Refer to Table 1 for results on selective media and diagnostic characteristics of the test microbes.
 From the TSA plates, select 3-5 isolated colonies of the test organism and re-suspend in 1 mL of TSB. For S. aureus, select only golden yellow colonies. For P. aeruginosa, select colonies from each of the two possible phenotypes present. Spread plate 0.1 mL of the suspension onto each of 6-10 TSA plates. Incubate the plates for 24+-2 h at 36+-1°C. If necessary, to obtain more frozen stock cultures, a larger suspension (e.g., 2 mL) may be prepared using the same ratio of TSB (1 mL) to number of colonies (3-5 colonies).
       Using the TSB suspension, perform a streak isolation of the suspension onto a BAP as a purity check, and streak on the appropriate selective media (refer to Table 1).
       Incubate all plates for 24+-2 h at 36+-1°C. Record results. Refer to Table 1 for results on selective media and diagnostic characteristics of the test microbes.
 After the incubation period, harvest growth from TSA plates by adding approximately 5 mL sterile cryoprotectant solution (TSB with 15% (v/v) glycerol) on the surface of each plate.  Re-suspend the growth in the cryoprotectant solution using a sterile spreader without damaging the agar surface. Aspirate the suspension from the plate with a pipette and place it in a sterile vessel large enough to hold about 30 mL.
 Repeat the growth harvesting procedure with the remaining plates and continue adding the suspension to the vessel (more than 1 vessel may be used if necessary). Mix the contents of the vessel(s) thoroughly; if more than 1 vessel is used, pool the vessels prior to aliquoting culture.
 Immediately after mixing, dispense 0.5-1.0 mL aliquots of the harvested suspension into cryovials; these represent the frozen stock cultures.
          For QC purposes, perform a streak isolation of the pooled culture onto a BAP as a purity check and streak on appropriate selective media (refer to Table 1).
          Incubate all plates for 24+-2 h at 36+-1°C.
          Record results. Refer to Table 1 for results on selective media and diagnostic characteristics of the test microbes.
          After incubation, perform a Gram stain on growth from the BAP; observe the Gram reaction by using brightfield microscopy at 1000X magnification (oil immersion).
          Conduct confirmation using an automated identification system (e.g., Vitek) or biochemical and antigenic analyses from growth taken from the BAP according to the manufacturer's instructions.
 Store the cryovials at approximately -80°C for a maximum of 18 months. These cultures are single use only.
 If the characteristics of the organism are not consistent with the information in Table 1 at any step in the process, or the Vitek profile is inconsistent with the organism, discard the cultures and re-initiate the process.
Table 1. Selective media and diagnostic characteristics for P. aeruginosa and S. aureus
                                    Aspect
                                P. aeruginosa*
                                   S. aureus
                              Gram stain reaction
                                   Negative
                                   Positive
                     Mannitol Salt Agar
(Selective medium)
                                      N/A
       Circular, small, yellow colonies, agar turning fluorescent yellow
                       Cetrimide Agar
(Selective medium)
Circular, small, initially opaque, turning fluorescent green over time; agar fluorescent yellowish green
                                      N/A
                               Blood agar (BAP)
Flat, opaque to off-white, round spreading (1), metallic sheen, slightly beta hemolytic
         Small, circular, yellow or white, glistening, beta hemolytic

                      Typical Microscopic Characteristics
                                Cell appearance
Straight or slightly curved rods, single polar flagella, rods formed in chains; 0.5-1.0 um in diameter x 1.5-5.0 um in length
Spherical, occurring singly, in pairs and tetrads, sometimes forming irregular clusters; 0.5-1.0 um in diameter
      *After 24+-2 h (1) P. aeruginosa may display two phenotypes.

Appendix 2
                             Neutralization Assay
The purpose of this section is to assess the effectiveness of the neutralization processes associated with this method. Perform the neutralization assay with both microbes for each carrier type prior to or concurrently with testing to demonstrate the neutralizer's ability to inactivate the chemical and determine if there is interference from the carrier itself.  Differences in performance (quality) between batches of media may lead to misleading neutralization results.
Select a neutralizing medium that is not inhibitory to the test microbe. The acceptance criteria for acceptable neutralization is <= 50% difference in colony counts between the neutralization effectiveness, neutralization toxicity control, titer control, carrier control, and test interference control.
 Refer to Section 3 in the preceding method for preparation of the test cultures. Conduct preliminary tests as necessary to determine appropriate dilution(s) of Test Suspension A (used to prepare Test Suspension B) to achieve the target challenge of 20-200 CFU per 10 uL or per carrier.
 Prepare Test Suspension A (without soil load). Serially dilute the microbial test suspension with PBS (e.g., through 10[-4] or 10[-5]). Select appropriate dilutions of Test Suspension A so that after the addition of the soil load, the Test Suspension B will achieve an average challenge of 20-200 CFU per 10 uL. Use Test Suspension A within 30 min of preparation.
 Prepare Test Suspension B (with soil load). Prepare the soil load: using a vortex, mix each component and combine 25 uL BSA, 35 uL yeast extract, and 100 uL of mucin; then vortex-mix the solution. Combine 340 uL of diluted Test Suspension A and the 160 uL of the soil load (SL) and vortex-mix for 10 seconds. Use Test Suspension B within 30 minutes of preparation.
 Ensure Test Suspension B provides an average challenge of 20-200 CFU per 10 uL.
 Two separate serial dilutions of Test Suspension A may be used to prepare two different concentrations of Test Suspension B to ensure at least one dilution with an average challenge of 20-200 CFU per 10 uL.
 A calibration curve (OD @ 650nm) may be used to estimate the number of viable organisms in Test Suspension A.
 Neutralization Treatments (see Appendix 2, Figure 2)
 Treatment 1: Neutralizer Effectiveness. Add 50 uL of the test substance to each of three reaction vessels. At timed intervals, add 10 mL neutralizer to each vessel and briefly swirl (by hand). After 10 s, gently add 10 uL of neutralizer test suspension using a micropipette to each vessel and briefly vortex. Proceed with section 4.
 Treatment 2: Neutralizer Toxicity Control. Add 10 mL neutralizer to each of three reaction vessels. At timed intervals, add 10 uL of Test Suspension B using a micropipette to each vessel and briefly vortex. Proceed with section 4.
 Treatment 3: Titer Control. Add 10 mL PBS to each of three reaction vessels.  At timed intervals, add 10 uL of Test Suspension B using a micropipette to each vessel and briefly vortex. Proceed with section 4.
 Treatment 4: Carrier Interference Control. Add one carrier to each of three reaction vessels. At timed intervals, add 10 mL neutralizer to each vessel and briefly swirl (by hand). After 10 s. gently add 10 uL of Test Suspension B using a micropipette to each vessel and briefly vortex.  Proceed with section 4. Assess one set of three carriers for each carrier type.
 Treatment 5: Test Interference Control. Add one carrier to each of three reaction vessels. At timed intervals, add 50 uL of test substance and 10 mL neutralizer to each vessel and briefly swirl (by hand). After 10 s gently add 10 uL of Test Suspension B using a micropipette to each vessel and briefly vortex. Proceed with section 4. Assess one set of three carriers for each carrier type.
 Hold the neutralization treatments for 10+-1 minutes at room temperature (21+-3°C).
 At the conclusion of the holding period, vortex each reaction and filter each mixture through a separate, pre-wetted 0.2 um PES membrane filter. Initiate filtration as soon as possible.
 Wash each reaction vessel with ~20 mL PBS and vortex; filter the wash through the same filter membrane. Finish the filtering process by rinsing the inside of the funnel unit with ~20 mL PBS and filter the rinsing liquid through the same filter membrane.
 Remove the membrane aseptically with sterile forceps and place it carefully over the surface of the recovery medium. Avoid trapping air bubbles between the filter and the agar surface.
 Incubate plates at 36+-1°C for 72+-4 h.
 It is recommended to monitor filters daily to optimize counting of colonies.

                          Titer Interference Control
Add 50 uL of test substance to each vessel. At timed intervals add 10 mL neutralizer and swirl by hand.
                           Neutralizer Effectiveness
Add 10 uL of Test Suspension B to each vessel containing 50 uL test substance and 10 mL neutralizer.
Wait 10 s
Vortex and hold for 10 min at 21+-3°C. Proceed to vortexing/filtering.
Treatment 1
 50 uL test substance
Add 10 uL of Test Suspension B to each vessel containing 10 mL neutralizer.
                         Neutralizer Toxicity Control
Vortex and hold for 10 min at 21+-3°C. Proceed to vortexing/filtering.
Treatment 2
Add 10 uL of Test Suspension B to each vessel containing 10 mL PBS.
                                 Titer Control
Vortex and hold for 10 min at 21+-3°C. Proceed to vortexing/filtering.
Treatment 3
Add 1 sterile carrier to each vessel. At timed intervals, add 10 mL neutralizer and swirl by hand.
Treatment 4
Add 10 uL of Test Suspension B to each vessel containing the carrier and 10 mL neutralizer.
Wait 10 s
Vortex and hold for 10 min at 21+-3°C. Proceed to vortexing/filtering.
                          Titer Interference Control
Add 1 sterile carrier to each vessel. At timed intervals, add 50 uL of test substance and 10 mL neutralizer and swirl by hand.
Treatment 5
Add 10 uL of Test Suspension B to each vessel containing the carrier, 50 uL test substance, and 10 mL neutralizer.
Wait 10 s
Vortex and hold for 10 min at 21+-3°C. Proceed to vortexing/filtering.
Figure 2: Neutralization Schematic

Appendix 3
                               Carrier Examples

                   Figure 3: Examples of Inoculated Carriers

Figure 4: Examples of Dried Inoculated Carriers with 50 uL of Test Substance Added


Prepare and submit a notice of termination of financial assurance 0.25 0.00 0.50 0.25 1.00 $ 63.25 $ 0.00 $ 0.38 5,093 5,093 $ 324,068 If necessary, prepare and submit notice of failure to obtain alternative coverage within 60 days 0.00 0.50 1.00 0.25 1.75 $ 103.25 $ 0.00 $ 0.38 509 891 $ 52,748 Reporting ( 280.110)

Gather and submit forms listed in Section 280.111( b)

to the implementing agency documenting current evidence of financial responsibility 0.00 0.00 0.00 0.25 0.25 $ 7.25 $ 0.00 $ 0.38 7,833 1,958 $ 59,766 Recordkeeping ( 280.111)

Obtain and keep on file proof of financial responsibility 0.00 0.00 0.25 0.10 0.35 $ 17.15 $ 0.00 $ 0.10 254,666 89,133 $ 4,392,989 Update certification if financial assurance mechanism is modified 0.00 0.00 0.50 0.25 0.75 $ 35.75 $ 0.00 $ 0.00 12,733 9,550 $ 455,205 Bankruptcy or Other Incapacity ( 280.114)

Prepare and submit notification of commencement of bankruptcy procedures 0.50 0.25 0.50 0.25 1.50 $ 110.25 $ 0.00 $ 0.38 5,093 7,640 $ 563,439 If necessary, prepare and submit notice of inability to obtain alternative coverage within 30 days 0.00 0.50 1.00 0.25 1.75 $ 103.25 $ 0.00 $ 0.38 509 891 $ 52,748 Subtotal varies varies varies varies varies varies varies varies varies 139,822 $ 7,763,246 TOTAL varies varies varies varies varies varies varies varies varies 6,131,127 $ 674,396,330 63 EXHIBIT 2 ANNUAL ESTIMATED RESPONDENT HOUR AND COST BURDEN STATE PROGRAM APPROVAL Hours and Costs Per Respondent Per Activity Total Hours and Costs Number Capital/ of Respon. Labor Startup O & M Respon. Total Total Legal Managerial Technical Clerical Hours/ Cost/ Cost per Cost/ or Hours/ Cost/

Information Collection Activity Hours Hours Hours Hours Activity Activity Activity Activity Activ. Year Year COMPONENTS OF A PROGRAM APPLICATION Reading the Regulations Read the regulations 4.00 8.00 8.00 0.00 20.00 $ 1,112.00 $ 0.00 $ 0.00 2 40 $ 2,224 Transmittal Letter ( 281.20( a))

Obtain a transmittal letter from the Governor's office 1.00 1.00 2.00 1.00 5.00 $ 244.00 $ 0.00 $ 0.00 2 10 $ 488 Submit letter to implementing agency 0.00 0.00 0.00 0.10 0.10 $ 2.30 $ 0.00 $ 0.38 2 0.2 $ 5 Description of State Program ( 281.20( b) and 2801.21)

Prepare and submit description of state program 6.00 10.00 30.00 0.00 46.00 $ 2,310.00 $ 0.00 $ 3.64 2 92 $ 4,627 Procedures for Adequate Enforcement ( 281.22 and 281.20( c))

Gather and submit information on compliance monitoring and enforcement procedures 6.00 4.00 30.00 5.00 45.00 $ 2,083.00 $ 0.00 $ 3.64 2 90 $ 4,173 Memorandum of Agreement ( 281.24 and 381.20( e))

Negotiate an MOA with EPA 10.00 6.00 15.00 5.00 36.00 $ 1,887.00 $ 0.00 $ 0.00 2 72 $ 3,774 Prepare and submit written MOA with application 4.00 6.00 8.00 2.00 20.00 $ 1,044.00 $ 0.00 $ 3.64 2 40 $ 2,095 Attorney General's Statement ( 281.25 and 2801.20( f))

Draft and submit statement from Attorney General 47.00 9.00 18.00 8.00 82.00 $ 5,213.00 $ 0.00 $ 3.64 2 164 $ 10,433 Copies of Statutes and Regulations ( 281.20( g))

Research and gather statutes and regulations 8.00 0.00 4.00 12.00 24.00 $ 1,084.00 $ 0.00 $ 0.00 2 48 $ 2,168 Submit copies as a part of the application to EPA 0.00 0.00 0.00 0.10 0.10 $ 2.30 $ 0.00 $ 3.64 2 0.2 $ 12 Subtotal varies varies varies varies varies varies varies varies varies 556 $ 29,999 ADEQUATE ENFORCEMENT AND COMPLIANCE Reading the Regulations Read the regulations 1.00 1.00 2.00 0.00 4.00 $ 221.00 $ 0.00 $ 0.00 2 8 $ 442 Sharing of Information ( 281.43( a))

Maintain files and current information on program administration 0.00 5.00 4.00 5.00 14.00 $ 568.00 $ 0.00 $ 0.00 39 546 $ 22,152 Submit information to EPA, if requested 0.00 0.00 0.00 0.10 0.10 $ 2.30 $ 0.00 $ 3.64 1 0.1 $ 6 Subtotal varies varies varies varies varies varies varies varies varies 554 $ 22,600 64 EXHIBIT 2 ( continued)

ANNUAL ESTIMATED RESPONDENT HOUR AND COST BURDEN STATE PROGRAM APPROVAL Hours and Costs Per Respondent Per Activity Total Hours and Costs Number Capital/ of Respon. Labor Startup O & M Respon. Total Total Legal Managerial Technical Clerical Hours/ Cost/ Cost per Cost/ or Hours/ Cost/

Information Collection Activity Hours Hours Hours Hours Activity Activity Activity Activity Activ. Year Year APPROVAL PROCEDURES Reading the Regulations Read the regulations 1.00 2.00 2.00 0.00 5.00 $ 278.00 $ 0.00 $ 0.00 0 0 $ 0 Collect and submit information on changes in regulations or shifts in responsibilities 2.00 10.00 5.00 5.00 22.00 $ 1,055.00 $ 0.00 $ 3.64 0 0 $ 0 Prepare and submit revised application,

if requested by EPA 20.00 15.00 25.00 6.00 66.00 $ 3,643.00 $ 0.00 $ 3.64 0 0 $ 0 Subtotal varies varies varies varies varies varies varies varies varies 0 $ 0 WITHDRAWAL OF APPROVAL OF STATE PROGRAMS Reading the Regulations Read the regulations 1.00 1.00 2.00 0.00 4.00 $ 221.00 $ 0.00 $ 0.00 0 0 $ 0 Gather information for program transfer 2.00 2.00 16.00 8.00 28.00 $ 1,130.00 $ 0.00 $ 0.00 0 0 $ 0 Prepare and submit transfer notification 0.00 0.50 1.00 1.00 2.50 $ 93.50 $ 0.00 $ 0.38 0 0 $ 0 Develop and submit plan for information transfer at least 90 days before the transfer 2.00 4.00 8.00 2.00 16.00 $ 770.00 $ 0.00 $ 3.64 0 0 $ 0 Subtotal varies varies varies varies varies varies varies varies varies 0 $ 0 TOTAL varies varies varies varies varies varies varies varies varies 1,110 $ 52,599 Procedures for Withdrawal of Approval of State Programs ( 281.61( a)( 1))

Revision of Approved State Programs ( 281.52( a)( b)) 65 EXHIBIT 3 ANNUAL ESTIMATED AGENCY HOUR AND COST BURDEN UST TECHNICAL AND FINANCIAL REQUIREMENTS Hours and Costs Per Respondent Per Activity Total Hours and Costs Number Capital/ of Respon. Labor Startup O & M Respon. Total Total Legal Managerial Technical Clerical Hours/ Cost/ Cost Cost or Hours/ Cost/

Information Collection Activity Hours Hours Hours Hours Activity Activity Activity Activity Activ. Year Year UST SYSTEMS: DESIGN, CONSTRUCTION, INSTALLATION AND NOTIFICATION Review and file notification forms 0.00 0.00 0.50 0.25 0.75 $ 21.81 $ 0.00 $ 0.00 33,333 25,000 $ 726,993 Enter information into a database 0.00 0.00 0.00 0.10 0.10 $ 2.03 $ 0.00 $ 0.00 33,333 3,333 $ 67,666 Subtotal varies varies varies varies varies varies varies varies varies 28,333 $ 794,659 RELEASE DETECTION Review demonstration of the effectiveness of an alternative method of release detection for piping 0.00 2.00 8.00 0.00 10.00 $ 362.84 $ 0.00 $ 0.00 0 0 $ 0 Review demonstration of the effectiveness of an alternative method of release detection for tanks 0.00 2.00 8.00 0.00 10.00 $ 362.84 $ 0.00 $ 0.00 0 0 $ 0 Subtotal varies varies varies varies varies varies varies varies varies 0 $ 0 RELEASE REPORTING, INVESTIGATION, AND CONFIRMATION Review and file information on suspected releases 0.00 0.50 2.00 0.50 3.00 $ 100.88 $ 0.00 $ 0.00 9,792 29,376 $ 987,817 Enter information into a database 0.00 0.00 0.00 0.10 0.10 $ 2.03 $ 0.00 $ 0.00 9,792 979 $ 19,878 Review and file information on major spills and overfills over 25 gallons or the reportable quantity 0.00 0.50 2.00 0.50 3.00 $ 100.88 $ 0.00 $ 0.00 7,833 23,499 $ 790,193 Enter information into a database 0.00 0.00 0.00 0.10 0.10 $ 2.03 $ 0.00 $ 0.00 7,833 783 $ 15,901 Review and file information on minor spills and overfills under 25 gallons or the reportable quantity that could not be cleaned up within 24 hours of the occurence 0.00 0.25 1.00 0.25 1.5 $ 50.44 $ 0.00 $ 0.00 1,000 1,500 $ 50,440 Enter information into a database 0.00 0.00 0.00 0.10 0.10 $ 2.03 $ 0.00 $ 0.00 1,000 100 $ 2,030 Subtotal varies varies varies varies varies varies varies varies varies 56,237 $ 1,866,259 66 EXHIBIT 3 ( continued)

ANNUAL ESTIMATED AGENCY HOUR AND COST BURDEN UST TECHNICAL AND FINANCIAL REQUIREMENTS Hours and Costs Per Respondent Per Activity Total Hours and Costs Number Capital/ of Respon. Labor Startup O & M Respon. Total Total Legal Managerial Technical Clerical Hours/ Cost/ Cost Cost or Hours/ Cost/

Information Collection Activity Hours Hours Hours Hours Activity Activity Activity Activity Activ. Year Year RELEASE RESPONSE AND CORRECTIVE ACTION FOR UST SYSTEMS CONTAINING PETROLEUM OR HAZARDOUS SUBSTANCES Receive and review initial response report 0.00 0.00 0.50 0.00 0.50 $ 16.72 $ 0.00 $ 0.00 7,833 3,917 $ 130,968 Enter information into a data base 0.00 0.00 0.00 0.50 0.50 $ 10.17 $ 0.00 $ 0.00 7,833 3,917 $ 79,662 Review and file summary report of initial abatement measures 0.00 1.00 4.00 0.25 5.25 $ 186.51 $ 0.00 $ 0.00 7,833 41,123 $ 1,460,933 Review and file information on initial site characterization 0.00 1.00 4.00 0.25 5.25 $ 186.51 $ 0.00 $ 0.00 7,833 41,123 $ 1,460,933 Enter information into a database 0.00 0.00 0.00 0.25 0.25 $ 5.09 $ 0.00 $ 0.00 7,833 1,958 $ 39,870 Review and file free product removal report 0.00 1.00 4.00 0.25 5.25 $ 186.51 $ 0.00 $ 0.00 1,567 8,227 $ 292,261 Enter information into a database 0.00 0.00 0.00 0.10 0.10 $ 2.03 $ 0.00 $ 0.00 1,567 157 $ 3,181 Review and file soil and groundwater investigation report 0.00 1.00 4.00 0.25 5.25 $ 186.51 $ 0.00 $ 0.00 3,133 16,448 $ 584,336 Enter information into a database 0.00 0.00 0.00 0.25 0.25 $ 5.09 $ 0.00 $ 0.00 3,133 783 $ 15,947 Review and file corrective action plan 1.00 2.00 8.00 0.25 11.25 $ 434.19 $ 0.00 $ 0.00 2,350 26,438 $ 1,020,347 Notify public of the release and the corrective action plan through an appropriate means 0.00 0.00 1.00 0.25 1.25 $ 38.53 $ 0.00 $ 0.00 2,350 2,938 $ 90,546 Hold a public hearing and consider comments on the proposed corrective action plan 0.00 2.00 8.00 0.00 10.00 $ 362.84 $ 0.00 $ 0.00 2,350 23,500 $ 852,674 Notify the owner or operator whether plan is approved or disapproved 0.00 0.25 0.00 0.25 0.50 $ 17.00 $ 0.00 $ 0.38 2,350 1,175 $ 40,843 Review and file the report on results of implementing the plan 2.00 4.00 16.00 0.25 22.25 $ 863.29 $ 0.00 $ 0.00 2,350 52,288 $ 2,028,732 Give public notice if the approved plan fails to achieve established cleanup levels, and termination of plan is being considered ( failure rate of 5%) 0.00 0.00 1.00 0.25 1.25 $ 38.53 $ 0.00 $ 0.00 118 148 $ 4,547 Subtotal varies varies varies varies varies varies varies varies varies 224,140 $ 8,105,780 OUT OF SERVICE UST SYSTEMS AND CLOSURE Review and file notification of closure or a change in service 0.00 0.00 0.50 0.25 0.75 $ 21.81 $ 0.00 $ 0.00 31,667 23,750 $ 690,657 Subtotal varies varies varies varies varies varies varies varies varies 23,750 $ 690,657 67 EXHIBIT 3 ( continued)

ANNUAL ESTIMATED AGENCY HOUR AND COST BURDEN UST TECHNICAL AND FINANCIAL REQUIREMENTS Hours and Costs Per Respondent Per Activity Total Hours and Costs Number Capital/ of Respon. Labor Startup O & M Respon. Total Total Legal Managerial Technical Clerical Hours/ Cost/ Cost Cost or Hours/ Cost/

Information Collection Activity Hours Hours Hours Hours Activity Activity Activity Activity Activ. Year Year FINANCIAL RESPONSIBILITY Review and file submitted financial assurance information 0.00 0.00 0.50 0.25 0.75 $ 21.81 $ 0.00 $ 0.00 7,833 5,875 $ 170,838 Enter financial assurance information into a database 0.00 0.00 0.00 0.10 0.10 $ 2.03 $ 0.00 $ 0.00 7,833 783 $ 15,901 Notify owners or operators if they no longer meet financial assurance responsibilities 0.00 0.25 0.00 0.25 0.50 $ 17.00 $ 0.00 $ 0.38 5,093 2,547 $ 88,516 Review and file notification of failure to obtain alternative financial assurance due to cancellation or non renewal of policy 0.00 0.25 1.00 0.25 1.50 $ 50.44 $ 0.00 $ 0.00 509 764 $ 25,674 Review and file notification of commencement of bankruptcy procedures 0.00 0.25 1.00 0.25 1.50 $ 50.44 $ 0.00 $ 0.00 5,093 7,640 $ 256,891 Review and file notification of failure to obtain alternative financial assurance due to bankruptcy 0.00 0.25 1.00 0.25 1.50 $ 50.44 $ 0.00 $ 0.00 509 764 $ 25,674 Subtotal varies varies varies varies varies varies varies varies varies 18,373 $ 583,494 TOTAL varies varies varies varies varies varies varies varies varies 350,833 $ 12,040,849 68 EXHIBIT 4 ANNUAL ESTIMATED AGENCY HOUR AND COST BURDEN STATE PROGRAM APPROVAL Hours and Costs Per Respondent Per Activity Total Hours and Costs Number Capital/ of Respon. Labor Startup O & M Respon. Total Total Legal Managerial Technical Clerical Hours/ Cost/ Cost Cost or Hours/ Cost/

Information Collection Activity Hours Hours Hours Hours Activity Activity Activity Activity Activ. Year Year COMPONENTS OF A PROGRAM APPLICATION Examine State program application and notify the State whether the application is complete 0.00 1.00 4.00 0.00 5.00 $ 181.42 $ 0.00 $ 0.00 2 10 $ 363 Review the complete State program application and tenatively determine approval or disapproval for State program 8.00 16.00 40.00 0.00 64.00 $ 2,630.24 $ 0.00 $ 0.00 2 128 $ 5,260 Issue public notice of the tentative decision 0.00 0.50 0.00 0.25 0.75 $ 28.92 $ 0.00 $ 0.00 2 2 $ 58 Receive and review public comments 0.00 1.00 4.00 0.00 5.00 $ 181.42 $ 0.00 $ 0.00 2 10 $ 363 Conduct public hearing, if necessary 0.00 2.00 2.00 0.00 4.00 $ 162.20 $ 0.00 $ 0.00 2 8 $ 324 Make a final decision on approval or disapproval for a State program 0.00 1.00 4.00 0.00 5.00 $ 181.42 $ 0.00 $ 0.00 2 10 $ 363 Issue notice of the final decision 0.00 0.50 0.00 0.25 0.75 $ 28.92 $ 0.00 $ 0.00 2 2 $ 58 Review the revision of approved State program and tentatively determine approval or disapproval 2.00 4.00 10.00 0.00 16.00 $ 657.56 $ 0.00 $ 0.00 0 0 $ 0 Issue public notice of the tentative decision 0.00 0.50 0.00 0.25 0.75 $ 28.92 $ 0.00 $ 0.00 0 0 $ 0 Notify State if decision receives negative public comment and either withdraw the decision or respond to the negative public comments 0.00 1.00 4.00 0.25 5.25 $ 186.51 $ 0.00 $ 0.38 0 0 $ 0 Subtotal varies varies varies varies varies varies varies varies varies 170 $ 6,789 WITHDRAWAL OF APPROVAL OF STATE PROGRAMS Review and file transfer notification and plan 1.00 2.00 8.00 0.25 11.25 $ 434.19 $ 0.00 $ 0.00 0 0 $ 0 Publish notice of the transfer 0.00 0.50 0.00 0.25 0.75 $ 28.92 $ 0.00 $ 0.00 0 0 $ 0 Conduct withdrawal proceedings in accordance with 40 CFR 271.23( b) and ( c) 2.00 4.00 16.00 0.00 22.00 $ 858.20 $ 0.00 $ 0.00 0 0 $ 0 Subtotal varies varies varies varies varies varies varies varies varies 0 $ 0 TOTAL varies varies varies varies varies varies varies varies varies 170 $ 6,789 69 6( e) Bottom Line Hour and Cost Burden ( 1) Respondent Tally Exhibit 5 summarizes the total annual hour and cost burden to UST owners and operators and to States ( as respondents seeking program approval). As shown in the exhibit, EPA estimates the annual respondent burden to be approximately 6.13 million hours and $ 674.45 million. The bottom line burden to respondents over three years is estimated to be approximately 18.39 million hours and $ 2,023.35 million.

EXHIBIT 5 TOTAL ESTIMATED RESPONDENT HOUR AND COST BURDEN SUMMARY Total Capital/ Total Hours/ Labor Startup O & M Cost/ Information Collection Activity Year Cost Cost Cost Year Technical and Financial Requirements 6,131,127 $ 330,889,322 $ 80,250,656 $ 263,256,350 $ 674,396,330 State Program Approval 1,110 $ 52,560 $ 0 $ 40 $ 52,599 TOTAL 6,132,237 $ 330,941,882 $ 80,250,656 $ 263,256,390 $ 674,448,929 ( 2) Agency Tally Exhibit 6 summarizes total annual Agency hour and cost burden associated with all the requirements covered in this ICR. As shown in the exhibit, EPA estimates the annual Agency burden to be approximately 0.35 million hours and $ 12.05 million. The bottom line burden to the Agency over three years is estimated to be approximately 1.05 million hours and $ 36.15 million.

EXHIBIT 6 TOTAL ESTIMATED AGENCY HOUR AND COST BURDEN SUMMARY Total Capital/ Total Hours/ Labor Startup O & M Cost/ Information Collection Activity Year Cost Cost Cost Year Technical and Financial Requirements 350,833 $ 12,038,021 $ 0 $ 2,828 $ 12,040,849 State Program Approval 170 $ 6,789 $ 0 $ 0 $ 6,789 TOTAL 351,003 $ 12,044,810 $ 0 $ 2,828 $ 12,047,638 70 ( 3) Tally of Key Data Table 5 summarizes key data examined in this document. This information can be used in completing the OMB 83 I Form for this ICR.

Table 5 Key Data Parameter Value Number of respondents 254,705 Number of responses 500,000 Total hours requested 6,132,237 Total annualized capital/ startup costs $ 80,250,656 Total annual O& M costs $ 263,256,390 Total annualized cost requested $ 343,507,046 6( f) Reasons for Change in Burden This ICR presents a comprehensive description of the total annual respondent burden for all information collection activities related to the UST program. In renewing this ICR, EPA has updated its respondent universe and burden estimates based on updated data from the Office of Underground Storage Tanks ( OUST) and the regulated community. Because of these updated data, the total annual hourly burden to respondents has increased from the previous ICR by 106,694 hours ( from 6,025,543 hours/ year to 6,132,237 hours/ year). The current OMB inventory reports $ 363,000 in total annualized costs; there was an error in the cost burden associated with the previous ICR ( 1360.06) and the cost estimate should have been $ 363,561,000. The total annualized cost requested in this renewal ICR is $ 343,507,046. In this renewal ICR, EPA is correcting this error in the cost burden estimate. The total annualized cost requested is now $ 343,507,046. This corrects the current OMB inventory and is based on updated data from the Office of Underground Storage Tanks ( OUST) and the regulated community. This new burden is $ 20.05 million less than the previous ICR submission.

6( g) Burden Statement Exhibit 7 presents the average annual respondent hourly burden for each UST facility and for each State applying for program approval. As shown in the exhibit, for UST facilities, the total hourly burden for conducting the activities covered in this ICR is estimated to average approximately 24 hours per respondent annually. The reporting burden is estimated to average 12 hours per respondent per year. This estimate includes 71 time for preparing and submitting notices, preparing and submitting demonstrations and applications, reporting releases, gathering information, and preparing and submitting reports. The recordkeeping burden for UST facilities is estimated to average 12 hours per respondent per year. This estimate includes time for gathering information and for developing and maintaining records.

EXHIBIT 7 TOTAL AVERAGE RESPONDENT BURDEN Information Collection Activity Average Reporting Burden ( Hours) Average Recordkeeping Burden ( Hours) Total Average Burden ( Hours) Technical and Financial Requirements 11.83 12.24 24.07 State Program Approval 13.24 15.23 28.47 For States applying for program approval, the total hourly burden is estimated to average approximately 28 hours per State annually. The reporting burden is estimated to average 13 hours per respondent per year. This estimate includes time for preparing and submitting an application and associated information. The recordkeeping burden is estimated to average 15 hours per respondent per year. This estimate includes time for maintaining application files.

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.

To comment on the Agency's need for this information, the accuracy of the provided burden estimates, and any suggested methods for minimizing respondent burden, including the use of automated collection techniques, EPA has established a public docket for this ICR under Docket ID No. UST 2004 0001, which is available for public viewing at the UST Docket in the EPA Docket Center ( EPA/ DC), EPA West, Room B102, 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 Reading Room is ( 202) 566 1744, and the telephone number for the UST Docket is ( 202) 566 0270. An electronic version of the public docket is available through EPA Dockets ( EDOCKET) at http:// www. epa. gov/ edocket. Use EDOCKET to submit or view public comments, access the index listing of the contents of the public docket, and to access those documents in the public docket that are available electronically. Once in the system, select " search," then key in the docket ID number identified above. Also, you can send comments to the Office of Information and Regulatory Affairs, Office of Management and Budget, 725 72 17th Street, NW, Washington, DC 20503, Attention: Desk Office for EPA. Please include the EPA Docket ID No. ( UST 2004 0001) and OMB control number ( 2050 0068) in any correspondence.


[Federal Register Volume 80, Number 167 (Friday, August 28, 2015)]
[Rules and Regulations]
[Pages 52177-52179]
[FR Doc No: 2015-21204]

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DEPARTMENT OF TRANSPORTATION

Federal Aviation Administration

14 CFR Part 39

[Docket No. FAA-2014-1130; Directorate Identifier 2015-NE-04-AD;
Amendment 39-18250; AD 2015-17-17]
RIN 2120-AA64

Airworthiness Directives; Pratt & Whitney Turbofan Engines

AGENCY: Federal Aviation Administration (FAA), DOT.

ACTION: Final rule.

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SUMMARY: We are adopting a new airworthiness directive (AD) for all
Pratt & Whitney (PW) PW4164-1D, PW4168-1D, PW4168A-1D and PW4170
engines, and certain PW4164, PW4168, and PW4168A turbofan engines. This
AD was prompted by fuel nozzle-to-fuel supply manifold interface fuel
leaks. This AD requires inspecting fuel nozzles for signs of leakage,
replacing hardware as required, and torqueing to specified requirement.
We are issuing this AD to prevent fuel leaks which could result in
engine fire and damage to the airplane.

DATES: This AD is effective October 2, 2015.
    The Director of the Federal Register approved the incorporation by
reference of a certain publication listed in this AD as of October 2,
2015.

ADDRESSES: For service information identified in this AD, contact Pratt
& Whitney, 400 Main St., East Hartford, CT 06108; phone:<PHONE_NUMBER>;
fax:<PHONE_NUMBER>. You may view this service information at the FAA,
Engine & Propeller Directorate, 12 New England Executive Park,
Burlington, MA. For information on the availability of this material at
the FAA, call<PHONE_NUMBER>. It is also available on the Internet at
http://www.regulations.gov by searching for and locating Docket No.
FAA-2014-1130.

Examining the AD Docket

    You may examine the AD docket on the Internet at http://www.regulations.gov by searching for and locating Docket No. FAA-2014-
1130; or in person at the Docket Management Facility between 9 a.m. and
5 p.m., Monday through Friday, except Federal holidays. The AD docket
contains this AD, the regulatory evaluation, any comments received, and
other information. The address for the Docket Office (phone: 800-647-
5527) is Document Management Facility, U.S. Department of
Transportation, Docket Operations, M-30, West Building Ground Floor,
Room W12-140, 1200 New Jersey Avenue SE., Washington, DC 20590.

FOR FURTHER INFORMATION CONTACT: Katheryn Malatek, Aerospace Engineer,
Engine Certification Office, FAA, Engine & Propeller Directorate, 12
New England Executive Park, Burlington, MA 01803; phone:<PHONE_NUMBER>;
fax:<PHONE_NUMBER>; email<EMAIL_ADDRESS>
SUPPLEMENTARY INFORMATION:

Discussion

    We issued a notice of proposed rulemaking (NPRM) to amend 14 CFR
part 39 by adding an AD that would apply to all PW PW4164-1D, PW4168-
1D, PW4168A-1D and PW4170 engines, and certain PW4164, PW4168, and
PW4168A turbofan engines. The NPRM

published in the Federal Register on April 21, 2015 (80 FR 22140). The
NPRM was prompted by reports of four fuel nozzle leaks in service and
an additional six fuel nozzle leaks found during shop visits. The root
cause is inadequate torque of the fuel nozzle-to-fuel supply manifold
B-nuts for the temperatures that the fuel nozzles experience. The NPRM
proposed to require inspecting fuel nozzles for signs of leakage,
replacing hardware as required, and torqueing B-nuts to specified
requirement. We are issuing this AD to prevent fuel leaks which could
result in engine fire and damage to the airplane.

Related Service Information Under CFR Part 51

    We reviewed PW Alert Service Bulletin (ASB) No. PW4G-100-A73-44,
Revision 1, dated February 12, 2015. This ASB describes procedures for
fuel supply manifold inspection and re-torque of the B-nut connection.
This service information is reasonably available because the interested
parties have access to it through their normal course of business or by
the means identified in the ADDRESSES section of this AD.

Comments

    We gave the public the opportunity to participate in developing
this AD. The following presents the comments received on the NPRM (80
FR 22140, April 21, 2015) and the FAA's response to each comment.

Request To Change Referenced Service Information

    Korean Air requested that this AD mandate following PW ASB No.
PW4G-100-A73-44 Revision 1, dated February 12, 2015 instead of PW ASB
No. PW4G-100-A73-44, dated October 10, 2014. Korean Air would like to
receive credit for service performed in accordance with the latest
revision of the ASB.
    We agree. We changed this AD to include PW ASB No. PW4G-100-A73-44
Revision 1, dated February 12, 2015 and added a Credit for Previous
Action section to provide credit when PW ASB No. PW4G-100-A73-44, dated
October 10, 2014 is followed, before the effective date of this AD.

Request To Add Service Information

    Korean Air requested that engines incorporating Special Instruction
(SI) 129F-14 meet the requirement for compliance with this AD since SI
129F-14 provides the same instructions as PW ASB No. PW4G-100-A73-44,
dated October 10, 2014 and PW ASB No. PW4G-100-A73-44 Revision 1, dated
February 12, 2015.
    We agree. We added SI 129F-14 to the Credit for Previous Action
section.

Request To Change Mandatory Terminating Action

    Korean Air requested that the Mandatory Terminating Action section
be changed to state that the actions listed are closing actions to the
repetitive inspections defined in the Compliance section.
    We agree. We changed the Mandatory Terminating Action section by
adding, ``As terminating action to the repetitive inspection
requirements in paragraph (e)(1) of this AD do the following:''.

Request To Change Applicability

    PW requested that engines incorporating PW Service Bulletin (SB)
No. PW4G-100-72-220, Revision 4, dated September 30, 2011 be added to
the Applicability section.
    We disagree. Engines incorporating PW SB No. PW4G-100-72-220,
Revision 4, dated September 30, 2011 are identified in the
Applicability section by model designation. We did not change this AD.

Request To Redefine ``Cycles''

    PW requested that the definition of cycles be changed from ``cycles
since new or cycles since the incorporation of PW SB No. PW4G-100-72-
214, dated December 15, 2011 or SB No. PW4G-100-72-219, Revision 1,
dated October 5, 2011'' to ``since new (1st run) or since last torque
application to the B-nuts on the fuel nozzle installation.'' The
justification for this request is that the B-nuts could have been
torqued subsequent to the incorporation of the service bulletins.
    We agree. We changed the Definition paragraph to define cycles as
``. . . since new or cycles since last torque application to the B-nuts
on the fuel nozzle installation.''

Request To Change Compliance Time

    Asiana Airlines requested that the compliance time listed in this
AD match the dates listed in the ASB. Asiana believes the compliance
time listed in this AD is more restrictive than the dates listed in the
ASB.
    We disagree. Using cycles since the effective date of this AD
instead of calendar dates provides greater fleet management flexibility
to the operator while acceptably resolving the unsafe condition.

Conclusion

    We reviewed the relevant data, considered the comments received,
and determined that air safety and the public interest require adopting
this AD with the changes described previously.
    We also determined that these changes will not increase the
economic burden on any operator or increase the scope of this AD.

Costs of Compliance

    We estimate that this AD would affect about 72 engines installed on
airplanes of U.S. registry. The average labor rate is $85 per hour. We
estimate that parts replacement will cost about $1,356 per engine.
Based on these figures, we estimate the cost of this AD on U.S.
operators to be $391,392.

Authority for This Rulemaking

    Title 49 of the United States Code specifies the FAA's authority to
issue rules on aviation safety. Subtitle I, section 106, describes the
authority of the FAA Administrator. Subtitle VII: Aviation Programs,
describes in more detail the scope of the Agency's authority.
    We are issuing this rulemaking under the authority described in
Subtitle VII, Part A, Subpart III, Section 44701: ``General
requirements.'' Under that section, Congress charges the FAA with
promoting safe flight of civil aircraft in air commerce by prescribing
regulations for practices, methods, and procedures the Administrator
finds necessary for safety in air commerce. This regulation is within
the scope of that authority because it addresses an unsafe condition
that is likely to exist or develop on products identified in this
rulemaking action.

Regulatory Findings

    This AD will not have federalism implications under Executive Order
13132. This AD will not have 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.
    For the reasons discussed above, I certify that this AD:
    (1) Is not a ``significant regulatory action'' under Executive
Order 12866,
    (2) Is not a ``significant rule'' under DOT Regulatory Policies and
Procedures (44 FR 11034, February 26, 1979),
    (3) Will not affect intrastate aviation in Alaska to the extent
that it justifies making a regulatory distinction, and
    (4) Will not have a significant economic impact, positive or
negative, on a substantial number of small entities under the criteria
of the Regulatory Flexibility Act.

List of Subjects in 14 CFR Part 39

    Air transportation, Aircraft, Aviation safety, Incorporation by
reference, Safety.

Adoption of the Amendment

    Accordingly, under the authority delegated to me by the
Administrator, the FAA amends 14 CFR part 39 as follows:

PART 39--AIRWORTHINESS DIRECTIVES

0
1. The authority citation for part 39 continues to read as follows:

    Authority:  49 U.S.C. 106(g), 40113, 44701.

Sec.  39.13  [Amended]

0
2. The FAA amends Sec.  39.13 by adding the following new airworthiness
directive (AD):

2015-17-17 Pratt & Whitney: Amendment 39-18250 ; Docket No. FAA-
2014-1130; Directorate Identifier 2015-NE-04-AD.

(a) Effective Date

    This AD is effective October 2, 2015.

(b) Affected ADs

    None.

(c) Applicability

    This AD applies to all Pratt & Whitney (PW) PW4164-1D, PW4168-
1D, PW4168A-1D and PW4170 engines; and all PW4164, PW4168, and
PW4168A turbofan engines that have incorporated either PW Service
Bulletin (SB) No. PW4G-100-72-214, dated December 15, 2011 or PW SB
No. PW4G-100-72-219, Revision 1, dated October 5, 2011.

(d) Unsafe Condition

    This AD was prompted by fuel nozzle-to-fuel supply manifold
interface fuel leaks. We are issuing this AD to prevent fuel leaks
which could result in engine fire and damage to the airplane.

(e) Compliance

    Comply with this AD within the compliance times specified,
unless already done.
    (1) Within 800 flight hours after the effective date of this AD,
and within every 800 flight hours since last inspection thereafter,
inspect all fuel nozzle-to-fuel supply manifold interfaces for
evidence of fuel leaks, soot, and coke formation. Use the
Accomplishment Instructions, Part A, of PW Alert Service Bulletin
(ASB) No. PW4G-100-A73-44, Revision 1, dated February 12, 2015 to do
the inspections.
    (2) Replace hardware that fails an inspection. Use the
Accomplishment Instructions, Part A, of PW ASB No. PW4G-100-A73-44,
Revision 1, dated February 12, 2015 to do the replacement.

(f) Mandatory Terminating Action

    As terminating action to the repetitive inspection requirements
in paragraph (e)(1) of this AD do the following:
    (1) Inspect all fuel nozzle-to-fuel supply manifold interfaces
for fuel leaks, soot, and coke formation, replace hardware that
fails inspection, and re-torque all fuel nozzle-to-fuel supply
manifold B-nuts as follows:
    (i) For engines with fewer than 1,500 cycles on the effective
date of this AD, before accumulating another 650 cycles, not to
exceed 1,900 cycles.
    (ii) For engines with 1,500 cycles or more, but less than 2,500
cycles on the effective date of this AD, before accumulating another
400 cycles, not to exceed 2,700 cycles.
    (iii) For engines with 2,500 cycles or more on the effective
date of this AD, before accumulating another 200 cycles.
    (2) Use the Accomplishment Instructions, Parts B through E, of
PW ASB No. PW4G-100-A73-44, Revision 1, dated February 12, 2015 to
do the inspection, replacement, and retorqueing.

(g) Credit for Previous Action

    This paragraph provides credit for the actions required by
paragraphs (e) and (f) of this AD, if the actions were performed
before the effective date of this AD, using the procedures specified
in PW ASB No. PW4G-100-A73-44, dated October 10, 2014 or Special
Instruction 129F-14.

(h) Definition

    For the purpose of this AD ``cycles'' is defined as cycles since
new or cycles since last torque application to the B-nuts on the
fuel nozzle installation.

(i) Alternative Methods of Compliance (AMOCs)

    The Manager, Engine Certification Office, FAA, may approve AMOCs
for this AD. Use the procedures found in 14 CFR 39.19 to make your
request. You may email your request to<EMAIL_ADDRESS>
(j) Related Information

    For more information about this AD, contact Katheryn Malatek,
Aerospace Engineer, Engine Certification Office, FAA, Engine &
Propeller Directorate, 12 New England Executive Park, Burlington, MA
01803; phone:<PHONE_NUMBER>; fax:<PHONE_NUMBER>; email:
<EMAIL_ADDRESS>
(k) Material Incorporated by Reference

    (1) The Director of the Federal Register approved the
incorporation by reference (IBR) of the service information listed
in this paragraph under 5 U.S.C. 552(a) and 1 CFR part 51.
    (2) You must use this service information as applicable to do
the actions required by this AD, unless the AD specifies otherwise.
    (i) Pratt & Whitney (PW) ASB No. PW4G-100-A73-44, Revision 1,
dated February 12, 2015.
    (ii) Reserved.
    (3) For PW service information identified in this AD, contact
Pratt & Whitney, 400 Main St., East Hartford, CT 06108; phone: 860-
565-8770; fax:<PHONE_NUMBER>.
    (4) You may view this service information at the FAA, Engine &
Propeller Directorate, 12 New England Executive Park, Burlington,
MA. For information on the availability of this material at the FAA,
call<PHONE_NUMBER>.
    (5) You may view this service information that is incorporated
by reference at the National Archives and Records Administration
(NARA). For information on the availability of this material at
NARA, call<PHONE_NUMBER>, or go to: http://www.archives.gov/federal-register/cfr/ibr-locations.html.

    Issued in Burlington, Massachusetts, on August 18, 2015.
Diane S. Romanosky,
Acting Directorate Manager, Engine & Propeller Directorate, Aircraft
Certification Service.
[FR Doc. 2015-21204 Filed 8-27-15; 8:45 am]
 BILLING CODE 4910-13-P


[Federal Register Volume 78, Number 109 (Thursday, June 6, 2013)]
[Notices]
[Page 34090]
[FR Doc No: 2013-13393]

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DEPARTMENT OF ENERGY

Federal Energy Regulatory Commission

[Project No. 5379-008]

New Hampshire Water Resources Board, Hydro Dynamics Corporation;
Notice of Transfer of Exemption

    1. By letter filed April 16, 2013, New Hampshire Water Resources
Board co-exemptee and the New Hampshire Department of Environmental
Services, as successor agency, and Hydro Dynamics Corporation as co-
exemptee, Goffstown Hydro Corporation, successor in interest informed
the Commission that the exemption from licensing for the Hadley Falls
Project, FERC No. 5379, originally issued January 19, 1982,\1\ has been
transferred to the New Hampshire Department of Environmental Service,
as sole exemptee. The project is located on the Piscataquog River in
Hillsborough County, New Hampshire. The transfer of an exemption does
not require Commission approval.
---------------------------------------------------------------------------

    \1\ 18 FERC ] 62,032, Order Granting Exemption From Licensing of
a Small Hydroelectric Project of 5 Megawatts or Less.
---------------------------------------------------------------------------

    2. New Hampshire Department of Environmental Services, located at
29 Hazen Drive, Concord, NH 03302 is now the sole exemptee of the
Hadley Falls Project, FERC No. 5379.

    Dated: May 30, 2013.
Kimberly D. Bose,
Secretary.
[FR Doc. 2013-13393 Filed 6-5-13; 8:45 am]
BILLING CODE 6717-01-P


[Federal Register: April 10, 2009 (Volume 74, Number 68)]
[Notices]
[Page 16433]
[DOCID:fr10ap09-115]

=======================================================================
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SECURITIES AND EXCHANGE COMMISSION

[File No. 500-1]

All Line, Inc.; Order of Suspension of Trading

April 8, 2009.
    It appears to the Securities and Exchange Commission that there is
a lack of current and accurate information concerning the securities of
All Line, Inc. (``All Line''), which is quoted on the Pink Sheets under
the ticker symbol ALIN. Trading in the securities of All Line appears
to be predicated on apparent misstatements. Certain persons appear to
have usurped the identity of a defunct or inactive publicly-traded
corporation by making false statements to a court and transfer agent,
in order to gain control of the corporation. A new CUSIP and ticker
symbol appear to have been obtained based on false representations
regarding the identity of the corporation. The accuracy and adequacy of
publicly disseminated information concerning, among other things, the
corporate history and identity of All Line are questionable.
    The Commission is of the opinion that the public interest and the
protection of investors require a suspension of trading in the
securities of the above-listed company.
    Therefore, it is ordered, pursuant to Section 12(k) of the
Securities Exchange Act of 1934, that trading of the securities of the
above-listed company is suspended for the period from 9:30 a.m. EDT,
April 8, 2009 through 11:59 p.m. EDT, on April 22, 2009.

    By the Commission.
Elizabeth M. Murphy,
Secretary.
[FR Doc. E9-8332 Filed 4-8-09; 4:15 pm]

BILLING CODE 8010-01-P