Public Comment and Responses for the National Primary and Secondary Drinking Water Regulations: Approval of Colitag   for Compliance Monitoring of Total Coliforms and E. coli in Finished Drinking Water U. S. Environmental Protection Agency Office of Water Office of Ground Water and Drinking Water December 2003 Docket ID No. OW 2002 0031 2 Comment No. Docket ID Docket Part Comment Response Notes 01 RECOMMENDATIONS REGARDING EPA APPROVAL OF COLITAG   1 ID1.001 06   In conclusion, we feel that the protection of public health is more than adequately guaranteed at present, with several diverse methods currently being approved for drinking water compliance testing. We further feel very strongly that the public interest deserves better than the inconsistent and haphazard approach exhibited in this proposal, and that public health in fact may be compromised if methods were employed based on data exhibited herein. We therefore request that the US EPA ask for CPI to complete a full ATP Comparability Study, following the accepted scientific practices, prepare a well documented study report and resubmit the data to the US EPA for public comments and for further consideration of approval.  The approval of alternate methods for detecting coliform bacteria is independent of the number of existing methods.

The protocol that guided the Colitag   method comparability testing is titled   Protocol for Alternate Test Procedures ( ATP) for Coliform Bacteria in Compliance With Drinking Water Regulations,

published in 1995. The protocol is not mandatory nor is it regulatory in nature. Rather, EPA established the guidelines in the protocol to encourage the collection of adequate information for the Agency s evaluation of a new method ( i. e., to allow the Agency to reach a reasoned judgement concerning the comparability between the new method and the reference methods).

Keeping that objective in mind, EPA notes that it has exercised a degree of flexibility in the application of the guidance. While EPA believes that those who follow the protocol guidelines increase the likelihood of the Agency having sufficient information on which to base an approval decision, EPA notes that following the guidelines precisely does not guarantee method approval.

Similarly, deviation from the guidelines does not preclude EPA from considering a method for approval.

EPA considers all information submitted and, when there is a question or concern ( e. g., when there is a suggestion that information was not collected precisely in accordance with the guidance), EPA generally considers the underlying issue that the protocol was designed to address. Where the Agency has concluded that adequate information is available to judge a particular issue, it has proceeded with the evaluation of the method; this approach has been reflected in EPA s past evaluation of numerous methods, including currently approved methods for the measurement of Comment No. Docket ID Docket Part Comment Response Notes 3 total coliforms and E. coli.

The commenter requests that a full, new ATP study be done following accepted scientific practices. A full ATP comparability study was in fact, performed. EPA reviewed the comparability study results and judged that the Colitag   method demonstrated comparability to the reference methods ( i. e., the comparability study did not identify a statistically significant difference in performance between the reference methods and Colitag). Specific details of the comparability study are discussed in responses to other comments.

In summary, EPA believes that CPI s Colitag   comparability study data represents an adequate basis for evaluating the method. Accordingly, it is not necessary to perform a new ATP study, as the commenter requests.

2 ID1.002 15   The original submission of testing data in support of the application for EPA approval of the Colitag   product was deficient in numerous areas under the ATP guidelines;

moreover, a number of questions concerning quality control of the supporting data remain unanswered. EPA has failed to respond to the original concerns raised by independent reviewers and most recently has solicited review comments on only a portion of the data submitted by the manufacturer. In light of the fact that new water testing products should be subjected to rigorous internal and external review, these unanswered questions suggest that the data submitted are inadequate to support the contention that the performance of the Colitag   product meets or exceeds that of the EPAapproved reference methods. Because it may be possible for the manufacturer and/ or the independent testing laboratory to respond to these concerns, EPA should request additional supporting data and should resubmit the data for external review.  The commenter states that the data submitted for approval of Colitag   was deficient under the ATP guidelines. See response to Comment 1 ( Docket ID ID1.001 Part 06) regarding use of the ATP protocol as guidance.

With respect to the concerns expressed by commenters about quality control, EPA notes that the comparability studies were performed by an independent, certified drinking water laboratory. As such, EPA can reasonably expect that the laboratory has adhered to QC practices established for analysis of drinking water samples.

With respect to EPA s response to comments raised previously, EPA notes that it is using this document to provide an integrated response to comments on both the original ( March 2002) proposed rule and the December 2002 Notice of Data Availability ( NODA); it was not EPA s intention to address the proposed rule comments via the NODA. Comment No. Docket ID Docket Part Comment Response Notes 4 Finally, the data submitted by CPI to demonstrate the comparability of Colitag   to the reference methods were subjected to a thorough review which addressed the questions from the commenter. EPA believes that these data demonstrate comparability between Colitag   and the reference methods, and that further testing is not needed.

3 ID1.005 02   Regarding the recent Federal Register notice that apparently indicates that the Colitag   test will be approved, this is a best premature. First, the it has been US EPA policy that any method approved to protect the public's health must recover damaged Escherichia coli. ( please see my letter of 3 May below,

with documentation). E. coli is the ONLY biological fecalspecific indicator for tap water. How long E. coli survives in the environment has been central to the Groundwater, drinking water, and other rules. To approve a method that is unproven in its ability to recover damaged E. coli is against the public interest.  Promulgation of the Colitag   method was proposed in a March 2002 Federal Register notice. EPA used its December 2002 NODA not to indicate that Colitag   would be approved, but instead to solicit public comment on additional Colitag   information available subsequent to the March 2002 proposal; the December 2002 NODA did not represent final action on the Colitag   method. Such action is being taken by EPA via today s notice, and only after having an opportunity to respond to all comments.

With respect to the comment that EPA s policy has been that coliform methods must recover damaged Escherichia coli, EPA notes that its ATP protocol has recommended that bacteria be exposed to chlorine so that there is an appropriate stressing of test bacteria prior to conducting the comparability study of the proposed and reference methods. The goal is to model the stress that bacteria experience in a water distribution system. Since knowledge of the degree of chlorine stress experienced by bacteria in a distribution system and its effect on bacterial physiology are not precisely known, the 3 to 4 log reduction recommendation in the protocol only represented a best estimate at the time of the appropriate level of chlorine stress, and in past ATP evaluations has only been regarded as an estimate ( For examples of past method evaluations where this approach has been used,

see comparability study data for the Colisure and Readycult methods).

When judging the Colitag   comparability study s test to Comment No. Docket ID Docket Part Comment Response Notes 5 determine if Colitag   could adequately recover damaged E. coli, EPA experts evaluated the chlorine stress that was applied to the test bacteria and concluded that the test bacteria had been adequately stressed, albeit under a wider range of log reduction than recommended in the protocol. The rationale for accepting a wider range is twofold: ( 1) EPA experts believe that a wider range ( 2 to 4 logs) may, in fact, be more representative of the wide variety of chlorine stress that occurs in realworld conditions; and ( 2) in their experience, a lower degree of log reduction ( 2 to 3 logs) may, in fact, subject the method being evaluated to an even greater challenge than the original 3 to 4 log reduction goal. Regarding the second point, their reasoning was that only the hardiest cells survive at higher levels of log reduction,

and, therefore, the surviving bacteria in high log reduction situations may be stronger ( i. e., less stressed)

than the collection of bacteria surviving stress at lower levels of log reduction. Therefore, EPA will generally accept a wider range of log reduction ( 2 to 4 logs).

4 ID1.005 04   Third, there are many other methods that have been approved by EPA  why rush to approve one that is suspect? At the minimum, EPA should defer its decision to allow informed public analysis. There is nothing lost by doing this ( there are many other methods) but the risk is great should this method fail ( missing a public health threat). Fourthly, while there are other questions regarding the ability of the Colitag method to recover E. coli that have not been resolved ( see my letter of 3 May to EPA below). While it is apparently not the purpose of this Federal Register notice to address other issues, these significant public health issues must be answered.

  It would be a tragedy to have the Colitag method approved and for it to fail in the field, and for human illness to occur.

There is no reason this should happen. Therefore, at the minimum please defer the final decision pending sufficient time to analyze the complete data set. I also add that deferral pending completion of analysis was exactly the course the EPA EPA has taken the time it believed necessary to adequately judge the method and notes that the multiyear review process for the Colitag   comparability study data has been extensive. EPA has sought,

considered, and responded to public comments not only via the March 2002 proposed rule, but also via the December 2002 NODA. EPA has come to the conclusion that the Colitag   method is comparable to the reference methods in its ability to initiate growth of chlorine stressed bacteria and does not believe that further testing is needed.

EPA performed a very thorough analysis of the CPI comparability study data and it is not clear what additional analysis the commenter is suggesting here.

EPA judged that Colitag   was comparable to the reference methods in its ability to recover injured E. coli with the data submitted because statistical analysis did Comment No. Docket ID Docket Part Comment Response Notes 6 took when the same question  the ability to recover injured E.

coli  arose regarding the MM0 MUG method. EPA undertook this re analysis including additional studies, and subsequently approved the method it has worked admirably. Conversely,

recently, the EPA approved total coliform methods that had unacceptably high false positives, prompting a re analysis of the data. This should not happen again.  not show a significant difference between the performance of the reference method and that of Colitag   .

This situation differs from the MMO MUG situation cited by the commenter in several important respects.

First, at the time that the MMO MUG method was being considered, the Agency had limited experience with chromogenic/ fluorogenic presence/ absence methods. As a result, the Agency approached the review of such methods in a more conservative manner.

Secondly, EPA notes that at the time it was evaluating the MMO MUG method the operative protocol for ATP methods was quite different ( e. g., the protocol at that time did not include log reduction guidelines). Hence,

the context in which EPA made decisions regarding MMO MUG differs significantly from that the Agency faces today.

5 ID2.001 05   Finally, as I have indicated, many other countries look to USEPA for guidance. Acceptance of a new method based on incomplete and inferior data does nothing to help such countries nor the credibility of USEPA. I would strongly urge you to look again at the data submitted and to investigate the other issues that I have raised before " approving" Colitag as an alternative test procedure.  It is not clear from this comment how the Colitag   data is   incomplete and inferior.  To the extent this comment has been elaborated upon elsewhere, EPA has responded to these assertions. See also the responses to Comment 2 ( Docket ID I D1.002, Part 15) and Comment 4 ( Docket ID I D1.005, Part 04) concerning the adequacy of the data and the extent of the Agency s evaluation.

6 I F. 004 08   Based on all the issues identified, we strongly object the approval of Colitag for use under the Total Coliform Rule as a presence/ absence liquid culture ( LC) method for detecting total coliforms and E. coli in drinking water and also in source water.

The Colitag evaluation did not follow the USATP protocol as required by the USEPA to evaluate a proposed method for the presence/ absence liquid culture ( LC) methods in microbiology.

Colitag should complete a full ATP Comparability Study,

prepare a well documented study report and resubmit the data to the USEPA for public comments and for further consideration of approval. The evaluation should strictly follow Concerning the comment that the Colitag   comparability study did not precisely follow the ATP protocol, EPA notes that it has historically applied a degree of flexibility in judging comparability study results relative to the protocol guidelines. EPA s policy concerning adherence to the ATP protocol is explained in the response to Comment 1 ( Docket ID I D1.001,

Part 06). Comment No. Docket ID Docket Part Comment Response Notes 7 all the requirements described in the " Protocol for Alternate Test Procedures for Coliform Bacteria in Compliance with Drinking Water Regulations   Presence/ Absence Liquid Culture Methods for Finished Water" ( Version 1.2 December 1995) and result in data addressing the issues raised in this report.

7 I F. 022 10   The test data provided indicate that the USEPA ATP Protocol was not followed, as noted above. USEPA should not approve Colitag until the appropriate ATP comparability studies and documentation are provided demonstrating that the method has been evaluated in accordance with USEPA ATP Protocol.

These studies must be reviewed by USEPA and also published for public comment prior to final approval of the method.

  In conclusion, it is very important for USEPA to ensure the scientific integrity of proposed methods, especially for total coliform and E. coli, by insisting and ensuring that the USEPA ATP Protocol is met. Data and documentation must be provided by proposers to demonstrate that the USEPA ATP Protocol is met.  See response to Comment 1 ( Docket ID I D1.001. Part 06) concerning adherence to the ATP protocol.

02 LENGTH OF COMMENT PERIOD/ RESPONSIVENESS TO COMMENTS 8 ID1.001 02   However, we point out, that out of a total of 8 distinct and very important issues submitted by various groups during the first round of comments, the USEPA chose to address only one   the log reduction. It is our hope that the other issues will be addressed by the USEPA in due course.

  Furthermore, no new data, but rather a recalculation of the old data was offered in the form of a power point presentation made by CPI during a meeting with the USEPA.

  Given the extremely limited and abbreviated   new information  at hand, we review the   old  data and   new information  and offer the following comments.  See response to Comment 2 ( Docket ID I D1.002, Part 15) concerning the manner in which the complete set of issues is being addressed.

The December 2002 NODA included supplemental data and a re evaluation of previously reported data. EPA notes that soliciting public comments on such via the NODA process was the appropriate course of action.

The   8 distinct  issues mentioned by commenter here are addressed elsewhere in this document.

9 ID1.002 03   Numerous deficiencies ( as determined under the ATP protocol) were identified in the original submission for the Colitag   product; these concerns were submitted during EPA s open solicitation for comments in April of 2002 and yet the EPA is responding to all comments received regarding the Colitag   test in this document. See also the responses to Comment 2 ( Docket ID I D1.002, Part 15);

Comment 3 ( Docket ID I D1.005, Part 02); and Comment No. Docket ID Docket Part Comment Response Notes 8 only response to reviewer concerns was to indicate that   EPA has received and is reviewing clarifying information concerning the evaluation of the Colitag   test. Thus, EPA is not taking final action on this method at this time. EPA will respond to all significant comments regarding this method in a future action.

To date, the response from EPA to reviewer comments on the Colitag   data has been limited to the recomputation of the log reductions presented in the original application and yet in the December 2, 2002 Federal Register EPA specifically states that reviewer    comments should be submitted only on the additional information presented in this notice and in Docket No. OW 2002 0031. EPA is not reconsidering any other drinking water issue in this notice nor will EPA respond to any comments on other issues. Comments should be limited to the additional information described herein and found in docket No. OW 2002 0031 and its applicability to the Agency s consideration of the Colitag   method.  In effect, EPA delayed providing a formal response to the initial review comments and is now restricting reviewer comment to only a small portion of the data submitted by the manufacturer. This lack of responsiveness to the original concerns brought up by the external reviewers is unacceptable; moreover, it entirely defeats the purpose of soliciting external review comments and trivializes the effort that reviewers invested in analyzing the data in the first place. If a key objective of the ATP is to promote the public health by encouraging the development of improved water testing methods, by all means EPA must provide a clear response to the concerns submitted by external reviewers and under no circumstances should restrict the review of data.

EPA s decision to revisit the original application for Colitag   is not justified adequately in the Federal Register notice of December 2, 2002, especially in light of the fact that EPA failed to address any of the reviewer concerns following the original application.  Comment 8 ( Docket ID I D1.001, Part 02) concerning the response to the comments on the March 2002 proposal and the purpose of the December 2002 NODA.

10 ID1.002 07   Moreover, EPA failed to address a number of additional departures from the Alternate Testing Protocol presented by external reviewers during the original March 2002 review of these data. These include concerns about missing data on See responses to Comment 1 ( Docket ID I D1.001, Part 06) concerning adherence to the protocol, and Comment 2 ( Docket ID I D1.002, Part 15) concerning QC data and the purpose of the December 2002 NODA. EPA Comment No. Docket ID Docket Part Comment Response Notes 9 product shelf life, questions regarding incubation temperatures,

concerns about the presentation ( or lack thereof) of supporting Quality Control data and inconsistencies in the reporting of sensitivity/ specificity data required under the ATP. To date EPA s primary response to these concerns has been to stipulate that the ATP guidelines are not legal requirements for manufacturers. As a consequence, it would appear that manufacturers are not compelled to conduct comparability testing under the carefully controlled conditions described in the EPA s own Alternate Testing Protocol. If this is the case, by all means EPA should disavow the utility of the ATP and should provide alternate, legally binding requirements to ensure that manufacturers develop and market products that are validated under realistic conditions for water testing. To do otherwise is to provide a disservice to the national public health and also to the manufacturers who in good faith attempt to follow the existing USEPA guidelines. Finally, failure to respond completely to reviewer comments is a disservice to the external reviewers themselves who will not be keen to share their time and/ or expertise in future evaluations of water testing data.  may consider imposing alternate legally binding requirements for analytical methods in the future.

Today s action, however, is limited to approval of a specific method.

11 ID1.005 03   Second, while the Federal Register notice presents a cursory summary of " recalculated" data, there are not enough specifics to make an informed analysis. Is this not the purpose of a public comment period? While I respect the statement that EPA expert( s) examined the data, it seems a mockery of the process to provide so little data and 30 days during a holiday period to make such an important decision.  See also the response to Comment 8 ( Docket ID ID1.001 Part 02) concerning the purpose/ scope of the December 2002 NODA. EPA believes that sufficient data were presented in the NODA and the associated public docket to conduct an informed analysis, and for EPA to make an informed decision.

EPA agreed with the request for a longer comment period and granted a 2 week extension to that period.

03 EPA'S ALTERNATE TEST PROCEDURES ( ATP) PROGRAM 03.01 Adherence to Specific ATP Guidelines 12 I F. 021 10 The following comments are drawn from review of the individual ATP worksheets submitted by the independent testing facility ( Biovir Labs) See also the responses to Comment 1 ( Docket ID ID1.001 Part 06) concerning use of the ATP protocol as guidance and Comment 3 ( Docket ID I D1.005, Part 02)

concerning a target chlorine stress level of 2 4 log Comment No. Docket ID Docket Part Comment Response Notes 10   Millbrae, CA # 1: Pretreatment of the seeded test water with chlorine (~ 1.0 mg/ L for 2 minutes) resulted in microbial reductions of 1.8 2.1 log10. The experiment failed to demonstrate the target reduction level of 3 4 log10 as required in the ATP protocol under section <IP_ADDRESS>. Test data for E. coli failed to meet the 25%: 75% criterion under the ATP protocol (> 15 for both total coliforms and E. coli). Should these test data be considered valid if they failed to demonstrate the degree of inactivation that might be considered an appropriate model of real world disinfection conditions? Should the E. coli data be considered valid? Only two dilutions were subjected to assay, however, the ATP protocol calls for a minimum of three dilutions to bracket the countable dilution. Should the selected dilution be considered valid?

  JEA Water Quality: Only two dilutions were subjected to assay, however, the ATP protocol calls for a minimum of three dilutions to bracket the countable dilution. Should the selected dilution be considered valid?

  Schaumberg, IL: The computed log10 reduction ( 2 log10) fails to meet the 3 4 log10 criterion. Should the data be considered valid? Only two dilutions were subjected to assay, however, the ATP protocol calls for a minimum of three dilutions to bracket the countable dilution. Should the selected dilution be considered valid?

  Watertown, WI: Only two dilutions were subjected to assay,

however, the ATP protocol calls for a minimum of three dilutions to bracket the countable dilution. Should the selected dilution be considered valid?

  Mission, KS # 1: The computed log10 reduction ( 1.2 log10)

fails to meet the 3 4 log10 criterion. Should the data be considered valid? The E. coli test data failed to meet the 25%: 75% criterion under the ATP protocol ( 3< 5). Should this data be considered valid? Only one dilution was subjected to reduction.

With respect to comments related to the degree of inactivation, EPA experts evaluated these data and concluded that the data were sufficient to judge the comparability between the Colitag   method and the reference methods. As stated in the response to Comment 3 ( Docket ID I D1.005, Part 02), EPA believes that a wider range for log reduction ( i. e., 2 to 4 logs) is acceptable in that it achieves an appropriate degree of chlorine stress. EPA notes that, per the data included in the December 2002 NODA, a log reduction between 2 and 4 was achieved in 10 of 10 total coliform studies and 9 of 10 E. coli studies.

To the extent that the commenter is basing its assertions on log reduction calculations performed in a manner other than that described in the December 2002 NODA,

see also the responses to Comment 24 ( Docket ID ID1.001 Part 03) and Comment 25 ( Docket ID I D1.002,

Part 04).

[ EPA notes that, consistent with the expression of the log reduction goal as one significant figure, it has evaluated the log reduction results based on one significant figure ( e. g., a 1.8 log reduction is viewed as meeting the 2 log reduction goal). EPA notes that 1.6 log represents a 97.5% reduction, whereas 2.0 log represents a 99% reduction. Considering the small difference in % reduction associated with rounding, and considering the inherent variability in bacterial density measurements ( likely impacting % reduction and log reduction precision to a greater degree than rounding),

the Agency is satisfied that this is a reasonable approach.]

In response to the comment suggesting deficiencies in the 25%: 75% split between positive and negative results, Comment No. Docket ID Docket Part Comment Response Notes 11 assay, however, the ATP protocol calls for a minimum of three dilutions to bracket the countable dilution. Should the selected dilution be considered valid?

  Ames, Iowa: The computed log10 reduction ( 1 log10) fails to meet the 3 4 log10 criterion due to inadequate seeding density ( 10 cfu/ 100 mL). Should the data be considered valid? The E.

coli test data failed to meet the 25%: 75% criterion under the ATP protocol ( 4< 5). Should the data be considered valid?

Only one dilution was subjected to assay, however, the ATP protocol calls for a minimum of three dilutions to bracket the countable dilution. Should the selected dilution be considered valid?

  Liberty, MD: The computed log10 reduction ( 0.7 log10) fails to meet the 3 4 log10 criterion. Should the data be considered valid? Only one dilution was subjected to assay, however, the ATP protocol calls for a minimum of three dilutions to bracket the countable dilution. Should the selected dilution be considered valid?

  Salem, OR: The computed log10 reduction ( 2.1 log10) fails to meet the 3 4 log10 criterion. Should the data be considered valid? The total coliform test data under the reference method failed to meet the 25%: 75% criterion under the ATP protocol ( 18> 15). Should the data be considered valid? Only one dilution was subjected to assay, however, the ATP protocol calls for a minimum of three dilutions to bracket the countable dilution. Should the selected dilution be considered valid?

  Mission, KS# 2: The computed log10 reduction ( 1.4 log10)

fails to meet the 3 4 log10 criterion. Should the data be considered valid? It appears that this sample was collected from the same source as an earlier Kansas submission ( KS# 1).

Should these two samples be considered geographically distinct?

Only one dilution was subjected to assay, however, the ATP protocol calls for a minimum of three dilutions to bracket the countable dilution. Should the selected dilution be considered and the three dilution recommendation used to achieve the split, EPA first notes that the underlying goal of this split is to confirm that very low numbers of bacteria are being introduced into the test media. Establishing conditions under which very low numbers of target bacteria are expected in the test sample ( i. e., the spiked drinking water sample used to innoculate the referencemethod and Colitag    method media) presents a greater challenge to the presence/ absence methods than would exist if high numbers of bacteria were present in the sample. This is intended to model conditions potentially found in distribution systems, where isolated contaminant bacteria could occur.

Under ideal ( i. e., the most challenging) test circumstances, half of 20 replicate cultures would yield a positive result and half would yield a negative result ( i. e.,

a   50: 50" split), representative of a situation where the lowest possible numbers of bacteria per culture were being inoculated. Recognizing the inherently imprecise nature of microbiological work, and recognizing that even the reference methods for total coliforms and E.

Coli are imperfect, EPA established a more flexible goal of a 25%: 75% split, such that when testing 20 replicate cultures, 5 15 of the replicates should yield positive results and 5 15 should yield negative results. Generally,

a 25%: 75% split will still be indicative of very low numbers of inoculated bacteria and should be far more practical for laboratories to achieve than a 50: 50 split.

The use of 3 serial dilutions per location is intended to increase the likelihood that one of the dilutions achieves a 25%: 75% split.

Significantly, the 25%: 75% split represents only one of two means described in EPA s ATP protocol to help ensure that the bacterial count is low. The other means by which to judge whether the bacterial count is low is to examine the post chlorination bacterial density Comment No. Docket ID Docket Part Comment Response Notes 12 valid?

  Millbrae, CA # 2: The computed log10 reduction ( 4.5 log10)

exceeds the 3 4 log10 criterion ( likely due to the excessive chlorine pretreatment of 4 mg/ L). Should the data be considered valid? It appears that this sample was collected from the same source as an earlier California submission ( CA# 1).

Should these two samples be considered geographically distinct?

The total coliform data under the reference method failed to meet the 25%: 75% criterion under the ATP protocol ( 17> 15).

Should these data be considered valid? The experimental data submitted include either one or two dilutions for each condition, however, the ATP protocol calls for a minimum of three dilutions in order to bracket the countable dilution.

Should the selected dilution be considered valid?  determination. Recalling that the goal ( per section 3.1.1 of the ATP protocol) is to achieve a post chlorination density of   1 10 organisms per 100 mL of sample,  EPA notes that all such measurements for total coliforms and E. Coli were, in fact, between 1 and 7, as reflected in Tables 1 and 2 from the December 2002 NODA.

Recognizing that a series of tests of both the reference methods and the Colitag   method were performed, for each of 10 test samples, for both total coliforms and for E. Coli, a total of 40 tests series ( 2x10x2) was performed.

Among those test series, 32 of 40 ( 80%) were within the 25: 75 goal. 1 EPA acknowledges that, in the following cases,

deviations from the 25: 75 goal occurred:

 17 positives for the reference method and 20 positives for the Colitag   method for total coliforms in sample 982305A  18 positives for the reference method for total coliforms in sample 990273A ( vs. 13 positives for the Colitag   method)

 16 positives for the reference method and 17 positives for the Colitag   method for E. Coli in sample 982084A  4 positives for the Colitag   method for E. Coli in sample 982305A ( vs. 6 positives for the reference method)

 3 positives for the reference method for E. Coli in sample 990217A ( vs. 5 positives for the Colitag   method)

 4 positives for the reference method for E. Coli in sample 990438A ( vs. 7 positives for the Colitag   method)

EPA considered the similarity between the above reference method and Colitag    method results.

Collectively, these results are quite similar. Even on an 1. The ATP protocol ( at section 4.6.5)

seeks comparison of test results wherein an acceptable split in positive and negative results for the reference method is achieved.

For the purpose of responding to comments on this issue, however, EPA has assessed results in which the 25%: 75%

split was not achieved in either the reference method or Colitag   . Comment No. Docket ID Docket Part Comment Response Notes 13 individual basis, nearly all of these dilutions yielded results that are quite close when comparing the performance of Colitag   and the reference methods.

EPA also considered the proximity between these results and the 25: 75 goal articulated in the ATP protocol.

Again, these figures are very similar; in only one instance do the results deviate from the 25: 75 goal ( i. e., 5: 15 to 15: 5 individual replicate results) by more than 3 individual replicate results.

Lastly, EPA considered the impact of the above deviations on the statistical analysis and concluded that such individual deviations did not impact EPA s ability to perform its comprehensive analysis, since the   chi squared  statistical test, used to determine method comparability, relied on a collective data set from all samples. [ Since individual test result deviations from the 25: 75 goal were few ( and small) and since the chisquared probability and Fisher s exact two sided probability ( both key indicators of comparability between Colitag   and the reference methods) were well above the minimum threshold of 0.05, this approach ( analysis of the data as a collective set) is statistically valid and EPA has confidence in the conclusion of the statistical analysis In a method evaluation where the individual test results deviate substantially from the 25: 75 goal, and where the computed statistical probability is much closer to 0.05, EPA would not have that same confidence The statistical results are discussed in greater detail in the response to Comment 24 ( Docket ID I D1.001, Part 03).]

Moreover, EPA reminds the commenter that the 25: 75 goal represents only one of two means by which to judge whether testing has taken place using a very low bacterial count. Referring to the other means by which to judge such count ( i. e., the post chlorination density determination), EPA notes that such measurement met Comment No. Docket ID Docket Part Comment Response Notes 14 EPA s goal of 1 10 organisms/ 100mL in 100% of the cases. Accordingly, EPA has concluded that the all of the above constitute valid results, with respect to demonstrating that a low bacterial count was employed in the studies.

Concerning the comment regarding the number of dilutions performed, EPA notes that where fewer than 3 dilutions were performed, but where at least one of those dilutions yielded reasonable splits between positive and negative results, additional dilutions would serve no useful purpose.

In response to the comment on the number of geographical locations ( in particular, Mission, KS and Millbrae, CA) used in the Colitag   comparability study,

EPA acknowledges that the protocol recommends that comparability data be generated   from a single finished water which has been spiked with different ( e. g., 10)

wastewaters or polluted surface waters containing the target organisms.  The goal underlying this provision is to use   biodiverse  spiked samples ( i. e., those with a range of coliform strains) to test the candidate method with a range of bacterial metabolic variation, and EPA s protocol recommends that the wastewater/ surface water come from 10 geographically dispersed sites.

EPA reviewed and supported CPI s plan to compare the Colitag   method to the reference methods based on 10 samples from 8 different locations. It is EPA s judgment that this particular sample set meets the goal of achieving substantial biodiversity and represents a sufficient degree of diversity to provide the Agency with confidence that the Colitag   method performs adequately over a wide range of bacterial strains. Comment No. Docket ID Docket Part Comment Response Notes 15 It is helpful to understand that substantial biodiversity generally exists even within a single wastewater sample,

and between multiple wastewater samples from a single location. Although it may be more obvious that spacial variation ( i. e., sampling over multiple locations) achieves an additional degree of diversity ( i. e., additional coliform strains), additional diversity can also reasonably be expected with temporal variation ( i. e., sampling over a period of time). Regarding the latter point, in both instances in which samples were taken from the same location, these samples were collected at least one month apart. The two samples from Millbrae, CA ( 98204A and 982305A) were collected on 11/ 10/ 98 and 12/ 14/ 98 and the two samples from Mission, KS ( 990217 and 990442A) were collected on 3/ 3/ 99 and 4/ 26/ 99.

Recognizing that significant biodiversity exists even with a single sample; recognizing that substantial spacial variation achieved further diversity for the 8 geographically dispersed Colitag   samples; and recognizing that substantial temporal variation achieved further diversity for the remaining 2 samples, EPA concluded that all 10 of the samples used in the comparability studies of Colitag   constitute valid samples, having met the goal of achieving substantial biodiversity.

Concerning the comment that the chlorine stress log reduction used with Millbrae, CA # 2 exceeded 4 logs of reduction, EPA notes that the recalculated log reduction for this sample was less than 4 in the data presented in the December 2002 NODA ( i. e., the data upon which EPA is basing its decision). Specifically, the log reduction for this sample was recalculated as 3.4.

03.01.01 Injury level guidelines ( 3 4 log goal; recovery rates; alleged math errors; applicability to natural samples)

13 ID1.001 04 4.   As shown in Tables II and III, per IDEXX calculation, only 10% of samples met the ATP guidelines for Log10 reduction See responses to Comment 3 ( Docket ID I D1.005, Part 02) and Comment 12 ( Docket ID I F. 021, Part 10) ` Comment No. Docket ID Docket Part Comment Response Notes 16 for both total coliform (# 990095A) and for E. coli (#

990025A). Please note, that since sample # 982305A 1 was not used for comparability statistics purposes, it cannot be used toward meeting the log reduction requirement either.  regarding the log reduction results.

Moreover, commenter appears to have made several assumptions in calculating ( or asserting an inability to calculate) individual log reductions. Some of these assumptions are either incorrect or depend upon assertions with which EPA disagrees. For example,

EPA did not use prechlorination density data ( i. e.,

measurements after the initial density determination and immediately preceding chlorination) to calculate the log reduction. Instead, as explained in EPA s December 2,

2002 NODA, the log reduction was calculated using the initial density determination divided by the sample dilution factor. Although the prechlorination density data was submitted by CPI for some samples, it is not necessary ( and was not used by EPA) to calculate the log reductions.

For the reasons discussed in response to Comment 24 ( Docket ID I D1.001, Part 03) and Attachment 1, EPA believes that the hold times are such that they support the procedure outlined in the NODA.

Responses concerning specific data presented in the commenter s attachments follow:

Total Coliforms ( Table II):.

Sample 982084A   The commenter has stated that this sample lacks pre chlorination density data. As discussed above, the fact that prechlorination density data was not available for this sample does not invalidate the sample;

such data were not used in EPA s analysis.

Sample 982305A 1   The commenter suggests that an inconsistent data set was used for the log reduction calculation and the statistical analysis. As described further in the response to Comment 24 ( Docket ID ID1.001 Part 03), EPA has repeated its statistical analysis Comment No. Docket ID Docket Part Comment Response Notes 17 using the data from the 1: 1000 dilution experiments ( sample 982305A 1, 12/ 23/ 98). As with the original analysis, the repeat analysis did not identify a statistically significant difference between the performance of Colitag   and the reference methods.

Sample 982305A 2  The commenter has stated that the data sheets for this sample are missing the postchlorination density. Since, as described in the response to Comment 24 ( Docket ID I D1.001, Part 03), EPA has repeated its statistical analysis excluding the results from sample 982305A 2 ( study results dated 12/ 29/ 98) and substituting the results from sample 982305A 1, and since comparability between Colitag   and the reference methods continued to be demonstrated, the missing post chlorination data for sample 982305A 2 has no impact on EPA s decision.

Sample 990025A   The commenter has suggested that the ATP worksheets refer to dilutions of 1: 500 and 1: 2000 for the replicate tests with total coliforms, yet the comparability study data used in EPA s statistical analysis appear to be associated with a 1: 1000 dilution.

EPA notes that it originally used data from the first ( 1: 500) dilution in calculating total coliform log reduction. Although the comparability data sheet indicates that the second set of replicates were diluted at a 1: 1000 ratio, EPA believes that this is likely a typographical error. This is because all of the previous references to dilutions were either 1: 500 or 1: 2000, as reflected in the ATP worksheet. In the absence of a clear explanation for this discrepancy, EPA believes that a typographical error is likely and that the dilution figure for the second set of comparability studies should be 1: 2000. Since EPA used the data associated with the second dilution ( originally stated as 1: 1000, but presumably 1: 2000) in its original statistical analysis,

EPA first recalculated log reduction, using that data set, Comment No. Docket ID Docket Part Comment Response Notes 18 for consistency. ( EPA notes that using a dilution factor of 1: 2000 in the log reduction calculation instead of 1: 1000 represents a more conservative approach; it yields a lower log reduction than would be obtained using the 1: 1000 factor). As described in the response to Comment 24 ( Docket ID I D1.001, Part 03), that recalculation produced a log reduction of 3.0, versus the original log reduction of 3.2; thus, relying on the figures used in the original statistical analysis and substituting a revised log reduction ( 3.0) has no impact on EPA s decision. In the alternative, the comparability study analysis does not suffer if the Agency relies instead upon the comparability data from the set of replicates diluted at 1: 500 ( i. e., the data set associated with the originally reported log reduction of 3.2). Although the reference method generated slightly more positive results ( 16 out of 20) than ideal to meet the 25%: 75% goal ( 5  15 out of 20), this is not vital to the overall analysis. See response to Comment 12 ( Docket ID I F. 021, Part 10).

Indeed, using the data for the 1: 500 dilution resulted in no impact on the   chi squared  probability figures that indicate comparability and, therefore, no impact on the conclusion of the analysis. Finally, as discussed in the response to Comment 24 ( Docket ID I D1.001, Part 03),

EPA lacks information that would conclusively demonstrate appropriate hold times for four samples;

sample 990025A is one of them. Even if this sample were discarded entirely, the outcome of the overall comparability analysis ( see response to Comment 24)

would not change.

Sample 990052A   Commenter appears to be using the prechlorination density data to calculate the log reduction. As noted above, this is not the procedure followed by EPA. The comment in Table II suggests that commenter believes that the log reduction should be based on the dilution factor 1: 2,500. Using this dilution factor, and the associated post chlorination enumeration Comment No. Docket ID Docket Part Comment Response Notes 19 data, the recalculated log reduction remains at 2.3 ( log (( 1,000,000/ 2,500)/( 1x2)) = 2.3) EPA notes that even if the commenter had appropriately calculated the log reduction as 1.7, this would appropriately fit the 2 4 log reduction range as discussed in response to Comment 3 ( Docket ID I D1.005, Part 02).

Sample 990438A   EPA has performed additional and repeat statistical analyses in response to public comments and has ensured that the correct data are being used. See also the response to Comment 33 ( Docket ID I D1.002, Part 05).

E. coli ( Table III):

Sample 982084A   The commenter has stated that this sample lacks pre chlorination density data. As discussed above, the fact that prechlorination density data was not available for this sample does not invalidate the sample;

such data were not used in EPA s analysis. See also EPA s response to Comment 25 ( Docket ID I D1.002,

Part 04).

Sample 982305A 1   Commenter incorrectly suggests that the post chlorination count for mFC is absent. This count appears in section 5 on page 2 of the ATP worksheet for this sample.

( EPA notes that it is no longer relying on the results from Sample 982305A 2. Thus, the missing data sheet the commenter refers to has no impact on EPA s decision.)

Sample 990095A   As reflected in the December 2, 2002 NODA, the E. coli log reduction for this sample cannot be determined using the standard calculation because the original density measurement is not available. This does not, however, preclude the use of the comparability Comment No. Docket ID Docket Part Comment Response Notes 20 5.   Even with the recalculation per the tables in the FR of 12/ 02/ 2002, only 50% of the total coliform data and 80% of study results from the sample in EPA s analysis; the key consideration is whether those results were generated using E. coli that had been subjected to adequate chlorine stressing. Based on the following facts, and absent information to the contrary, EPA believes that it is reasonable to consider the comparability study results for this sample in its statistical analysis ( i. e., EPA can reasonably conclude that the E. coli used in the comparability study were subjected to adequate chlorine stressing):

 100% ( 19 of the 19) of the samples for which total coliform and E. coli log reduction could be calculated yielded acceptable results ( i. e., log reductions between 2

4).

 Among the 10 total coliform samples used in the studies, the subject sample had one of the highest levels of total coliforms ( at 16,000,000 CFU/ 100mL). One can reasonably postulate that the source water had high E. coli levels as well ( among the other 9 source waters, E.

coli was measured at an average of 42% of the total coliform measurements).

 If the pre chlorination sample contained even moderate E. coli levels, and since it is clear that the postchlorination E. coli level was low ( 7 CFU/ 100mL), one can be confident that a substantial degree of chlorine stressing occurred. The fact that a consistent chlorination procedure was used in the studies lends further credence to this predictive approach.

 The total coliform log reduction for this sample, at 2.8,

was among the higher results.

All of these facts suggest that the E. coli in this sample were subjected to substantial chlorine stressing; EPA is not aware of any facts that contradict this position.

Referring to the discussion of EPA s log reduction goal in the response to Comment 3 ( Docket ID I D1.005, Comment No. Docket ID Docket Part Comment Response Notes 21 the E. coli data satisfies US EPA s chosen interpretation of the 3 4 log10 reduction requirement, i. e. rounding up from 2.5 onwards. Plus, as stated earlier, the data selected for comparability statistics was not consistent with the dilutions used for the Log10 reduction.

6.   Data using ATCC strain 25922 is irrelevant since the strain does not allow the method to detect the diversity of total coliforms and E. coli that is present in the natural environment.

Furthermore, the strain was used without the presence of any background flora and background sample matrix. Data generated from this type of experiment does not prove that the method will perform adequately with natural samples and therefore, if approved, may compromise public health. The US EPA s own ATP however, if followed, was designed to address exactly the critical natural sample aspects cited above.  Part 02), EPA notes that such goal was met in 100% of the cases for which log reduction results were available ( 10 of 10 total coliform results and 9 of 9 E. coli results).

As discussed above in the responses to sample specific comments [ and as discussed further in the response to Comment 24 ( Docket ID I D1.001, Part 03)], EPA has repeated its statistical analysis using comparability data and log reductions associated with the same dilutions. As with the original analysis, the repeat analysis did not identify a statistically significant difference between the performance of Colitag   and the reference methods.

The data presented where ATCC 25922 was used represent additional unsolicited information presented by CPI together with re calculated log reduction data:

the former information was not used in EPA s evaluation of the Colitag   method.

14 ID1.004 02   The data provided still is inadequate to determine if the Colitag product can in fact recover chlorine stressed organisms. The ability of a test to recover chlorine stressed organisms is the most important aspect of the EPA required alternative test protocol. The test principle is straight forward. A complex wastewater sample with at least 100,000 of the target organism / 100mL ( E. coli or total coliforms) is exposed to a dose of chlorine for 10 to 30 minutes which results in a 3 to 4 log reduction. This disinfection stressing protocol is intended to mimic the stress experienced by a naturally occurring organism introduced from a sanitary sewer cross connection into a chlorinated public water supply. The procedure for preparing stressed organism samples for use in the ATP is clearly outlined by EPA and is based on published literature.( Evaluating a Commercially Available Defined Substrate Test for Recovery of E. coli. S. C. McCarty, J. H. Standridge and M. C. Stasiak. See responses to Comment 3 ( Docket ID I D1.005, Part 02) and Comment 12 ( Docket ID I F. 021, Part 10)

concerning recovery of chlorine stressed bacteria.

Details of how the log reduction was calculated were included in the public docket and relevant information on recalculation of log reductions was published in the December 2002 NODA.

EPA is unclear as to why this commenter purports to lack information on how stressing was conducted. This information was included in CPI s study plan, which was included in the docket at proposal. Moreover, stressing procedures were described in Biovir s log sheets which are also in the docket. Similarly, EPA does not understand why this commenter purports to lack information on the log reductions. The methodology, Comment No. Docket ID Docket Part Comment Response Notes 22 JAWWA: 84: 5. May 1992. Pp. 91 97) The tables presented in the Dec. 2nd additional information still do not make it clear how the stressing procedures were done and exactly what the log reduction was. The before and after disinfection concentrations as measured by an accepted standard method must demonstrate at least a 3 log reduction and result in a detectable number of organisms. The result of subtracting the base 10 log of the post disinfection from the base 10 log before chlorination must be greater than 3. If this number is not greater than three, the stressing of the organisms is insufficient. The numbers presented in the Dec. 2nd tables do not demonstrate a sufficient log reduction, and therefore the ATP has not been followed and we have no data that tells us whether or not the product is capable of detecting stressed organisms.

  I strongly disagree that the methods and data presented are consistent with previous product approvals. Approval must be denied until further evaluation can be completed.  the individual figures used in the calculations and the results were published in the Federal Register itself, and the worksheets from which the individual figures were derived have been in the docket since approval of this method was proposed. Other commenters do not seem to have had the difficulty expressed by this commenter.

EPA disagrees that it lacks data indicating that Colitag   is capable of detecting stressed organisms. As discussed in response to Comment 3 ( Docket ID I D1.005, Part 02), EPA believes that this method has successfully demonstrated its ability to recover organisms stressed at levels resulting in 2 4 log reductions   levels which the Agency believes reflects a high degree of stress.

With respect to EPA consistency in evaluating methods,

EPA notes that there is a history of using expert judgement to supplement the ATP protocol guidance; a review of past method evaluations confirms that the log reduction guideline and other guidelines have not been applied as rigid requirements. The commenter has not specified any decision EPA has made in the past that is inconsistent with today s decision.

15 I F. 004 02 1. Colitag's ATP Comparability Study did not meet the Injury Level Requirements   As stated in the USEPA ATP Section <IP_ADDRESS> and 4.5.5, the chlorination must reduce the 105 CFU/ 100 ml in the spiked water samples by 3  4 Log10 ( i. e. 99.9 99.99%) in order to assure the presence of stressed organisms. This is the most important criterion for the USEPA ATP protocol for the evaluation of a proposed method to detect total coliforms and E. coli in drinking water as this is directly relevant to public health since the potability of drinking water is assessed by the presence or absence of total coliforms and E. coli in the water sample.

  The comparability data submitted by CPI International for the The commenter states that the comparability study data presented by CPI for Colitag   does not meet the 3 to 4 log reduction criterion for demonstrating that Colitag   is able to recover chlorine stressed organisms. See responses to Comment 3 ( Docket ID I D1.005, Part 02)

and Comment 12 ( Docket ID I F. 021, Part 10)

concerning log reduction and recovery of chlorinestressed bacteria, and the acceptability of a wider range of log reduction.

EPA disagrees that   there was no documentation on the injury levels for . . . sample # 982305A.  The log reduction for sample 982305A and the other samples was computed based on the original density determination and the post chlorination cell counts, as Comment No. Docket ID Docket Part Comment Response Notes 23 approval consideration under the Safe Drinking Water Act did not meet this critical requirement ( see Appendix) Strikingly,

none of the data for E. coli met this most critical requirement ( the reduction levels only ranged from 1 2 Log10). With the exception of two samples [ I. D. #: 990025A & 990095A] where a 3.3 Log10 reduction of target coliforms was achieved, 8/ 10 samples did not achieve a 3  4 Log10 reduction for total coliforms ( ranged from 0.7 to 2.1 Log10). In addition, there was no documentation on the injury levels for either coliforms or E. coli for sample # 982305A. The average Log10 reduction in cell number ( excluding one unreported data by Colitag) for total coliforms was 1.89 and for E. coli was 1.51. Clearly,

Colitag did not meet such requirements in this study to evaluate its ability in detecting stressed bacteria.  described in the December 2, 2002 NODA. The data used for these calculations is included in the ATP worksheets, which was in the docket.

Using the revised data, the average log reduction for total coliforms is 2.5 and the average log reduction for E. coli is 2.9. EPA surmises that commenter has either made errors in calculating the log reductions or has employed a method other than the one described by the Agency in the December 2, 2002 NODA.

16 I F. 006 02 o Detection of Escherichia coli ( E. coli) [ both Colitag   and ReadyCult]

  To protect the public's health, it is essential that a procedure accurately detect both native and chlorine stressed E. coli. E.

coli is the indicator of fecal pollution, upon which rests each of these test's protection of the public health. In fact the detection of E. coli was considered so important for the protection of public health that, when a question arose regarding the accuracy of the MMO MUG test to detect stressed E. coli,

approval of the method was put in abeyance pending resolution.

Additional studies needed to be conducted by third parties to resolve this issue ( see references 1 and 2 below. To highlight the seriousness of this concern, reference number 2 is from the USEPA's own laboratory.). Neither Colitag   nor ReadyCult followed the procedure and sample requirements to demonstrate recovery of oxidant stressed E. coli. The Colitag   product did not meet this basic requirement in 80% of its samples and the ReadyCult product in 60% of its samples. This deficit is particularly disturbing since the injury protocol is clearly delineated by USEPA and has been successfully used, and described, in two peer reviewed papers ( 1,2). Accordingly, this glaring deficit must be remedied and the data made available for public comment prior to approval.  Concerning questions about accurately detecting native and chlorine stressed E. coli, whereas such questions existed in the case of MMO MUG and warranted additional analysis, EPA is satisfied that its analysis of the Colitag   data demonstrated comparability with the reference methods and did not feel that more testing was needed for evaluation of the performance of Colitag   .

This is further explained in the responses to Comment 3 ( Docket ID I D1.005, Part 02) and Comment 12 ( Docket ID I F. 021, Part 10), concerning recovery of chlorine stressed bacteria, and Comment 4 ( Docket ID ID1.005 Part 04) concerning the approach to the MMOMUG review. References:

1. McCarty, S. C.,

Standridge, J. H., and Stasiak, M. C. 1992.

Evaluating a commercially available definedsubstrate test for recovery of E. coli. J.

American Water Works Association 84( 5): 91 97.

2. Covert, T. C., Rice,

E. W., Johnson, S. A.,

Berman, D., Johnson,

C. H., and Mason, P. J.

1992. Comparing defined substrate coliform tests for the detection of Escherichia coli in water. J. American Water Works Comment No. Docket ID Docket Part Comment Response Notes 24 Association 84( 5): 98

104.

17 I F. 015 01   I also note that another product, Colitag   is also being considered for regulatory approval. As with the ReadyCult study, there are some deviations from the ATP. In particular, in the majority of samples used, the 3 4 log reduction by disinfection was not adhered to. As stated above, this is a most important criterion for deciding on the suitability of a method for assessing the quality of drinking water. It is clear therefore that the Colitag   product should not be approved for assessing the quality of drinking water in the United States.  See responses to Comment 3 ( Docket ID I D1.005, Part 02) and Comment 12 ( Docket ID I F. 021, Part 10)

concerning recovery of chlorine stressed bacteria.

18 I F. 021 03 ( 2)   During comparability testing, it is critical to establish that chlorine injured microorganisms can be recovered using the proposed method( s) in order to model ' real world' scenarios,

with specific reference to drinking water distribution systems where chlorination is practiced. The ATP protocol requires establishment of 3 4 log10 reductions of test microorganisms following chlorine pretreatment, however, in all but two of the tests conducted, the data failed to meet this criterion ( in 7 experiments the log10 reductions were significantly below this target level, and in one experiment the log10 reduction exceeded the target level of 3 4 log10). If only 20% of the submitted data met the criterion defined under the ATP protocol, it is inappropriate to consider the data set representative of the conditions that might be found in field samples.  See responses to Comment 3 ( Docket ID I D1.005, Part 02) and Comment 12 ( Docket ID I F. 021, Part 10)

concerning recovery of chlorine stressed bacteria stressed to less than 3 to 4 logs of reduction.

The commenter also suggests that the log reduction of total coliforms in one sample ( 982305A, Millbrae, CA)

was 4.5 in the proposal of Colitag   , exceeding the 3 4 log goal. EPA notes, however, that this number was recalculated in the December 2002 NODA, and the recalculated number is 3.4.

19 I F. 022 03 1 .  The injury requirements were not met by Colitag   ' s ATP comparability study. Eight out of 10 samples did not achieve a 3 to 4 log reduction for total coliforms, and none of the data for E. coli met the 3 to 4 log reduction requirement.  See responses to Comment 3 ( Docket ID I D1.005, Part 02) and Comment 12 ( Docket ID I F. 021, Part 10)

concerning recovery of chlorine stressed bacteria. Comment No. Docket ID Docket Part Comment Response Notes 25 03.01.02 Sample source guidelines ( spiked samples;

10 geographically dispersed sites)

20 ID1.002 12   EPA has revised the log reduction following chlorine pretreatment for sample number 990442A. It should be noted that this sample was drawn from the same source as sample number 990217A ( Mission, KS), in violation of ATP section 4.1.1. which calls for samples from   ten or more geographically dispersed sites . The concern regarding two samples being taken from Mission, Kansas is addressed in the response to Comment 12 ( Docket ID I F. 021, Part 10). As explained, EPA concluded that an acceptable degree of bacterial diversity was achieved in this particular case using the 10 samples from 8 sites.

21 I F. 004 03 2.   Colitag   ' s ATP Comparability Study did not meet Sample Sources Requirement

  As stated in the USEPA ATP protocol section 4.1.1, the recommended spiked samples should be non chlorinated secondary sewage effluents or polluted surface waters. And these samples must come from at least 10 or more geographically dispersed sites.

  It appears that there were 2 sets of sewage effluent samples that came from identical sources ( see Appendix). Samples ( I. D # 990442A & 990217A) were from Johnson County Environmental Laboratory [ 4800 Nall, Mission, KS] and samples ( I. D.# B982305A & 982084A) were collected from City of Millbrae WWTP, CA]. This means that samples used in Colitag   ' s ATP study were ONLY collected from 8 different geographically dispersed sites and the requirements for sample sources laid out in ATP protocol section 4.1.1 were not met.  See response to Comment 12 ( Docket ID I F. 021, Part 10) concerning geographic distribution of samples.

22 I F. 021 02 ( 1)   A major requirement for comparability testing under the USEPA Protocol for Alternate Test Procedures for Coliform Bacteria in Compliance with Drinking Water Regulations is to validate proposed methods using water samples collected from geographically diverse regions of the U. S. Although it is not entirely clear from the ATP worksheets submitted by the independent laboratory precisely where each sample was collected, it appears that at least two pairs of samples were collected from the same source ( Mission, Kansas and Millbrae,

California), with the end result of a data set containing only 8 geographically diverse samples. Hence, the submitted data fails to comply with the criteria established in the ATP protocol.  See response to Comment 12 ( Docket ID I F. 021, Part 10) concerning geographic distribution of samples. Comment No. Docket ID Docket Part Comment Response Notes 26 23 I F. 022 04 2.   Two sewage effluent samples were repeat samples. Samples 990442A and 990217A were both from Johnson County Environmental Laboratory, Mission, KS. Samples 982305A and 982084A were both from the City of Millbare, CA. Hence, only 8 sites were actually sampled. The USEPA ATP Protocol calls for at least 10 or more geographically dispersed sites to be sampled.  See response to Comment 12 ( Docket ID I F. 021, Part 10) concerning geographic distribution of samples.

03.01.03 Replicate and data selection guidelines ( ability to detect 1 10 TC or achieve 25%/ 75% split; replicate analysis on 3 dilutions; lag time/ inferred bacteria densities)

24 ID1.001 03 1.   Recalculation of Log10 reduction by the US EPA based on the dilution factor and a measurement of the source water prior to dilution, as described in the December 2, 2002 Federal Register, is inappropriate. Through detailed review of Colitag     s ATP worksheets, we found that in a number of samples there was a significant lag time for bacterial density determination between the source sample and the diluted sample prior to chlorination ( in 6 instances per Table I,

boldened for emphasis). Bacterial density and microflora in the source samples may significantly be altered during this lag time.

In addition, contrary to standard microbiological laboratory practices, chlorination injury experiments were performed from the diluted source samples rather than from the original source samples. Therefore, the   Log10 Reduction  calculation for the pre chlorination bacterial density should be based on the membrane filter analysis of the diluted source water samples,

which was originally reported by BioVir Laboratories. Also per Table 1. ( boldened for emphasis), in one instance there is a time lag between dilution and chlorination, plus in four instances,

there is a time reversal between the two ( indicated by a question mark).  With respect to the comments concerning   hold time

between the initial density determination and the postchlorination measurement used to calculate log reduction, EPA notes that it has not established guidelines for such in its ATP protocol. The Agency has not asked that such be documented, nor has it applied a standard for such in previous method evaluations.

Further, a review of previously evaluated methods reveals that the public record for some ( e. g., Readycult,

Colisure) does not include sufficient data to determine hold time.

EPA s presumption, for this and for previous method evaluations, is that the certified drinking water laboratories performing the comparability studies will employ a reasonable hold time. Nonetheless, EPA conducted a thorough review of the Colitag   hold times, pursuant to these comments. In doing so, EPA considering dates identified for sample collection;

sample receipt at the laboratory; original density determination; and comparability study completion, as reflected in the worksheets, comparability study data sheets, and chain of custody documentation in the record. EPA further considered the chronology and duration of the steps associated with the various tests performed. Based on all of this information, EPA concluded that a reasonable hold time could be documented for the majority of the tests, but that clear hold times could not be determined for four of the tests Comment No. Docket ID Docket Part Comment Response Notes 27 2.   The dilutions used for the comparability data statistics do not match the dilutions used for the Log10 reduction calculation as indicated in Table II for total coliforms and Table III for E. coli. For total coliforms this is the case in four instances and for E. coli in five instances ( boldened for emphasis in the respective tables).  ( samples 990025A, 990052A, 990217A, and 990273A).

See Attachment 1   Description of CPI Comparability Study Log Sheet Hold Times for Colitag     for information on how the hold times were calculated.

EPA s inability to conclude with certainty that the hold time for those four tests was reasonable is not tantamount to concluding that the hold times were unreasonably long. Rather, it is possible that a reasonable hold time was achieved for each of the aforementioned tests. As noted above, these samples were evaluated by a certified drinking water laboratory and EPA presumes a reasonable hold time for all samples.

Under a worst case scenario, however, EPA repeated its statistical analysis of the Colitag   data set, excluding the aforementioned four tests. The conclusion ( i. e., that the comparability study did not identify a statistically significant difference in performance between the reference methods and Colitag   ) did not change, nor was the strength of the conclusion substantially different with the limited ( 6 test) data set as compared to the full ( 10 test) data set. ( See Attachment 2,   Statistical Impact of Relying on a 6 Test Data Set Versus a 10 Test Set

for additional discussion.)

Accordingly, EPA stands by its decision to use the log reduction data presented in the December 2002 NODA.

In response to the comment that the dilutions used for the comparability data statistics do not match the dilutions used for the log reduction calculations, the commenter has not indicated the relevance of this comment. EPA surmises that the commenter intended to suggest that the log reductions presented in the NODA should be corrected to match the dilutions that were used for the comparability studies or the data set used in Comment No. Docket ID Docket Part Comment Response Notes 28 3.   Based on the observations above, IDEXX recalculated the Log10 reduction using the bacterial density of the actual diluted source sample which was used to injure the bacteria, rather than the statistical analysis should be modified. To the extent that such is the case, EPA agrees and has taken steps to ensure that the log reduction calculations and statistical analysis rely on the same data set ( i. e, data associated with the same dilution).

Specifically, the data sets have been reviewed and the statistical analysis has been repeated by EPA using logreduction and comparability study data associated with the same dilutions. Under such approach, total coliform log reductions remain as presented in the NODA, with the following exceptions: the log reduction associated with the 1: 10,000 dilution for sample 982084A would change from 2.1 to 1.8, and the log reduction associated with the 1: 2000 dilution for sample 990025A would change from 3.2 to 3.0. Under such approach, E. coli log reductions remain as presented in the NODA, with one exception: the log reduction associated with the 1: 200 dilution for sample 990438A would change from 2.7 to 3.4. None of these changes indicate that the log reductions for the samples analyzed were potentially improper and none of these changes had an effect on EPA s conclusion.

In the case of total coliforms for sample 982305A,

sufficient data were not available to recalculate log reduction for the data set associated with the 1: 2000 dilution used in the 12/ 29/ 98 comparability study ( the comparability study results originally used in EPA s statistical analysis); instead, therefore, the statistical analysis was repeated using the data set associated with the 1: 1000 dilution used in the 12/ 23/ 98 comparability study ( i. e, the same data set used to calculate the 3.4 log reduction.) The conclusion with respect to reference method/ Colitag   method comparability did not change. Comment No. Docket ID Docket Part Comment Response Notes 29 extrapolating from the original source sample. In addition,

IDEXX used the bacterial density from the same dilution that was used for the comparability statistics.  The commenter s recalculated log reduction values are not relevant to today s action because these calculations were done differently than those used for the log reduction determinations presented in the Dec. 2, 2002 NODA and used as the basis for EPA s evaluation of method comparability. ( As described in the NODA, the approach used by EPA to calculate log reduction is consistent with previous method evaluations. It is also consistent with EPA s ATP protocol.). Also, as described above, EPA analyzed the data using the same dilutions that were used in the comparability study samples, and found that these did not change EPA s conclusions about the comparability study results.

25 ID1.002 04   The ATP protocol devised by USEPA requires the testing laboratory to collect wastewater isolates from geographically diverse regions of the country and to conduct   chlorine injury

experiments to model pathogen intrusion events that may occur in distribution systems. For these experiments, the independent testing laboratory performed a series of enumerative assays on the wastewater isolates. The initial   range finding  assay was performed to identify the concentration of total coliforms and E. coli in each wastewater so that the appropriate dilution could be identified that would yield 1 10 CFU/ 100mL following chlorine treatment. The second assay identified the densities of microorganisms in the actual dilution used for the chlorine injury experiments. The final assay was performed to enumerate test microorganisms following chlorine injury so that microbial reductions could be computed. Chlorine treated samples were then subjected to comparability studies using both the proposed and reference methods. For some samples, these assays were performed over a span of a few days. For others, the time span between the initial characterization of the microbial densities and the densities following chlorine treatment was a month or more. In its December 2 revision of the comparability data,

EPA relied exclusively upon the data from the initial rangefinding assay to identify the   pre chlorination  concentration of test microorganisms rather than using the data reported by the testing laboratory on the actual, diluted samples used in the In response to the commenter s suggestion that the ATP protocol   requires  particular practices, see response to Comment 1 ( Docket ID I D1.001, Part 06).

In response to commenter s suggestion that the time lag between the initial bacterial density determination and the dilutions of samples preceding chlorination were as long as 6 weeks, see response to Comment 24 ( Docket ID I D1.001, Part 03) concerning   hold time  between initial density determination and post chlorination measurement, and also Attachment 1 for specific information about how hold times were determined.

The approach of performing Membrane Filtration analyses on the spike source to determine original density of target microorganisms and using this information to determine the necessary dilution with drinking water to achieve the target density of microorganisms is the procedure described in sections <IP_ADDRESS> and <IP_ADDRESS> of the ATP protocol. Moreover, this procedure is consistent with previous method evaluations by EPA.

EPA experts included the procedure of determining bacterial density followed by serial dilutions in the ATP Comment No. Docket ID Docket Part Comment Response Notes 30 disinfection tests. EPA concluded in the December 2, 2002 Federal Register notice that this approach was    sound and provides consistency with previous method evaluations by EPA . In fact, this approach is inappropriate in that it assumes that the microbial densities in the working dilutions used for the chlorine exposures can simply be extrapolated from the results of the initial assay after accounting for the dilution factor ( e. g.

that a one thousand fold dilution of a solution of 105 bacterial cells will result in a solution containing 102 cells). This assumption fails to account for inaccuracies that may result from dilution errors and more importantly it completely fails to account for the fact that the initial densities of test microorganisms were in numerous instances measured several weeks earlier than the actual disinfection experiment. Hence,

microbial reductions were computed using numbers from assays that were spaced up to six weeks apart. This approach also assumes that the concentration of test microorganisms in wastewater is stable over time. It should be noted that total and fecal coliform densities in wastewater are not constant over time, regardless of the storage condition. Hence, measurements of microbial densities that occur 2 6 weeks prior to disinfection treatment are not valid for computing the true response to disinfection. The revised table presented in the December 2,

2002 Federal Register notice is a misrepresentation of the experimental data and should not be considered adequate to support manufacturer claims.  protocol because they felt that this was a sound procedure. The use of serial dilutions to achieve a desired bacterial density is a standard microbiological practice, and if done with care gives results that are generally within the accepted range of variability inherent in microbiological enumeration methodologies.

EPA decided to use the methodology for calculating bacterial density it has used to evaluate other analytical methods. Moreover, actual post dilution enumeration was not performed for every sample in this study. ( See,

e. g., Sample 982084A.) The Agency s inability to employ actual post dilution enumeration figures consistently across all Colitag   samples further supports EPA s decision to use figures derived from an initial density determination and a dilution factor.

For the above reasons, EPA believes that the December 2002 NODA included an appropriate representation of the CPI experimental data.

See also response to Comment 24 ( Docket ID I D 1.001, Part 03).

26 ID1.002 08 E. coli data   EPA has revised the log reduction following chlorine pretreatment for sample number 982084A. For this experiment the pre disinfection concentration of E. coli was determined by using a different dilution of the original titer established from the initial evaluation of the wastewater isolate rather than enumerating the 1,000 fold diluted sample used for the experiments; hence, the   seed  titer for the disinfection experiment is an inferred number rather than a direct measurement. The absence of actual data for the diluted solution used in the disinfection experiment makes it impossible In reference to the comment on the use of an inferred density of bacteria based on sample dilution and the density of bacteria in the undiluted sample, see response to Comment 25 ( Docket ID I D 1.002 Part 04)

concerning the use of inferred versus measured data for log reduction calculations. EPA experts who wrote the ATP protocol felt that this was a reasonable and reliable approach to determine log reduction. EPA believes that it is reasonable to compute log reduction from chlorine exposure with this approach.

See response to Comment 12 ( Docket ID I F. 021, Part Comment No. Docket ID Docket Part Comment Response Notes 31 to compute the true log reduction that resulted from chlorine exposure. Regardless, the microbial reduction for E. coli in this experiment failed to achieve the required 3 4 log10 reduction.  10) concerning log reduction.

27 ID1.002 09   EPA has revised the log reduction following chlorine pretreatment for sample number 982305A. For this experiment the pre disinfection concentration of E. coli was determined using the 10,000 fold dilution of the original wastewater isolate.

However, the 1,000 fold dilution of this isolate provided no colonies in mFC agar so the testing laboratory expressed the concentration as a detection limit of  < 1.0x105 CFU . Because no bacteria were observed in the dilution used by EPA in the revised data ( Table 2), it is inappropriate to assume that the initial concentration of test microorganisms was identical to the concentration observed in the   original sample  reported on the bench sheets. It is also puzzling that both enumerative experiments were performed on the same day ( 12/ 23/ 98) since the initial experiment ( identification of the concentration of E.

coli in the original wastewater isolate) was presumably used to identify the specific dilution to be used for the subsequent chlorination experiment that presumably would have been conducted 24 hours later. In the methods comparability analysis, 4 of 20 samples were positive according to the proposed method; however, the chi square tables for this sample indicate that 6 of 20 samples were positive for E. coli under the 1: 1000 dilution used in the disinfection experiment.

This inconsistency must be explained. Data generated from sample 982305A should not be considered in support of product approval.  As the commenter notes, the log reduction calculation was based on the number of bacteria found in the original sample. The 1: 10,000 dilution that the commenter mentions was a dilution used during enumeration of bacteria in the original sample. The 1: 000 dilution that the commenter mentions was a dilution that was made of the original sample preceding chlorination for the purpose of achieving the desired density of bacteria for the chlorination experiment. The measured < 1.0 X 10 5 CFU value the commenter mentions, from the 1: 000 dilution from the prechlorination enumeration, was not used to determine prechlorination bacterial density, and instead the value was inferred from the original bacterial density, adjusting for the dilution. EPA notes the difference in bacterial numbers observed between original density determination of the sample, where 11 colonies were observed on mFC medium at a 10,000 fold dilution, but no colonies were observed in the 1000 fold dilution of the prechlorination density determination. For the purpose of the data presented in the NODA, this difference is not relevant because data were not used from the prechlorination density determination. The 1000 fold dilution factor cited in Table 2 of the revised data that was associated with the sample used for the chlorination experiment was used, along with the initial density determination, for the log reduction calculation.

As such, there is no conflict between use of E. coli data from the 10,000 fold dilution of the original waste water sample( for the initial density determination), and use of the subsequent 1000 fold dilution used prior to the chlorination experiment. See also the response to Comment 25 ( Docket ID I D 1.002 Part 04) concerning the use of calculated bacterial density versus directly measured bacterial density. Comment No. Docket ID Docket Part Comment Response Notes 32 In response to the commenter s observation that both the initial sample density determination and the density determination of the diluted sample were done on the same day ( Dec. 23), the commenter should also note that the results of the comparability study were also dated Dec. 23. As discussed in Attachment 1, however, the Colitag   method takes 24 hours to complete and the reference methods each require a minimum of 48 hours.

Hence, the dates entered in the worksheets cannot possibly reflect all of the actual dates for the corresponding steps in the evaluation of the Colitag   method. Instead, EPA surmizes that data may have been entered into laboratory notebooks on a real time basis and subsequently transfered to the ATP worksheets,

with the dates in the worksheets reflecting the dates of transcription rather than those corresponding to the underlying lab procedures. For more information concerning the timing and documentation of studies, the commenter is referred to the response to Comment 24 ( Docket ID I D1.001 part 03) and to Attachment 1.

The commenter has observed that four Colitag   E. coli replicates were positive in the comparability study, but that six replicates were positive for data used in the chi squared analysis. The explanation for this apparent inconsistency is that two different comparability experiments were done for this sample: one with four positives; the other with six. The log reduction was originally reported for the experiment with four positive Colitag   E. coli replicates, but the chi squared analysis was run on the experiment with six positive Colitag   E.

coli replicates. For the reasons explained in the response to Comment 24 ( Docket ID I D1.001, Part 03), EPA repeated the statistical test with the data with four positives, and found that there was no impact on the conclusion concerning comparability between the proposed and reference methods. Comment No. Docket ID Docket Part Comment Response Notes 33 28 ID1.002 13   Again, the log reduction presented in Table 2 of the December 2 Federal Register notice was computed using the assay results generated from the initial analysis of the wastewater isolate [ for sample 990442A]. Instead, the data presented on the bench sheet from the actual dilution employed in the disinfection experiment should have been used ( 1 CFU in 10 mL from the 1: 1000 dilution for an effective concentration of 10 CFU/ 100 mL). The log reduction that results from use of this number is 0.7 log10 rather than the 3.2 presented in Table 2. The bench sheet is missing several items and should be corrected by the testing laboratory and resubmitted to external review. This data fails the ATP criteria and should not be considered in support of product approval.

  EPA has revised the log reduction following chlorine pretreatment for sample number 990443A. If the data from the actual dilution used in the disinfection experiments are used instead of the data from the original analysis of the wastewater sample, the log reduction that would result is 0.7 log10 rather than the 3.0 log10 reported in Table 2. This level of inactivation fails to satisfy the 3 4 log10 criterion specified in ATP section <IP_ADDRESS>. Data generated from sample 990443A should not be considered in support of product approval.  In response to the comment on the use of bacteria densities determined from the initial sample density determination versus the measured value from the sample after dilution to the desired pre chlorination density, EPA notes that the ATP protocol provides for calculation of bacterial density based on the dilution figures and on the original density determination; such procedure has been followed, as is discussed in the response to Comment 25 ( Docket ID I D 1.002 part 04).

While EPA agrees that some bench sheets appear to be missing some information, none of this information is critical to EPA s evaluation of the method and did not interfere with EPA s ability to evaluate the test results in the statistical analysis.

In response to the comments suggesting that the log reductions from samples 990442A and 990443A did not meet the 3 to 4 log reduction criteria, see response to Comment 13 ( Docket ID I D1.001, Part 04) and Comment 15 ( Docket ID I F. 004, Part 02) concerning EPA s calculation of log reduction results.

29 I F. 004 04 3. Colitag   ' s ATP Comparability Study did not meet the number of Replicates and Data Selection Requirements   These two criteria are related. The intent of these criteria is to demonstrate the ability of the proposed method to detect 1 10 total coliforms and E. coli per test volume ( i. e. 100 ml) or to achieve a 25%/ 75% split in positive and negative results in either direction ( Ideally, a 50%/ 50% split). The USEPA ATP protocol Section 3.1.2 states that a minimum of 20 replicate analyses must be performed by each method on the dilution selected for each of the 10 spiked samples. Replicate analyses on the three or more dilutions of each spiked sample must be performed on the same day for both the reference and proposed method ( i. e. a total of at least 60 replicates must be done for each method). Therefore, three dilutions will bracket an ideal The subject comments outlines concerns that ( 1) for some samples, the comparability study was not performed using three separate sets of replicates ( i. e., at three dilutions), and ( 2) the recommended 25%/ 75%

split was not achieved by the replicate analysis for one or more of the samples tested. Each of these issues is addressed in detail in the response to Comment 12 ( Docket ID I F. 021, Part 10); as noted in that response,

EPA concluded that the studies met the underlying goal of detecting low numbers of bacteria and produced results that supported the Agency s statistical analysis.

EPA concluded that the reference methods and Colitag   perform comparably. Comment No. Docket ID Docket Part Comment Response Notes 34 set of 20 replicates each in which there is at most a 25%/ 75%

split ( i. e. 5 minimum and 15 maximum positive observations out of 20) in positive & negative responses. Out of the 60 replicates in the three dilutions, only one set of 20 is expected to satisfy the split and count toward the result requirements.

  It is evident that in the study performed by BioVir Laboratories on Colitag   only 1 2 dilutions were used for each spiked sample ( in most cases, only one dilution was used). As a result, much of the data is invalid based on this 25%/ 75% rule.

  The USEPA ATP protocol section 4.6.5 clearly states " if one of the dilutions does not produce an acceptable split in positive and negative results for the reference method, the proposer must return to the original spiked sample and begin again for the Comparability Study". In this study, the data set for total coliforms from samples [ I. D. # B982305A and 990273A] and the data set for E. coli from samples [ I. D. #: 982084A,

982305A, and 990217A] did not meet the data selection criteria ( see Appendix). This means that these data are not valid and do not meet the requirements as described in the USEPA ATP protocol.

30 I F. 006 03 o Sensitivity of the Method to Detect 1 CFU/ ml [ Colitag   ]

  The ATP, and the U. S. regulations, clearly state that a method must be able to detect 1 CFU of both total coliforms and E. coli per 100 ml sample. The ATP contains a protocol so that the applicant can perform sufficient data points to demonstrate statistically this required level of sensitivity. However, the Colitag   test does not meet the basic criteria set forth in the ATP. Its data submission provides insufficient number of Replicates and Data Selection. It clearly violates the USEPA's 25%/ 75% rule. The ATP directly states that if there is not an acceptable split in the positive and negative splits from the samples, then " the proposer must return to the original spiked sample and begin again for the Comparability Study."  In response to the comment that the Colitag   comparability study data did not meet the 25%: 75% split guideline, and therefore did not demonstrate Colitag     s ability to detect very low numbers of target bacteria, the commenter is referred to the response to Comment 12 ( Docket ID I F. 021, Part 10); as noted in that response,

EPA concluded that the studies met the underlying goal of detecting low numbers of bacteria and produced results that supported the Agency s statistical analysis.

EPA concluded that the reference methods and Colitag   perform comparably.

EPA is not aware of the regulatory requirement the commenter refers to and reminds the commenter that the ATP protocol constitutes guidance. That protocol recommends that comparability studies be done using Comment No. Docket ID Docket Part Comment Response Notes 35 chlorine stressed bacteria in low numbers ( 1 10 CFU/ 100mL); Colitag   studies met this goal.

31 I F. 021 04 ( 3)   The ATP protocol calls for the performance of serial dilutions to bracket microbial densities that can be compared directly using robust statistical methods. The use of bracketed dilutions is aimed at ensuring that the presence/ absence data is not skewed in one direction ( 100% absent) or the other ( 100%

present). However, the independent laboratory failed to bracket the test samples using the required minimum of 3 dilutions for all experiments. The result, not surprisingly, is that a number of the tests failed to comply with the 25%: 75% ' split' guideline defined under the ATP protocol. Fully half of the submitted data ( 5 of 10 experiments) failed to fall within these boundaries for either total coliforms, E. coli, or both, rendering the statistical analysis of the data set somewhat questionable. In light of the failure of the independent laboratory to comply with the established testing guidelines, it is inappropriate to accept the data in support of the proposed method  In response to the comment that the ATP protocol calls for serial dilutions to bracket microbial densities that can be compared using robust statistical methods, EPA has indicated that the data as submitted are sufficient to permit a valid statistical analysis. See also responses to Comment 12 ( Docket ID I F. 021, Part 10) and Comment 29 ( Docket ID I F. 004, Part 04) for additional explanation.

32 I F. 022 05 3.  The ATP comparability results did not meet the replicate and data selection requirements of Section 3.1.2 and 4.6,

respectively. The BioVir Laboratories data sheets indicated that only 1 to 2 dilutions were used for each spiked sample. In most cases, only 1 dilution was used. Hence, this data should not have been included and the experiment should have been repeated.  See responses to Comment 12 ( Docket ID I F. 021, Part 10) and Comment 29 ( Docket ID I F. 004, Part 04) for additional explanation.

03.01.04 Calculation of Log10/ Chi Squared Analysis 33 ID1.002 05 Specific comments:

  For sample number 982084A, USEPA revised the data presented in the original application from the manufacturer.

The original bench sheet for this sample indicates that the diluted wastewater used for the actual disinfection experiment was not subjected to enumerative assay; rather, the   original

sample was assayed upon receipt, and the testing laboratory then assumed that dilutions of 1: 1,000 or 1: 10,000 would produce adequate numbers of coliforms/ E. coli to meet the ATP criteria requiring 3 4 log10 reductions following chlorination and final See response to Comment 25 ( Docket ID I D1.002 part 04) regarding the use of inferred bacterial densities.

See response to Comment 24 ( Docket ID I D1.001, Part 03) for a discussion of a consistent data set ( i. e., one associated with the same dilution) being used for both log reduction and comparability study purposes.

See response to Comment 28 ( Docket ID I. D1.002, Part 13) concerning incomplete bench sheets. Comment No. Docket ID Docket Part Comment Response Notes 36 densities of 1 10 CFU/ 100 mL. Hence, the log reduction for this experiment was computed using inferred rather than actual data. Even in the case that this approach were correct, the bench sheets indicate that the final concentration of test microorganisms post disinfection was in excess of 104 CFU/ mL, more than a thousand times greater than the required level of 1 10 CFU/ mL ( the mEndo count of 6 was multiplied by the dilution factor of 1,000 and then again by the chlorine solution dilution factor of 2 to produce a concentration of 1.2x104 CFU/ mL). It may be that this computation was in error, since the post disinfection samples presumably were not diluted 1,000 fold as indicated on the bench sheet.

Nonetheless, if the data and/ or computations are in error, the bench sheets should be replaced with corrected data and reviewed again. Also, the chi square analysis on the method comparability study data following this disinfection experiment relied upon the 1: 10,000 dilution; however, the original and revised computations for the log reductions relied upon the 1: 1,000 dilution. This inconsistency should be corrected and the data reviewed again. Several items are missing from the bench sheet ( dates of assays, initials of technicians, etc.).

Data generated from sample 982084A should not be considered in support of product approval under ATP guidelines.

  For sample number 982305A, EPA has recomputed the log reductions for coliform bacteria arising from chlorine pretreatment and revised the reduction originally reported by the manufacturer ( 4.5 log10) to a lower level ( 3.4 log10). This revision is in error because of an incorrect value used for the source water concentration ( Msource) of coliform bacteria.

Table 1 indicates a source water concentration of 30,000,000 CFU/ 100 mL based on the assay of the original wastewater sample received in the laboratory on 12/ 16/ 98. This value was See response to Comment 12 ( Docket ID I F. 021, Part 10) concerning the 25: 75 split guideline and the selection of geographically dispersed sites.

See response to Comment 24 ( Docket ID I D1.001, Part 03) concerning   hold time  between initial density determination and post chlorination measurement.

See response to Comment 12 ( Docket ID I F. 021, Part 10) concerning log reduction results.

Concerning the comment about the post disinfection microorganism concentrations for sample 982084A being   in excess of 10 4 CFU/ 100 mL , the commenter appears to be basing such calculation on an errant ( and insignificant, with respect to EPA s analysis) note written on the bottom of the subject worksheet. The 1: 1000 dilution factor was used by EPA to calculate the initial density determination, not the post chlorination density,

as suggested by the commenter. EPA has reviewed its calculations and confirmed that they were performed correctly. As previously discussed, EPA did not rely on the log reduction calculations included in the worksheets.

Having considered all of the commenter s points concerning this sample, and in light of the above responses, EPA is satisfied that it is appropriate to use sample 982084A in its analysis.

Concerning the comment that it was incorrect to change the log reduction value for sample 982305A from 4.5 to 3.4, data on bacterial density before chlorine stressing were calculated as described in sections <IP_ADDRESS> and <IP_ADDRESS> of the ATP protocol. This method of calculating of bacterial density and log reduction of bacteria due to chlorine stress is consistent with EPA s historical approach to method evaluation and is the basis of the Comment No. Docket ID Docket Part Comment Response Notes 37 later divided by the dilution factor of 1,000 to infer the number of viable microorganisms present prior to chlorine injury.

However, the testing laboratory s bench sheets indicate that the actual 1,000 fold diluted   working  solution was assayed,

producing a coliform density of 200,000 CFU/ 100 mL. This value should be used to compute the log reductions rather than the inferred value used by EPA in the revised table presented in the December 2, 2002 Federal Register notice. The corrected log reduction for the total coliform experiment should be 4.2 log10.

  It is also unclear how the comparability data from this experiment were used for the chi square analysis performed by the subcontractor providing the statistical analysis. The bench sheets indicate that the 1,000 fold dilution used for the disinfection experiments resulted in 20 out of 20 positive samples for the proposed method and 17 out of 20 positives for the reference method, a failure of the   25: 75" rule under ATP section 4.6.5, where greater than 75% of the test samples under the proposed method were positive ( 17 of 20 positive, for an actual percentage of 85%). The chi square analysis indicated that 15 of 20 samples were positive under the proposed method and 17 of 20 were positive for the reference method. This inconsistency warrants explanation. Consequently, data generated from sample 982305A should not be considered in support of product approval under ATP guidelines. Finally,

note that samples 982305A and 982084A both arise from the same source ( Millbrae, California). Under ATP guidelines ( section 4.1.1), wastewater isolates   must come from ten or more geographically dispersed sites  in order to proceed with the chlorine injury experiments. One of these samples should be replaced with an isolate from another site; the other should not be considered in support of product approval under ATP guidelines.

  A third chlorine injury experiment was conducted using the water from Millbrae, California ( presumably from sample 982084A but the sample number is missing from the bench calculations of log reduction presented in the Dec. 2,

2002 Federal Register notice. See also the responses to Comment 24 ( Docket ID I D1.001, Part 03), Comment 13 ( Docket ID I D1.001, Part 04) and Comment 25 ( Docket ID I D1.002, Part 04).

Concerning the suggested inconsistency in the use of data to support the statistical analysis and log reduction calculations for sample 982305A, EPA refers to the responses to Comment 24 ( Docket ID I D1.001, Part 03) and Comment 27 ( Docket ID I. D. 1.002, part 09) and notes that two different comparability studies were done,

producing the two sets of results. For consistency, EPA repeated its statistical analysis using the second set of data and the conclusion concerning comparability did not change; the repeat analysis did not identify a statistically significant difference between the performance of the reference methods and Colitag   .

See response to Comment 12 ( Docket ID I F. 021, Part 10) concerning the selection of geographically dispersed sites.

The additional subject experiment that the commenter presumes to have been conducted from sample 982084A was actually a repeat study for sample 982305A. Log Comment No. Docket ID Docket Part Comment Response Notes 38 sheets). It appears that this experiment was a repeat of the total coliform experiment, possibly due to the failure of the previous experiment to meet the ATP   25: 75" criterion. The assays from this experiment resulted in concentrations of 1.8x107 CFU/ 100 mL for the   original  sample and 2.6x107 CFU/ 100 mL in the   diluted  sample. Unfortunately, a page is missing from the bench sheets ( the page including the results of the postchlorination assay) so it is not possible to compute the true log reductions that occurred following chlorine injury. What is most troubling about this third experiment relates to the fact that these data were the ones accepted for the comparability study and also used in the ensuing chi square analysis ( where 15 of 20 samples were positive by the proposed method and 17 of 20 were positive by the reference method). Hence, EPA used the results of the second experiment on this sample to compute the log reductions but the results of the third experiment to compute the chi square analysis. This is absolutely incorrect and the data from this sample are invalid.

  For sample number 990025A, EPA has recomputed the log reductions for coliform bacteria arising from chlorine pretreatment and revised the reduction originally reported by the manufacturer from 3.3 log10 to 3.2 log10. This revision is in error because of an incorrect value used for the source water concentration ( see previous comment). Table 1 indicates a source water concentration of 11,000,000 CFU/ 100 mL based on an extrapolation of the bacterial concentration in the diluted solution used in the experiments ( this value was later divided by the 500 fold dilution factor to compute the log reduction caused by chlorine pretreatment). However, the testing laboratory s bench sheets indicate that the actual 500 fold and 2,000 fold   working  solutions were assayed, producing coliform densities of 21,000 CFU/ mL and 6,500 CFU/ mL, respectively. It is unclear why EPA has elected to exclude the data originally reported but again it must be emphasized that the initial concentrations of test microorganisms were inferred rather than measured to deduce the initial bacterial concentrations for the chlorine injury experiments. If the data from the 500 fold reduction was computed consistent with the ATP protocol, and as described previously in this document.

As also discussed above, a consistent data set was used for both the log reduction calculation and the statistical analysis in EPA s reassessment of the data. EPA s analysis did not identify a statistically significant difference between the reference methods and Colitag   .

As the commenter noted, the comparability study data from the experiment used for log reduction determination did not meet the 25%: 75% criterion, but this did not preclude the use of this individual test result since the chi squared test considered results from all sites collectively. This issue is also discussed in the responses to Comment 12 ( Docket ID I F. 021, Part 10) and Comment 29 ( Docket ID I F. 004 Part 04).

The commenter states for sample 990025A that the bacterial density determined after dilution of the sample,

but before chlorination should have been used to determine log reduction determination rather than relying on the original density determination and dilution factor to compute log reduction. As explained in the response to Comment 24 ( Docket ID I D1.001, Part 03)

and Comment 25 ( Docket ID I D1.002, Part 04), the approach used by EPA is consistent with the ATP protocol and with past practice. The use of a consistent data set in the chi squared test and in the log reduction determination for sample 990025A are also discussed in the response to Comment 24 ( Docket ID I D 1.001 part 03). Allowing for the possibility of a typographic error ( as the commenter presumed), EPA used a dilution factor of 1: 2000 in recomputing the total coliform log reduction. As noted by the commenter, the log reduction changed from 3.2 to 3.0. This change had no impact on EPA s analysis or the conclusion that Comment No. Docket ID Docket Part Comment Response Notes 39 dilution is used to compute the log reductions, it would result in a 3.2 log10 reduction; if the 2,000 fold dilution were used instead, the log reduction would be 3.0 log10. Following the chlorine injury experiments, the method comparability study was conducted   albeit ten days later. Here again, the chi square analysis relied upon the data drawn from the 1,000 fold dilution ( presumably a typographic error? The disinfection experiments were carried out at 1: 500 and 1: 2,000) and yet the disinfection experiment considered by EPA was drawn from the experiment where the sample was diluted 1: 500. Hence, the chi square analysis is invalid in that it relied upon data from a different experiment. Data generated from this sample should not be considered in support of product approval.

  For sample number 990052A, EPA has recomputed the log reductions for coliform bacteria arising from chlorine pretreatment and revised the reduction originally reported by the manufacturer from 2.0 log10 to 2.3 log10 ( see previous comments). Again, the initial coliform concentrations used to compute log reductions are inferred by assuming a volumetric dilution from the original stock concentration. Since the laboratory actually measured the concentration of test microorganisms in the diluted samples used for chlorine pretreatment studies, these actual numbers should be used in place of the inferred numbers ( as was done in the original submission). The correct log reduction for the experiment relying upon the 1: 1,250 dilution would be 1.9 log10, which fails to meet the criterion of 3 4 log10 required under section <IP_ADDRESS> of the ATP. EPA should also consider that its decision to use the data drawn from the initial range finding experiment is inappropriate in light of the fact that over one month elapsed from the time that the initial assay was conducted and the date of the methods comparability study for this sample. Hence,

EPA s recomputation used two data points from assays spaced almost 5 weeks apart to compute the log reductions. This is certainly not standard practice and invalidates the data generated from this experiment. Also, the chi square analysis was performed using data from the 1: 2,500 dilution   which was not Colitag   is an acceptable method.

Concerning the commenter s claim that the method comparability study for sample 990025A was conducted 10 days after chlorine injury, the commenter is referred to the response to Comment 24 ( Docket ID I D1.001,

Part 03).

The commenter notes that the log reduction for sample 990052A was determined from an inferred bacterial density derived from the initial coliform concentration in the original sample and the dilution used to prepare the prechlorination sample. EPA refers the commenter to its response Comment 24 ( Docket ID I D1.001, Part 03)

and Comment 25 ( Docket ID I D1.002, Part 04)

concerning calculation of log reduction. In response to the comment suggesting the possibility that over one month passed between the initial density determination and chlorination, EPA refers the commenter to the response to Comment 24 ( Docket ID I D1.001, Part 03)

for a discussion of this time interval for sample 990052A. Concerning the comment on the use of different dilutions for log reduction determination and for the comparability study and chi squared analysis,

EPA again refers the commenter to the response to Comment 24 ( Docket ID I D1.001, Part 03). Log reduction for this sample was recomputed, for consistency with the data set used in the statistical analysis; the figure ( 2.3) did not change. Thus, there was no impact on EPA s analysis. Comment No. Docket ID Docket Part Comment Response Notes 40 the consistent with the 1: 1,250 dilution used to generate the data for the disinfection experiments. Data generated from sample number 990052A should not be considered in support of product approval under ATP guidelines.

  For sample number 990095, log reductions for coliforms were inferred rather than measured, as stated for previous samples.

The actual measured concentration of the diluted stock coliforms was reported as 72 CFU in a 1 mL sample of the 1: 7,000 dilution. This number should be used as the initial density of test microorganisms rather than the 16,000,000 CFU/ 100 mL number reported in table 1   note also that the bench sheet for this sample is missing the derived concentration of test microorganisms. Table 1 should be revised to reflect the actual, measured bacterial densities and the correct log reductions ( 3.3 log10 rather than 2.8 log10). In addition, the independent testing laboratory should provide the missing items on the bench sheet ( microbial concentrations, dates of assays,

and technicians responsible for performing and documenting assay results). The log reduction for coliform bacteria in this sample was computed using the   diluted sample  plate counts   this is actually the correct approach but is not the approach EPA reports using in the December 2, 2002 Federal Register notice to modify the data presented in Table 1 during its reconsideration of the original data submitted and reviewed in March of 2002. The revised bench sheet and table should be resubmitted for reviewer comment.

  For sample 990217, again the diluted sample ( 1: 600) subjected to chlorine pretreatment indicates a coliform density of 100 CFU/ 100 mL. However, the logarithmic reduction presented in Table 1 was generated using the bacterial density from the initial determination of coliforms in the stock wastewater isolate ( 5,000,000 CFU/ 100 mL). Therefore, the actual logarithmic determination was again inferred rather than measured. If the actual data were used instead ( 100 CFU per 100 mL), the computed log reduction would be 0.9 log10 rather than the reported 2.8 log10. This reduction fails to meet the ATP The commenter states that the log reduction data from sample 990095 was inferred from the initial density determination for the original sample rather than directly measuring the bacterial density in the diluted sample that was prepared for chlorination. EPA refers the commenter to its responses to Comment 24 ( Docket ID I D1.001, Part 03) and Comment 25 ( Docket ID ID1.002 Part 04) concerning calculation of log reduction for data as presented in the Dec. 2, 2002 Federal Register notice. The bench sheet for sample 990095 contained the critical information needed by EPA to assess the results. EPA used the approach reported in the December 2002 NODA to calculate log reduction.

The commenter states that the log reduction for sample 990217 was determined by inferring a bacterial density from the initial determination of coliforms in the stock wastewater. EPA refers the commenter to its responses to Comment 12 ( Docket ID I F. 021, Part 10), Comment 24 ( Docket ID I D1.001, Part 03) and Comment 25 ( Docket ID I D1.002, Part 04) concerning calculation of log reduction. In response to the comment on the time lapse between initial count of bacteria in the wastewater sample and chlorination, EPA refers the commenter to Comment No. Docket ID Docket Part Comment Response Notes 41 criterion of 3 4 log10. Also, the logic behind the use of the range finding data rather than the actual diluted samples to compute the log reductions is flawed due to the time that elapsed between the two assays ( approximately two weeks judging by the dates on the comparability study sheets). Table 1 should be revised to reflect the actual, measured bacterial densities and should be resubmitted for reviewer comment.

  For sample 990273A, again the diluted sample ( 1: 800)

subjected to chlorine pretreatment indicates a reported coliform density of 800 CFU per 100 mL based on the original bench sheets submitted by the testing laboratory. However, EPA has used the coliform density reported from the original assay of the wastewater isolate ( 2,000,000 CFU per 100 mL) in recomputing the log reductions in Table 1 of the Federal Register notice.

The use of the inferred data rather than the actual data reported by the testing laboratory results in a significant error ( the true log reduction should be 1.8 log10 rather than the 2.3 indicated in Table 1. Regardless, the level of inactivation fails to meet the 3 4 log10 reduction requirement under section <IP_ADDRESS> of the ATP. Following chlorine pretreatment, application of the reference method resulted in 18 of 20 positive samples, failing the   25: 75" rule under section 4.6.5 of the ATP. And again,

EPA s use of the initial assay data creates an artifact due to the period of time that elapsed between this date and the date of the assay on the post chlorination sample ( three weeks later). Note that the comparability assay bench sheets indicate that a dilution of 1: 600 was used for these samples rather than the 1: 800 indicated on the disinfection bench sheets. Which number is correct? The log reductions reported for sample 990273A should not be considered in support of product approval under ATP guidelines.  the response to Comment 24 ( Docket ID I D1.001, Part 03). The commenter states that the log reduction for sample 990273A used an inferred bacterial density derived from the original wastewater assay rather than using the actual value determined from the diluted waste water spike sample before chlorination. EPA refers the commenter to its responses to Comment 24 ( Docket ID I D1.001,

Part 03) and Comment 25 ( Docket ID I D1.002, Part 04)

concerning calculation of log reduction. In response to the comment on the log reduction criterion, see the response to Comment 12 ( Docket ID I F. 021, Part 10)

concerning application of the 3 to 4 log reduction as guidance. Concerning the comment on the 25%: 75%

split guideline, EPA refers the commenter to its response to Comment 12 ( Docket ID I F. 021, Part 10) and Comment 29 ( Docket ID I F. 004, Part 04). Concerning the comment on the time lapse between cell counts and chlorination, EPA refers the commenter to its response to Comment 12 ( Docket ID I F. 021, Part 10) and Comment 24 ( Docket ID I D1.001, Part 03). The commenter observed that a 1: 600 dilution was identified in connection with the comparability study results and a 1: 800 dilution was identified in connection with the log reduction calculation, per the disinfection bench sheets.

Since one of these figures appears to be an error, and since it is not clear which figure is correct, EPA examined the potential influence of this difference on log reduction. EPA did so by substituting a 600 fold dilution into the log reduction calculation in place of the 800 fold dilution originally used; doing so resulted in a log reduction of 2.4 ( versus the 2.3 log reduction associated with a dilution of 1: 800). This minor change Comment No. Docket ID Docket Part Comment Response Notes 42   For sample 990438A, again the log reductions presented in Table 1 of the Federal Register notice of December 2, 2002, are based upon an extrapolation of the initial coliform densities prior to chlorine treatment. Since the testing laboratory measured and reported the actual starting concentrations of coliforms in the diluted wastewater isolate ( 1 CFU in the 1: 13,000 dilution), this number should have been used rather than the number reported following the initial determination of coliform densities in the wastewater isolate. The correct log reduction should be 0.7 log10 rather than the 2.3 reported in the revised table; this reduction fails to meet the ATP criterion indicated in section <IP_ADDRESS>. More troubling than the computational errors is the fact that the data used in the chi square analysis are not those presented in the bench sheets from the disinfection experiments and the ensuing methods comparability study. The bench sheets indicate a split of 10: 10 ( positive: negative) for the proposed method under the 1: 13,000 dilution used for the disinfection experiments; the reference method produced a split of 9: 11. And yet the chi square tables indicate a split of 13: 7 and 9: 11 for the proposed and reference methods, respectively. What is the origin of this data and how can it be reconciled with the analysis performed to support the contention that the proposed and reference methods are equivalent? Moreover, the bench sheets indicate dates that are not logical. If the density determination for the original sample was produced on 5/ 5/ 99 as indicated on the bench sheets, how is it that the   subsequent  comparability study was conducted on 4/ 30/ 99??? In the absence of verification from the testing laboratory, these data should be excluded.  in log reduction has no impact on EPA s assessment.

The commenter notes that the log reduction for sample 990438A is based on an extrapolation of the initial coliform densities prior to chlorine treatment. EPA refers the commenter to its responses to Comment 24 ( Docket ID I D1.001, Part 03) and Comment 25 ( Docket ID I D1.002, Part 04) concerning calculation of log reduction, and to Comment 12 ( Docket ID I F. 021,

Part 10) concerning application of the 3 to 4 log reduction as guidance. In response to the comment that the total coliform data used in the chi squared analysis for sample 990438A are not those presented in the bench sheets and ensuing comparability study, EPA has reviewed the raw data and confirmed that the correct values are 10 positives: 10 negatives for Colitag   and 9 positives: 11 negatives for the reference method. EPA has performed a number of repeat analyses/ reanalyses,

as discussed elsewhere in this document, and has confirmed that the correct comparability study results are being used. None of those analyses identified a statistically significant difference between the performance of the reference methods and Colitag   .

EPA agrees that the dates for the density determination and comparability studies documented on the log sheets are inconsistent and may be the result of the density data being entered on the log sheet post data collection.

Based on the chain of custody sheet ( showing that the original sample was received in the lab on 4/ 27/ 99) and the comparability study sheet ( showing Colitag   test results dated 4/ 30/ 99), EPA has concluded that it is unlikely that the samples were held for an excessive period. See also the response to Comment 24 ( Docket ID I D1.001, Part 03) for additional discussion concerning this   hold time  question.

Concerning the comment about spiking sources from 10 Comment No. Docket ID Docket Part Comment Response Notes 43   Sample number 990442A originates from the same source as sample number 990217A ( Mission, KS). Under section 4.1.1 of the ATP, spiking sources   must come from ten or more geographically dispersed sites . One of these two samples must be excluded from consideration in the study data and should not be considered in support of product approval. The manufacturer/ independent testing laboratory should collect at least one additional sample and subject it to testing under ATP guidelines for it to be included in the comparability data in Tables 1 and 2.

  Following EPA s reexamination of the original manufacturer data, the log reductions following chlorine pretreatment of sample 990442A were revised from 1.4 log10 to 2.6 log10 ( based upon the use of the extrapolated initial concentration of test microorganisms as previously described). Again, the actual,

measured concentrations of test microorganisms recorded on the bench sheets should be employed to compute the log reductions. The coliform density reported on the bench sheet for this sample is 3 CFU per 10 mL in the 1: 9,000 dilution. The correct log reduction for this experiment should be 0.9 log10 which fails the ATP criteria. In the absence of additional clarification and external review, data generated from sample 990442A should not be considered in support of product approval.

  Similarly, sample number 990443A was reexamined to revise the log reduction originally reported by the manufacturer. An extrapolated bacterial density was used to compute a revised reduction of 2.4 log10. This number should instead be 0.4 log10. Regardless of the computational approach the reported log reduction failed to meet the ATP criterion.  geographically dispersed sites, EPA refers the commenter to its response to Comment 12 ( Docket ID I F. 021, Part 10).

The commenter notes that the log reduction for sample 990442A was revised based on the use of log reduction calculations based on the initial density determination.

EPA refers the commenter to its responses to Comment 24 ( Docket ID I D1.001, Part 03) and Comment 25 ( Docket ID I D1.002, Part 04) concerning calculation of log reduction, and to its response to Comment 12 ( Docket ID I F. 021, Part 10) concerning application of the 3 to 4 log reduction as guidance The commenter notes that the log reduction for sample 990443A was revised based on the use of log reduction calculations based on the initial density determination.

EPA refers the commenter to its responses to Comment 24 ( Docket ID I D1.001, Part 03) and Comment 25 ( Docket ID I D1.002, Part 04) concerning calculation of log reduction, and to its response to Comment 12 ( Docket ID I F. 021, Part 10) concerning application of the 3 to 4 log reduction as guidance. The commenter is Comment No. Docket ID Docket Part Comment Response Notes 44 referred to the response to Comment 1 ( Docket ID I D 1.001, Part 06) concerning the use of the ATP criteria as guidance.

34 ID2.001 02   Firstly the data supplied demonstrates that when the testing was carried out using disinfection to determine if Colitag   is able to recover stressed organisms, the 3 log reduction was not met in many cases. Furthermore, the method for calculating a 3 log reduction was incorrect. The correct procedure is to take the log of the initial number of bacteria and the log of the final number and subtract one from the other. It would appear that the mechanism used for the Colitag   study was to take the difference between the initial and final concentration of bacteria and use the log of that number to indicate log reduction. This is incorrect.

  For example, if the initial concentration of bacteria were one million and the final concentration was one thousand, the correct calculation is log one million ( 6) minus log one thousand ( 3) equals 3. Thus a three log reduction has been achieved.

  If however the method used by CPI is used one million minus one thousand is 999000. The log of 999000 is 5.999. Might I suggest that the data is re evaluated and the correct log reductions are used. It is extremely important that this part of the testing procedure is carried out and evaluated correctly. A " near enough" approach is just not acceptable. This is particularly true in the case of Colitag   since the manufacturers claim to have improved performance with respect to recovery of chlorine stressed organisms over existing methods.  In response to the comment suggesting that a 3 log reduction was necessary for each sample, the commenter is referred to the response to Comment 12 ( Docket ID IF 021, Part 10). In response to the comment suggesting that EPA calculated the log reduction figures incorrectly,

EPA believes that the log reduction data were correctly calculated. Referring to the December 2002 NODA,

the formula in the EPA s approach ( log[ A/ B]) is mathematically equivalent to that advocated by the commenter. ( logA logB). Using sample 990442A as an example, where Msource= 1.4 x 10 7; DF= 9000;

Mchlorinated= 2; and CF= 2, Log reduction =

log 10 [( 1.4x10 7/ 9000)/( 2x2)]= 2.6 EPA agrees that it is important to correctly calculate log reduction, and assures the commenter that the log reductions have been correctly calculated as indicated above.

03.02 General Comments on Adherence to ATP Guidelines ( e. g., claim that protocol must serve as benchmark)

35 ID1.002 02 General comments:

  A detailed review of the original data submitted in support of the Colitag   test for coliforms and E. coli was submitted in April, 2002. Although EPA responded to reviewer comments concerning some of the products being considered for approval, In response to the comment on EPA s use of the ATP protocol as guidance, see response to Comment 1 ( Docket ID I D1.001, Part 06).