° If the water quality allows a full 10 L to be filtered without clogging, the PWS can simply ship the filter to the laboratory and save the remaining materials for subsequent events.

° If the first filter clogs after 5 L or more have been filtered, and the volume is not anticipated to yield 2 mL of packed pellet volume, the PWS should be able to filter the remaining volume through the second filter and ship both filters to the laboratory for processing.

6.1.2 E. coli Samples PWSs should analyze up to 100 mL of sample for LT2 monitoring. EPA recommends that the PWS collect and ship more than 100 mL of sample to ensure sufficient volume for sample analysis is available in the event of spillage at the laboratory. If spillage or leakage occurs during shipment, there is an opportunity for sample contamination to occur and the sample should not be analyzed ( see Section 8.3.1). Additional details on sample collection procedures are provided in Section 6.4.3. The capacity of sample containers should be 120 mL ( 6 oz.) or 250 mL ( 8 oz.) to allow for sufficient sample volume and at least a 1 inch head space to facilitate mixing of the sample by shaking prior to analysis. Section 6: Collecting and Shipping Source Water Samples 37 Draft June 2003 6.2 Sample Collection Location LT2 rule monitoring is intended to assess the mean Cryptosporidium level in the influent to drinking water plants that treat surface water or ground water under the direct influence ( GWUDI) of surface water. Generally, monitoring is required for each plant that treats a surface water or GWUDI source. However, where multiple plants receive all of their water from the same influent ( e. g., multiple plants draw water from the same pipe), the same set of monitoring results may be applied to each plant. E. coli samples should be collected at the same location as Cryptosporidium samples.

PWSs are required to collect source water samples for the LT2 rule from the plant intake prior to any treatment [ 40 CFR part 141.704 ( a)]. Guidance on sampling at plants where this may not be feasible, or where other factors, such as the use of multiple sources, need to be addressed, is provided below, in Sections 6.2.1 through 6.2.5.

6.2.1 Plants That Do Not Have a Sampling Tap Located Prior to Any Treatment Plants in this situation should pursue one of the following options:

° Manually collect source water samples as close to the intake as is feasible, at a similar depth and distance from shore.

° Establish a sampling location prior to treatment ° 6.2.2 Plants That Use Different Water Sources at the Same Time This includes multiple surface water sources and blended surface water and ground water sources. Plants in this situation should pursue one of the following options:

° If there is a sampling tap where the sources are combined prior to treatment, the sample should be collected from the tap.

° Samples can be manually collected at each source near the intake on the same day and composited into one sample. The volume of sample from each source should be weighted according to the proportion of that source used by the plant. For example, if a plant has two sources and 75% of the drinking water is from Source A and 25% is from Source B, then for a 10 L sample, 7.5 L would be collected from Source A and combined with 2.5 L collected from Source B. Compositing of samples should reflect plant operation at the time the sample is collected and may change during the monitoring period.

° Separate samples can be manually collected at each source near the intake on the same day and analyzed independently. The results would then be used to calculate a weighted average of the analysis results. The weighted average would be calculated by multiplying the analysis result for each source by the fraction of the source contribution to total plant flow at the time the samples were collected, and then summing these values. For example, if a plant has two sources and 75% of the drinking water is from Source A and 25% is from Source B, then one sample would be collected from each source and analyzed independently. If the concentration of oocysts for the sample from Source A was 5 oocysts/ L and the concentration of the sample from Source B was 2 oocysts/ L, the final result for the plant for this sampling event would be 4 oocysts/ L ([ 5 oocysts/ L x 0.75] + [ 1 oocyst/ L x 0.25]).

6.2.3 Plants That Use Presedimentation For these plants, source water samples must be collected after the presedimentation basin but before any other treatment [ 40 CFR 141.704 ( b)]. Use of presedimentation basins during monitoring should be consistent with routine operational practice and should be documented. For systems taken samples after Section 6: Collecting and Shipping Source Water Samples 38 Draft June 2003 presedimentation basin, no " Microbial Toolbox" credits will be allowed for presedimentation, if the plant is classified into a bin that requires additional treatment [ 40 CFR 141.726 ( a)].

6.2.4 Plants That Use Raw Water Off Stream Storage For these plants, source water samples must be collected after the off stream storage reservoir [ CFR 141.704 ( c)]. Use of off stream storage during monitoring should be consistent with routine operational practice and should be documented.

6.2.5 Plants That Use Bank Filtration The correct sampling location for PWSs with plants using bank filtration differs depending on whether the bank filtered water is treated by subsequent filtration for compliance with the Surface Water Treatment Rule ( SWTR) [ 40 CFR 141.704 ( c)].

° PWSs using bank filtered water that is treated by subsequent filtration for compliance with the SWTR must collect source water samples from the well ( i. e., after bank filtration) but before any other treatment. Use of bank filtration during monitoring should be consistent with routine operational practice and should be documented. Systems collecting samples after a bank filtration process may not receive microbial toolbox credit for the bank filtration [ 40 CFR 141.726 ( c)].

° PWSs using bank filtered water without additional filtration must take source water samples in the surface water source ( e. g., the river). Use of bank filtration during monitoring should be consistent with routine operational practice and should be documented.

Before monitoring begins, all plants must establish a source water monitoring schedule, as discussed in Section 6.3.

6.3 Source Water Monitoring Schedule PWSs are required to collect samples at least monthly and in accordance with a schedule established by the PWS prior to initiation of monitoring. PWSs may collect samples more frequently ( e. g., twice permonth weekly), provided the same frequency is maintained throughout the monitoring period [ 40 CFR part 141.701 ( e)].

Water treatment plants that use surface water or ground water under the direct influence ( GWUDI), but are operated only seasonally ( e. g., during times of high water demand) should monitor at least monthly during the period when the plant is in operation.

Systems regulated under the LT2 rule are required to submit source water monitoring schedule to EPA within 3 months of rule promulgation [ 40 CFR part 141.703 ( a)]. The schedule is entered using the scheduler function within the LT2 Data Collection System. Details on the use of the scheduler are provided in the Users' Manual for the Long Term 2 Enhanced Surface Water Treatment Rule ( LT2 Rule) Data Collection System. Systems are required to collect samples within 2 days before or after the dates indicated in their sampling schedules [ 40 CFR part 141.703 ( b)].

The scheduler function will be available for PWSs to establish their LT2 monitoring schedule for a 3 month period, beginning on the date of final rule publication. The use of a predetermined monthly or semimonthly sampling schedule at each PWS during LT2 is designed to capture storm events and other factors that affect water quality on a periodic basis. Because a PWS can potentially bias the results of the monitoring by avoiding sample collection during periods of low water quality, the submission of prescheduled sampling dates will be used to assess compliance. Section 6: Collecting and Shipping Source Water Samples 39 Draft June 2003 6.4 Sample Scheduling Compliance Issues Permissible exceptions to the sampling schedule are noted as follows:

° If extreme conditions or situations exist that may pose danger to the sampler, or which are unforeseen or cannot be avoided and which cause the system to be unable to sample in the required time frame, the system should sample as close to the scheduled date as feasible and submit an explanation for the alternative sampling date to EPA concurrent with shipment of the sample to the laboratory.

EPA will evaluate the explanation and update the schedule in the LT2 Data Collection System, if acceptable, to permit the analytical result to be submitted through the system ( results with sample collection dates that do not comply with the schedule entered by the PWS before monitoring began will be rejected from the system).

° Systems that are unable to report a valid Cryptosporidium analytical result for a scheduled sampling date due to failure to comply with the analytical method quality control requirements ( e. g., sample is lost or contaminated; laboratory exceeds analytical method holding time) must collect a replacement sample within 14 days of being notified by the laboratory that a result cannot be reported for that date. Systems must submit an explanation for the replacement sample with the analytical results. Systems should collect an E. coli sample at the same time as the Cryptosporidium replacement sample.

Alternative sample collection dates should be timed so as not to coincide with another scheduled Cryptosporidium sample collection date. Documentation of alternate sample collection, including the reason, should be provided with the grandfathered data package.

6.4 Sample Collection Guidance Large plants must begin collecting source water samples 6 months after rule promulgation and small plants must begin 30 months after rule promulgation. Because the LT2 monitoring program is designed to assess source water Cryptosporidium and E. coli concentrations, not the concentrations of these organisms at points after any treatment, samples must be collected prior to any treatment and where the water is no longer subject to surface runoff during LT2 monitoring ( 40 CFR part 141.704).

During each of the scheduled sampling events, several actions must be performed in addition to collecting the sample. These actions, and an indication of which plant types each applies to, are summarized in Table 6 2. Section 6: Collecting and Shipping Source Water Samples 40 Draft June 2003 Table 6 2. Sample Collection Activities Required for Each Plant Type Action Large filtered plants Large unfiltered plants All small plants Small plants that exceed the E. coli trigger level Document sample collection information T T T T Collect Cryptosporidium sample T T T Collect E. coli sample T T Measure turbidity T Monitor sample temperature during sample transport Ta Ta Tb T a Those utilities with on site Cryptosporidium analytical capabilities will not need to transport samples unless the laboratory is not located in close proximity to the sample collection location b Those small plants with on site E. coli analytical capabilities will not need to transport samples unless the laboratory is not located in close proximity to the sample collection location Guidance and procedures for each of these sample collection activities is provided in Sections 6.4.1  6.4.5, below.

6.4.1 Sample Collection Documentation The information in Table 6 3 should be recorded during sample collection to link the monitoring result to the plant, and to provide information required for development of the microbial index.

Table 6 3. Minimum Data Elements to Record During Sample Collection Sampling Information Required Recommended PWS name T Public Water System Identification ( PWSID) numbera T Facility name T Facility IDa T Sample collection point name T Sample collection point IDa T Sample collection datea T Source water typeb T Requested analysis T Sample collection time ( start time for field filtered samples) T Meter readings ( for field filtered samples only) T Section 6: Collecting and Shipping Source Water Samples 41 Draft June 2003 Sample collection stop time ( for field filtered samples only) T Turbidityb T a The combination of these elements constitute the unique sample identifier for LT2 monitoring samples b This information should be recorded with the E. coli sample collection information, as it will be entered into the LT2 data collection system with the E. coli sample results, for use in reassessing the microbial index. It does not need to be reported with the Cryptosporidium sample collection information For samples that are shipped off site, this information should be documented on an LT2 sample collection form ( Appendix C), or similar form provided by your contract laboratory. For samples analyzed on site by your utility's laboratory, this information can be documented in a sampling log book or other standard form used by your utility; the LT2 sample collection form can also be used.

The source water type for the sample will be used to reassess the relationship between Cryptosporidium and E. coli concentrations ( the microbial index discussed in Section 1.5). Sample collection personnel must select from four source water types on the LT2 sample collection form:

° Flowing stream ( defined under the LT2 rule as " a course of running water flowing in a definite channel")

° Reservoir/ lake ( defined under the LT2 rule as " a natural or man made basin or hollow on the Earth's surface in which water collects or is stored that may or may not have a current or single direction of flow")

° Ground water under the direct influence ( GWUDI) of flowing stream surface water ° GWUDI of reservoir/ lake surface water The source water type should be selected based on the type of source water that accounts for the majority of the surface water used as source water at the time of sample collection. For example, if the plant uses a mix of approximately 55% reservoir/ lake water and 45% flowing/ stream water, the " reservoir/ lake" option should be circled on the LT2 sample collection form.

The majority of source water for plants that use GWUDI is ground water. However, as noted above, the selection of source water type under the LT2 rule is based on the majority of surface water used as source water. As a result, the selection of source water type is based on the type of surface water that accounts for the majority of the influence of the ground water source.

The turbidity of the source water also needs to be measured. Cryptosporidium sample collection procedures are discussed in Section 6.4.2; E. coli sample collection procedures and turbidity measurement procedures are discussed in Section 6.4.3 and 6.4.4, respectively.

6.4.2 Cryptosporidium Sample Collection Several options are available to the PWS in collecting untreated surface water samples for Cryptosporidium analysis, including the following:

° Collection of bulk water samples for shipment to the laboratory for filtration and analysis. A detailed protocol for collecting, packing, and shipping bulk samples is provided as Appendix D.

° On site filtration of water samples using the Pall Gelman Envirochek   or Envirochek   HV capsule filter. A detailed protocol for filtering samples on site from pressurized or unpressurized sources is provided as Appendix E.

° On site filtration of water samples using the IDEXX   Filta Max foam filter. A detailed protocol for filtering samples on site from pressurized or unpressurized sources is provided as Appendix F. Section 6: Collecting and Shipping Source Water Samples 42 Draft June 2003 Regardless of the procedure used to collect Cryptosporidium samples, the sample must be eluted from the filter within 96 hours of sample collection, per EPA Method 1622/ 1623 ( Section 8.2). If this holding time is violated, the laboratory will reject the sample, and your PWS will be required to recollect and reship the sample.

LT2 rule requirement: Each sample must meet the QC criteria for the methods [ 40 CFR part 141.705 ( a) ( 3)]. Per EPA Method 1622/ 1623, samples must be processed or examined within each of the holding times specified by the method ( Section 8.2).

<IP_ADDRESS> Matrix Spike Samples Method 1622/ 1623 requires matrix spike ( MS) samples to be analyzed at a frequency of 1 MS sample for every 20 monitoring samples from each plant. This frequency translates to the following, for each plant category:

° For large PWSs that perform monthly monitoring for 2 years ( resulting in 24 monitoring samples), 2 MS samples must be collected and analyzed ° For large PWSs that perform semi monthly or more frequent monitoring for 2 years ( resulting in 48 or more samples), a minimum of 3 MS samples will be collected and analyzed ° For small PWSs that are triggered into Cryptosporidium monitoring and collect semi monthly samples for 1 year ( resulting in 24 samples), 2 MS samples must be collected and analyzed The MS sample and the associated unspiked sample must be analyzed by the same procedure and the MS sample must be the same volume as the associated monitoring sample. If the volume of the MS sample is greater than 10 L, the system is permitted to filter all but 10 L of the MS sample in the field, and ship the filtered sample and the remaining 10 L of source water to the laboratory to have the laboratory spike the remaining 10 L of water and filter it through the filter used to collect the balance of the sample in the field.

Utilities collecting and shipping bulk water samples for filtration and analysis at the laboratory should split their sample stream and collect the monitoring sample volume and MS sample volume simultaneously.

° The sample stream should be split using flow controllers on both sides of the split to regulate the pressure difference between the side being subjected to filtration ( resulting in higher pressure) and the side flowing into a bulk sample container. A mixing chamber ( filter housing without filter) can be added immediately upstream from the Y to aid in equalizing the distribution of sample particulates to either side.

° If splitting the sample stream is not practical, the utility should collect the MS sample immediately before or after the monitoring sample.

MS sample results would not be used to adjust Cryptosporidium recoveries at any individual source water; but MS results would be used collectively to assess overall recovery and variability for EPA Method 1622/ 1623 in source water. No resampling would be necessary for MS samples that do not meet Method 1622/ 1623 recovery guidelines. Section 6: Collecting and Shipping Source Water Samples 43 Draft June 2003 LT2 rule requirements: ( 1) The MS and field sample must be collected from the same sampling location by splitting the sample stream or collecting the samples sequentially. ( 2) The volume of the MS sample analyzed must be within 10% of the volume of the field sample analyzed. ( 3) The MS and field sample must be analyzed by the same procedure [ 40 CFR part 141.705 ( a) ( 2) ( i)].

<IP_ADDRESS> Purchasing Filters If one of the field filtration options is used, you may want to consider purchasing filters in bulk from the manufacturer ( or the manufacturer's local distributor), as it may be cheaper than purchasing the filters from your Cryptosporidium contract laboratory as part of the sampling kit. This approach also provides your PWS with a ready supply of extra filters on site, if a filter clogs during a sampling event. Plants wishing to explore this option should call one of the contacts in Table 6 4.

Table 6 4. Contacts for Filters Approved for Use in EPA Method 1622/ 1623 Pall Life Sciences ( Envirochek   and Envirochek   HV capsule filters) IDEXX ( Filta Max   foam filters)

www. pall. com/ gelman 600 South Wagner Road Ann Arbor, MI 48103 Sales: Phone: ( 800) 521 1520 ext. 2 Fax: ( 734) 913 6495 Technical Support: Phone: ( 800) 521 1520 ext. 3 Fax: ( 734) 913 6495 www. idexx. com Sales: Phone: ( 800) 321 0207 ext. 1 Fax: ( 207) 856 0630 Technical Support: Phone: ( 800) 321 0207 ext. 2 Fax: ( 207) 856 0630 E mail: water@ idexx. com The PWS also can purchase and assemble the entire sampling kit and maintain this kit on site, rather than shipping it back and forth between the Cryptosporidium laboratory and the plant. If the filters you use have associated shelf lives and storage conditions, ensure that the filters are stored according to the manufacturers' directions and are not used past the specified shelf life.

The components and part numbers for the sampling kit are specified in the individual protocols for each filter. If the sampling kit is maintained on site by the utility, the utility should use disposable materials wherever possible to mitigate the risk of cross contamination between samples or sampling events, and must disinfect the non disposable sampling equipment between uses ( if the laboratory provides the sampling kit, this disinfection step is performed at the laboratory.)

Sampling kit cleaning should consist of the following:

° Cleaning equipment by scrubbing with warm detergent solution and exposing to hypochlorite solution ( minimum of a 5% solution of bleach and water) for at least 30 minutes at room temperature ° Rinsing the equipment with reagent water and placing the equipment in an area free of potential Cryptosporidium contamination until dry <IP_ADDRESS> Filter Clogs and Highly Turbid Water Samples PWSs with highly turbid source waters are likely to generate larger packed pellet volumes after centrifugation and to clog filters than PWSs with low turbidity waters. As noted in Section 6.1, at least 2 mL of packed pellet volume must be analyzed ( for samples in which 10 L is filtered), or as much volume Section 6: Collecting and Shipping Source Water Samples 44 Draft June 2003 as two filters can accommodate before clogging. ( If more than 10 L is filtered, then less of the packed pellet volume needs to be analyzed.)

PWSs with highly turbid water may be able to collect the required minimum packed pellet volume by avoiding filtration altogether, and shipping a bulk water sample to the laboratory for centrifuging. The laboratory can centrifuge 250 mL or greater aliquot volumes sequentially, until a packed pellet volume of 2 mL is generated.

6.4.3 E. coli Sample Collection For most large systems, E. coli analyses will be conducted on site, so samples will not be shipped in most cases, unlike Cryptosporidium samples. However, many small systems will collect E. coli samples and ship them off site for analysis. Regardless of whether the samples are analyzed by the utility's own laboratory or by a commercial laboratory, laboratories analyzing E. coli samples for the LT2 rule must use an E. coli method approved for use under the rule and must be certified under the drinking water certification program for the general coliform analysis technique corresponding to the method the laboratory plans to use for LT2 rule monitoring [ 40 CFR part 141.705 ( b) and 141.706 ( b)]. Approved E. coli methods and their corresponding drinking water certification program coliform techniques are discussed in the Microbial Laboratory Guidance Manual for the Long Term 2 Enhanced Surface Water Treatment Rule ( LT2 Rule). Summary information on these methods is also provided in Section 4 of this document.

Collect E. coli samples in sterile, non toxic, plastic, or glass containers with a leak proof lid. The capacity of sample containers should be 120 mL ( 6 oz.) or 250 mL ( 8 oz.) to allow for sufficient sample volume and at least a 1 inch head space to facilitate mixing of the sample by shaking prior to analysis A detailed protocol for collecting source water samples for E. coli analysis, as well as packing and shipping guidance for utilities that transport samples off site for analysis, is provided as Appendix G.

EPA strongly encourages laboratories to analyze samples as soon as possible after collection. E. coli samples must be analyzed within 24 hours of sample collection [ 40 CFR part 141.705 ( b)( 1)]. Note: This is a longer time period than currently permitted in Standard Methods and the Manual for the Certification of Laboratories Analyzing Drinking Water, and is based on data demonstrating that surface water samples could be held, chilled, for up to 24 hours and still yield valid results ( Reference 9.5).

Samples should be maintained above freezing and below 10 ° C in a refrigerator or in a cooler with wet ice, blue ice, or gel packs, etc. Additional guidance on monitoring sample temperature is available in Section 6.4.5 of this manual.

6.4.4 Measuring Turbidity PWSs must measure the turbidity of the source at the time of Cryptosporidium and E. coli sample collection during LT2 rule monitoring. Turbidity must be measured by a party approved by the State [ 40 CFR part 141.706 ( c)] using methods for turbidity measurement approved at 40 CFR part 141.74 [ 40CFR part 141.705 ( c)]. These methods include:

° Method 2130B, published in Standard Methods for the Examination of Water and Wastewater ( 19th or 20th Edition). The full text of the 19th Edition is provided as Appendix H.

° Great Lakes Instrument ( GLI) Method 2. The full text of this method is provided as Appendix I.

° Revised EPA Method 180.1, approved in August 1993 in Methods for the Determination of Inorganic Substances in Environmental Samples ( EPA 600/ R 93 100). The full text of this method is provided as Appendix J. Section 6: Collecting and Shipping Source Water Samples 45 Draft June 2003 Systems must use turbidimeters that conform to one of the approved methods for measuring turbidity, such as Hach Turbidimeter 1720D with EPA Method 180.1, GLI Turbidimeter Accu 4 with GLI Method 2, or equivalents ( Note: These examples do not constitute an endorsement of specific instrumentation. Approved methods provide specifications that turbidimeters must meet, and conformance of instruments with these particular specifications must be determined prior to analysis.). For regulatory reporting purposes, either an on line or a benchtop turbidimeter may be used, and systems must comply with all quality control requirements specified in methods and regulations. If a system chooses to utilize on line units for monitoring, the system must validate the continuous measurements for accuracy on a regular basis using a protocol approved by the State [ 40 CFR part 141.74 ( c) ( 1)].

<IP_ADDRESS> Measuring Sample Turbidity During LT2 Monitoring When measuring turbidity, cuvettes must be clear, colorless glass or plastic. The tube must be kept clean, both inside and out, to provide accurate readings. If a sample tube is scratched, it must be discarded.

° Measuring Sample Turbidity Using SM 2130B. Measure turbidity immediately after sample collection to prevent temperature changes, particle flocculation, and sedimentation from changing sample characteristics. Shake sample well before pouring into cuvette. Gently agitate to remove air bubbles from the inside of the sample before pouring the sample into cell. Wait until all the air bubbles disappear and remove all moisture from the outside of the sample cell before placing it into the instrument. If fogging occurs, warm the sample by warm water bath for a short time, then reagitate the sample before placing it in the turbidimeter. Read turbidity directly from instrument display. Note: Measurements should be within the calibration range.

° Measuring Sample Turbidity Using GLI Method 2 or Revised EPA Method 180.1. Different procedures should be followed, depending on the turbidity of the sample:

° For turbidities estimated to be less than 40 NTU. Shake the sample thoroughly to disperse the solids. After waiting for the air bubbles to disappear, pour the sample into the turbidimeter tube and read directly from the instrument scale.

° For turbidities estimated to be greater than 40 NTU. Dilute the sample with turbidity free water and compute the turbidity with the dilution factor included.

<IP_ADDRESS> General Quality Control for Turbidity Measurements Utilities performing environmental sample measurements must be approved by the State ( or EPA Region, for states that do not have primacy) under the drinking water laboratory certification program [ 40 CFR part 141.706 ( c)]. Each utility laboratory is required to operate a formal quality control ( QC) program and to maintain performance records that define the quality of the data generated. Two types of calibration are required for turbidity measurements:

° A primary suspension standard. The primary suspension standard should be used to calibrate the turbidimeter initially and at least every four months in order to prevent instrument drift. The calibration should be documented. The standards should be replaced when they exceed the expiration date.

Acceptable primary suspensions include Formazin ( a recipe for preparation can be found at EPA Method 180.1 and Standard Method 2130B), AMCO AEPA 1 ( available from Advanced Polymer Systems), and Hach StablCal Stabilized Formazin Standards ( available from Hach Company). Please note that Formazin standards are relatively unstable, particularly at low concentrations. Therefore, dilutions used for calibration need to be prepared on the day they will be used. Stock solutions may be stable for a month ( at 400 NTU) to 1 year ( at 4000 NTU). Consult an approved method for more information. Section 6: Collecting and Shipping Source Water Samples 46 Draft June 2003 ° A secondary suspension standard. The secondary suspension standard is used daily to check the calibration of the instrument. The calibration should be documented, and should not vary by more than 10% from the initial calibration values ( if they do vary by more than 10%, the system should be corrected so that performance is acceptable). The standards should be replaced when they exceed the expiration date.

Acceptable secondary standards include all primary standards, or other materials that are suggested by instrument manufacturers   such as sealed sample cells filled with a labeled suspension or metal oxide particulates in a polymer gel, or a turbid glass cube. The purpose of the secondary standard is to provide a quick check of calibration. The secondary standards should have a fixed turbidity that does not vary from use to use.

6.4.5 Monitoring Sample Temperature Source water samples are dynamic environments and, depending on sample constituents and environmental conditions, Cryptosporidium oocysts present in a sample can degrade and E. coli present in a sample can grow or die off, biasing analytical results. Cryptosporidium and E. coli samples for LT2 rule monitoring that are not analyzed the same day they are collected must be maintained below 10 ° C to reduce biological activity. This is specified in Section 8.0 of the June 2003 versions of EPA Method 1622/ 1623 for Cryptosporidium samples and at 40 CFR part 705 ( b) ( 1) and Chapter V, Section 6.3, of the Laboratory Certification Manual ( Reference 5.2) for E. coli samples.

Samples for all analyses should remain above freezing at all times. This is a requirement in Section 8.0 of the June 2003 versions of EPA Method 1622/ 1623. Although not a significant concern for 10 L water samples, this is a real concern for Cryptosporidium filters and 120 or 250 mL E. coli samples that are shipped off site with coolant materials, such as wet ice, blue ice, or gel packs. E. coli holding time studies performed in support of the LT2 rule ( Reference 9.5) demonstrated that E. coli samples can freeze under these conditions if samples are not packed properly.

The sample collection protocols procedures in Appendices D, E, F, and G provide sample packing procedures for E. coli and Cryptosporidium samples. Utility personnel should follow these procedures to ensure that samples remain at acceptable temperatures during shipment.

Because Cryptosporidium samples collected for the LT2 rule must meet the QC criteria in the methods [ 40 CFR part 705 ( a) ( 3)], and because these QC criteria include receipt of samples at < 10 ° C and not frozen, laboratories must reject LT2 Cryptosporidium samples received at > 10 ° C or frozen ( this is discussed further in Section 3.3.12 in this manual). In these cases, the PWS must re collect and re ship the sample.

LT2 rule requirement: Each sample must meet the QC criteria for the methods [ 40 CFR part 141.705 ( a) ( 3)]. Per EPA Method 1622/ 1623, samples not processed on the day of collection must be received at the laboratory at < 10 ° C and not frozen ( Section 8.1)

The sample collection protocols discussed in Section 6.4.2 for Cryptosporidium samples and Section 6.4.3 for E. coli samples provide guidance on packing samples to maintain appropriate temperatures. Utility personnel should follow these procedures to ensure that samples remain at acceptable temperatures during shipment. Section 6: Collecting and Shipping Source Water Samples 47 Draft June 2003 Several options are available to measure sample temperature upon receipt at the laboratory and, in some cases, during shipment:

° Temperature sample. One option, for Cryptosporidium filtered samples ( not for 10 L bulk samples) and E. coli 120 and 250 mL samples, is for the PWS to fill a small, inexpensive sample bottle with water and pack this " temperature sample" next to the field sample. The temperature of this extra sample volume is measured upon receipt to estimate the temperature of the field sample. Temperature sample bottles are not appropriate for use with bulk samples because of the potential effect that the difference in sample volume may have in temperature equilibration in the sample cooler. Example product: Cole Parmer cat. no. U 06252 20.

° Thermometer vial. A similar option is to use a thermometer that is securely housed in a liquid filled vial. Unlike temperature samples, the laboratory does not need to perform an additional step to monitor the temperature of the vial upon receipt, but instead just reads the thermometer. Example product: Eagle Picher Sentry Temperature Vial 3TR 40CS F or 3TR 40CS.

° iButton. Another option for measuring the sample temperature during shipment and upon receipt is a Thermocron ® iButton. An iButton is a small, waterproof device that contains a computer chip to record temperature at different time intervals. The information is then downloaded from the iButton onto a computer. The iButton should be placed in a temperature sample in the cooler, rather than placed directly in the cooler, where it may be affected by close contact with the coolant. Information on Thermocron ® iButtons is available from http:// www. ibutton. com/. Distributors include http:// www. pointsix. com/, http:// www. rdsdistributing. com, and http:// www. scigiene. com/.

° Stick on temperature strips. Another option is for the laboratory to apply a stick on temperature strip to the outside of the sample container upon receipt at the laboratory. This option does not measure temperature as precisely as the other options, but still mitigates the risk of sample contamination while providing an indication of sample temperature to verify that the sample temperature is acceptable. Example product: Cole Parmer cat. no. U 90316 00.

All temperature measurement devices should be calibrated routinely to ensure accurate measurements. See the U. S. EPA Manual for the Certification of Laboratories Analyzing Drinking Water ( Reference 9.3) for more information. 48 Draft June 2003 SECTION 7: REVIEWING CRYPTOSPORIDIUM DATA When Cryptosporidium samples are processed and analyzed by the laboratory, data on sample measurements, sample processing times, and slide examination results are recorded at the laboratory and reported to the PWS through the LT2 Data Collection System and via hardcopy forms. This section provides an overview of the data recording and reporting processes and discusses the significance of the examination results reported by the laboratory. This section also provides guidance to those PWSs interested in reviewing laboratory data.

7.1 Cryptosporidium Data Recording at the Laboratory The Cryptosporidium laboratory records LT2 rule monitoring data using the following standardized forms:

7.1.1 LT2 Sample Collection Form This form ( an example of which is provided as Appendix C) is initiated at the plant upon sample collection and is completed at the laboratory. The following information is recorded on this form by the Cryptosporidium laboratory:

° Date and time of sample receipt ° Laboratory personnel receiving the sample ° Sample temperature upon receipt ° Sample condition upon receipt Although none of this information is entered into the LT2 data collection system, it provides documentation for the utility, the laboratory, and EPA or State officials on sample receipt information relevant to LT2 rule requirements regarding sample temperatures and sample holding times.

7.1.2 Method 1622/ 1623 Bench Sheet The laboratory uses the bench sheet to record all information associated with sample processing, up to, but not including, sample examination. Information on filtration ( if performed in the laboratory), elution, concentration, immunomagnetic separation, and sample staining are documented on this form. These data include:

° Sample ID ° Dates and times for all steps associated with method required holding times ° All primary measurements used to calculate sample volume analyzed, if less than 100% of the volume filtered was analyzed. This information includes the following: ° The volume of the sample after the concentrate ( packed pellet) has been resuspended ° The volume of this resuspended concentrate that was actually analyzed ( These two values are used to calculate the percent of the sample volume analyzed, if less than 100% of the volume filtered was analyzed.) Section 7: Reviewing Cryptosporidium Data 49 Draft June 2003 ° Filter clog and packed pellet information, which may need to be provided to demonstrate compliance with LT2 rule sample analysis requirements if less than 10 L was analyzed ° Cryptosporidium spiking information for OPR and MS samples ° Analyst names or initials for each step ° Reagent and filter lot information 7.1.3 Method 1622/ 1623 Cryptosporidium Slide Examination Form The laboratory uses the slide examination form to document detailed information on slide examination. This information includes the following: ° Sample ID ° Date and time the examination was completed ° Positive and negative staining control results ° Detailed information on the characteristics of each object on the slide that the analyst determined was a Cryptosporidium oocyst, including the following: ° Size of the oocyst ° Shape of the oocyst ° Whether the DAPI stain applied to the sample revealed the presence of nuclei, and, if so, how many were observed by the analyst ° Whether the analyst observed internal structures during DIC examination 7.2 Submitting Cryptosporidium Data through the LT2 Data Collection System During the LT2 rule, laboratories will report Cryptosporidium data to their PWS clients electronically through EPA's LT2 Data Collection System. The LT2 Data Collection System is a web based application that allows laboratory users to enter or upload data, then electronically " release" the data to the PWS for review, approval, and submission to EPA and the State. Although ownership of the data resides with the PWS throughout this process, the LT2 Data Collection System increases the ease and efficiency of the data entry and transfer process from one party to another by transferring the ability to access the data from the laboratory to the PWS to EPA and the State, and ensuring that data cannot be viewed or changed by unauthorized parties. A summary of the data entry, review, and transfer process through the LT2 Data Collection System is provided in Table 7 1, below. Section 7: Reviewing Cryptosporidium Data 50 Draft June 2003 Table 7 1. LT2 Data Collection System Data Entry, Review, and Transfer Process Laboratory actions ° Laboratory posts analytical results to the LT2 Data Collection System ° LT2 Data Collection System reduces data and checks data for completeness and compliance with LT2 rule requirements ° Laboratory Principal Analyst confirms that data meet quality control requirements ° Laboratory " releases" results electronically to the PWS for review ° Laboratory user cannot edit data after it is released to the PWS EPA does not have access to data PWS actions ° PWS cannot edit data  only review data and either return to laboratory to resolve errors or submit to EPA ° PWS reviews electronic data through LT2 Data Collection System ° PWS " releases" data back to the laboratory if questions ° If no questions, PWS submits data to EPA as " approved" or " contested" ( indicating that samples have been correctly analyzed, but that the PWS contends are not valid for use in LT2 binning)

EPA and State actions ° EPA and State users cannot edit data  only review data ° EPA and State review data through LT2 Data Collection System The data reporting process is discussed in more detail below, in Sections 7.2.1 through 7.2.3, and discussed in detail in the Users' Manual for the Long Term 2 Enhanced Surface Water Treatment Rule ( LT2 Rule) Data Collection System. The LT2 data system users' guide also provides detailed information on the PWS user registration process. Information on the LT2 Data Collection System, as well as a downloadable users' manual, is available at http:// www. epa. gov/ safewater/ lt2/ index. html.

7.2.1 Data Entry/ Upload The analyst or another laboratory staff member enters a subset of the data recorded at the bench ( Section 7.1) into the LT2 Data Collection System, either by entering the data using web forms or by uploading data in XML format. This information includes the following:

° PWS ID ° Facility ID ° Sample collection point ° Sample collection date ° Sample type ( field or MS)

° Sample volume filtered ( L), to nearest ¼ L ° Was 100% of filtered volume examined?

° Number of oocysts counted ° For samples in which less than 10 L is filtered or less than 100% of the sample volume is examined, the laboratory also must enter or upload the number of filters used and the packed pellet volume.

° For samples in which less than 100% of sample volume is examined, the laboratory also must report the volume of resuspended concentrate and volume of this resuspension processed through immunomagnetic separation. Section 7: Reviewing Cryptosporidium Data 51 Draft June 2003 ° For matrix spike samples, the laboratory also must report the sample volume spiked and estimated number of oocysts. These data are not required for field samples.

The laboratory must verify that all holding times and other QC requirements were met.

After the information has been entered or uploaded into the system, the system will reduce the data to yield final sample results, in oocysts/ L, verify that LT2 rule Cryptosporidium sample volume analysis requirements were met for samples in which less than 10 L were analyzed ( see Section 6.1), and calculate MS recoveries.

The laboratory's Primary Analyst under the Lab QA Program is responsible for verifying the quality and accuracy of all sample results in the laboratory, and is required to review and approve the results before they are submitted to the PWS for review. If inaccuracies or other problems are identified, the primary analyst discusses the sample information with the analyst or data entry staff and resolves the issues before the data are approved for PWS review.

If no inaccuracies or other issues are identified, the Primary Analyst approves the reported data for " release" to the PWS for review ( EPA does not receive the data at this point). When the data are approved, the rights to the data are transferred electronically by the system to the PWS, and the data can no longer be changed by the laboratory.

7.2.2 PWS Data Review After the laboratory has released Cryptosporidium data electronically to the PWS using the LT2 Data Collection System, the PWS will review the results. The PWS user cannot edit the data, but if the PWS has an issue with the sample result, such as if the PWS believes that the sample collection point ID or collection date is incorrect, the PWS can release the results back to the laboratory for issue resolution. In addition to noting the reason in the LT2 Data Collection System for the return of the data to the laboratory, you also should contact the laboratory verbally to discuss the issue.

If the PWS determines that the data are accurate, the PWS releases the results to EPA ( and the State, if applicable) as " approved" results. If the PWS determines that the data are accurate, but believes that the data are not valid for LT2 binning purposes, the PWS can release the results to EPA and the State as " contested." Contested samples are those that have been correctly analyzed, but that you contend are not valid for use in LT2 binning, and have submitted to EPA for evaluation.

7.2.3 EPA/ State Review After the PWS has released the results as approved or contested, they are available to EPA and State users to review through the LT2 Data Collection System. EPA and State users cannot edit the data.

7.3 What Do the Sample Examination Results Mean?

As noted in Section 3.1, the laboratory applies two stains to a sample slide, and then examines the sample using three different techniques to determine whether objects that cannot be ruled out as Cryptosporidium oocysts are on a sample slide. A description of these stains and techniques   and how each is used to evaluate objects examined by the analyst, is provided below.

7.3.1 Immunofluorescent Assay ( IFA)

One of the two stains added to the sample before examination is a fluorescent antibody stain that reacts with Cryptosporidium. The antibodies in this stain, which exhibit an intense apple green fluorescence Section 7: Reviewing Cryptosporidium Data 52 Draft June 2003 when the slide is examined using ultraviolet light, will attach to Cryptosporidium oocysts that may be present in the sample. During IFA, the analyst scans the entire well at relatively low magnification ( 200X) for apple green fluorescing objects the size and shape of oocysts. If such an object is located, the analyst proceeds to the next step in the examination process. The analyst cannot conclude at this stage that an apple green fluorescing organism the size and shape of a Cryptosporidium oocyst is indeed an oocyst because the object may be another organism that has cross reacted with the antibody stain. Additional examination procedures are used to determine whether this is the case.

7.3.2 4', 6 diamadino 2 phenylindole ( DAPI) Examination The second stain added to the sample before examination is DAPI, a dye that interacts with nucleic acids and stains nuclei that may be present within the oocyst. The DAPI stain fluoresces when the slide is examined using ultraviolet light. During the DAPI examination, the analyst observes the object at medium magnification ( 400X) to determine whether it contains stained nuclei. Cryptosporidium oocysts contain four nuclei.

Although looking for four nuclei during DAPI examination, if the object has less than four nuclei, the analyst cannot rule out the possibility that the organism is a Cryptosporidium oocyst. For example, if less than four stained nuclei are observed, the object may actually have four nuclei, but some may not be visible in the plane of focus. Similarly, objects in which no stained nuclei are observed may be organisms other than Cryptosporidium, may be dead Cryptosporidium oocysts, or may even be live oocysts that are resistant to DAPI staining.

The DAPI examination is one of several tools for the analyst to use to determine whether an object is an oocyst. The analyst cannot conclude whether the object is an oocyst based on this examination alone, nor can the analyst conclude, based on negative results, that the organism is non infectious. As a result, the analyst must proceed to the next step in the examination process, even if less than four nuclei are observed.

7.3.3 Differential Interference Contrast ( DIC) Examination The third evaluation performed by the analyst is an examination of the object at high magnification ( 1000X). Using DIC, the analyst looks at the object's external or internal morphological characteristics ( this does not require the use of a stain). The analyst looks for characteristics atypical of Cryptosporidium oocysts ( e. g., spikes, stalks, appendages, pores, one or two large nuclei filling the cell, crystals, spores, etc.). If atypical structures are not observed, and the object cannot be ruled out as an oocyst based on the results of the IFA and the DAPI examination, the analyst reports this object as a Cryptosporidium oocyst.

Based on the DIC examination, the size of the object is determined and compared to the acceptable range for the target organism. If the size and shape of the object is within the acceptable range, the analyst records the size and shape and characterizes the Cryptosporidium oocyst in one of three ways: ( 1) an oocyst with internal structures, i. e., those having recognizable structures consistent with Cryptosporidium, ( 2) an oocyst with amorphous structures, or ( 3) an empty oocyst. Assignment of these characterizations is dependent on analyst judgement and none of these characterizations is a direct indicator of whether oocysts are viable and infectious.

7.5 Reviewing and Validating Raw Cryptosporidium Data ( Optional)

If your PWS plans to review the raw data generated by the laboratory, you should request from the laboratory the hardcopy data needed to verify the electronic results ( see Section 5.1.5). However, this step is not required. However, for those PWSs interested in taking this extra step, Sections 7.5.1 through 7.5.3 provide guidance on how to review and validate hardcopy data and verify accuracy. Section 7: Reviewing Cryptosporidium Data 53 Draft June 2003 7.5.1 Data Completeness Check Upon receipt of the hardcopy sample results for a monitoring sample, verify that the laboratory has submitted the following materials:

° Sample result summary sheet, which should include the following:

° Monitoring sample identification information ° Monitoring sample result, in oocysts/ L ° Laboratory quality control batch associated with the sample ° Result for the ongoing precision and recovery ( OPR) sample analyzed for this QC batch ° Result for the method blank sample analyzed for this QC batch ° LT2 sample collection form initiated by your utility and completed with sample receipt information by the laboratory ° Method 1622/ 1623 Bench Sheet with raw data associated with the monitoring sample ( and MS sample, if applicable)

° Method 1622/ 1623 Cryptosporidium Slide Examination Form with raw data for the monitoring sample ( and MS sample, if applicable)

° Laboratory comments. If the laboratory provided comments on the sample analyses or results that require follow up, contact the laboratory to discuss, if necessary. Comments may include any applicable data qualifiers. The following is a list of potential data qualifiers:

° The recovery for the associated ongoing precision and recovery ( OPR) sample did not met method requirements ° Oocysts were detected in the method blank ° Positive and negative staining controls were not acceptable or not examined ° Method holding times were not met ° Sample arrived at the laboratory in unacceptable condition Any of the above data qualifiers would result in the sample being considered invalid for LT2 use and the laboratory should not report the results for the sample to EPA. The PWS may be required to resample.

If forms are missing, incomplete, or incorrect, contact the laboratory immediately to discuss and request resubmission of the missing forms and/ or spreadsheets.

7.5.2 Evaluation of Data Against Method Quality Control Requirements To verify that the laboratory analyzed your monitoring sample within the analytical controls specified by the method, check the following information:

° Sample condition upon receipt. Verify on the completed LT2 sample collection form that your sample was received in acceptable condition ( not leaking, etc.), and at a temperature between 0 ° C and 10 ° C, and not frozen.

° Method blank. Verify that the laboratory analyzed a method blank with the monitoring sample's QC batch and confirm that the method blank did not contain any oocysts. Section 7: Reviewing Cryptosporidium Data 54 Draft June 2003 ° Ongoing precision and recovery sample. Verify that the laboratory analyzed an OPR sample with the monitoring sample's QC batch and that the OPR sample recovery was between 11% and 100%, as required by EPA Methods 1622 and 1623.

° Holding times. Using the sample collection date and time on the LT2 data collection form and the dates and times of the method steps recorded by the laboratory on the Method 1622/ 1623 bench sheet and report form for the monitoring sample, verify the following:

° The laboratory began elution no more than 96 hours from sample collection ° The laboratory performed the elution, concentration, purification, and slide preparation ( application of the purified sample to the slide) within 1 working day ( the date of the elution step should be the same as the date of the slide preparation step)

° The laboratory stained the slide within 72 hours of application of the purified sample to the slide ( generally, the date next to the slide staining step should be no more than 3 days later than the date next to the slide preparation step)

° The laboratory examined the slide within 7 days of staining ( the examination date should be no more than 7 days from the slide staining date)

° Positive and negative staining controls. Based on the information at the top of the Method 1622/ 1623 Cryptosporidium reporting form, verify that the laboratory performed positive and negative staining controls, and that the results of these controls were acceptable.

7.5.3 Calculation Verification The laboratory does not directly report the final concentration of oocysts/ L in the sample to EPA. Instead, they report a series of primary measurements that are used by the LT2 data system to automatically calculate the final concentration. The volume filtered, the total volume of resuspended concentrate, and the volume transferred to IMS are used to determine the volume analyzed. The laboratory also records the total count of oocysts detected, which is divided by the volume analyzed to determine the final concentration of oocysts/ L. Although the final results are automatically calculated by the LT2 data collection system using the primary measurements supplied by the laboratory, the plant still may wish to verify them. Guidance on recalculating and verifying final results using primary measurements is provided below.

<IP_ADDRESS> Field Sample Calculations To calculate the concentration of Cryptosporidium in your field sample, reported as oocysts/ L, the following information is needed:

° Number of oocysts detected in the sample ( recorded as a primary measurement from the examination results form)

° Volume analyzed Using these two data elements, the final concentration is calculated as:

final concentration = oocysts detected in the sample volume analyzed ( L)

If 100% of the sample volume filtered is examined, then the volume analyzed equals the volume filtered. This applies whether one filter or more than one filter was used; if more than one filter was used, and all Section 7: Reviewing Cryptosporidium Data 55 Draft June 2003 of the volume filtered through the multiple filters is processed through the remainder of the method, then the volume examined is simply the sum of the volumes filtered through each of the filters used.

If < 100% of the volume filtered was processed through the remainder of the method, then additional calculations are needed to determine the volume analyzed. This is discussed below.

Determining Volume Analyzed when Less than 100% of Sample Was Examined When < 100% of the sample filtered is processed through the remainder of the method and examined ( such as when the volume filtered yields > 2 mL of packed pellet volume after centrifugation), then the volume analyzed must be determined using the following equations to determine the percentage of the sample that was examined.

percent examined = total volume of resuspended concentrate transferred to IMS ( see Section 7.1.2)

total volume of resuspended concentrate produced volume analyzed ( L) = percent examined × sample volume filtered Determining the Volume of Resuspended Concentrate to Use for Packed Pellets > 5 mL Packed pellets with a volume > 0.5 mL must be divided into subsamples. Use the formula below to determine the total volume of resuspension required in the centrifuge tube before separating the concentrate into two or more subsamples and transferring to IMS.

total volume of resuspended concentrate ( mL) required = pellet volume ( mL) after centrifugation × 5 mL 0.5 mL Example. A 10 L field sample was filtered and processed, producing a packed pellet volume of 2.7 mL. The laboratory transferred 20 mL of the total resuspended concentrate to IMS and examination. The laboratory detected 20 oocysts during examination. The following calculations were performed to determine the volume analyzed and final concentration.

total volume of resuspended concentrate ( mL) required = 2.7 mL × 5 mL = 27 mL 0.5 mL percent examined = 20 mL = 0.74 ( 74%) 27 mL volume analyzed ( L) = 0.74 × 10 L = 7.4 L final concentration ( oocysts/ L) = 20 oocysts = 2.7 oocysts/ L 7.4 L <IP_ADDRESS> Matrix Spike Sample Calculations For matrix spike ( MS) samples, the laboratory records all of the same information that is recorded for field samples, in addition to information specific to matrix spike samples. The sample volume spiked and estimated number of oocysts spiked into the sample are used to calculate the concentration of spiked Section 7: Reviewing Cryptosporidium Data 56 Draft June 2003 organisms in the sample. To correct for background concentration, the number of organisms detected in the unspiked field sample is subtracted from the number of oocysts detected in the MS sample.

To determine the percent recovery for a matrix spike ( MS) sample, the following information is needed:

° The number of oocysts detected in the MS sample ° The true value of the oocysts spiked into the MS sample ° The number of oocysts detected in the unspiked field sample ( to correct for background concentration)

percent recovery = oocysts counted in MS sample  oocysts counted in unspiked field sample × 100% oocysts spiked into MS sample 7.6 Data Archiving Requirements LT2 rule monitoring data must keep monitoring results until 36 months after source water monitoring has been completed. Although it is the PWS's responsibility to meet LT2 rule data storage requirements for compliance monitoring samples, the PWS may designate this responsibility to the laboratory.

Although not required, laboratories also can archive slides and/ or take photographs of slides to maintain for clients. As noted in Section <IP_ADDRESS>, this may be considered an extra service and result in extra costs, as these steps may not be routinely performed by the laboratory. Slides should be stored in a humid chamber in the dark at 0 ° C to 10 ° C. An alternative mounting medium also may be used, which may potentially preserve slides longer. Details are provided in the Microbial Laboratory Guidance Manual for the Long Term 2 Enhanced Surface Water Treatment Rule ( LT2 Rule). 57 Draft June 2003 SECTION 8: REVIEWING E. COLI DATA When E. coli samples are processed and analyzed by the laboratory, data on sample measurements, sample processing times, and slide examination results are recorded at the laboratory and reported to the PWS through the LT2 Data Collection System. This section provides an overview of the data recording and reporting processes and provides guidance on how to review the data you receive from the laboratory.

8.1 E. coli Laboratory Data Recording at the Laboratory The laboratories performing E. coli analyses during the LT2 rule record the following general types of information:

° Sample identification information ° All primary measurements used to calculate the final E. coli concentration for each sample ° The incubation start and read times for each method to verify that method requirements were met ° The name of the analyst performing the sample analysis ° Quality control ( QC) analysis results ( e. g., positive/ negative controls, blanks, etc.)

8.1.1 Sample Identification Information Sample identification information is used to track the sample through sample collection, analysis, and data reporting. At a minimum, the laboratory records the sample ID, the target parameter ( E. coli), and the method being used ( e. g., Membrane Filtration: SM 9222D/ SM 9222G).

8.1.2 Primary Data The laboratory records all primary measurements needed to calculate the final concentration of E. coli per 100 mL. Primary measurements for membrane filtration methods will include the volumes filtered and the plate counts for each volume filtered. The multiple well and multiple tube formats will include the volumes or dilutions of samples analyzed and the number of positive wells or tubes per each volume analyzed.

8.1.3 Sample Processing and Quality Control Information The laboratory records information on the bench sheet on how they processed and analyzed the sample, including incubation start/ end date and times and temperature, and the analyst performing each step of the method. The lot numbers of reagents, media, and materials used to process the sample and the results of QC analyses should be recorded in a media log book or QC checklist. In addition to being used to resolve questions regarding validity of results, this information may be used by the laboratory to determine the source of any problems the laboratory is having with method performance. Section 8: Reviewing E. coli Data 58 Draft June 2003 8.1.4 Sample Results The final E. coli concentration for field samples will be reported as CFU/ 100 mL or MPN/ 100 mL depending on the method used for analysis. If no E. coli are detected in the sample, a low censored value based on the volume of sample analyzed must be reported ( e. g. < 1CFU / 100 mL or < 1.8 MPN/ 100 mL). E. coli concentration will never be reported as a zero.

8.2 Submission of E. coli Data through the LT2 Data Collection System During the LT2 rule, laboratories will report E. coli data electronically through EPA's LT2 Data Collection System to the PWS staff responsible for approving and submitting monitoring results to EPA. The LT2 Data Collection System is a web based application that allows laboratory users to enter or upload data, then electronically " release" the data to the appropriate PWS staff for review, approval, and submission to EPA and the State. Although ownership of the data resides with the PWS throughout this process, the LT2 Data Collection System increases the ease and efficiency of the data entry and transfer process from one party to another by transferring the ability to access the data from the laboratory to the PWS to EPA and the State, and ensuring that data cannot be viewed or changed by unauthorized parties. A summary of the data entry, review, and transfer process through the LT2 Data Collection System for both Cryptosporidium and E. coli samples is provided in Table 7 1, in Section 7.2, above.

The data reporting process is summarized below, in Sections 8.2.1 through 8.2.3, and discussed in detail in the Users' Manual for the Long Term 2 Enhanced Surface Water Treatment Rule ( LT2 Rule) Data Collection System. The LT2 data system users' guide also provides detailed information on the laboratory registration process. Information on the LT2 Data Collection System, as well as a downloadable users' manual, is available at http:// www. epa. gov/ safewater/ lt2/ index. html.

8.2.1 Data Entry/ Upload The analyst or another laboratory staff member enters a subset of the data recorded at the bench ( Section 8.1) into the LT2 Data Collection System either by entering the data using web forms or by uploading data in XML format. This information includes the following:

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

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

° E. coli/ 100 mL ( see note below)

Note: The laboratory may then enter the final result for the sample that was calculated at the laboratory or may enter the primary measurements recorded at the bench, and have the LT2 Data Collection System automatically calculate the final sample concentration. Because this information is specific to method type ( membrane filtration, multiple tube fermentation, 51 well, and 97 well), the system provides Section 8: Reviewing E. coli Data 59 Draft June 2003 different entry screens for each method type. The laboratory staff entering the data should verify that all holding times and other QC specifications were met.

The laboratory's official contact is responsible for verifying the quality and accuracy of all sample results in the laboratory, and is required to review and approve the results before they are submitted to the PWS for review. If inaccuracies or other problems are identified, the official contact discusses the sample information with the analyst or data entry staff and resolves the issues before the data are approved for PWS review.

If no inaccuracies or other issues are identified, the laboratory's official contact approves the data for " release" to the PWS for review ( EPA does not receive the data at this point). When the data are approved, the rights to the data are transferred electronically by the system to the PWS, and the data can no longer be changed by the laboratory.

8.2.2 PWS Data Review After the laboratory has released E. coli data electronically to the PWS using the LT2 Data Collection System, the PWS will review the results. The PWS user cannot edit the data, but if the PWS has an issue with the sample result, such as if the PWS believes that the sample collection point ID or collection date is incorrect, the PWS can release the results back to the laboratory for issue resolution. In addition to noting the reason in the LT2 Data Collection System for the return of the data to the laboratory, you also should contact the laboratory verbally to discuss the issue.

If the PWS determines that the data are accurate, the PWS releases the results to EPA ( and the State, if applicable) as " approved" results. If the PWS determines that the data are accurate, but believes that the data are not valid for other reasons, the PWS can release the results as " contested."

8.2.3 EPA/ State Review After the PWS has released the results as approved or contested, they are available to EPA and State users to review through the LT2 Data Collection System. EPA and State users cannot edit the data.

8.3 Reviewing and Validating E. coli Data ( Optional)

If the PWS staff responsible for submitting data to EPA plans to review the raw data generated by the laboratory, the original, hardcopy records ( whether generated by an in house laboratory or a contract laboratory) should be compared to the electronic results. However, this step is not required. Sections 8.3.1 through 8.3.3 provides guidance on how to review and validate the hardcopy data and verify accuracy.

8.3.1 Data Completeness Check Upon receipt of hardcopy sample results for a monitoring sample, verify that the following information is included:

° Sample result summary sheet, which should include the following:

° Monitoring sample identification information ° Monitoring sample result, in E. coli/ 100 mL ° Laboratory quality control checklist ( or other verification from the laboratory that all QC specifications were met) Section 8: Reviewing E. coli Data 60 Draft June 2003 ° LT2 sample collection form initiated at the time of sample collection and completed with sample receipt information by the laboratory ° E. coli Method Bench Sheet completed by the laboratory with primary sample processing and analysis data associated with the monitoring sample ° Laboratory comments. If the laboratory provided comments on the sample analyses or results that require follow up, contact the laboratory to discuss, if necessary. Comments may include any applicable data qualifiers. The following is a list of potential data qualifiers:

° Sample arrived at the laboratory in unacceptable condition ( i. e., leaking)

° Sample holding time exceeded ° Sample holding temperature not within acceptable range ° Unacceptable blank sample result ° Unacceptable positive or negative control result ° Media sterility checks were not acceptable ° Method incubation times or temperatures were not within acceptable range ° Membrane filtration: Too much sediment on the filter ° Membrane filtration: Confluent growth of non target organism ° Membrane filtration: Colonies too numerous to count ( TNTC)

° Membrane filtration: Pre or post filtration series sterility check not acceptable ( e. g., contamination with E. coli organism)

° Quanti Tray ® was damaged or leaked ° Sample was not distributed to all wells in Quanti Tray ® ° All rows of tubes were not inoculated ° Positive presumptive tubes were not transferred into the appropriate confirmatory medium Any of the above data qualifiers would result in the sample being considered invalid for LT2 use and the results for the sample should not be entered into the LT2 data collection system. If the laboratory enters the results into the LT2 data collection system, the PWS should not submit the results to EPA.

If forms are missing, incomplete, or incorrect, contact the laboratory immediately to discuss and request resubmission of the missing forms and/ or spreadsheets.

8.3.2 Evaluation of Data Against Method Quality Control Requirements To verify that the laboratory analyzed your monitoring sample within the analytical controls specified by the method, check the following information:

° Sample condition upon receipt. If the sample was shipped to the laboratory, verify on the completed LT2 sample collection form that your sample was received in acceptable condition ( e. g., not leaking, etc.), and at a temperature below 10 ° C, but not frozen.

° QC samples associated field samples. The frequency of analysis of quality control samples including method blanks, positive and negative controls, etc. varies according to method requirements Section 8: Reviewing E. coli Data 61 Draft June 2003 and specifications in the Certification Manual. Verify that the required QC samples were run with the field sample. A summary of these QC specifications is provided in Section 4.2 of this document.

° Holding time. Using the sample collection date and time on the LT2 data collection form and the date and time of the first method step recorded by the laboratory on the E. coli method bench sheet, verify that the laboratory began sample analysis within 24 hours of sample collection.

° Incubation times and temperatures. Using the dates and times of the method steps recorded by the laboratory on the E. coli method bench sheet, verify that the method specified incubation times and temperatures, specified in Table 8 1 were met.

Table 8 1. Incubation Times and Temperatures for Approved E. Coli Methods Method Media Incubation Time/ Temperature Standard Methods 9223B Colilert ® 24 to 28 hours at 35 ° C ± 0.5 ° C Colilert 18 ® 18 to 22 hours at 35 ° C ± 0.5 ° C Standard Methods 9221B/ F LTB 24 ± 2 and 48 ± 3 hours at 35 ° C ± 0.5 ° C EC MUG 24 ± 2 hours at 44.5 ° C ± 0.2 ° C Standard Methods 9222B/ 9222G mENDO ! NA MUG 24 ± 2 hours at 35 ° C ± 0.5 ° C ! 4 hours at 35 ° C ± 0.5 ° C LES ENDO ! NA MUG 24 ± 2 hours at 35 ° C ± 0.5 ° C ! 4 hours at 35 ° C ± 0.5 ° C Standard Methods 9222D/ 9222G mFC ! NA MUG 24 ± 2 hours at 44.5 ° C ± 0.2 ° C ! 4 hours at 35 ° C ± 0.5 ° C Standard Methods 9213D mTEC agar 2 hours at 35 ° C ± 0.5 ° C ! 22 to 24 hours at 44.5 ° C ± 0.2 ° C EPA 1603 Modified mTEC 2 hours at 35 ° C ± 0.5 ° C ! 22 to 24 hours at 44.5 ° C ± 0.2 ° C EPA 1604 MI medium 24 hours at 35 ° C ± 0.5 ° C Other Membrane Filter Method m ColiBlue24 ® ® Broth 24 hours at 35 ° C ± 0.5 ° C 8.3.3 Calculation Verification Method specific data to record for each of the individual method types as well as standardized calculations for each method type are discussed in Sections <IP_ADDRESS> through <IP_ADDRESS>.

<IP_ADDRESS> Calculations for Determining the E. coli Concentration Using the Colilert ® Quanti Tray 2000 ® ( 97 well)

A. Select appropriate dilution to yield countable results. If multiple dilutions are used, the tray exhibiting positive wells in the 40% to 80% range ( 39 to 78 total positive large and small wells) should be used to determine MPN value.

B. Determine MPN. Using the number of large positive wells and small positive wells from the appropriate dilution, identify the corresponding MPN/ 100 mL in the table provided by the vendor. Large well values are located in the left column; small well values are located in the top row. For example, if a 100 mL sample was analyzed, and there were 29 large positive wells and 5 small positive wells, the corresponding MPN would be 49.6 MPN/ 100 mL. Section 8: Reviewing E. coli Data 62 Draft June 2003 C. Adjust for dilution factor. Because the MPN/ 100 mL values in the table are based on 100 mL samples, the MPN value should be adjusted if less than 100 mL of sample volume was analyzed. Use the following calculation to adjust the MPN to account for the dilution:

Analytical result = MPN value mL of sample analyzed Example:

Volume analyzed = 10 mL of sample ( in 90 mL of dilution water) Large wells positive = 39 Small wells positive = 5 The MPN value calculated based on the intersection of 10 and 2 in the table. MPN = 81.3 Analytical result = 81.3 × 100 = 813 E. coli MPN/ 100 mL 10 <IP_ADDRESS> Calculations for Determining the E. coli Concentration Using the Colilert ® Quanti Tray 51 ® ( 51 well)

A. Select appropriate dilution. If multiple dilutions are used, the tray exhibiting positive wells around the 80% range ( 41 positive wells) should be used to determine MPN value.

B. Determine MPN. Using the number of positive wells from the appropriate dilution, identify the corresponding MPN/ 100 mL in the table provided by the vendor. For example, if a 100 mL sample was analyzed, and there were 26 positive wells, the corresponding MPN would be 36.4 MPN/ 100 mL C. Adjust for dilution factor. Because the MPN/ 100 mL values in the table are based on 100 mL samples, the MPN value should be adjusted if less than 100 mL of sample volume was analyzed. Use the following calculation to adjust the MPN to account for the dilution:

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

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

83.1 × 100 = 831 E. coli MPN/ 100 mL 10 Section 8: Reviewing E. coli Data 63 Draft June 2003 Note: Results that are TNTC or CNFG are not appropriate for LT2 microbial data analysis, and cannot be entered into the LT2 Data Collection System. <IP_ADDRESS> Calculations for determining the E. coli concentration using membrane filter data ( adapted from Reference 9.4)

A. E. coli counts should be determined from the volume( s) filtered that yielded 20 to 80 E. coli colonies ( 20 60 for mFC NA MUG), and not more than 200 total colonies per plate. ( Guidance for samples that do not yield countable plates is provided in Sections E and F)

Note: The analytical result can be automatically calculated using the LT2 Data Collection System. See Section 8.2.1 for additional information.

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

C. If colonies are not sufficiently distinct for accurate counting, the result is recorded as " confluent growth" ( CNFG). To prevent CNFG from occurring, smaller sample aliquots should be filtered. For example, if sample volumes of 100, 10, 1 and 0.1 mL are analyzed and even the 0.1 mL plates results in CNFG, then potentially 0.01 mL should be analyzed during the next sampling event. The 100 mL volume can be eliminated. Note: If growth is due to high levels of total coliforms but low E. coli then another method should be chosen for analyses that does not rely on total coliform determination prior to or simultaneously with E. coli determination.

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

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

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

40 E. coli CFU × 100 = 400 E. coli CFU/ 100 mL 10 mL E. If no E. coli colonies are present, the detection limit is calculated as < largest volume filtered per 100 mL. Section 8: Reviewing E. coli Data 64 Draft June 2003 Example 2:

Filter 1 volume ( mL) = 100 mL CFU = 0 Filter 2 volume ( mL) = 10 mL CFU = 0 Filter 3 volume ( mL) = 1.0 mL CFU = 0 Detection limit = 100 mL = E. coli CFU/ 100 mL Largest volume filtered 100 mL = < 1 E. coli / 100 mL 100 mL Example 3:

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

100 mL = < 10 E. coli CFU / 100 mL 10 mL F. If there are no filters with E. coli counts in the 20 80 colony range ( 20 60 for mFC NA MUG), sum the E. coli counts on all filters, divide by the volume filtered and report as number per 100 mL.

Example 4:

Filter 1 volume ( mL) = 50 mL CFU = 15 Filter 2 volume ( mL) = 25 mL CFU = 6 Filter 3 volume ( mL) = 10 mL CFU = 0 The analytical result is calculated as:

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

Example 5:

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

( 105 + 92 + 85) × 100 = 332 E. coli CFU/ 100 mL ( 50 + 25 + 10) Section 8: Reviewing E. coli Data 65 Draft June 2003 Example 6:

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

( 82 + 18 + 0) × 100 = 90 E. coli CFU/ 100 mL ( 100 + 10 + 1)

Example 7:

Filter 1 volume ( mL) = 50 mL CFU = TNTC Filter 2 volume ( mL) = 25 mL CFU = TNTC Filter 3 volume ( mL) = 10 mL CFU = 83 The analytical result is calculated as:

83 × 100 = 830 E. coli CFU/ 100 mL 10 <IP_ADDRESS> Calculation of E. coli Concentrations Using Multiple Tube Methods ( adapted from Reference 9.6):

The guidance and examples for determining E. coli concentrations using multiple tube methods are based on the revision of Standard Methods 9221C included in the 2001 Supplement to the 20th Edition of Standard Methods, approved by the Standard Methods Committee in 1999.

Note: The analytical result can be automatically calculated using the LT2 Data Collection System. See Section 8.2.1 for additional information.

A. For each sample volume ( e. g., 10, 1, 0.1, and 0.01 mL or additional sample volumes as necessary), determine the number of positive tubes out of five.

B. A dilution refers to the volume of original sample that was inoculated into each series of tubes. Only three of the dilution series will be used to estimate the MPN. The three selected dilutions are called significant dilutions and are selected according to the following criteria. Examples of significant dilution selections are provided in Table 8 2, below.

° Choose the highest dilution ( the most dilute, with the least amount of sample) giving positive results in all five tubes inoculated and the two succeeding higher ( more dilute) dilutions. ( Table 8 2, Example A).

° If the lowest dilution ( least dilute) tested has less than five tubes with positive results, select it and the two next succeeding higher dilutions ( Table 8 2, Examples B and C).

° When a positive result occurs in a dilution higher ( more dilute) than the three significant dilutions selected according to the rules above, change the selection to the lowest dilution ( least dilute) that has less than five positive results and the next two higher dilutions ( more dilute) ( Table 8 2, Example D). Section 8: Reviewing E. coli Data 66 Draft June 2003 ° When the selection rules above have left unselected any higher dilutions ( more dilute) with positive results, add those higher dilution positive results to the results for the highest selected dilution ( Table 8 2, Example E).

° If there were not enough higher dilutions tested to select three dilutions, then select the next lower dilution ( Table 8 2, Example F).

C. MPN values need to be adjusted based on the significant dilutions series selected above. Because the MPN/ 100 mL values in the table are based on the 10 mL, 1 mL, and 0.1 mL dilution series, per method requirements, the MPN value must be adjusted if these are not the significant dilution series selected. Use the following calculation to adjust the MPN when the 10 mL, 1 mL, and 0.1 mL dilution series are not the significant dilution series selected:

Analytical result = MPN value = E. coli MPN/ 100 mL # of mL in middle dilution Table 8 2. Examples of Different Combinations of Positive Tubes ( Significant Dilution Results Are in Bold and Underlined)

Example Least dilute Most dilute ( Lowest) ( Highest) Combination of positives MPN Index from Standard Methods E. coli/ 100 mL ( after adjustment) 10 mL 1 mL 0.1 mL 0.01 mL 0.001 mL A 5 5 1 0 0 5 1 0 33 330 B 4 5 1 0 0 4 5 1 48 48 C 0 0 1 0 0 0 0 1 1.8 1.8 D 5 4 4 1 0 4 4 1 40 400 E 5 4 4 0 1 4 4 1 40 400 F 5 5 5 5 2 5 5 2 540 54,000 Example A: The significant dilution series for the 5 1 0 combination of positives includes the 1 mL, 0.1 mL, and 0.01 mL dilutions. Since the 10 mL, 1 mL, and 0.1 mL dilutions were not selected, an adjustment is necessary to account for the dilutions selected:

Analytical result = 33 = 330 E. coli / 100 mL 0.1 Example B: Since the 10 mL, 1 mL, and 0.1 mL dilutions are the significant dilutions, no adjustment is necessary and the result is 48 E. coli/ 100 mL.

Example C: Since the 10 mL, 1 mL, and 0.1 mL dilutions are the significant dilutions, no adjustment is necessary and the result is 1.8 E. coli/ 100 mL.

Examples D and E: The significant dilution series for the 4 4 1 combination of positives includes the 1 mL, 0.1 mL, and 0.01 mL dilutions. Since the 10 mL, 1 mL, and 0.1 mL dilutions were not selected, an adjustment is necessary to account for the dilutions selected: Section 8: Reviewing E. coli Data 67 Draft June 2003 Analytical result = 40 = 400 E. coli / 100 mL 0.1 Example F: The significant dilution series for the 5 5 2 combination of positives includes the 0.1 mL, 0.01 mL and 0.001 mL dilutions. Since the 10 mL, 1 mL, and 0.1 mL dilutions were not selected, an adjustment is necessary to account for the dilutions selected:

Analytical result = 540 = 54,000 E. coli / 100 mL 0.01 8.4 Data Archiving Requirements Under the LT2 rule, monitoring data must keep until 36 months after source water monitoring has been completed [ 40 CFR part 141.731 ( a)]. Although it is the PWS's responsibility to meet LT2 rule data storage requirements for compliance monitoring samples, the PWS may designate this responsibility to the laboratory. 68 Draft June 2003 SECTION 9: REFERENCES 9.1 Connell, Kevin, et al. 2000. ICRSS  Building a Better Protozoa Data Set, J. AWWA. 91( 10): 30  43.

9.2 Pope, Misty, et al. 2003. " Using E. coli To Indicate Source Water Susceptibility to High Concentrations of Cryptosporidium," in Information Collection Rule Data Analysis. AWWARF, Denver, CO.

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

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

9.5 Pope, M., et al. 2002. Assessment of the effects of holding time and temperature on E. coli concentrations in surface water samples. Appl. Environ. Micro. ( submitted).

9.6 2001 Supplement to the 20th Edition of Standard Methods 9221 C: Explanation of Bacterial Density. This supplement is available for download at http:// www. techstreet. com/ cgibin detail? product_ id= 923645. 69 Draft June 2003 SECTION 10: ACRONYMS CFU Colony forming unit CNFG Confluent growth DAPI 4, 6 diamidino 2 phenylindole DIC Differential interference contrast EPA U. S. Environmental Protection Agency FA Immunofluorescense assay FITC Fluorescien isothiocyanate GWUDI Ground water under the direct influence of surface water ICR Information Collection Rule IFA Immunofluorescence assay IMS Immunomagnetic separation IPR Initial precision and recovery IPT Initial proficiency testing L Liter LT2 rule Long Term 2 Enhanced Surface Water Treatment Rule LT2ESWTR Long Term 2 Enhanced Surface Water Treatment Rule MPN Most probable number MS Matrix spike MS/ MSD Matrix spike/ matrix spike duplicate µ m Micrometer NA MUG Nutrient agar ( NA) with 4 methylumbelliferyl beta D glucuronide ( MUG)

nm Nanometer NPDWR National Primary Drinking Water Regulations NTU Nephelometric turbidity unit OPR Ongoing precision and recovery OPT Ongoing proficiency testing PBMS Performance based measurement system PT Proficiency testing PWS Public water system QA Quality assurance Section 10: Acronyms 70 Draft June 2003 QAP Quality assurance plan QC Quality control RSD Relative standard deviation SDWA Safe Drinking Water Act TNTC Too numerous to count UV Ultraviolet


 Coal Units FGD Existing ( MW) FGD Projected ( MW) No FGD ( MW) Total ( MW)

BART 42,658 18,478 87,786 148,922 Not BART 51,979 6,333 126,028 184,340 All Units 94,637 24,811 213,814 333,262 Existing and Planned SO2 Controls ( FGD) at Coal Fired Electric Generating Units ( Pre BART)

0 50,000 100,000 150,000 200,000 250,000 300,000 350,000 BART Not BART All Units Coal EGUs Total Nameplate Capacity (

MW) No FGD ( MW)

FGD Projected ( MW)

FGD Existing ( MW) Coal Units FGD Existing ( MW) FGD Projected ( MW) No FGD ( MW) Total ( MW)

BART 42,658 106,264 0 148,922 Not BART 51,979 6,333 126,028 184,340 All Units 94,637 112,597 126,028 333,262 Existing and Planned SO2 Controls ( FGD) at Coal Fired Electric Generating Units ( Post BART)

0 50,000 100,000 150,000 200,000 250,000 300,000 350,000 BART Not BART All Units Coal EGUs Total Nameplate Capacity (

MW) No FGD ( MW)

FGD Projected ( MW)

FGD Existing ( MW)


[Federal Register Volume 77, Number 63 (Monday, April 2, 2012)]
[Notices]
[Page 19657]
[FR Doc No: 2012-7777]

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DEPARTMENT OF ENERGY

Federal Energy Regulatory Commission

Combined Notice of Filings 1

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

    Docket Numbers: EC12-84-000.
    Applicants: AER NY-Gen, LLC, Alliance NYGT, LLC.
    Description: AER NY-Gen, LLC and Alliance NYGT, LLC submits their
Application for Approval under Section 203 of the Federal Power Act and
Request for Expedited Consideration.
    Filed Date: 3/20/12.
    Accession Number: 20120320-5171.
    Comments Due: 5 p.m. ET 4/10/12.

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

    Docket Numbers: ER12-629-001.
    Applicants: Westar Energy, Inc.
    Description: Westar Energy, Inc. submits tariff filing per 35:
Compliance Filing, Oklahoma Municipal Power Authority Letter Agreement
to be effective N/A.
    Filed Date: 3/20/12.
    Accession Number: 20120320-5163.
    Comments Due: 5 p.m. ET 4/10/12.

    Docket Numbers: ER12-1296-000.
    Applicants: ResCom Energy LLC.
    Description: Amended Tariff Filing to be effective 2/3/2012.
    Filed Date: 3/20/12.
    Accession Number: 20120320-5154.
    Comments Due: 5 p.m. ET 4/10/12.

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

    Docket Numbers: ES12-23-000.
    Applicants: Wolverine Power Supply Cooperative, Inc.
    Description: Second Amendment to Application for Authorization of
the Assumption of Liabilities and the Issuance of Securities under
Section 204 of the Federal Power Act of Wolverine Power Supply
Cooperative, Inc.
    Filed Date: 3/16/12.
    Accession Number: 20120316-5102.
    Comments Due: 5 p.m. ET 3/26/12.

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

    Dated: March 21, 2012.
Nathaniel J. Davis, Sr.,
Deputy Secretary.
[FR Doc. 2012-7777 Filed 3-30-12; 8:45 am]
BILLING CODE 6717-01-P


[Federal Register Volume 82, Number 151 (Tuesday, August 8, 2017)]
[Rules and Regulations]
[Pages 37010-37011]
[FR Doc No: 2017-16645]

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DEPARTMENT OF HOMELAND SECURITY

Coast Guard

33 CFR Part 100

[Docket No. USCG-2017-0640]

Special Local Regulation for Marine Events; Back River, Hampton,
VA

AGENCY: Coast Guard, DHS.

ACTION: Notice of enforcement of regulation.

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SUMMARY: The Coast Guard will enforce special local regulations for the
Poquoson Seafood Festival Workboat Race held on the Back River on
October 15, 2017, with a rain date of October 29, 2017. This action is
necessary to provide for the safety of life on navigable waterways
during the power boat race. Our regulation for recurring marine events
in Captain of the Port--Sector Hampton Roads zone identifies the
regulated area for this regatta. During the enforcement period, no
vessel may transit this regulated area without approval from the
Captain of the Port or a designated representative.

DATES: The regulations in 33 CFR 100.501 will be enforced for the
location listed at (c)(8) in the Table to Sec.  100.501, Coast Guard
Sector Hampton Roads--COTP Zone, from 1 p.m. through 4 p.m. on October
15, 2017, with a rain date of October 29, 2017.

FOR FURTHER INFORMATION CONTACT: If you have questions about this
notice of enforcement, call or email LCDR Barbara Wilk, Waterways
Management Sector Hampton Roads, U.S. Coast

Guard; telephone<PHONE_NUMBER><EMAIL_ADDRESS>
SUPPLEMENTARY INFORMATION: The Coast Guard will enforce special local
regulations in 33 CFR 100.501 from 1 p.m. until 4 p.m. on October 15,
2017, with a rain date of October 29, 2017, for the Poquoson Seafood
Festival Workboat Race at Poquoson, VA. This action is being taken to
provide for the safety of life on navigable waterways during the power
boat race. Our regulation for recurring marine events within the Fifth
Coast Guard District, Sec.  100.501, specifies the location for this
special local regulation; Race area: The area is bounded on the north
by a line drawn along latitude 37[deg]06'30'' N., bounded on the south
by a line drawn along latitude 37[deg]06'15'' N., bounded on the east
by a line drawn along longitude 076[deg]18'52'' W. and bounded on the
west by a line drawn along longitude 076[deg]19'30'' W. Buffer area:
The waters of Back River extending 200 yards outwards from east and
west boundary lines, and 100 yards outwards from the north and south
boundary lines described in this section. As specified in Sec.
100.501(c), during the enforcement period, no vessel may transit this
regulated area without approval from the Captain of the Port (COTP)--
Sector Hampton Roads or a COTP designated representative.
    This notice of enforcement is issued under authority of 33 CFR
100.501 and 5 U.S.C. 552(a). This notice of enforcement will be
published in the Federal Register, Local Notice to Mariners, and
conveyed in marine information broadcasts.

    Dated: August 1, 2017.
Richard J. Wester,
Captain, U.S. Coast Guard, Captain of the Port, Hampton Roads, VA.
[FR Doc. 2017-16645 Filed 8-7-17; 8:45 am]
 BILLING CODE 9110-04-P