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             REPORT TO THE




   MOBILE BAY ENFORCEMENT CONFERENCE




                  ON




BACTERIOLOGICAL CRITERIA FOR SHELLFISH




              HARVESTING
                  by




    MOBILE BAY TECHNICAL COMMITTEE




          MOBILE BAY,  ALABAMA




              AUGUST 1972

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                   MOBILE BAY TECHNICAL COMMITTEE
     Member

Robert F. Schneider, Chairman



W. F. Anderson


Arthur N. Beck


J. David Clem



James H. Coi1, Jr.

C. B. Kelly


F. James Silva
          Representing

Environmental Protection Agency,
 National Field Investigations
 Center-Denver, Colorado.

Alabama Department of Conservation
 and Natural Resources.

Alabama Water  Improvement Commission
 and Alabama State Health Department.

Department of Health, Education
 and Welfare, Public Health Service,
 Food and Drug Administration.

Scott Paper Company, Mobile, Alabama.

Environmental Protection Agency,
 Water Supply Programs Division.

Environmental Protection Agency
 Region IV, Atlanta, Georgia.
                            -1-

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                          TABLE OF CONTENTS






                                                              Page




MOBILE BAY TECHNICAL COMMITTEE 	    1




INTRODUCTION 	    3



THE MOBILE BAY SHELLFISH HARVEST PROBLEM 	    8




A SUBSTITUTE BACTERIOLOGICAL CRITERION 	    9




TECHNICAL COMMITTEE RECOMMENDATION 	   11




LITERATURE CITED 	   12




APPENDIX A




     Request for the Establishment of the Mobile




       Bay Technical Committee 	   13




APPENDIX B




     Progress Report 	   28



APPENDIX C




     Mobile Bay Technical Committee Meeting, Dauphin Island,




       Alabama - March 2k, 1972	33




APPENDIX D




     Correspondence Concerning a Proposed




       Recommendation  	   56



APPENDIX E




     Rapid Recovery of Escherichia coli  from Estuarine




       Waters	68
                                -2-

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                            INTRODUCTION






     A Conference on the Matter of Pollution Affecting Shellfish in




Mobile Bay, Alabama was called by the Secretary of the Interior, and




was held in the City of Mobile on January 27-28, 1970.  The Conference




Proceedingsl/contained a recommendation (No. 6) that emphasized




the need for reevaluation of the bacteriological criterion for




shellfish harvesting areas.  The recommendation read:  "In determin-




ing acceptability of areas in Mobile Bay for shellfish harvesting,




a more realistic criterion than the present method be employed to




more effectively evaluate the probability of pathogen presence.  A



coordinated effort by the U.  S. Department of Health, Education and




Welfare and the U. S. Department of the Interior, in cooperation with




the States, should be undertaken immediately to develop such a




criterion with respect to approval  of areas for shellfish harvesting.




A technical committee shall be established by the Conferees to




assist in the development of such a criterion.  This committee should




report to the Conferees no later than one year from the date of the first




session of conference."




     After a lengthy delay, the Conference summary was approved;




on November 30, 1971 the EPA Enforcement Division, Region IV, Atlanta,




Georgia requested that the National  Field Investigations Center-Denver




establish a technical committee in  compliance with the Mobile Bay
                                   -3-

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Enforcement Conference Recommendation No. 6.  Subsequently, through




the Conferees the Technical Committee membership was established




in February, 1972.  (Appendices A and B)




     This report summarizes the considerations used by the Mobile




Bay Technical Committee for proposing a substitute bacteriological




criterion to evaluate the probability of pathogen presence in Mobile




Bay shellfish harvest areas.






        HISTORICAL REVIEW OF THE COLIFORM BACTERIA CRITERION
     The Mobile Bay Technical Committee reviewed the historical




and current rationale behind the present bacteriological criterion




for shellfish growing areas.




     Near the turn of the century, investigators found that Escherichia




coli and other related bacteria normally occurred in the intestinal




tracks of mammals.—  This assemblage of intestinal organisms became




known as the coliform bacteria group.  Later, it was discovered




that the coliform bacteria were consistently present in sewage,




polluted water, and contaminated soil,—  —  —  and the coliform




organisms became accepted as indicators of fecal pollution.  Furthei—




more, it was found that water containing coliform bacteria often




contained enteric pathogens; thus the results of the "coliform test"




have become important considerations in judging the sanitary quality




of water in shellfish harvesting areas.




     In 1936— , Dr. C. A. Perry proposed that Escherichia coli  rather




than other coliform group bacteria be the index of pollution for both
                                  -k-

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shellfish and shellfish waters.  Five years later, a report titled,


"Public Health Aspects of Clamming in Raritan Bay" concluded that


"70 coliforms/100 ml, on the basis of average results from waters


overlying the shellfish beds,  is recommended as the limiting allow-


able standards for the taking of hard clams to be eaten raw."


     The 19^6 Manual  of Recommended Practice for Sanitary Control


of the Shellfish IndustryZ/  presented the bacteriological criteria


for growing areas as follows:  "The median bacteriological content


of samples of x^ater . . . shall not show the presence of organisms
                      i

of the coliform group [total coliform bacterial density]  in excess


of 70/100 ml of water."


     At the 5th and 6th National Shellfish Sanitation Workshops, par-


ticipants were in disagreement with the 70/100 ml  criterion.


Studies—' 9/ had shown that the fecal coliform bacterial group was a


more sensitive index of fecal pollution than the total  coliform


group.  This conclusion was based on the fact that, in most instances,


the presence of fecal coliform organisms could be correlated with


the presence of fecal wastes from humans or other warm blooded


animals; conversely,  investigators found that large numbers of non-


fecal coliforms could be recovered from waters and soils that were


remote from any known sources of warm blooded animal pollutants.


     The Workshop participants reviewed these study findings and


discussed coliform standard for shellfish meats and a coliform


standard for the waters in which the shellfish are grown.   An attempt
                                 -5-

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was made by some participants to convince the Workshop that the




fecal coliform organisms were a better indicator of water pollution




than total coliform bacteria.  Indicator levels of fecal coliform




bacteria were suggested for approved shellfish growing areas by




those who favored changing the bacteriological criterion.  After




considerable consternation among the 1961* and 1968 Workshop repre-




sentatives, they recommended continued use of coliform organism




criteria and further study of the fecal coliform group in shell-




fish harvesting waters.




     The substitution of the fecal coliform indicator group for the




total coliform group in judging shellfish harvest area sanitation




came under discussion again at the 7th National Shellfish Sanitation




Program (NSSP) in 1971.12/  During the Microbiology Task Force




deliberations at the Workshop, median fecal  coliform values of 7-8




and 1*3 were recommended as growing area standards by representatives




of Maryland and Virginia, respectively.  A spokesman from Lousiana




remarked that in his State a 23 fecal coliform MPN median standard



would close a high percent of "approved" shellfish-growing areas




which could not be shown to be affected by known sources of pollu-




tion.  Delegates from Rhode Island proposed  that the words "not




more than 10 percent of the samples ordinarily exceed an MPN of




230 per 100 ml" be omitted from the coliform standard.  These actions




indicate the diversity of opinions expressed by scientists who were



actively trying to Devaluate bacteriological criteria for approved




shellfish-growing areas.  After considerable discussion, all but two




State representatives in the 1971 NSSP Task  Force voted to support




the following FDA recommendation:





                                  -6-

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     "The Food and Drug Administration  (FDA) recommends to the 7th




National Workshop that the bacteriological criteria for shellfish




harvest areas be maintained as stated in the 19&5 Revision of the




NSSP Manual of Operations [not to exceed a median total coliform




value of 70/100 ml— ].   It is also recommended that the NSSP




continue to use the fecal coliform test as defined in Recommended




Procedures for the Examination of Seawater and Shellfish, APHA 1970,




as an adjunct to the coliform test to provide additional information




relative to water quality of shellfish harvest areas."




     The FDA based this recommendation on five conclusions.




1.  The present bacteriological criteria for approved growing areas




    has been tested for many years and has demonstrated its effective-



    ness in providing safe shellfish.




2.  The 70/100 ml value of the coliform standard is well above the




    lower limits of sensitivity of the multiple tube fermentation




    procedure.  Acceptable levels for a fecal coliform standard




    would be in the range of the lower limits of sensitivity of the



    multiple tube fermentation test resulting in more indeterminate




    values of water quality data.




3.  The coliform indicator group is a broad spectrum indicator of




    pollution compared to the species of organisms recovered by the




    fecal  coliform test.




k.  The relationship of numbers of fecal coliform organisms to direct




    fecal  sources has not been established.
                              -7-

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5.  At the present time, there is no universal agreement among public



    health experts as to the advisability of accepting a fecal



    coliform growing area criterion for shellfish growing areas.





                 THE MOBILE BAY SHELLFISH HARVEST PROBLEM




     The Proceedings of the Mobile Bay Enforcement Conference of 1970



contain a discussion of the problems affecting shellfish harvesting



in Mobile Bay.il/  It was reported that during the spring, flood



runoff (532,000 cfs) from the Tombigbee-Mobile River system trans-
                 i


ported large numbers of coliform bacteria into Mobile Bay.  The



sanitary significance of these coliform densities was unknown;



yet, shell fishing areas in Mobile Bay were closed by the State of



Alabama during portions of 8 of the past 16 years because the bacterial



densities over the shellfish beds exceeded the 70/100 ml total coli-



form criterion for harvesting.  The region has suffered substantial



economic losses because of these closures.



     The problem was reiterated at a Mobile Bay Technical Committee



meeting at Dauphin Island, Alabama on March 2k, 1972 (Appendix C).



Committee spokesmen for the State of Alabama contended that the



application of the total coliform criterion unduly penalized the



Gulf Coast of the Southeastern Unived States.   Coastal  areas have a



potential for the occurrence of certain bacteria of the coliform group



in sources of no sanitary sgnificance without  concomitant growth of



enteric pathogens.   The Committee was reminded by representatives from
                                -8-

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the State of Alabama that during the past decade, portions of




Mobile Bay oyster reefs were closed on nine occasions for average




periods of 150 days.  As of March 2^, 1972, a closure (effective




February k, 1972) resulted  in a dockside economic loss of nearly




$100,000.  This was equivalent to a total economic loss to the




Mobile area of almost $^00,000.






              A SUBSTITUTE BACTERIOLOGICAL CRITERION
     During the Technical Committee deliberations, opinions were




unanimous that a substitute bacteriological criterion for the total




coliform test was needed for evaluating shellfish harvest areas in




Mobile Bay.  The Committee agreed that representatives of EPA and




FDA who are responsible for criteria development should consider the




following characteristics in the design of a substitute bacteriological



cri terion.




     The substitute bacterial indicator should:  (1) be more specific




for fecal pollution than the currently used indicator group; (2) stand




on its own merit as an indicator of actual sources of fecal  pollution




without a dependency upon a specific relationship with the coli-




form group; (3) be more indicative of potential pathogen contamination;



(k) consistently indicate the sanitary quality of shellfish-growing




waters in diverse geographical  locations; and  (5) have a survival




time in the estuary consistent with that of bacterial pathogens




regardless of nutrient value of the water.
                                -9-

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     After considering several avenues of approach, the Technical




Committee agreed that the two indicator tests most promising for




evaluating bacteriological contamination in Mobile Bay shellfish




harvest areas were  (1) fecal coliform or (2)  isolation of Escherichia




coli.  Major benefits and weaknesses of these substitute criteria




were outlined by Committee members as follows:




     (1)  Presently, the fecal coliform test  requires less time and




          effort than the E_. col i measurement; therefore, results




          can be obtained sooner, at less expense, and by fewer




          microbiologists.  However, studies  by the EPA, FDA and




          the State of Alabama have failed to demonstrate a consistent




          total coliform/fecal coliform ratio, or determine a fecal




          coliform value that can be directly related to known




          sources of fecal pollution and consistently provide public




          health protection to consumers of raw shellfish.



     (2)  Sanitary surveys and published documentation confirm that




          the presence of E_. coli consistently indicates fecal



          pollution and potential contamination by pathogenic




          bacteria.




     By letter to the Technical  Committee on April 2k, 1972, the




Chairman proposed that the criterion for judging sanitary conditions




in Mobile Bay shellfish areas be based on the measurement of Escher ichia




coli (Appendix D).   A majority of the Committee agreed with the




recommended E_. coli substitute criterion.  Furthermore, the spokesman




for FDA cited a study on the rapid recovery of E_. col i from estuarine




waters (Appendix E).  The study describes new methodology and concludes
                                -10-

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that £. col i can be  isolated from raw seawater within 21* hours




without significant  loss of accuracy.   If the method accuracy can




be verified by collaborating studies, it will provide public health




protection  to consumers of raw shellfish by rapidly and consistently




confirming  fecal pollution and indicating potential contamination




by pathogenic bacteria.






                 TECHNICAL COMMITTEE RECOMMENDATION
     In compliance with Recommendation No. 6 of the Mobile Bay




Enforcement Conference, the Technical Committee submits the follow-




ing statements to the Conferees:  "To assist in the development of



a more realistic guideline than the present method for evaluating



the probability of pathogen presence in Mobile Bay, it is recommended




that the bacteriological criterion be based on the measurement of




Escherichia coli in waters overlying shellfish harvest areas.  Further-




more, the Technical Committee recommends that the Environmental




Protection Agency, Food and Drug Administration and State shellfish




sanitation control agency combine their efforts to support the




research needed to develop a rapid, reliable method for measuring signifi'




cant numbers of E_. coli and to compare these values with other pollu-




tion indicator bacterial densities in estuarine waters used for




shellfish harvesting."
                                 -11-

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                           LITERATURE CITED
 1.  Mobile Bay Enforcement Conference Proceedings.

 2.  Escherich, T.   1885.   Die Darmbakterin des Neugeborenen und
        Saughlings.   Fortsch.  Med.  3,  515 and 5^7.

 3.  Rogers, L. A.,  W.  M.  Clark,  and A.  C.  Evans.   1915.
        The characteristics of bacteria  of the colon type found
        in bovine feces.   Jour. Infect.  Dis. 15i 99.

 4.  Bards ley, D. A.  193**.  The  distribution and sanitary signi-
        ficance of E.  coli, B.  lactus  aerogenes and  intermediate
        types of coliform bacilli in water,  soil,  faeces  and ice cream.
        Jour. Hyg.  3**,  38.

 5.  Chen, C. C. and L.  F. Rettger.   1920.   A correlation study of the
        colon-aerogenes  group of bacteria with special reference
        to the organisms occurring in  the soil.   Jour. Bact.  5, 253.

 6.  Perry, C. A.  1936.   Examination  of shellfish for fecal pollution.
        Committee Report,  APHA Yearbook  1935-1936,  pp 111-117.

 7.  USPHS, 19^6. Manual of recommended practice for sanitary  control
        of the shellfish industry.   U.  S. Department of Health, Edu-
        cation and  Welfare, Washington,  D.C.

 8.  Beck, W. J.  1964.   Bacteriological criteria for shellfish
        growing areas.   Proc.  5th National  Shellfish Sanitation Workshop.

 9.  Strebel, G. A.   1968.  Coliform-fecal  coliform  bacteria in tidal
        waters.  Jour.  San. Eng.  Div.,  Proc. Amer.  Soc. Civil Eng.
        August 1968.  pp.  6M-655.

10.  Slanetz, L. W., C.  H. Bartley,  and  K.  W. Stanley.  1971.
        Comparison of numbers of coliform and fecal  coliform bacteria
        in shellfish growing waters  in relation to bacteriological
        standards.   Proc.  7th National  Shellfish Sanitation Workshop.

11.  USPHS, 1965. national shellfish,  sanitation program,  manual of
        operations,  Part III Public  Health  Service appraisal of state
        shellfish sanitation programs.   U.  S. Department  of Health,
        Education and  Welfare,  Washington,  D.C.

12.  Gallagher, T.  P., F.  J. Silva,  L. W. Olinger and R.  A.  Whatley.
        1969.  Pollution affecting shellfish harvesting in Mobile
        Bay,  Alabama.   FWPCA,  U.  S.  Department of the Interior, South-
        east Water Laboratory,  Athens, Georgia (mimeo).
                                      -12-

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          APPENDIX A






 REQUEST FOR THE ESTABLISHMENT




            OF THE




MOBILE BAY TECHNICAL COMMITTEE
            -13-

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                      ENVIRONMENTAL PROTECTION AGENCY
 Dalt:  November 30, 1971

     -  Actine Director, Enforcement Division
       Region IV
Subjut:  Mobile Enforcement Conference Recommendation, In Re Shellfish
       Technical Committee
   To:  Director, DFI-Denver Center
       In accordance with our telephone conversation sometime back and
       the follow-up conversation we had during our visit to Memphis
       in September, I was directed by Mr. Stein to advise you that it
       was your responsibility to see that recommendation 6 of the
       enclosed conclusions and recommendations for the Mobile Bay
       Enforcement Conference is followed.

       As I indicated to you, when the project summary was approved on
       March 2 of 1971 after a lengthy delay, the Regional Office had
       moved to establish a technical committee in compliance with the
       above referenced recommendation.  However, in contacting personnel
       in Mr. Stein's office, I was advised that this committee was to
       be established at the national level a.nd that it would be
       coordinated in Washington.  Vic Lambou started working on this
       but never got further than preliminary discussions with HEW.
       After the standards section was removed from the Enforcement
       Office, Lambou ceased working on the project.

       Also as indicated to you, Jim Silva at my request prepared a
       charter and developed a list of prospective committee members
       (copy enclosed).

       You will note in the recommendation that the committee is to
       report to the conferees within a year.  Because of the delay in
       getting approval of the conference summary, the due date for the
       committee's report is March 2, 1972.
                                                         o
       Your early attention to this matter will be appreciated.

                                                           ^
                                                          ^1-i..-C-^—•
       Enclosures:  Conclusions & Recommendations
                    Charter & Member List

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                             YIRONMENTAL PROTECTION AGE
                                OFFICE OF  ENFORCEMENT
                    DIVISION  OF FIELD  INVESTIGATIONS-DENVER CENTER
                      BUILDING   22  , ROOM  'MO  , DENVER FEDERAL CENTER
                                 DENVER. COLORADO 80225


10    :   Mr.  John White, Acting  Director,                   DATE:  December 6,  1971
        Enforcement  Division, Reg.  IV, EPA
        Atlanta

FROM  :   Director,  DPI  - Denver  Center


SUBJECT:   Establishment  of Shellfish Technical Committee -
         Mobile Bay
        I  have  received your memorandum of November 30,  1971,  requesting establish-
        ment of a committee to  recommend acceptable bacteriological criteria for
        Mobile  Bay.  This  request  from your office  is made to  assure compliance
        with Recommendation No. 6  of  the Mobile Bay Enforcement Conference.

        I  am sure you  recognize the delay in transmitting this request effectively
        precludes completion of the required report by March 2, 1972.  I am asking
        Mr. Robert Schneider, Chief,  Biology Branch, to  serve  as representative
        from this office on the prospective coiiirni ttee.   We have also requested
        that Mr. David Clem, Director, Division of Shellfish Sanitation, serve as
        the DHEW representative.   At  this time  I would like to request the services
        of Jim  Silva of the Atlanta Regional Office, EPA, as the regional repre-
        sentative.   It is  my intention that these people should meet at the
        earliest mutual opportunity to designate an industrial and state repre-
        sentative.   Recommendations of this committee should be based upon con-
        siderations  relevant to Mobile Bay only.  It is  presently anticipated that
        the committee will submit  a progress report to the Conferees by March 1972.

        Your early attention to this  proposal will be appreciated and we look forward
        to working with you and your  excellent staff in  completing this assignment in
        a  timely manner.   You will be kept fully informed of committee activities.
        If you  have  any further questions, please do not hesitate to contact me.
                                               Thomas P. Gallagher
        cc:  Adm. ,  Reg.  IV, EPA
            Dir.,  Enf.  Proceedings Div., EPA, w/cy
               memo, Act. Dir., Enf. Div., Reg.  IV
               11/30/71 , same subject
            Chief, Bio. Br., DPI, w/memo 6 encl
            Dir.,  Div.  of Shellfish San., DHEW
                                        -15-

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                   22          'llO
                                       December  6,  1971
 Mr.  David  Clem,  Director
 Division of  Shellfish  Sanitation
 DHEW,  200  "C"  St.,  S.V,'.
 Washington,  D. C. 2020*i
.Dear  Mr.  Clem:

As  you  know,  Recommendation  Ho.  6 of  the  Mobile  Boy  Enforcement
Conference  directed  that  a technical  committee be  established  to
develop a more  realistic  criterion  than the  present  method  to  nore
effectively evaluate the  probability  of pathogen presence  in Mobile
Bay.  This  responsibility has  been  assigned  to Division  of  Field
Investigations  Center - Denver,  EPA.

I have  designated Mr.  Robert Schneider as  this Division's  repre-
sentative on  this corrr.'ii ttee  and  I would like to  solicit  you as a
representative  also.   I have asked  Mr. Schneider to  be  in  touch  with
you to  arrange  a rceeting  at  the  earliest  convenience.  A copy  of my
letter  to the Regional Office  is also enclosed.  I believe  that  your
participation on this committee  is  absolutely essential  to  the de-
velopment- of  relevant recommendations to  the Conferees.

Your  early  attention to this matter v/Ill  be  greatly  appreciated.

                                      Sincerely,
                                      Thomas  P.  Gallagher
                                      Di rector
encl.  (1)
  Cy memo  12/6/71  to  Act.  Dir.
    Enf. Div.,  Reg.  IV
                               -16-

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            DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE

                        PUBLIC HEALTH SERVICE

                    FOOD AND DRUG ADMINISTRATION
                        WASHINGTON. D.C. 20204
                     December  14,  1971
Mr. Thomas P. Gallagher
Director
Environmental Protection Agency
Office of Enforcement
Division of Field Investigations
Building 22, Room 410
Denver, Colorado 80225

Dear Tom:

This replies to your letter of December  6  regarding the
establishment of a technical committee to  consider recommendation
No. 6 of the Mobile Bay Enforcement  Conference.   I would be
pleased to participate on this committee and  to  attend a
meeting of the committee in early  1972.  1 would also like
to request that Mr. Daniel A. Hunt,  Assistant Director,  be
appointed as my alternate to the committee.

I will look forward to hearing from  you  or Mr. Robert Schneider
regarding the time and place of the  meeting.
                         Sincerely yours,
                         J. David  Clem
                         Director
                         Division  of  Shellfish  Sanitation (BF-230)
                         Office of Food  Sanitation
                         Bureau of Foods
                               -17-

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                          ENVIRONMENTAL PROTECTION AGEN'"'
                                                    i

  Date:  December 23, 1971

Reply to  Director, Enforcement Division
Ann of:  Region !v

Subject:  Establishment of Shellfish Technical Committee--Mobile Bay

   To:  Director, DFI - Denver Center
        The proposal set forth in your memorandum of December 6  concerning the
        above subject is satisfactory.

        Mr. Traina has authorized Mr.  Jim Silva to serve  as  regional  representa-
        tive on the committee.
                                       -18-

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                NVIRONMENTAL PROTECTION AGE^ ^Y
                     OFFICE  OF ENFORCEMENT
        DIVISION OF FIELD  INVESTIGATIONS-DENVER CENTER
          BUILDING   22 • ROOM  ^10 • DENVER FEDERAL CENTER
                     DENVER, COLORADO 80225
                                       k January 1972
J. David Clem, Director
Division of Shellfish Sanitation
Office of Food Sanitation
Bureau of Foods
Washington, D.C.

Dear Dave:

The first meeting of the Technical  Committee to consider
recommendation No; 6 of the Mobile Bay Enforcement  Conference
wi11 be held in:

                     Environmental  Protection Agency
                     Region IV Office
                     3rd Floor
                     1A21 Peachtree Street,  NE
                     Atlanta,  Georgia

We will meet in Mr. Jim Silva's office at  1:00 p.m. on
Tuesday, January 18, 1972.   I  have prepared  a charge  that
we will discuss at this first  meeting.

1 will look forward to seeing  you in Atlanta.

                                  Sincerely,
                                  Robert  F.  Schneider, Acting Chief
                                  Biology Branch
RFS:ls
cc:  T. P.  Gallagher, Director,  DFI-DC
     F. J.  Silva, Atlanta Regional  Office
                        -19-

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                          ENVIRONMENTAL PROTECTION AGENCY
                                OFFICE OF ENFORCEMENT
                   DIVISION OF  FIELD INVESTIGATIONS-DENVER CENTER
                     BUILDING   22 , ROOM   ^10 , DENVER FEDERAL CENTER
                                DENVER, COLORADO  80225
COPY

10    :   Mr. F. J. Silva                                   DATE:  12 January 1972
        Atlanta Regional Office


FROM  :   Chief, Biology Section


SUBJECT:   Postponement of Technical Committee to Study Mobile Bay Enforcement
        Conference Recommendation No. 6.
        Following our telephone conversation of January 10,  1972 I  contacted
        Mr. Clem to advise him of the need to postpone our Technical  Committee
        Meeting until February.  He has arranged his calendar accordingly.

        As  it is set up now, we will meet with you in your Atlanta  Office
        at  1:00 p.m. on Tuesday, February 1, 1972.  There will  be five of
        us at this first meeting as follows:

                            Comm i 11ee Membe r s

                     Robert Schneider - DFI-DC
                     Jim Silva        - Atlanta Region - EPA
                     Dave Clem        - FDA Headquarters

                                  Others
                     Bobby Carroll    - SEWL - EPA
                     Roger Olmsted    - Atlanta Region - FDA

        Thanks for letting me know of your January calendar conflict.   I  am
        looking forward to our February meeting.
                                           R. F.  Schneider

        RFS/lms

        cc:  T. P. Gallagher, DFI-DC
             J. D. Clem, FDA, Washington, D.C.
             B. J. Carroll, SEWL, Athens, GA
                                      -20-

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 MEMORANDUM       DEPARTMENT OF HEALTH, ED'  VTION, AND WELFARE
                                            PUBLIC HEALTH SERVICE
                                        FOOD AND DRUG ADMINISTRATION

       Regional Food  ft  Drug  Director
10    :  (ATL-F1)                                 DATE:  January 17,  1972

       ATTN:  Chief,  Special Programs Branch  (ATL-F15)


       Director
 ROM  :  Division of Shellfish Sanitation  (BF-230)
       Office of Food Sanitation/Bureau  of  Foods

 Jlill^CT1
       First. Meeting  of the  Technical Committee on Recommendation 6
       of the Mobile  Bay Enforcement Conference


       1.  This is to confirm  my plans to visit the  Regional
       Office, February 1, in  connection with a meeting of the
       technical committee organized to  consider  recommendation
       No. 6 of the Mobile Bay Enforcement  Conference.   I  suggest
       that Mr. Roger Olmsted  accompany  me  to the meeting  that
       has been scheduled for  1:00 p.m.  in  Mr.  Jim Silva's office.
       I plan to visit your  office, opening of business, Feb.  1.
                              JX David  Clem
       cc=  R. Schneider, EPA, Colorado
            T. Gallagher, DFI-DC
            F. Silva, ATL Regional Office
            R. Dimsted, ATL Regional Office
                                   -21-

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                                          I
                  ENVIRONMENTAL PROTECTION AGENCY
                        OFFICE  OF ENFORCEMENT
           DIVISION OF FIELD  INVESTIGATIONS-DENVER CENTER
             BUILDING   22 . ROOM  /no , DENVER FEDERAL  CENTER
                        DENVER. COLORADO 80225
                                       10 February 1972
Mr. James McDermott
1921 Jefferson Davis Highway
Arlington, Virginia   202^2

Dear Mr. McDermott:

Through the Environmental Protection Agency - Office of Enforcement,
I  have been requested to establish a committee to recommend acceptable
bacteriological criteria for Mobile Bay shellfish harvesting areas.
I  would Hke to solicit the services of Mr. C. B. Kelly of your staff
to join this committee as a representative for Water Hygiene.

As a way of giving you some background, I  will briefly describe the
history behind this request.  On January 27 and 28,  1970 a Conference
called by the Secretary of the Interior was held in  Mobile, Alabama
concerning the economic effects of pollution in Mobile Bay upon the
Alabama shellfish  industry.  Recommendation No. 6 of the "Proceedings--
Conference in the Matter of Pollution Affecting Shellfish Harvesting
in Mobile Bay, Alabama" deals with shellfish growing water criteria.
The recommendation reads as follows:  "In  determining acceptability
of areas in Mobile Bay for shellfish harvesting, a more realistic
criterion than the present methods [total  coliform density of  70 per
100 ml] be employed to more effectively evaluate the probability of
pathogen presence.  A coordinated effort by the U. S.  Department of
Health, Education and Welfare and the U. S. Department of the  Interior
(now E.P.A. responsibility) in cooperation with the  states should be
undertaken immediately to develop such a criterion with respect to
approval of areas for shellfish harvesting.  A technical  committee
shall be established by the Conferees to assist in the development
of such a criterion."
                                -22-

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Page 2
Mr. James McDermott
Arlington, Virginia
10 February 1972
Currently, the technical committee membership consists of:  Robert F.
Schneider - Committee Chairman; F. James Silva - E.P.A. Regional
Representative; J. David Clem - DHEW Representative.

At a recent committee organization meeting in Atlanta, Georgia
Mr. Kelly's name was suggested as a committee member to represent
Water Hygiene.  Because of his vast experience in microbiology and
knowledge of shellfish problems I  believe  his participation on the
technical committee is essential to successful development of relevant
recommendations to the Conferees.

Arrangements are being made at the Water Hygiene Dauphin  Island
facility in Mobile, Alabama for a technical committee meeting place.
The first meeting of the technical committee  is scheduled for March
23i 1972.  All committee members will be provided with a specific
schedule as soon as arrangements are final.

Your early attention to this matter will be greatly appreciated.

                                         Sincerely,
                                         Robert F. Schneider
                                         Chief, Biology Branch
RFS/ls

be:  Mr. Kelly
                                  -23-

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              ENVIRONMENTAL PROTECTION AGENCY
                      WASHINGTON,  D.C.   20460
                                                   MR  2 -  1972
Mr. Robert F. Schneider
Chief, Biology Branch
Division of Field Investigations  — Denver Center
Office of Enforcement
Environmental Protection Agency
Denver, Colorado

Dear Mr. Schneider:

    I shall be pleased to assign  Mr. C. B. Kelly  to serve on the
Technical Committee  to develop  criteria of quality for shellfish
growing areas.  He will be prepared  to attend a meeting of the
committee to be held at Dauphin Island, Alabama on March 24, 1972

                                    Sincerely yours,
                                    James H. McDermott, P.E.
                                    Director
                                    Water Supply Programs Division

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                   22         410
                                          15 February 1972
Mr. C. B. Kelly
Special Assistant for Research
Division of Criterion Standards
Uater Supply Program Division
fcoom 503. Crystal 1^11 Buildin.R 2
Washington, D.C.   20460

Dear Mr. Kelly:

On behalf of the Technical Committee I would like to velcone you
as a member.  I an looking forward to eeeting with you and others
of the Technical Coraeftttee to disenss our task of developing a
Eoro realistic criterion for shellfish growing areas in Mobile,
Alabana.

The Technical Coranittee membership is as follows:
      1) J. David Clem, PDA Representative;
      2) F. Janes Silva, EPA Regional ReprcBcntative;
      3) Arthur Bock, Alabama Water Improvement and Health
          Departncint Repreaentat ivc;
      4) James Coil, Pulp and Paper Industry Representative; and
      5) VT. B. Anderson, Alabana Conservation and Natural
          ResourccB Representative.

A Technical Corsnittea neetln^ in bcln?, planned and will be held
at the E.P.A. Kater Hygiene Laboratory, Dauphin Island, Mobile,
Alabama on Itorch 24, 1972.  You will be provided with a specific
standard when arrangements are final.

                                 Sincerely,
                                 Robert F. Schneider
                                 Chief, Biology Branch
RFS/ls
                                 -25-

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                     22          410
                                          February 1972
Mr. Arthur 11. Brick, Technical  Secretary
Water Improvement Centals s ion
State Office Building
Ifontgoiaery, Alabaua   3G104

Dear JJr. Beck:

On behalf of the Technical Copsnittee  I would lite to wclcarui
you SB a cessbor.  I as loaltiag forward to neccln^ with you aad
others of the Technical Contralttac  to  discucs o-.ir task of
developing a eoro realistic criteria  for  shellfish growing
areas la libblle Bay.

A Technical CcKnrf.ttee tisctinq  Is boinj; planned and vill be
held in the E. ?. A. Water Hygiene laboratory. Dauphin Island,
Mobile, Alahana on March 24, 1972.  You will be provided with a
specific schedule &G soon as arrsugerasnts are final.

                                       Sincerely,
                                      Robert  F.  Schneider
                                      Chief,  Biology Branch
BFS/la
                                -26-

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                   22          410
                                      14 February 1972
Hr. James Coil
lianager of Adainistrative Services
Scott Paper Conpany
P. 0. Box 2447
Mobile, Alabama   36601

Dear Hr. Coll:

Pursuant to our telephone conversation of February 11, 1972 rsay
I take this opportunity to welcoTjO you to the Mobile Bay Tech-
nical Coiarcittce.  I an looking forward to meeting with you and
others of tho Ccraaittae to discusc onr challenging task cf
rcconasendlng acceptable bacteriological criteria for shellfifih
areas in Mobile Bay.

A Technical Cornaitteo Kocting is beinR plamied and will be held
at the S. P. A. Wattr Hygiene Laboratory, Dauphin Island, Mobile,
Alabana on March 2h, 1972.  You will be provided Kith specifics
as soon as &rrangeaent8 arc final.

                                 Sincerely,
                                 Robert F. Schneider
                                 Chief, Biology Branch
RFS/lo
                             -27-

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  APPENDIX B




PROGRESS REPORT
    -28-

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                             PROGRESS REPORT




                     MOBILE BAY TECHNICAL COMMITTEE




                             R. F. Schneider






     On January 27-28, 1970 in the City of Mobile, Alabama, a




Conference  in the Matter of Pollution Affecting Shellfish in Mobile




Bay, Alabama was called by the Secretary of the Interior.  Recommen-



dations contained in the Conference Proceedings included one that




emphasized  reevaluation of the bacteriological criterion for shell-




fish harvesting areas.



     Recommendation No. 6 reads:  "In determining acceptability



of areas in Mobile Bay for shellfish harvesting, a more realistic




criterion than the present method be employed to more effectively



evaluate the probability of pathogen presence.  A coordinated effort




by the U. S. Department of Health, Education and Welfare and the U. S.



Department of the Interior, in cooperation with the States, should be



undertaken  immediately to develop such a criterion with respect to



approval of areas for shellfish harvesting.  A technical committee



shall be established by the Conferees to assist in the development of



such a criterion.  This committee should report to the Conferees no




later than one year from the date of the first session of conference."



     In compliance with Recommendation No. 6 of the Mobile Bay Enforce-



ment Conference the following persons agreed to serve on the Technical



Committee:     Mr. Robert Schneider, NFIC-Denver



              Mr. J. David Clem, DHEW, Washington, D.C.



              Mr. F. James Silva, EPA, Atlanta, Georgia
                                -29-

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     On February 1, 1972, the members listed above met in Atlanta,




Georgia with Mr. Bobby Carroll, Chief Bacteriologist, EPA, Southeastern




U. S. Region and Mr. Roger Olmstead, FDA, Southeastern U. S. Region.




This meeting had a multi-purpose:  (1)  recommend prospective representa-



tives from the pulp and paper industry, fishery industry and the State



of Alabama; (2) review the highlights of the 1970 Mobile Bay Enforcement




Conference; (3) discuss the rationale behind the present bacteriological



criterion for shellfish growing areas and (1») jointly review the charge




to the technical committee.



     Suggested additions to the Technical Committee membership included:



Mr. C. B. Kelly of EPA to represent the Water Hygiene Program; Mr. James



Coil of Scott Paper Company to represent the pulp and paper industry;



Mr. Arthur Beck, the Chief Sanitary Engineer of Alabama to represent



the Alabama Water  Improvement Commission and the Alabama Health Depart-



ment; and Mr. W.  F. Anderson, Chief of  the Alabama Marine Resources to




represent the Alabama Department of Conservation and Natural Resources.




These individuals have agreed to serve  on the Technical Committee in



the capacity noted above.



     Highlights of the 1970 Enforcement Conference Proceeding and the



rationale behind the total coliform standard for shellfish growing



areas were reviewed by attendees at the meeting in Atlanta, Georgia on




February I, 1972.  Discussions centered around tne fact that during tne




late winter or spring, flood runoff from the Tombigbee-Mobile River



system transports  large coliform concentrations into Mobile Bay.   The
                                 -30-

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sanitary significance of these total coliform densities is unknown; yet,

shellfishing areas in Mobile Bay have been closed by the State of Alabama

because the bacterial densities over the shellfish beds exceeded the

70/100 ml  total coliform standard for harvesting.  The region has suffered

substantial economic.losses because of these closures.

     The most recent closure is in effect, curreitly.  On January 28,

1972 the State of Alabama closed all areas of Mobile Bay east of Dauphin

Island Bridge because the total coliform density exceeded the 70/100 ml

criterion.   This closure means that some of the most productive shell-

fish areas  of the Bay (Bon Secour Bay) are closed to harvesting for at

least a portion of the 1972 season.  Economic loss to the region because

of past closures (excluding the 1972 closure) is estimated to exceed

200 thousand dollars.

     After  reviewing and discussing Recommendation No. 6 and other

shellfish related problems in Mobile Bay the following charge to the

Technical  Committee was developed:

     "The Technical Committee  is requested to assist in the development
                                                      •
of a more realistic bacteriological criterion than the present method

[total coliform density of 70/100 ml] for determining the probability

of pathogen presence in Mobile Bay shellfish harvesting waters.  To

accomplish  this goal the Committee may form such sub-committees as

needed to review various avenues of approach, provide technical infor-

mation or conduct studies.  The Committee will select an approach and

will set into motion a scheduled program to fulfill  its charge.  Further-

more, the Committee may exercise such latitude as required in fulfil ling


                                  -31-

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its charge and shall meet at such times and places as necessary."




     In response to this charge the Technical Committee has agreed to




meet in Mobile, Alabama on March 2*t, 1972.  Various bacteriological




criteria for determining the acceptability of Mobile Bay waters for




shellfish harvesting will be discussed.  Consideration will be given to



the sanitary significance of fecal coliform, fecal streptococci,



FC/FS ratio, or other parameters that could be used for evaluating




shellfish harvesting waters.  The Committee will appoint a subcommittee



to review technical reports concerning the "State of the Art" and



sanitary significance of the various bacteriological criteria.  The




subcommittee should be prepared to submit a written report of its




findings to the Technical Committee by August 1, 1972.  After review-



ing the subcommittee findings,  the Technical Committee will meet at




their earliest opportunity (no later than September 15, 1972) to formu-



late a  recommendation.   The Committee chairman will prepare and submit



the final  recommendation to the Conferees by October 1, 1972.
                                 -32-

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              APPENDIX C









MOBILE BAY TECHNICAL COMMITTEE MEETING




       DAUPHIN ISLAND, ALABAMA




            MARCH 2k, 1972
                 -33-

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                    22
                                               I March 1972
Mr. James Coil
Manager of Administrative Services
Scott Paper Company
P.O. Box 2^7
Mobile, Alabama  36601

Dear Mr. Coll:

The technical conijnltteo to consider recommendation no. 6 of the
Mobile Bay Enforcement Conference will r.eet in Mobile, Alabama
on March 2*1, 1972.  The meeting Is scheduled for H:00 a.m. In
the library (conference room) of the V.'ater Hycilene laboratory
located on Dauphin Island.   If you need specific directions,
please contact:  Richard J. Hammerstro:n, Director
                 Gulf Coast Water Supply Laboratory
                 EPA
                 P.O. Box 15-3
                 Dauphin island, Alabama  3&52S
                 Phone:  205/861-2962 (not an FTS number)

At the meetInn we plan to discuss several bacteriological criteria
for determining the acceptability of Mobile Bay waters for shell-
fish harvesting.  It v«uld be helpful to the committee If you
would present your views on the use of fecal coliform tests (tempera-
ture elevated bacteria) versus E. col I (IMVIC) tests or other alter-
nate methods to determine sanitary conditions In shellfish harvest-
Ing areas.

I am looking forward to x^orklng with you on this technical
committee.

                                   Sincerely,
                                   Robert F. Schneider
                                   Chief, Biology Branch
RFS/ls

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                    22
                                         I March 1972
Mr. J. David Clean
Division of Shellfish Sanitation
Bureau of Foods
Washington, D.C.

Dear Dave:

Plans have been finalized for our technical committee meeting.
We will meet at the Water Hygiene Laboratory on Dauphin Island
at 9:00 a.m. on March 2-'», 1972.

Because four of the committee members were absent from the
Atlanta meeting, there wl11 be a need for repeating some of
our previous discussions.  Would you discuss the rationale
behind the present bacteriological criterion for shellfish
growing areas as you did for us in Atlanta?

I will be looking forward to working with you again on March 2*

                                   Sincerely,
                                   Robert F. Schneider
                                   Chief, Biology Branch


RFS/ls
                                 -35-

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                    22
                                   1 March 1972
Mr. F. J. Sllva
EPA, Region IV
Suite 300
U21 Pcachtroe Street, NE
Atlanta, Scorpio  30309

Dear Jim:

Our technical  meeting is set for 9:00 a.m. on March 2h at the
Water Hygiene Laboratory, Dauphin Island, Alolxv-ia.  Thinks for
all your special efforts as I mentioned in our recent telephone
conversation.   I do have one more favor to ask of you.  Because
four of the coffinIttee nnnbers were absent fron the Atlanta
meeting, there will be a need for repeating some of our previous
discussions,  l.'ould you review the highlights of the 1970
Mobile ?ay Enforcement Conference?  This would 31 ve the ricmbers
of our full connittee a common background.

I'll be looking forvwrd to our Dauphin Island meeting \vith the
opportunity to work with you on tills long overdue shellfish
standard revision.

                                   Sincerely,
                                   Robert F. Schneider
                                   Chief, Blolociy Branch
RFS/ls
                          -36-

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                     22


                                                1 March  1972
Mr. Arthur H. Beck, Technical Secretary
Water Improvement Co-Tirclsslon
State Office Building
Montgomery, Alabaraa   3610*»

Dear Mr. Deck:

The technical committee to consider recommendation no. 6 of the
Mobile !3ay Enforcement Conference will meet  In Mobile, Alabama
on March 24, 1372.  The meeting  is scheduled for 9:00 a.m.  In
tho library  (conference room) of the l/ater liyglcnc laboratory
located on Dauphin Island.   If you need specific directions please
contact:  Richard J. l-te-r.ierstro.T?, Director
          Gulf Coast Water Supply Laboratory
          EPA
          P.O. Box 153
          Dauphin island, Alabama  36528
          Phone:  205/361-2962 (not an FTS number)

Some of the corral ttee members are not fatal liar with the recent
closure of Mobile Bay.  At our March 24 meeting would you brief
the controlttee on the Bay closure and discuss the State of Alabama
views on the sanitary significance of the present bacteriological
criterion?

I asi looking forward to working with you on this special technical
committee.

                                   Sincerely,
                                   Robert F. Schneider
                                   Chief, Biology Branch
RFS/ils
                            -37-

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                     22          410
                                                1  March 1572
Mr. C. B. Kelly
Special Assistant  for jJese.irch
Division of Criterion Standards
Water Supply Program Division
ROOM 509, Crystal  ttall  Suilulnn  2
Washington O.C.    20'»GO

Dear Mr. Kclly:

The technical convnlttee to  consider rcconricntlntlon no. 6 of the
Mobile Bay Enforcement  Conference  will  .-.icct in .Mobile, Alabama
on March 2'j, 137-2.  The Kieetinq  Is scheduled for 3:^0 a.m. In
tho library (conference roo-~>) of the. Water Hygiene laboratory
located on Dauphin Island,   if yo:i nccsd specific directions
please contact:  Richard J.  Narwujrstrom, Director
                 Gulf Coost  V/ator  Supply Laboratory
                 EPA
                 P.O. Cox 1!#
                 Douphln Island, Alabama   36523
                 Phone:  205/561-2562 (not an FT5 number)

At the. laeetln? \:o.  pl«5n  to discuss  several bacteriol or, lea I criteria
for determining the acceptability  of Mobile Say waters for shell-
fish harvesting,   it would  be helpful  to th& coi.TU:!ttGc If you
would present your views on  the  sanitary stqnlfIcanee of various
bacteriological indicators  such  as fecal collforni.

I am looking forward to meeting  you and having the opportunity
to worlclaogcther on developing a rccorraenciatlon for the Conferees.

                                    Sincerely,
                                    Robert  F.  Schneider
                                    Chief,  Biology Oranch
RFS/ls
                              -33-

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                    22         410
                                      2 March 1572
Mr. W. G. Anderson
Department of Conservation and Natural
 Resources
Marine Resources Division
P.O. Box 186
Dauphin Island, Alabama  36523

Dear Mr. Anderson:

The technical corns taca to consider recoracndatlcn no. 6 of the
Mobile Bay Enforcement Conference will fncet In Mobile, Alabama on
March ?.•'», 19/2.  Th2 meeting Is schctJuled for 5:00 a.ci. In the
library (conference! room) of the Water Hygiene laboratory located
on Dauphin Island,  tf you need specific directions please contact:
           Richard J. Mannerstram, Director
           Gulf Coast Water Supply Laboratory
           EPA
           P.O. Box 158
           O.iuphfn Island, Alabama 36523
           Phone:  205/2&1-22&2 (not an FTS number)

Recently, 1 lesrneJ that Mobile Day Is closed to shellfish harvest-
ing again this season.  It would be helpful to the co~rclttce If
you would present soi:»e Inforrnatlon concerning the economic loss or
other problems to the Mobile area because of this closure.

I'n lookInq forward to having the opportunity of working with you
on this technical committee.

                                 Sincerely,
                                 Robert F. Schneider
                                 Chief, Biology Sranch
RFS/ls
                            -39-

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                                    State of Alabama
                           Department of Public  Health
                                   State Office Building
                                Montgomery, Alabama
IRA L. MYERS, M. D.
STATE HEALTH OFFICER
                                      March  6,  1972
      Mr. Robert F. Schneider
      Chief, Biology Branch
      Environmental Protection Agency
      Water Quality Office
      Bldg. 22, Room 410
      Denver Federal Center
      Denver, Colorado  80225

      Dear Mr. Schneider:

                Thank you for your  letter  of  March  1,  1972 advising me of
      the meeting date and place of  the newly formed Technical Committee to
      implement the recommendations  of No.  6  of  the Mobile Bay Enforcement
      Conference.

                I will be happy to make any contribution that I can to the
      Committee members at the March 24th.  meeting.
                                      Yours ver
      ANB/iw
 fief Engineer & Director
Bureau of Environmental Health

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             Status of Bacteriological Criteria
                      for Shellfish Areas
     The fecal coliform indicator group has been under

consideration by the National Shellfish Sanitation Program

(NSSP) .as a substitute for the coliform group in the

bactcr.iologicn] criteria for shellfish-growing areas for a

number of years.  A fecal coliform standard of 7.8 was

proposed at the Fifth National Workshop in 1964 which
         *
represented a coliform/fccal coliform ratio of approximately

10/1 when compared to the present coliform standard.  There

appeared to be an assumption by the Fifth Workshop that any

new criteria or standard must have a consistent correlation

with the existing coliform standard.  The proposed standard

was rejected on the basis of insufficient data and additional

studies were recommended.

     The Microbiology Task Force of the Sixth National Workshop

again concluded that there was insufficient evidence for

recommending a fecal coliform standard and requested that

further work be done.  .In response to this request, shellfish

sanitation personnel hnvc analyzed growing area data from

Louisiana, Mississippi, Alabama, Florida, Georgia, Virginia,

New York, Rhode Island, Massachusetts, and Maine.  These data


Prep;M-cIl~Fy~ tiTe" Division of Shellfish Sanitation, Rurc.iu of Foods,
Food and Dru«> Administration, Washington, D. C. 20204 for prcsenta
tion ;it Mobile; Hay Technical Committee Meeting, Dauphin Island,
Al.-ibamu, March 21, 1972.

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                                2
 indicate  that  there  is  no  consistent  coliform/fecal  coliform
 .relationship  in  cstuarine  waters.   The  coliform/fecal  coliform
 ratio  was  found  to vary from 1/1  to 10/1  depending upon such
 factors  as  geographic  location, meteorological  conditions,
 season,  pollution source,  distance  of sampling  station from
 pollution  source (dilution effect)  and  salinity,  nutrients,
 or bacterial  inhibitors of receiving  waters.  We  have  not
 been able  to  correlate  these factors  with the actual
 bacteriological,  data nor determine  how  these  factors relate
 to the differences in  coliform/fecal  ratios.  Tlie Division
 of Shellfish  Sanitation concluded that  any new  indicator
 criteria  should  be evaluated according  to the sanitary
 significance  of  the  specific indicator  and not  according
 to the relationship  between the indicator group being  evaluated
 and tiie  present  criteria.   In review  of the fecal coliform
 water  quality  data obtained from  State  shellfish  control
 programs,  very few of  these data  had  been correlated with
 known  pollution  sources.   It has  been very difficult to make
 concise  public health  appraisal of  these  data for lack of
 in depth  sanitary survey information.
      During the  Microbiology Task Force deliberations  at the
 Seventh  National Workshop  in October  1971,  median fecnl coliform
• values o (:  7.8  and 
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                               3



A spokesman from Louisiana stated that a 23 fecal coliform



MPN median would close a high percent of "approved" shellfish-



growing areas in his State which could not be shown to be



affected by known sources of pollution.  Delegates from



Rhode Island proposed that for the present coliform standard



that "not more than 10 percent of the samples ordinarily



exceed an MPN of 230 per 100 ml." be omitted from the present



coliform standard.   These actions may give you some indication



as to the diversity of opinions expressed in this task force.



Certainly, one must conclude that there is a iNational n'eed



to reconsider and evaluate bacteriological criteria for



approved shel]fish-growing areas.  After considerable



discussion, all but two State representatives present in the



task force voted to support FDA's proposal to retain the
                                                             \


present growing area standards based upon the coliform indicator



group.



     On February 17, 1972, a representative of the Division of



Shellfish Sanitation participated in an Ad Hoc Committee



meeting on bacteriology, which included representatives of



the Environmental Protection Agency (EPA), Oregon Dept. of



Environmental Quality, the academic community, the medical



profession, the National Council on Stream and Air Improvement,
                                -43-

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                               4


and the pulp and paper mill industry.   The significance of
 t-

Klcbsiclla pneumonia was discussed.  This organism, a member


of the coliform and in part the fecal  coliform groups was


reported found in high numbers in paper mill wastes.  It


may also be found in other waste effluents high in reducing


sugars and other carbohydrates such as the sugar industry,


food-processing plants, and some domestic effluents.  IMViC


reactions will not separate Klebsiella pneumoniae from


Entero_b_acter aerogcnes.


     In attempting to interpret existing data on Mobile Bay


waters, there is no way to determine the percentage of the


reported coliform or fecal coliform valuesi that are actually


K. pneumoniae.  Such information would be valuable to the


agency responsible for the classification of shellfish-growing


areas in the interpretation of sanitary survey data considering


that EFA has reported that 51% of the  total coliform waste


source contribution to Mobile Bay is from paper mill wastes.


     During the Ad Hoc Committee meeting, a number of


characteristics of this organism were  discussed which are


significant to agencies using coliform or fecal coliform


criteria in the classification of environmental water quality.


These are listed as follows:

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                               5
          a.  K. pneumoniae is a member of the coliform group.
          b.  A large percentage ('10-60S) of the K. pneumoniae
isolated from environmental samples have been reported to
ferment lactose at 44.5°C., and, therefore, vould be reported
in the indicator system as a fecal coliform type.
          c.  K. pneumoniae has the same IMViC reactions as
Enterobacter aerogenes.

          d.  K. pneumoniae is heavily encapsulated and," has
                                                 f
a longer survival potential in environmental waters than
enteric pathogens or E.  co1i.

          e.  K_.	pneumoniae can "colonize" environmental
waters rich in carbohydrates and reducing sugars (such as
paper mil] wastes in Mobile Bay).
          f.  K. pneumoniae is a normal inhabitant of the
intestinal tract of warmblooded animals, but is generally
found in low numbers compared to F..  co 1 i populations .
          g.  K. pneumoniae is highly resistant to antibiotics
and is regarded as a "secondary" pathogen by the medical
profession.  It usually appears as a pathogen following
treatment of other enteric bacterial infections after normal
populations of bacterial flora have been modified by
antibiotic treatment.
                                -45-

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                               6
          h.  The presence of F.. co 1 i in the intestinal
tract of healthy individuals may restrict the growth of
K. pneumoniac and help prevent "colonization" of the
individual.
          i.  The presence of effluents enriched with
carbohydrates such as paper mill wastes in shellfish harvest
areas can have an adverse affect on the classification of
such areas, especially if aftergrowth or "colonization".
occurs.
          j.  Aftergrowth of K. pneumonias can result in
high counts of both coliform and focal coliform groups
which may be totally unrelated to actual pollution sources,
representing real health hazards to consumers of shellfish.
     In view of the confusion surrounding an "acceptable"
value of fecal coliform organisms to be used as a substitute
for the present coliform standard, FDA proposed to the
Seventh National Workshop that NS>SP take a closer look at
the more specific 45.5°C test for E. coli.  This was first
proposed to the American Public Health Association Committee
on shellfish methodology by its chairman, Dr. C. A. Perry,
in 1936.. Gcldrcich states that the fecal coliform test
at 4-1.5  (i-0.5°)C represents a, ". . . compromise between
acceptable sensitivity and specificity for the fecal types."

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                               7
For regulatory purposes, the FDA has been successful in the
courts in using E. coli as an indicator of fecal pollution.
Perhaps, the time has come in the history of the NSSP, that
we must re-evaluate our concepts and be willing to lose
sensitivity for an increase in specificity in the promulgation
of both bacteriological criteria and standards for "approved"
shellfish harvest areas.  Currently, neither the FDA nor the
NSSP has been convinced of the wisdom of substituting the
         *                                         /
narrow spectrum fecal coliform test for the broad spectrum
coliforin group.
     If we make errors in standard setting, let it be on
the side of safety.  On the other hand, FD.A'and cooperating
State agencies responsible for growing area control have
no desire to prevent utilization of a natural resource by
closing harvest areas where indicator levels exceed present
standards,  unless these indicator levels, according to the
best professional knowledge and experience, constitute a
real health hazard.
     In conclusion, we realize the need for a more specific
indicator and more realistic standards for use as an adjunct
to the sanitary survey in defining "approved" growing areas.
Any substitute fnr the present criteria should:  (1)  be more
specif.ic for fecal pollution than the current indicator group,
                              -47-

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                               8


 (2) stand on its own merit as an indicator of actual sources


 of  fecal pollution without a dependency upon a specific


 relationship with the coliform group,  (3) be more representative


 of  the potential pathogen load, (4) consistently indicate the


 sanitary quality of shellfish-growing waters in diverse


 geographical locations,  (5) and have a survival time ratio


 in  the estuary consistent with that of other bacterial pathogens


 regardless of nutrient value of the water.


     As previously stated, the Seventh National Workshop, the
                                                       V-
                                                 f      \
 NSSP supported the FDA position by a large majority that the


 fecal coliform has not adequately fulfilled the qualifications


 necessary for the promulgation of a fecal coliform standard

                                          i
 for "approved" shellfish harvest areas.  The need for a re-


 evaluation and renewed effort to develop new criteria is


 recognized.


     We recommend that 1-. coli be considered as a promising


 bacteriological indicator for classifying shellfish harvest


 areas and that necessary research be initiated to develop and/or


 evaluate rapid tests for the recovery of this organism.  The


 long-term goal would be new bacteriological  criteria and


.standards- for the classification of growing areas.  It is
                              -48-

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our considered opinion that this  effort  should  receive



immediate attention and research  investment  by  l-'cderal and



State agencies concerned with shellfish  sanitation  control,
March 22, 1972
                            D.. A. Hunt
                              -1*9-

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                                             -   March  24,  1972

COPY

                 Economic  Loss  of  Oyster  Reef Closure

                              1971  -  1972
 This  season  Alabama's  oyster  reefs  were  closed  on  January  30  and

 February  4  (Productive reefs)  and have  remained closed  for the  past

 50 days.



 The average  dockside value  of  the oyster catch  is  $1,671 or a total

 loss  of $83,550.   However,  production this  yearvas  above average

 and was worth  $1,916 per  day  in  December 1971.   At  this figure  the

 total  dockside loss has been  $95,800 during the current season.



 The dockside value represents  only  loss  to  the  fisherman.   It

 has been  shown that the total  economic value of oyster  production

 in Alabama  is  four times  the  dockside value.  Thus,  the total

 economic  loss  to  the fisheries community as a result of the current

 pollution closure is $382,400.
 Prepared  by  the Alabama  Department of Conservation and Natural
 Resources  for  presentation at Mobile Bay Technical Committee Meet1"
 ing,  Dauphin  island, Alabama, March 24,  1972.
                                -50-

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Donald W. Lear, 1962, Growth of bacteria in estuarine waters.  Chesa-
       peake Science 3 (3):  160-165-

—Organisms used were Escher ichia col i,  E_. freundi i ,  and Aerobacter
  aerogenes.

—Nutrients were added to filtered distilled water and to filtered
  estuarine water (1^.1 ppt).

--Growth was better in the saline waters than in the distilled
  waters.

--Cites papers by Kelly and Arcise  (195*0 which show increase in
  coliform MPN in oysters shipped--indicat ing growth in saline
  conditions utilizing food sources within the oyster.

--Implication is that  if nutrient sources are available in saline
  waters total coliform count will  increase.

Gallagher, T.P. and D.F. Spino, 1968, The significance of numbers
       of coliform bacteria as an indicator of enteric pathogens.
       Water Research, Vol. 2, 169-175.
       The traditional concept of using levels of total coliform

densities to describe bacteriological acceptability has been ques-

tioned by many because coliform may result from causes other than

fecal pollution.  Because fecal coliform densities result from

pollution by man and other warm blooded animals, they are more

directly indicative of the probable presence of the enteric

associated pathogens.  However, summarized data from several

stream surveys over the past few years show little apparent

correlation between quantities of total or fecal coliform and

the probable isolation of salmonellae.  Salmonellae were isolated

at total coliform densities of less than 1000/100 ml  and fecal

coliform densities of less than 150/100 ml.  The total coliform

density of 1000/100 ml has traditionally be«_.i acceptable for recrea-

tional waters.   It is probable that salmonellae will  be isolated

at high fecal  coliform densities but observed data has indicated that

isolation is also probable at low coliform densities  if there is an
Prepared by the Alabama Department of Conservation and Natural
Resources for  presentation at Mobile Bay Technical Committee Meeting
Dauphin Island, Alabama, March 2*1, 1972.

-------
inadequately disinfected v/aste source which may affect the area of




concern.  In one instance cited, the area was 73 miles and 4 days




travel time downstream from sources of waste.  This may be because




salmonellae are persistent under conditions which may be adverse




to survival  of fecal coliform.  Large percentage reductions of




coliform without chlorination will not of itself guarantee bacter-




iological acceptability.  Data show that salmonellae have been



isolated after a 99 percent reduction in fecal coliform below unchlorinated




waste sources.



       Although salmonellae at present are the only pathogens which




may be isolated from water by means of a routine test with any




degree of reliability, it is probable that methods for other




pathogens will be perfected in the near future.  Until that time,




the coliform will still be valuable as indicators of pollution.




However, low values of total and fecal coliform are not, of them-




selves, sufficient to indicate bacteriological safety from patho-




genic organisms.  A control program to more effectively evaluate



bacteriological safety in recreational waters should include:  (l) a




sanitary survey to delineate possible sources of pollution;




(2) effective disinfection of all  waste sources which may affect the




area; (3) use of the salmonella test in addition to total and fecal




coliform as  a monitoring device.
                             -52-

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Rubin, A. J., et al, 19&9, Coagulation of Escherichia coli  by neutral
       salts.  Water Research.  Vol. 3, 8^3-852.
       Shows £. col i  to coagulate at about 23 ppt NaCl (.^tlSN) at

a pH of 6.  Which  is beyond the desirable salinity range for oyster

production and indicates only dilution reduces the number of

coliforms.  This dilution is made by decrease in freshwater flow

in relation to saline waters within the bay.   pH's in the range

of the bay water cause no increase in aggregation or coagulation.
                                 -53-

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                           ENVIRONMENTAL PROTECTION AGENCY
                                OFFICE OF  ENFORCEMENT
                    DIVISION OF FIELD  INVESTIGATIONS-DENVER CENTER
                      BUILDING    22 , ROOM   410 , DENVER FEDERAL CENTER
                                 DENVER, COLORADO 80225


TO    .-    Files                                             DATE:  29 March  1972
FROM  :    Robert  F.  Schneider
         Chief,  Biology  Branch

SUBJECT:    Mobile  Bay Technical Committee Meeting, Dauphin  Island, Alabama
         March 24,  1972.
        Attendees  of  the March  24,  1972 Technical Committee Meeting to
        consider Recommendation No.  6  of  the Mobile Bay Enforcement
        Conference were:
        Committee  members:                     Representing
        W. F. Anderson              Alabama Department of Conservation and
                                     Natural Resources
        James Coil, Jr.             Scott  Paper Company, Mobile, Alabama
        Arthur  Beck                 Alabama Water Improvement Commission
        J. David Clem              FDA, Washington office
        C. B. Kelly                 EPA, Washington office
        Robert  Schneider            Chairman
        F. J. Silva  (absent         EPA, Atlanta office
         because of illness)

        Others:
        Albert  Story                FDA, Dauphin Island, Alabama
        Eddie May                   Alabama Marine Resources, Dauphin Island,
                                    Alabama
        Wayne Swigle                Alabama Marine Resources, Dauphin Island,
                                    Alabama

        Committee member spokesmen  for the State of Alabama discussed the
        present status of shellfish harvesting areas in Mobile Bay.  They
        informed the  Committee  that the Bay oyster production in December,
        1971 was nearly $300.00 per day higher than it had been for the
        past several  years.  On February  4, 1972 all productive reefs in
        Mobile Bay were closed and have remained closed for the past 50
        days.  Closure has resulted in an economic loss (total dockside)
        of $95,800.   The dockside value represents only loss to the fisherman.
        Alabama State Officials explained that the total economic value
        of oyster production in Alabama is four times the dockside value;
        thus, the  total economic loss to  the fisheries community as a
        result of  the current pollution closure is $382,400.

-------
Page 2
Memo to Files
Re:  Mobile Bay Technical Committee Meeting
March 29, 1972
Discussions during the remainder of the meeting dealt with the
various bacteriological criteria that might be substituted for the
total coliform criterion presently used in evaluating shellfish
harvesting waters.  The fecal coliform indicator group was the
principal substitute criterion considered.  The FDA spokesman
told the Committee that neither the FDA nor the MSSP is convinced
of the wisdom of substituting the narrow spectrum fecal coliform
test for the broad spectrum coliform group.  Contrasting views
were expressed by other attendees.  They pointed out to the
Committee that fecal coliform densities result from pollution by
man or other warm blooded animals; therefore, the fecal coliform
test was more directly indicative of the probable presence of enteric
associated pathogens than the "broad spectrum" coliform group.

Although the Committee did not reach an agreement (during this session)
on a more specific indicator or more realistic standards for Mobile
Bay, we did establish the qualifications any substitute criterion
must have.  Based upon a prepared statement by the FDA representative,
the Committee agreed that the substitute for the present criterion
should:  (1) be more specific for fecal pollution than the current
indicator group, (2) stand on its own merit as an indicator of
actual sources of fecal pollution without a dependency upon a
specific relationship with the coliform group, (3) be more represen-
tative of the potential pathogen load, (4) consistently indicate
the sanitary quality of shellfish-growing x%Taters in diverse
geographical locations, and (5) have a survival time ratio in the
estuary consistent with that of other bacterial pathogens regardless
of nutrient value of the water.

Within the Committee, a working committee was established.  Committee-
men Clem and Kelly agreed to jointly prepare a statement suggesting
an approach to assist regulatory agencies in the development of a
meaningful bacteriological criterion for Mobile Bay shellfish harvest-
ing waters.  The statement will be submitted to the Committee Chairman
by April 14, 1972 and subsequently distributed by the Chairman to
Committee members for their review and comment.

On May 5, 1972 the Technical Committee will meet at Dauphin Island,
Alabama to discuss the statement prepared by the working committee
and to deliberate a relevant recommendation to the Conferees.
                                  Robert F. Schneider
RFS/ls
                               -55-

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                    APPENDIX D
CORRESPONDENCE CONCERNING A PROPOSED RECOMMENDATION
                     -56-

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                           ENVIRONMENTAL PROTECTION AGEN^V
                                OFFICE OF  ENFORCEMENT
                    DIVISION  OF FIELD  INVESTIGATIONS- DENVER CENTER
                      BUILDING    22, ROOM   /»10 , DENVER FEDERAL CENTER
                                 DENVER, COLORADO 80225
TO    =                                                     DAiE:2A April  1972
          Mobile Bay Technical  Committee Member
FROM  :     Robert F.  Schneider,  Chairman


SUBJECT:     Technical  Committee to Consider Recommendation No.  6 of the Mobile
          Bay Enforcement Conference.
          Following our deliberations in Dauphin Island,  I  have had the oppor-
          tunity to talk with some of you by telephone concerning the Mobile
          Bay shellfish harvesting problem.   From these conversations and from
          the materials presented to the Committee during our March 2*», 1972
          meeting I  have developed the opinions contained herein.

          There is a Committee consensus that a substitute criterion for the
          total coliform test is needed for  evaluating shellfish harvest
          areas in Mobile Bay.  Those who are responsible' for criteria develop-
          ment (i.e. FDA shellfish Sanitation Branch and  EPA Office of Water
          Programs)  should consider the following characteristics in the design
          of a substitute bacteriological criterion.  The "new" bacterial indi-
          cator should:  (1)  be more specific for fecal  pollution than the
          current indicator group, (2) stand on its own merit as an indicator of
          actual  sources of fecal pollution  without a dependency upon a specific
          relationship with the coliform group, (3) be more representative
          of the potential  pathogen load, (*») consistently indicate the sanitary
          quality of shellfish-growing waters in diverse  geographical  locations,
          (5) and have a survival time ratio in the estuary consistent with that
          of other bacterial  pathogens regardless of nutrient value of the water.

          The Committee considered two alternative indicators as the most promising
          for evaluating bacteriological contamination in Mobile Bay shellfish
          harvest areas, (l)  fecal coliform  or (2)  Escherichia coli.

          Committee members and other attendees at the March 2k Dauphin Island
          meeting pointed out certain benefits and weaknesses of these substitute
          indicators.   The major points were:

          Presently, the fecal coliform test requires less time and effort than
          the E_.  col i  measurement; therefore, results can be obtained sooner,
          at less expense,  and by fewer microbiologists.   However,  studies by
          the Gulf Coast Laboratory and others have failed to demonstrate a
                                     -57-

-------
Page 2
Memo to Mobile Bay Tech Comm Mbrs
2*» April 72
consistent total coliform/fecal coliform ratio or arrive at a fecal
coliform value that can be directly related to known sources of
fecal pollution and provide public health protection to consumers
of raw shel1fi sh.

Sanitary surveys and published documentation confirm the fact that
the presence of E_. col? consistently indicates fecal pollution and
the potential contamination by other pathogenic bacteria.

Based upon these considerations and in compliance with Recommenda-
tion No. 6 of the Mobile Bay Enforcement Conference, the Technical
Committee submits the following statements to the Conferees:  "To
assist in the development of a more realistic criterion than the
present method for evaluating the probability of pathogen presence
in Mobile Bay,  it is recommended that the criterion be based on the
measurement of Escherichia coli in waters overlying shellfish harvest
areas.  Furthermore, the Technical Committee recommends that the
Federal and State shellfish sanitation control agencies combine their
efforts to support the research needed to develop a rapid, reliable
method for enumerating E_. col i in estuary waters used for shellfish
harvesting."

At present, t suggest we postpone the tentative meeting scheduled at
Dauphin Island on May 5i 1972.  I  would like to give each of you the
opportunity to respond to the recommendation contained herein.  If
possible, I would like your written comments by May 10, 1972.  If
you feel further Technical Committee meetings would be fruitful, please
advise me and I  will set up a mutual meeting place and time.
                                Robert F. Schneider

RFS/ls

cc:  Mr. John White, Atlanta, GA
     Mr. Murray Stein, D.C.
                              -58-

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                                     STATE OF ALABAMA

          **     DEPARTMENT OF CONSERVATION AND  NATURAL RESOURCES

                                f, 0. Boi 188 - Dauphin Island, Alabama 5652S
G.  !GE C. WALLACE
   GOVERNOR
 '• '.UDE P. KELIEY
   3MMISSIONER
 :  1JEY D. BLtDSOE
SISTANT COMMISSIONER
April 28, 1972
   ^
-*1 -V'.T

                      .-   ..v 1-.  '    &\
                      *   W.\%  -
                                •-..    W
                             'O. :-,->  CV/
                                      DIVISION OF MARINE RESOUR
                                       WIUIAM f. ANDERSON. DIRECTC
        Mr. Robert F. Schneider,  Chairman
        Mobile Bay Technical Committee
        Environmental Protection  Agency
        Office of Enforcement
        Division of Field Investigations
        Building 22, Room 1±10
        Denver Federal Center
        Denver, Colorado 80225

        Dear Mr. Schneider:

             We concur with your  opinions 100 percent.  Originally ire felt  that fecal
        coliform would be a better standard.  However, after learning that  Klebsiella
        is detected in the test for fecal coliform and that it also is frequently
        associated with paper mill effluent, we certainly agree that E. coli would be
        a much better indicator organism.  We feel this way because the discharge of
        paper mill effluent at the head of Mobile Bay has a population equivalent in
        excess of 1*00,000.

             It was suggested by  some of the FDA research group here on the island that
        a salinity level of 10 ppt be used as the closure point.  However,  on examination
        of some of the data compiled by them (attached) there seems to be little corre-
        lation between this level and high coliform levels.  Also their supposition is
        still based on the 70 MPH/10 cc of total coliform in relation to salinity.  We,
        therefore, feel that the  criterion you have suggested would be much better.

             We are sending under a separate cover some of our publications on Mobile
        Bay which might be helpful to you for background information.

                                         Sincerely,
                                         W. F. Anderson, Director
                                         Marine Resources Division
        WESrjb

        cc:  Mr. Claude Kel3ey/w copy of Mr. Schneiders letter
             Mr. Arthur N. Beck
                                             -59-

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                    22
                                           9 May  1?72
Mr. W, F. Anderson
Marine Resources
Dcpartnont of Conservation
and Natural Resources
P.O. Box 135
Dauphin  Island, Alabama   3^523

Dear Mr. Anderson:

Thank you for your prompt reply to my letter of April 2*:,  1972.
I have not received opinions fron all the Corr.ii tts?. ncnbers
yet, but the responses fron FDA rand Scott Paper arc in agreement
with the "E. ceil? indicator'1 recommendation.  V/hen  ! receive  the
others I wTl 1 draft a final recon-.nendation and send it to  you-
alonn with the letters I've received from the Coraiittee  for your
review and comment.

Aijaln, thank you for your help and for sending no the series  of
Alabama Marine Bulletins and oyster resource atlas.

                                   Sincerely,
                                   Robert F. Schneider
                                   Chief, Clolooy Branch


RFS/Is
                               -60-

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               TMENT OF HEALTH. EDUCATION, AND WELFARE

                       PUBLIC HEALTH SERVICE
                   FOOD AND DRUG ADMINISTRATION
                       WASHINGTON. D C 20204
                       May  5,  1972
Mr. Robert F. Schneider
Chairman
Mobile Bay Technical Committee
Room 410, Building 22
Denver Federal Center
Denver, Colorado 80225

Dear Bob:

I concur with your letter of April  24 but would  like  to
make the observation that one study conducted by our
staff indicates that E. coli can be recovered within
24 hours with results comparable to present methods.
Dr. Wallace H. Andrews' paper on this study is attached
and has also been published in Applied Microbiology,
Vol. 23, No. 3, March 1972 issue.  Of course, their
work needs further collaboration but it does look
promising.

If I can be of any further assistance, please call.
                             Since/ely ,
                             J. David Clem
                             Director
                             Division of Shellfish Sanitation
                             BF-230
                             Office of Food Sanitation
                             Bureau of Foods
Enclosure
                           -61-

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                           SCOTT  PAPER COMPANY
                                SOUTHERN OPERATIONS
                                   P. O. BOX 2447
                              MOBILE, ALABAMA  36601
JAMES H COIL. JR.
   MANAGER OP
   AOMINIS1 H At 1 VC SERVICFS
                                             May 5, 1972
    Mr. Robert F. Schneider
    Environmental Protection Agency
    Office of Enforcement
    Division of Field Investigations-Denver Center
    Building 22,  Room 410, Denver  Federal Center
    Denver, Colorado  80225

    Dear Mr.  Schneider:

                     Subject:   Technical Committee to Consider Recommendation
                                No.  6 of the Mobile Bay Enforcement Conference.

    I would suggest consideration of altering your statement to the conferees to
    read as follows:

          "To  assist in the development of a more  realistic criterion
          than the present method for evaHiating the probability of pathogen
          presence in Mobile Bay, it is recommended that the criterion be
          based on the measurement of Escherichia coll in waters overlying
          shellfish harvest areas.  The Technical Committee further
          recommends that the agencies concerned, i. c, , FDA Shellfish
          Sanitation Branch,  EPA Office of Water Programs and the Alabama
          State Department of Health, initiate as soon as possible stirveys of
          the E.  coli counts—in addition to total coliform—in the waters overlying
          the shellfish areas and on  the shellfish proper.  Furthermore, the
          Technical Committee recommends that these  aforementioned federal
          and state shellfish  sanitation control agencies combine their efforts
          to support the research needed to develop a rapid, reliable method
          for enumerating E. coli in estuary waters xised  for shellfish harvesting. "

                                           t Very truly yours,
                                           '   '           •'••'/
                                          /' James H.  Coil, Jr."
                                            Paper Industry Representative
                                       ,'   /Mobile Bay Technical Committee

    JHCtgor
    cc:  Mr. John White, Atlanta, GA
         Mr. Murray Stein,  D. C.       ,

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                     22           MO
                                           11  Hay 1972
Mr. James Coil
Manager of Administrative Services
Scott Paper Company
P.O. Eox 2^7
Mobile, Alabama  3&&01

Dear Mr. Coil:

To date I have received  replies  frcn the State,  FDA and you con-
cernlnu the recoitiT.erKbtion  !  prepared In sr.y letter of April 2J»,
1972.  Generally, these  Cor.inilttec members concur with the "E_. coH_
Indicator" aoproach.  When  I  receive comments frora the entire
CoiTiKittec t v;ill amend the  recor.n?endatlon, prepare ii final clraft
and nail a copy to you For  cerements.

May  I take this opportunity to thank you for your help on this
technical coraalttee.

                                    Sincerely,
                                    Robert F'.  Schneider
                                    Chief, Biology Branch
RFS/l!
                                  -63-

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                ENVIRONMENTAL PROTECTION AGENCY
                      Office of Water Programs
                       Washington, D.C. 20460
                           May 15, 1972
Mr. Robert F. Schneider
Office of Enforcement
Division of Field Investigations
Building 22, Room 410
Denver Federal  Center
Denver, Colorado  80225

Dear I'ir. Schneider:
   This is in response to you<" letter of 24 April  requesting
comments on a proposal to recommend that Federal  and State
Agencies combine their efforts to support the research needed
to develop a rapid, reliable method for enumerating E. coli
in estuary waters used for shellfish harvesting.   On this
issue, I have the following comments:

        1.  I have the sincere opinion that the Technical
Committee is not in a position to recommend the choice of any
one indicator system.

        2.  The matter of choice of indicator system properly
should be made only after careful study of the available data
and further cu*ta gathered in future investigations.

        3.  In my opinion, further study should not be conducted
only on E. coll.  To be meaningful, other coliforms, including
total  coliforms and fecal coliforms should be included in the
investigations.  Including these others would provide a bridge
from past experience and data on the other organisms and, if
contraindication for E. coli are found, more supporting
information for coliforms or fecal  coliforms would become
available.
                                               7
                              C. B.  Kelly
                              Special  Assistant for
                               Criteria and Standards
                              Water Supply Programs Division

-------
                         EN'"RONMENTAL PROTECTION AG[   :Y
                       Program Grants Section                  DATE:   May 2U, 1972
SUBJECT
TO:        'Mr7Itobert F. Schneider
          Chief, Biology Branch
          Denver Center

               From your memorandum and attached paper by Clem and Hunt, I
          presume that the direction for seeking a suitable indicator organism .
          to aid in the sanitary classification of shellfish grov/ing areas lies
          toward the utilization of the organism, E. Coli.

               The course which is recommended represents a progressive step
          toward our refining the bacteriological spectrum of the general coliform
          organism group for use in sanitary classification of shellfish growing
          areas .  First, the coliform test was used for classification purposes,
          then in the early 1960's - and after a lot of research - the "fecal
          coliform" test was accepted as a supplementary test for classifications.
          The test, which was widely used to obtain supplementary information, was
          never accepted as an alternate to the coliform test.  Now going to the
          E. Coli test represents a narrowing of the spectrum.

               The proposal in your memorandum suggests that a stxidy be made to
          determine the suitability of using E. Coli as an indicator organism
          This may be a good realistic approach to establish an organism with
          fecality integrity which will provide a true index of the sanitary quality
          of shellfish growing waters.  There has been, however, a great deal of
          experience utilizing the fecal coliform group in shellfish work and a
          lot of research too.  First, I suggest that the committee not be hasty
          in turning away from this accumulated experience and, therefore, recommend
          that a hard look be taken at the vast amount of F.C. information which
          has been collected and applied.  Secondly, if the committee elects to
          move forward toward encouraging the development of a rapid and accurate
          E. Coli test, then such development must move forward with all deliberate
          speed.

               It would be interesting to have C. B. Kelly's and Dr. Hosty's
          comments on the suggested course of action before a firm commitment to
          move ahead with the E. Coli study is made.  It would also be interesting
          to look into the standards and procedures used by the Eglish, French,
                                          -65-

-------
Portuguese, Spaniards, and Japanese in utilizing bacterial indices for
classifying shellfish growing areas.  While it may not be necessary,  I
hope we don't get involved in a protracted study - which includes that
more study is needed before definite recommendations can be put forth.
                                            J.  Silva
                                 -66-

-------
                                     State of Alabama
                           Department of  Public Health
                                   State Office Building
                                Montgomery, Alabama
          36104
IRA L. MYERS, M. D.
STATE HEALTH OFFICER
                                    May  22,  1972
    Mr.  Robert F.  Schneider
    Chief,  Biology Branch
    Environmental  Protection Ag.-ncy
    Office  of  Enforcement
    Bldg. 22,  Room 410,  Denver Federal Center
    Denver,  Colorado    80225

    Dear Mr. Schneider:


    to o-      z have asked  our Director of Laboratories, Dr. Thomas S. Hosty
    to give me hj.s  ideas  and recommendations in regard to proposals to evaluate
    measurements of pollution in Mobile Cay harvesting areas           evaluate
                                         Yours^yery truly, _
                                             "'"
    AMB/.
f rthur tf. Beck
 Chief Engineer & Director
 Bureau of Environmental Health
    cc:   Dr.  Thomas S.  Hosty
                                   -67-

-------
IRA L. MYERS. M. D.

STM E HEALTH OFFICER
                                    State of Alabama
                           Department of  Public Health
                                   State Office Building
                                Montgomery, Alabama  ism
August 7,  1972
 lir. Murrny Stein
 Chief  Enforcement Officer - Tteter
 Crystal  Hall Dldg.  2
 KoOal lilG
 U&Bhington, B.  C.  20460

 Dear Iftr. Stein:

            I apologize for not edvleing I.'r.  Robert F. Schneider,  Chairman,
 llobllc !5ny Trtchxiical Cj-.ui.lt toe before  now of r™ srecoa^cndRtion In regard to
 an indicator tost ;:."»r pollution  control. oi: oystc-r j;ra:;inj; waters.

            ProbcbLy wore work hcs bcon  done 0:1 labile 2=y waters  with  retard
 to oyscor 2>:o-.-:i«2 v:;t.crs th:
-------
Mr. Murray Stein
                        August 7, 1972
                                     -2-
Schr.eider.
          Again I apologize for being  late with my  report  to you  and  Mr.
ANB/iw
end.
                                     Yours very  truly,
   mr M. Beck
Chief Engineer f* Director
Bureau of Environmental Health
cc:  Mr. Robert F. Schneider

      Dr.  Thomas S.  Kooty
                                           -69-

-------
                                     State of Alsbssna
                                  .'. t*-, „. r. .J. —A .}•>•.lm  ».?i'
                                  ImJiU 0) /:.,,•,;b  ji'
                                  Bi!rc?u of l.r.!;oni'jriHs
                                    434 Monroa Strest
                                 Hontgonisry, Aiaisma 3G1C4
IRA L. MYEPS. M.
STATE HF.ALTH
 MEMORANDUM
TO:
FR011:
SUBJECT:
                                August  4,  1972
               Mr. Arthur Beck

               Thomas S. Hosty,  Ph.D., Director
               Bureau of Laboratories

               Mobile Bay
 THOMAS S. HCSTY. PH.D.
DIRECTOR LABORATORIES
       I  did not write  to Mr.  Schneider as I tolcl you  I  would since I felt
 that  the meeting we had in  Virginia would yivc a  satisfactory explanation
 of Alabama's stand.  All of the  testimony given in Virginia was transcribed
 and should be readily available  to FDA.  Many of  the representatives
 of FDA  were present at this  meeting.  I think it  is worthy of note chat  the
 opposition of the FC  cest was  soru or taKcn apart very nicely.  I was
 extrofuely interested  Ln the  facu thct psrhans two of the nost knowledgeable
 people  in the country on cvstcrs and seawater were in  co-./iplete agreement;
 that  the fecal col it'ovn tcse should be u:,£d rath or than coliLor-.Ti.  L sm, of

 that  every ncetin^ I  50 to  I ani  ccid there is not sufficient data co justify
 focal col i forms.  Wa ware advised before t;oip.:; to Virginia that we should
 be able, to document w'.at we  were savins.  Through the  years the opposition
 remains tree to say any;ning they please - mostly without adequate
 documentation.

       Recent developments in this area have left me completely confusod.
 Certainly we are not  rgainst tiie development of a better test.  1 .r.«cle
 a statcnent in Virginia chat '.-.ion they speak cf "E.coli'1 test "hat do
 they  wean:'    1 stated that  none or the tests we use  can be considered
 as an E.coli test unless yoj IMViC the strains and no  one was in
 opposition, to this sentiment.  Mr. Kelly very beautifully folLoved
 the history of th-? feccu coliform test and pointed out that even the
 author  of the E.coli  lost did  not consider this to be  a  specific test
 for tt.coli.
                                                                               £'
       A  considerable amornt  of  discussion li.is been made relative to the    ,-"
 growing of E.acrR»rc nos in aifluence from paper mills.   Certainly it is   '.'
 proba'uly lorjic.il to assume  that  with tiic waste products chat are present  '•
 and ;i tc-.iiporaturc c:i iiro-mci 32"  tha:. growth wouJd tr,
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                                    Stats of A
                                       y  rv?  •.iiiV.j-
                                       .,  OS  i .i...i
                                  Bureau of Ui'orator.
                                   434 Monroo Sinst
                                f.l&nlscir.sry, Aiibuiia J
IRA L. MYCRS. M. 0.
STATC HEALTH OFFICER

 TO:  Mr.  Arthur Beck
                                THOMAS S. HOSTY. PH.D.
                               DIRECTOR LABORATORIES
August 4,  1972
Page 2
 and  subsequent tests.  It was  found  later that: sewerage from  the  plant
 was  discharged into paper mill waste with the F.C. responding very  nicely.

      Mr.  Salinger from Maryland presented a very nice study on  the
 relationship of fecal coliform results  to polluted, clean and intermediate
 typos  of  pollution and that study  has become part of the record of  the
 Virginia  meeting.  A copy of Mr. Salinger's report is included with this
 memo as well as the study on the new Scott Paper mill.

      I am also enclosing a part of our  study of station  119 and  it shows
 rather dramatically not only the ability cf the fo.c^l coliform  test to
 detect pollution but coinc Ldcs quite well with the dropping of  salinity.
 These  studies were duplicated on at  least 25 other stations during  the
 course ol three years.

      May  1 refer you to the Denver,  Colorado reefing of Xay 10, 1972
 with microbiologisus of iiPA unu one  of  uhi:ir co7iciuijj.c-.ir,:  "The fecal
 coliform  is a valid indicator of fecal" pollution in ail waters."

      I was very interested in the  continuous referral of a test
 developed by the Uauvjnin Island workers which was entitled
 "The Rapid Recovery of E.coli  fror> Estuarine i.'aters."
 The  article appears to have considerable premise but rcaliv my  qood
 friend Mr. Prcsncli •..••xiLd agree that the only difference between  the
 E.coli and his test va.*. a matter of  24  hours or a quicker test:.   We
 are  at present doing a limited study of the tost on oysters but a^ain
 T aii certain Mr. Pres^oil would not  advoc.itc thar tnis test be  adopted
 immediately without confirming tests on  n much wider area since  the  area
 where  these studies wore made  is not generally of tne same character as
 the  areas over th*. ovsccr beds.  At  any rate we will continue our limited
 studies as time and help will allow.

      For  many years the cooperative  agreement between states  and  shellfish
 people has benefited the whole shellfish program and may I ask  now  is
 this fifty years of cooperative agreement to be nullified by  FDA  calling
 the  fhots.'  Our data ovf.-r three years showed the FC test could  be used  in
 Alabama with a cut of! MPN of about  9.0.  This JlFN would still  -ivc .adequate
 warning OL possible unLiution and  yet would allow the beds to be  reopened
 sometimes even four to live weeks  before the col i torv.is returned to  normal.
 Certainly our firr.t ou! :".ation r.iiouu! be the neaicli urobLcm buL ;it  the  same
 tima wo should bn a-./.-u-1.- uiat n-nnv  pconic arc nur. out of work  i r t!;c indicator
 is «'i  ; '.o'.i i u-.-'ii:; i L i \ i- r-.-iuiiro  i.n.'ii, it  ; i'-nai-j r-»si iiicion VIMMI  r.iu-ro  ronllv
 i..  • :'.-'.   ii !i!.l'.!:! L'.I.: .:<.: .'"..'li av.lli.ii)lo \-;.- vi : i ! ,,..!,»•.• ..Ill 1. • 11.1.' n.-. 1 '. C1  ••'.••.I'.y
 again  OL  the uxmciu;1. LOJC and aLLcr.ipt  to dcvciup new
                                  -71-'

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            APPENDIX E
RAPID RECOVERY OF ESCHERICHIA COLI
        FROM ESTUARINE WATERS
                -72-

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COPY






   ABSTRACTS OF RAPID METHODS FOR THE RECOVERY OF Escherichia coli






     Three rapid methods for the recovery of Escherichia colis which




appear applicable to shellfish and shellfish waters, are abstracted.




METHOD A




     Fishbein et al.  investigating the recovery of E. coli from




marine products and other foods found that the best recovery was




obtained from lauryl sulfate tryptose (1ST) broth incubated at kb C.




The method is a fermentation tube technic in which the samples are




inoculated directly into LST broth and incubated at bk C in a water




bath.  Since 96 percent of the E.  coli recovered by the method




were recovered with 2k hours it is essentially a 2^-hour test.




Results obtained by the method were comparable to those obtained by




the Association of Official Analytical Chemists (AOAC) test.  The




latter test is much longer, requiring from two to four days.




     A complete discussion of the method appears in Applied Microbiology3




Vol. 15, No.  2, pp 233-238, under the title, "Coliform Behavior in




Frozen Foods."




METHOD B




     Andrews  and Presnel1  of the Food and Drug Administration, Gulf




Coast Technical  Services Unit,  have developed and evaluated a 2A-hour,




elevated temperature,  tube fermentation, most probable number test.




The medium, designated as  A-l,  contains  concentrations of lactose,




tryptone, and sodium chloride found in most commercial media.  In
                               -73-

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addition the medium contains trition X-100, a surfactant, and salicin,




a carbohydrate  readily utilized by E.  coli.  This method involves the




direct inoculation of the sample, in appropriate dilutions,  into the




medium which is incubated for 2k hours at M.5 C in a water  bath.




The method has been compared with the procedure contained in the fourth




edition of APHA Recommended Procedures for the Bacteriological




Examination of Sea Water and Shellfish and the 1ST elevated  temperature




(M C) test described by Fishbein.  The APHA recommended method




requires a 2A-to-48-hour presumptive test at 35 C followed by a




2^-hour confirmatory test at kk.S C.  The three tests were compared



using samples of freshwater, estuarine water, and a limited  number




of shellfish samples.  Results of the comparative study demonstrated




that E.  coli recovery by A-l medium at kk.$ C and LST at M  C were




comparable and that both methods recovered more E.  coli than the APHA




recommended procedure.  The report pertaining to the development and




evaluation of the method is included in this Appendix (pp 72  to 82 ).




METHOD C




     Cabelli and Heffernan have described a rapid,  elevated  tempera-




ture plate count method for assaying the E. coli content of  seawater




and shellfish.   The medium used is MacConkey's agar modified by




decreasing the oile salts concentration from 0.15 percent to 0.075




percent.  The sodium chloride eliminated from the medium is  replaced,




to varying extents, by the addition of shellfish and seawater samples




mixed with the medium.  In using the method, known weights of

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shellfish meats or known volumes of seawater are mixed with a known




volume of the medium and distributed into petri plates.  The plates




are incubated at ^5-5 i. 0.5 C for 2k hours.  E. coli appear as brick



red subsurface colonies.  A description of the method  is published in




Applied Microbiology, Vol. 19, No. 2, pp 239-2^.
                                 -75-

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RAPID RBCOVERY OP Bscherichia coli FROM ESTUARINB WATERS
                  WALLACE H. AHDREBS
                  MATOARD W0 PRESHBLL
   U.S. DBPARTMBOT 0? HEALTH, EDUCATION, AHD WBLFARB
                 Public Health Service
             Food and Drug Adesiaiotration
                    Bureau of Foods
               Office of Food Sanitation
           Division of Shollfiah Sanitation
          Gulf Coast Technical Services Unit
                Dauphin Island, Alabaaa
                         -76-

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                            ABSTRACT








   The efflciences of two 24-hr elevated-teaperature tests to



recover E. coll froa estuarine water vere coapared siaultaneously



with the 72-hr standard ncthods procedure of the American Public



Health Association (APHA).  Froa 1,710 tubes, E^ cgli waa recovered



222 tiaes in lauryl tryptose aiedium incubated at 44 + 0.2 C for 24



hr, 261 tiaea in an expericantal aediua incubated at 44.5 + 0.2 C



for 24 hr, and 257 times by the 72-hr APHA oethod.  The muaber of



false positives enunerated was sieilcr in all three tests.  The data



indicated that E. coli in raw seawater could be deteriaincd in 24 hr



without a significant loss of accuracy.
                               -77-

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                             INTRODUCTION






   The need for a rapid, reliable method for determining the bacterial




quality of ohellfish-growlng waters is an important aspect of the con-




tinuing search for methods improvement.  The standard methods procedure




for the examination of water recosaended by the /uaerican Public Health




Association (APHA) requires a maximum 48-hr enrichment period in lac-




tose broth or lauryl sulfate tryptose broth (LST) to obtain optimum



recovery of fecal coliforas.  Positive tubes are then transferred to




EC Tsedium to determine the most probable number (KPN) of fecal coli-




foras.  Gas production with 24 hr in the fomentation vial of a tube




of EC medium incubated in a water bath at 44.5 +0.2 C is considered




a positive reaction Indicating an organism of fecal origin.  Tubes




that do not produce goo indicate that that the organisms are derived from



sources other than the tract of a warm-blooded animal (1).  Because of




the reported interference by non-fecal colifore biotypes, notably




Ettterobacter oerogenen and Eachorichle internediua types, with the




specificity of the EC mediua even at teaiperatures of 44 C (7,8) and




the APHA-adopted 44.5 ± 0.2 C (3,4,6), it is suggested that a determina-




tion of the E. coll density would give a wore accurate estimate of




the degree of fecal pollution of a water sample.  Working with frozen




foods, Fishbein et al.  (5) reported that LST incubated directly at 44 C



gave E. coli recovery similar to that of the standard methods procedure




of the APHA.  A preliminary investigation in our laboratory demonstrated




that a newly-formulated medium also gave E. coli recovery comparable to
                                  -78-

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                                                                 (2)


that of the APHA tsethod.  The purpose of this study was to compare,

simultaneously, the reliability of E. coll recovery from field oeawater

sasples by use of (i) Fishbein'a elevated-tceperature test,  (ii) an

experimental 24-hr elevated-tecperature teat with the newly  formulated

median, and (iii) the 72-hr APHA standard sethods procedure.


                         MATERIALS AND METHODS

   Sixty-eight saeples of seavater froa various sites in Mobile  Bay were

collected, iced, end transported to the laboratory.  All sacples were

tested within 24 hr. after collection.  Five-tube decimal dilutions

were perforead in phosphate buffered water (pH 7.2) ea far as necessary

to obtain a determinant KPN of colifores.  Test IA (LSI incubated at

35 + 0.5 C in a walk-in Incubator for 24 to 48 hr) end Tect  IB (positive

tubes froa Test IA transferred to EC cediusa and incubated in a water

bath at 44.5 j; 0.2 C for 24 hr) together comprise tha APHA standard

methods procedure for the examination of water (1).  In Test II, LST,

and in Test III, Kediua A-l (the newly forculated ctediun) were inoculated

and incubated directly in water baths at 44 ± 0.2 C and 44.5 + 0.2 C,

respectively.  Medium A-l included 5 sleecoce 20 g tryptone, and 5 g NaCl

found in most rf the coasaercially prepated dehydrated media; and, in

addition, 1 ml Triton X-100 (Roha and Haas Co.)*, a surfactant,  and

0.5 g salicin (Difco), a carbohydrate readily utilized by Bacherichia

species (2), added to 1,000 ml of distilled water.
  Mention of co*baerlcal products does not  iotply  endorsement by the
  Food and Drug Adainiatration.
                                  -79-

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                                                                (3)






   All positive tubes from Teats IB, II, and III were streaked  on




Levine's eosin nethylene blue agar and incubated at 35 + 0.5 C  for




24 hr.  One representative colony of each type was picked to lactose




broth and incubated at 35 C for 24 and 48 hr.  All positive tubes




(gas formation) were inoculated into 1% tryptone, M.R.-V.P. medlua,




and Koser citrate medium and subjected to indole, rcethyl red, Voges-




Froskauer, and citrate (IMViC) determinations at 35 + 0.5 C.  All




positive lactose broth tubes were also reinoculated into EC medium




and incubated at 44.5 j; 0.2 C fox 24 to 48 hr to confirm the ability



of the pure culture to grot; and produce ges at the elevated tempera-



ture.




   To facilitate a comparison with the d&ta of Fishbein et si.(5),




we decided to use their terminology, which in cs follows:  positive




tube, a tube that ferments lactose with the production of gas within




24 to 48 hr; negative tube, a tube that does not produce gas within 24




to 48 hr; B. coli , a tube frosa which E. coli Type I (-H- —) or Type




II (-+--) is isolated; false-positive tube (FP), a tube that gasscs




within 24 to 48 hr, but frosn which E. coli+ is not isolated; false pos-




itive (group), a tube that gasses within 24 to 48 hr, but from which a




coliforn organise other than E. coli* is isolated; false positive (syner-




gistic), a tube that gassea within 24 to 48 hr, but from which  neither



E. coll* nor any coliforn is isolated.





                        RESULTS AND DISCUSSION




   In Table I, the positive tube reactions of the four tests are compared,




LST incubated at 35 C (Test IA) produced the largest number of  positive





                                 -80-

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                                                                 (4)






tubes; all the elevated-temperature teats (IB, II, and III) produced




considerably fever positive tubes.  LSI incubated directly at 44 C



(Test II) produced the fewest positive tubes (631), whereas EC medium



(TEST IB) and Mediua A-l (Tect III) incubated at 44.5 C produced a



similar mu&ber of positive tubes (665 end 674).



   E. coll recovery data ore given In Table 2.  Not as nany B. coll



were recovered in Teat II ao in ?G8ts IB and III. The productivity




ratios (coiutsn 3) were obtained by dividing the number of tubes from



which E. coli+ waa isolated by the total nuober of gassing tubes.



The observed differences in productivity ratios of the threo tests



were not considered significant.



   Data for the recovery of coliforns; exclusive of E. coll Types I



and II, are given in Table 3.  These data indicate no significant



difference in the nusher of faloe-pocltive tubes enumerated by the



three elevated-temperature testa.




   The results of this ctudy are consistent with those of Fiohbein



et al. (5) who found that LST incubated at 44 C gave B. colt recovery



comparable to that obtained by the APHA standard icethods procedure.



Although with LST incubated at 44 C we did not recover as many coliforaa



(631 contrasted to 665) and B. coll (222 contrasted to 257) as were



recovered with the standard methods procedure, the differences were not



significant.



   We fcioh to emphasize that 44 C, rather than 44.5 C, was used as the




temperature of direct incubation of the LST in the water bath, inaaouch



as this was the temperature used by Fiohbein et al. (5).  Furthermore, we
                                      -fti-

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                                                                (5)
                    Table 1.  Positive tube reactions
(1)
Test
LST, 35 C, presumptive (IA)
EC, 44.5 C, confirmatory (IB)
LST, 44 C (II)
Kediuta A-l, 44.5 C (III)
(2)
Nusber of
positive tubes
1011
665
631
674
(3)
Percentage of
Cubes positive*
59.12
38.89
36.91
39.62
•=Based on the nuober of pocitive tubes divided by the total number



 of tubes (1,710).
                                -82-

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                                                                     (6)
                      Table 2.  Recovery of  K. coll
      (1)
    .  Test
   (2)

B. coli*
 tubes
                                                     E.  colf+Vtotal
                                                     no.  of gassing
EC, 44.5 C, confireatory  (IB)

LST, 44.C  (II)

Medium A-l, 44.5 C  (III)
  257

  222

  261
38.65

35.18

38.72
lEjrpriissed as a percentage.
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                                                                    (7)
                  Table 3.  Coliform group recoveries -
(1)
Test
EC, 44.5 C, con-
firmatory (IB)
LST, 44 C (II)
Medium A-l, 44.5
C (III)
-All Enter ohocter
(2)
Total
no. Ppb
406
407
405
(3)
No. FP
(group)
385
389
380
(4)
No. FB
(synergiatic)
21
18
25
and Eecheriebia species exclusive of
(5)
FP tubes/total
no. of gassing
tubes£
61.06
64.50
60.09
E. coli Types
 I and II.



£ FP-faloe positives; gassing  tubes  frost which  E.  coli  was not recovered.



£ Expressed as a percentage.
                                   -8*1-

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                                                                  (8)






believe the uae of 44 C is Justified since the recovery of E. coli and




the enumeration of false positives by the LSI at this temperature




were not significantly different from those by the APHA method.  With




Medium A-I, however, a tesperature of 44.5 C was used because this is




the most acceptable teisperature for the incubation of the fecal coli form




group (1).  The effects of the direct incubation of LSI and Mediua A-l




over a range of teaperatures will be the subject of a subsequent report.




When the overall performance of Medium A-l was compared with that of




the APHA procedure, results by both methods were aidlor in onuaerating




coliforas  (674 and 665) and E. coli (261 and 257).  When the tvo rapid




methods were compared to each other, Medium A-l recovered eiore coli-



fonns (674 compared to 631) and E. coli (261 compared to 222) than did




LSI incubated at 44 C.  Both rapid tests norit further inventigation




because of the savings in tine and cost of media, without an accompany-




ing loss in accuracy.  It would be particularly significant if a rapid,




reliable method could be adopted as a standard cathode procedure for




determining the bacterial quality of shellfish-growing waters as well as




the bacterial content of oysters, claaa, and other potentially narketable




seafoods.



                            ACKNOWLEDGMENT




   We wish to express our appreciation to Jack Gaines and Merrill




McPhearson for collecting saaples and to Bob Huntley and Clark Jones




for cheir  very capable technical assistance.
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                                                               (9)

                           LITERATURE CITED

1.  Aaerican Public Health Association.  1965.  Standard methods for
     the examination of water and wastewater, pp. 594-600.  12th ed.
     American Public Health Association, New York.
2.  Edwards, P.R., and W.H. Ewing.  1962.  Identification of Enterobac-
     teriaceae.  Burgess Publishing Co., Minneapolis.
3.  Fishbein, M. 1962. The aerogenic response of Escherichia coli
     and strains of Aerobacter in EC broth and selected sugar broths
     at elevated temperatures.  Appl. Microbiol.  10:79-85.
4.  Fishbein, M., and B. F. Surkiewicz.  1964.  Comparison of the re-
     covery of Escherichia coli from frozen foods and nuteasats by con-
     firmatory incubation in EC mediua at 44.5 and 45.5 C.  Appl.
     Microbiol.  12:127-131.
5.  Fishbein, M., B.F. Surkiowicz, E.F. Brown, H.M. Oxley, A.P. Pcdron,
     and R.J. Groomss.  1967.  Col if or ra behavior in frozen foods.  I.
     Rapid test for the recovery of Escherichia coli frost frozen foods.
6.  Raj, H., and J. Lieton.  1961.  Detection and enumeration of fecal
     indicator organisms in frozen seafoods.  I. Escherichia coli.
     Appl. Microbiol.  15:233-238.
7.  Tennant, A.D., '-.nd J.E. Reid.  1961.  Co 1 if or u bacteria in sea water
     and shellfish.  I. Lactose fernantotion at 35.5° and 44° C.
     Canad. J. Microbiol.  7:725-731.
8.  Tennant, A.D., J.E. Reid, L.J. Rockwell, and E.T. Bynoe.  1961.
     Colifora bacteria in sea water and shellfish.  II.  The E.C.
     confirmation test for Escherichia coli.  Canad.  J. Microbiol.
     7:733-739.
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