600184019
                                                 SRC TR-81-620
WATER DISTRIBUTION SYSTEM AS  A  POTENTIAL SOURCE
         OF MUTAGENS IN DRINKING WATER
                 Dipak K.  Basu
                  Jeet Saxena
              Frederick W. Stoss
              Joseph Santodonato
                Micheal W.  Neal
         Syracuse Research Corporation
     Center for Chemical Hazard  Assessment
        Syracuse,  New York   13210-4080
              Grant No. R 806413
                Project Officer

             Frederick C. Kopfler
          Exposure Evaluation Branch
      Health Effects Research Laboratory
           Cincinnati, Ohio   45268
      Health Effects Research Laboratory
      Office of Research and Development
     U.S. Environmental Protection Agency
           Cincinnati, Ohio   U5268

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                                    CONTENTS
DISCLAIMER	i
FOREWARD	ii
ABSTRACT	iii
LIST OF FIGURES	v
LIST OF TABLES	vi
LIST OF ABBREVIATIONS	vii
ACKNOWLEDGEMENTS	viii

     1 .   INTRODUCTION	1
     2.   CONCLUSIONS	3
     3.   RECOMMENDATION	5
     4.   LITERATURE REVIEW	7
     5.   MATERIALS AND METHODS	12
               MATERIALS	12
               EXPERIMENTAL PROCEDURES	15
     6.   RESULTS AND DISCUSSION	45
               RESULTS OF CHEMICAL ANALYSIS FOR PAH	48
               RESULTS OF MUTAGENICITY ASSAYS	68
     7.   REFERENCES	83

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                                  DISCLAIMER



     This report has  been  reviewed  by the Health Effects  Research Laboratory,




U.S. Environmental Protection Agency,  and approved for publication.   Approval




dose not signify that the contents necessarily reflect the views and policies of




the U.S. Environmental Protection Agency, nor does  it  mention trade  names  or



commercial products constitute endorsement or recommendation for use.

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FOREWARD
    11

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                                   ABSTRACT








     The primary objective of this study was to examine the changes in polycyclic



aromatic hydrocarbon concentrations and the possibility of changes in mutagenic




potential of treated waters  as a result of leaching during their passage through



commonly  used  distribution  pipes  in the  U.S.    A  method  developed in  our




laboratories  was  used for  the  collection and  analysis  of  water samples  for




polycyclic aromatic  hydrocarbons.   The polycyclic  aromatic  compound benzo(a)-




pyrene and  benzo(ghi)perylene  were among  the most  frequently  occurring  poly-




cyclic aromatic hydrocarbons in raw and treated water  and in waters travelling



through the  distribution  lines.    With  the exception  of Wheeling,  WV treated




water, which showed  an  unusually high total   polycyclic aromatic  hydrocarbon



concentration of 138.5 ppt,  the total  polycyclic aromatic hydrocarbon concentra-




tion in all  other treated  water ranged from 0  to  13.4  ppt.   The corresponding




total polycyclic aromatic hydrocarbon  concentration  in water after their passage



through the distribution pipes  varied  from  0 to 61.6 ppt.  This indicates a small



increase of polycyclic aromatic hydrocarbon concentration in waters as a result



of their passage through  the asphalt-lined distribution pipes.  The increase in



polycyclic  aromatic  hydrocarbon  concentration  was   maximum  with  recently



installed  asphalt-lined pipes.   Waters  from a few distribution  lines  showed



reduction  in polycyclic  aromatic  hydrocarbon  concentrations  possibly due  to



sorption of PAH on the surface of these distribution pipes.






                                      iii

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     Mutagenic activity was also detected in many of the water samples treated,



however, the levels of this activity did not correlate with either the transit of




water through the distribution system or the levels of polycyclic aromatic hydro-




carbon  in  the water.   There  was  some  evidence  to  indicate  that the  water



treatment process itself may have contributed to the mutagenicity observed in the




finished water and  that compounds  responsible  for the observed activity were



different from the  low molecular weight chlorinated compounds  produced during




chlorination.  This  study  also provides evidence that mutagenic activity is less



likely to be found in  the  raw water supply if this supply is from  ground water



rather than surface  water.
                                      IV

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                                LIST OF FIGURES

Number

  1        Portable sampling suitcase used to collect organic
            contaminates from drinking waters	22

  2        Locations and configurations of sampling points
            after the passage of potable waters through
            distribution pipes	25

  3        Experimental Design of Bioassays	33

  4        Emission spectra of an unidentified compound that
            recurred in several samples	6?

  5        Gas chromatograms of Elkhart, IN samples collected
            at the four sampling points	69

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                                LIST OF TABLES


Number

  1        Levels of Some PAH in Fossil  Fuel Derivatives	9

  2       Some Characteristics of the Water Supply System
            Used for Sampling	16

  3       Distance, Type and Configuration of the Distribution
            Pipes in the Supply Systems Used for Sampling	26

  4       Results of PAH Analyses of Water Samples	49

  5       Detection Limit for Six PAH by Thin Layer and
            Capillary Gas Chromatographic Methods	60

  6       Water supplies with no detectable mutagenic activity	70

  7       Variation in Mutagenicity in Raw and Distribution
            Water as Compared with the Finished Water	74

  8       Comparison of Mutagenic Activity in Raw Water  with
            the Type of Raw Source	80
                                      vi

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                             LIST OF ABBREVIATIONS
BaP   =  benzo(a)pyrene
BghiP =  benzo(ghi)prylene
BjF   =  benzo(j)fluoranthene
FL    =  fluoranthene
IP    =  indeno(1,2,3-cd)pyrene
PAH   =  polycyclic aromatic hydrocarbons
TLC   =  thin layer chromatography
U.V.  =  ultra violet
                                      vii

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                               ACKNOWLEDGEMENTS




     Syracuse Research  Corporation wishes  to acknowledge  the assistance  and




cooperation provided by the staff members of all the twenty-four water treatment




plants  during  our  sampling  trips.   Special  acknowlegements  are extended  to




Mr. Conrad Teufel and Mr.  James Morgan for assisting us with the field samplings.




We  are  also grateful to  Drs.  David  Schwartz  and  Lawrence  Mason  for  their




assistance  with  some of  the Ames  Assay  work.    The  generous  guidance  and




assistance  of  the  EPA Project  Officer,  Dr.  Frederick  C.  Kopfler,  is  also




appreciated.




     Cost sharing by the American Petroleum Institute at the level of 5%  of the




overall  cost  is  appreciated.    Special thanks  are  extended   to API Project




Officers, Dr. David D. Disbennet  and Dr. David Chen.
                                     viii

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                                   SECTION 1




                                 INTRODUCTION






     The  presence  of   trihalomethanes   and   other  potentially  carcinogenic




compounds in  drinking  waters has  caused  considerable public concern  over  the




potential effects on health  arising  from  the  consumption of these waters.  The




two generally recognized sources  of chemical contamination of drinking water are




pollutants present in raw water  sources of  potable waters and compounds formed




during treatment of raw  water, such as chlorination of finished waters.   A third




and little investigated  source is the possibility of contribution  of mutagenic or




carcinogenic substances  in drinking waters by the pipes used in the distribution




of potable waters.   The  level of exposure to chemicals through the consumption of




pipe-leachable compounds in potable waters may  be low but of long-term duration.




In the absence of a general  agreement  that  a  completely safe exposure level of




biologically  active  chemicals  does  exist,  it  is  important  to identify  the




suspected chemicals and  evaluate  the  possible health effects associated with the




use of potable waters  after their passage through the distribution pipes.




     German workers have shown that significant amounts of polynuclear aromatic




hydrocarbons (PAH),  many of which are suspected or proven carcinogens, are added




in the distribution system (Borneff,  1977).   In  one case, the concentration of




PAH at the end of the water  supply network  was about 10  times higher  than  the




concentration at the treatment site.   Increases  in the concentration of PAH at




the end of the supply network are also indicated from our preliminary investiga-






                                       1

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tion on three water supplies  within the United States; the asphalt-based coating



material used within the pipes was suspected  to be the cause  of  this  problem.



Microbial  mutagenicity  assays   utilizing  histidine  dependent   strains   of




S. typhimurium  (Ames  et  al.,  1973)  were  also performed  on drinking  waters



obtained from  the  distribution system at  three selected water supplies.   The



results revealed a  significant increase in the  mutagenic activity upon passage of



the treated water through distribution pipes.   Some of the compounds introduced



required mammalian  metabolic  activation (i.e., S-9 mix), whereas others elicited




mutagenic responses without the activation system.



     The present  investigation is directed at  examining  the contribution  of



mutagenic compounds from the  supply network to selected water supplies within the



United States.  The overall  mutagenic potential will be assessed  by utilizing



non-specific bioassay systems.   Furthermore, in view of the  potential  of  the



distribution pipes  for addition of PAH, the  presence of PAH in these waters will




also be examined.



     The study  proposed is a  screening  type  investigation and is designed to



provide information concerning the intensity  and frequency  of the problem with




water distribution processes.  The results  of  this investigation will be helpful



to EPA in setting priorities for in-depth and more specialized work, and in the



epidemiological investigation  currently  underway at EPA to derive a relation-



ship, if any, between the quality of drinking water and the occurrence of certain



types of cancer.

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                                   SECTION 2




                                  CONCLUSIONS






     The primary objective of this study  was  to investigate the possibility of




enhancement of PAH concentration and mutagenicity of treated water as a result of




its passage through distribution pipes.   The results indicate that in 67% of the




total water supplies studied, a measurable increase in PAH concentration in water




does  occur as  a consequence  of  treated water  travelling through  petroleum




asphalt-lined distribution lines.  The increases in PAH concentration,  however,




were small and amounted to a maximum of  61 ppt in water.  It was also determined




that  the  maximum leaching  effect occurred with  distribution  pipes that  had




recently been  coated  with  asphalt  linings.   The  recently  installed  asphalt-




coated pipes predominantly added  the more water-soluble PAH, whereas the older




pipes predominantly leached the less water-soluble PAH.  In older pipes this is




probably due to depletion of the more water-soluble PAH from the surface of the




lining.  Waters from a few distribution lines showed reductions in PAH concentra-




tion instead of the expected enhancements of PAH concentration.   This reversing




trend can be explained by assuming sorption of PAH on the surface of the distri-




bution pipes.   Distribution  pipes that are depleted  of the leachable PAH may act




as sorbents for these compounds.  Thus,  depending on the age of  the pipes, it is




possible to get both desorption and sorption  effects.  In  addition  to  the pre-




selected six PAH identified and  quantified in this study,  many  other compounds

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were detected  in the  water  samples.   No attempt  was  made to  identify  these




compounds.



     The results  of bacterial mutagenicity  assays  (Ames assay)  indicated  the



presence of  low levels of mutagenic  activity in many  of the waters  sampled.




There appeared to be no correlation between the level of biological activity and



the passage  of  water through distribution pipes.   Also there was  no  apparent



correlation  between the  type of  pipe  used and  the   presence  or absence  of




mutagenic activity.   The specific compounds that produced the observed mutagenic




response were not identified, however, there was no correlation between the PAH



levels of water and  the  observed mutagenicity.   In most  cases where  mutagenic




activity was observed in water at the treatment plant this activity was the same



or greater  than  the activity in the  raw water.  This  indicates that the treatment



plant was generally  ineffective in removing this low  level of mutagenic activity,



and that some of the processes employed in water treatment may actually contri-




bute to the presence of mutagens in the distribution system.  In considering the




source  of  water  for potable use,  ground waters  generally demonstrated  less



mutagenic activity as compared to  surface waters.

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                                   SECTION 3

                                RECOMMENDATION


     On the basis of our experience with  the  present project,  we recommend the

following areas for further investigations:


     (1)  Samples  that  showed mutagenicity  should be  fractionated  to
          isolate  and  identify  the  component(s)  responsible  for  the
          mutagenic effect.

     (2)  Since no  dramatic  PAH leaching was observed  with  the petroleum
          asphalt lined pipes,  further study should be directed towards the
          leaching effects from coal  tar-based  distribution  pipes.   It is
          expected that the leaching may be more dramatic with these pipes
          than asphalt based distribution pipes.

     (3)  In the present investigation,  the age  of the installed pipes was
          found to be one of the most critical factors in the leachability
          of PAH from the pipe  linings.  In future studies, the age of pipes
          should be given one  of the  primary  considerations  in the selec-
          tion of sampling  sites.   Recently installed pipes are expected to
          provide more  dramatic enhancement of  PAH  concentraton  than the
          older pipes.

     (4)  Factors, such as  pH,  residual  chlorine, aggressive index of water
          and conditioning  of the pipes  after application of coatings—all
          the factors that  may affect the leaching of PAH— should be more
          thoroughly studied.

     (5)  The  effect  of chlorination of  drinking  water on  some  of the
          leached compounds should be investigated.  A few recent investi-
          gations suggest that  the  leached compounds  are susceptible to the
          formation  of chlorinated  or  other  oxidized  products  in  the
          presence of chlorine.

     (6)  The  apparent  increase   in  mutagenic activity  resulting  from
          conventional  water  treatment  processes  should  be  studied  in
          greater detail.    This  should  include  an investigation  of the
          production of high molecular weight compounds  having biological
          activity as a result chlorination.

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(7)   The  mutagenic  activity  of  trace  contaminants  in  treatment
     chemicals used  for  the  production of  finished water should  be
     investigated  as a  possible  source  of mutagenic  activity  in
     drinking water.

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                                   SECTION 4



                               LITERATURE REVIEW






     The  presence  of   trihalomethanes   and  other  potentially  carcinogenic




compounds in  drinking  waters has caused  considerable public concern  over  the



potential health effects arising from the consumption of these waters.   The two




generally recognized sources of chemical  contamination  of drinking water  are




pollutants  present  in  the  raw  water sources  of potable  waters  and  compounds




formed during treatment of raw water, such as chlorination and ozonation used for




disinfecting water prior to human consumption.  A third and little investigated



source  of  contamination  is the   possibility  that  the  pipes  used  in  the



distribution  of  potable  waters  may  contribute  mutagenic  or  carcinogenic



substances to drinking waters.




     Finished waters from the treatment  sites  are transported to the  consumers




through a variety of pipelines.   In the United States, three kinds of  pipes are



commonly used as transmission distribution lines for transporting treated water




to consumers:  cast iron/ductile iron, abestos/cement, and steel  pipes.   Other



materials occasionally  used as water distribution lines are reinforced concrete,



a variety of  plastic materials,  iron, copper,  and rarely,  lead.  Of  the total



installed length of approximately 685,000  miles  of  water  distribution  lines in



the United  States,  about  75% consists  of cast  iron/ductile  iron pipes,  13?



consists of  asbestos/cement pipes,  and  6%  consists  of  steel  pipes  (Goldfarb




et al.,  1979).

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     With the exception of  plastic  pipes,  coating materials are often used for




the interior  of  the water pipes to  prevent  corrosion or to aid  in  curing the



pipes.   For  example,  cast  iron/ductile  iron  pipes  are sometimes  lined with




asphalt,  cement,   or  asbestos/cement  coatings;  concrete  pipes  with  asphalt



coatings; and steel pipes with cement or coal-tar coatings.  Depending upon the



characteristics, manufacturing conditions,  thickness,  and age of the  pipe  or its



lining,  some  leaching of  materials can be  expected  to occur  as  water passes




through the pipes.  McFarren £t _al.  (1977) have demonstrated  that galvanized iron




pipes may  leach  into  drinking water, lead,  zinc,  and cadmium;  asbestos/cement




pipes some  asbestos  fibers;  and PVC pipes some residual vinyl chloride.   The



leaching of volatile organic  compounds  from  different types of water pipes has




also been studied in Europe (Eklund at _§!., 1978).  Cast iron pipes coated with



polyurethane have been shown to add volatile contaminants such as chlorobenzene



and aromatic compounds in drinking waters.




     Both asphalt  and  coal-tar are known to contain  high levels of polycyclic




aromatic hydrocarbons (PAH),  some  of which are  known  or suspected carcinogens.



Table 1  presents  the  levels of a  few PAH  in coal-tar and petroleum asphalt as



determined by Radding et _al. (1975).

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 Table  1.   Levels  of Some  PAH  in Fossil Fuel Derivatives  (Radding et al.,  1975)
PAH Content in Fossil Fuel Derivatives, u.g/g
PAH Identified
Benzo(a)pyrene
Chrysene
Benzo ( a ) anthracene
Coal-Tar
30,000
up to 2,860
up to 6,980
Coal-Tar Pitch
12,500
up to 10,000
up to 12,500
Petroluem Asphalt
0.1-27.
O.M-34.
up to 34.
     It is thus  possible  that the  coating  of coal tar-based  or asphalt-based




paint or sealant on iron,  steel or concrete pipes could cause leaching of small




amounts of PAH into drinking water,  determined  partly  by their solubilities in




water.  Borneff (1976) reported a ten-fold increase in PAH concentration at the




end of water supply pipes resulting from the  coating used  inside the water pipes.



In a study in  Pascagoula, Mississippi, more than 9 PAH,  including  fluoranthene at



concentration as  high as  9.7  (ig/1,  were reported  in  water  stored  in  a  tank




freshly coated with coal-tar pitch (McClanahan,  1979).  Similarly, potable water



obtained from a terminal point  of  a water distribution pipe coated with coal-tar




showed the presence of PAH.   The fluoranthene and phenanthrene concentrations in



this  Portland,   Oregon,  water  were  reported  to be  0.6 u.g/1  and  3-2 u.g/1,



respectively (Roebeck, 1979).



     Although the leaching  of coating materials  from the pipes can account for



the presence of many compounds including PAH, it may not be  the  only mechanism



which could contribute to the deterioration of the water quality resulting from




passage of drinking waters  through  supply networks.   The  reaction of residual



chlorine with organics leached  from pipes,  oxidation of leached materials,  or




microbiological  synthesis   of  compounds on pipe  walls  and  their  subsequent

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release into the water may  cause  contamination  of the water with compounds not




originally present in the finished waters.



     The  examination  of changes  in  water quality resulting  from distribution




pipes  can be accomplished  by determining changes  in  the  levels  of  specific



chemicals.   Because of  their reported presence  in some coating materials used




inside the distribution lines, and because of  the known carcinogenic potential of



many of the compounds  in this class, PAH were  chosen as a class  of chemicals to be




monitored  in order to determine  possible adverse effects  of the distribution




system on water quality.  To monitor  the  effects of other harmful compounds that



may be leached from the pipes or  produced in the distribution system,  an assay



was also  employed  that  was  not  specific for a  particular  class of chemicals.




This general assay was an in vitro microbiological mutagenicity assay which has



the  capability  of , detecting a  large  variety  of   compounds  with  genotoxic




potential.   Therefore,  the present  investigation  was  primarily  oriented  to




provide  information  on two aspects  of  water  quality:   PAH content and  the




presence of mutagens in drinking waters  as a result  of  passage of those  waters



through the distribution pipes.



     The monitoring of the  expected low levels of PAH  in  drinking waters required



the selection of  a method (Basu and Saxena, 19?8a) that was capable of concentra-



ting PAH  from large volumes of water.  The method must also include a chemical



separation procedure capable of eliminating interfering impurities, and a quan-



tification method  sensitive enough to detect  small  amounts  of  PAH.   A  method



developed in a previous study (Basu and Saxena,   19?8b) was found to be suitable




and was used for the monitoring of PAH levels in waters.






                                       10

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     The  overall  mutagenic  potential   was   assessed  by  utilizing  the  Ames




Salmonella assay system.   This assay employs a mammalian  metabolic activation




system, thus allowing  the  detection of compounds that are biologically active




only after having  been metabolized.   The system  was chosen primarily  for two




reasons.   First,  it provides  a quick  screening  method for  testing mutagenic



potential of compounds.  Second, a high correlation between positive results in



the Ames assay and positive results in in vivo animal carcinogenicity assays has




been established  for  a large  number  of compounds  (Ames  et al.,  1973;  McCann



et al., 1975).
                                       11

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                                   SECTION 5




                             MATERIALS AND METHODS








MATERIALS




PAH Standards




     Fluoranthane, Benzo(ghi)perylene,  Indeno-(1,2,3-cd)pyrene,  and  Benzo(a)-




pyrene were obtained from Aldrich Chemical Co.  Each compound was  at  least  99%




pure  as  verified  by  GC-FID and  TLC-fluorimetric  analysis.    Benzo(j)-  and




Benzo(k)fluoranthene were  obtained  from Community  Bureau of Reference  (BCR),




Brussels, Belgium.  These were certified reference materials of  better than  99%




purity and used as received.




Materials for Capillary Column (WCOT) Chromatographic Analysis




     Gases:  He  (carrier gas) was of  ultrahigh purity grade, N, (detector make-up




gas) and H  were  both  prepurified medical  grade,  Linde Division,  Union Carbide




Corp.




     Aluminum foil-backed septa(Metasep) were from Altech Associates




     WCOT Columns were  30 m x 0.25 mm i.d.  and  15 m x 0.25  mm i.d., and both were




coated with  SE-51* obtained from Supelco,  Inc.   Analysis  of samples  collected




after 8-1-80 were performed with the shorter column.  The gas chromatograph with




dual  flame  ionization  detector was  a  Hewlett-Packard  Model 5730A  with  18740B




capillary column control accessories, operated in  the  splitless  mode,  and used




with a Fisher Recordall Series 5000.
                                       12

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Materials for Thin Layer Chromatography



     Acetylated  cellulose  (40^), aluminum  oxide G  (type E) ,  8  x 8 cm  glass



plates, TLC tank,  Desaga  template,  and Desaga-Brinkman Spreader were  obtained



from Brinkman Instrument, Inc.   Spectrophotofluorimeter with thin  film scanner



was from American Instrument Co., and the U.V.  Lamp (Mineralight) for visualiza-




tion of TLC spots was from Scientific Products,  Inc.



Materials for Radiolabeling Work


                            14
     Radiolabeled  7,   10    C-benzo(a)pyrene   was   obtained  from  California




Bionuclear Corp.  and  a Packard Tri-Carb Scintillation Spectrometer with Teletype



Printer  was used  for  determination  of  radioactivity.   Scintillation  fluid



contained  2,5-diphenyloxazole   (PPO),  2,2'-phenylene  bis-(S-phenyl)   oxazole



(POPOP) which was purchased from Packard Instrument  Co.



Materials for Sampling




     Cyclindrical flexible polyurethane foam tube or flask plugs (45 mm x 45 mm)




with  a trade  name  of  "Identi-Plug"  were  obtained  from VWR  Scientific  Co.



Extender  type  chromaflex columns of 25 mm  i.d. with "0" rings,  adapters and



clamps were from  Kontes  Glass Co.  The Masterflex pump, controller and pumpheads



were  made  by  Cole-Parmer  Instrument Co.,  Chicago,   and  the  Haake  Model  FE



Thermostated Circulator  with 1000 W Heater was purchased from VWR Scientific Co.



Chemicals, Reagents and  Apparatus for Sample Treatment



     All solvents were from Burdick  Jackson Lab.,  Inc. and distilled in glass.



     Chromatographic  grade  Florisil  (60-100 mesh),  was obtained from  Floridin



Co.
                                       13

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     The Buchler Rotary  Vacuum evaporator with  thermo-regulator  and  immersion




heater  was  purchased  from  Scientific  Products,  Inc.  and  calibrated  tubes



(6.5 ml), with each of  the last four-tenth ml calibrated to one-hundredth ml were




purchased from Fisher Scientific Co.



Standard PAH Solution



     Stock solutions of each of the  six PAH were prepared by dissolving 100 mg in




100 ml benzene in a volumetric flask (1000 ppm).



     In order to prepare working PAH standard mixtures for GC analysis, 1 ml each




of the six stock solutions were diluted in a 10 ml volumetric flask and brought



up to the final volume with benzene  (100  ppm).   Standard  PAH mixture for TLC was




prepared by mixing  5 ml of the  standard (100 ppm) fluoranthene solution and 1  ml



each of the five other PAH (100 ppm) in a glass stoppered bottle, (final concen-



tration:   50 ppm fluoranthene, 10 ppm others).




Materials for Bioassays




     Test  organisms utilized   in   the  rautagenicity  bioassays  were  specially



constructed strains  of Salmonella typhimurium designated TA98, TA100,  TA1535,




TA1537,  and  TA1538, all supplied  by  Dr. Bruce  Ames  (Berkeley,  California).



During  the  course  of  the  study  it  was  necessary  to  obtain subcultures  of



S. typhimurium strains TA100 and TA98 from Dr.  Edmond J.  LaVoie at the American



Health  Foundation.   Male  Sprague-Dawley  rats weighing  150-200  gm each  were



obtained from a commerical  supplier, Taconic  Farms,  Inc.   (Germantown,  NY) and



were used in the preparation of liver homogenates for the S-9 metabolic activa-



tion system.






                                       14

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     DIFCO Bacto-Agar and DIFCO  Nutrient  Broth (Scientific Products)  were used




for media preparations.   Aroclor  1254  (Monsanto Co.) and U.S.P. corn oil (Fisher




Scientific) were used in the  induction of rat liver enzymes.  Diagnostic mutagens




routinely used  for  positive  controls were:   4-nitro-o-phenylenediamine  (Sigma




Chemical), methylmethane  sulfonate (Aldrich  Chemical),  9-aminoacridine  (Sigma




Chemical), 2-aminoanthracene  (Sigma  Chemical).   The DMSO  (dimethyl sulfoxide)




was  obtained from  Burdick  and  Jackson  Laboratories.   All  salts,  and  other




chemicals were of reagent grade.




     Unconeentrated water samples were filter sterilized using a 90 mm standing




filter  holder  (Millipore  Corporation),   with 90 mm Gelman  filters  (0.20 um,




0.45 pjn, 1.20 MJH, and glass prefilters).   Revertant  colonies were counted with a




BioTran II automatic colony counter.




EXPERIMENTAL PROCEDURES




Sampling Procedures




Selection of Sampling Sites—




     The sampling locations for this  study represent the largest water supplies




within the 24  selected  counties in the United States.   The  areas  these  water




supplies serve  represent  communities with  both  high and  low cancer  incidence




rates.  In an ongoing epidemiological study,  the U.S.  Environmental Protection




Agency is making an effort to determine  if any  correlation exists between the




occurrence of certain types of cancer  with certain water quality parameters such




as  total  organic carbon,  halogenated hydrocarbons  and asbestos.   The present




investigation will  provide  an additional  parameter, PAH,  for correlation with




EPA's epidemiological data.   Table  2 identifies the raw water source, type of raw






                                       15

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water pollution, treatment provided to generate finished water,  and the date of



sampling for the water supplies investigated in this study.




Sampling Procedure



     Earlier  studies  (Basu  and  Saxena,  1978b;  Saxena _et  al.,   1977a;  Saxena




gt al.,  1977b) showed that water passing through foam plugs  at 62 + 2°C at a flow



rate of 250 ħ 10  ml/min  can  effectively concentrate PAH from water.  Four foam



plugs two  in  each column and connected  in  series,  were adequate  for sampling




20 liters  of  finished  water and  10  liters  of  polluted  raw water.   A maximum




volume of  75  liters of finished water and 60  liters of raw water were sampled



with a portable sampling unit assembled in our laboratory from a combination of



off-the-shelf hardware available commercially.



     With a minor modification, the sampling unit described in our earlier work



(Basu and Saxena,  1978a)  was  employed  for  the collection of water samples in the




field.  To facilitate air transportation, the components of the sampling equip-




ment  were assembled  inside suitcases.   The  overall  unit consisted of  the



following five parts:   (a) variable water pumping unit consisting of a Masterflex



pump with  appropriate  pump heads  and flow  controller, (b) Haake Thermostated




Water Circulator, (c) unit containing the column systems,  (d)  thermometer,  and



(e) flowmeter.



     The variable pumping unit allowed water from the sample source to be pumped



to a column system  (containing the foam  plugs)  at  a controlled rate  of  250  ħ



10 ml/min.  The water prior to reaching the  column passes through the stainless



steel coil of a Haake heating bath and was maintained  at 62 ħ 2°C.   The column




system consisted of  two 25 mm i.d.  Chromaflex extender-type columns connected in






                                       20

-------
series with the help of  tapered glass adaptors and double-bent pyrex tubing.  The




temperature of the water passing through the  columns was monitored by introducing



a water-trap equipped a thermometer.  A Gilmont flowmeter calibrated to measure



the flow rate was placed at the end of the sampling unit.  The  connections were




made with glass  tubing as  much as  possible  and only where necessary with Tygon




tubing.



     For convenience of transportation, the variable pumping unit with the flow



controller and pump heads was carried in one suitcase and  the  rest  of the unit




containing the Haake  Thermostated  Water Circulator, the clamps  for  the column



system, water trap for the thermometer, and the  flowmeter  were all fixed in place



in another suitcase.   The two suitcases were  transported  to the sampling site



along with an electric timer, 20 liter Jerrican and other detachable items such



as, extender columns,  thermometers,  clamps, "0" rings, and connecting tubes.  The



purpose of the calibrated  20  liter Jerrican was to collect the effluent from the




sampling unit  and  thereby keep an  exact  record of the  volume  of  water passed




through the  columns.   The timer was  used to  indicate the running time  of the



sampler.




     At the  sampling location, the unit was  assembled  as shown  in  Figure 1.



Water from a  1 liter  beaker  placed  under a continuously running water tap was



pumped through two foam columns under the previously described conditions.  Since



the residence time for water  in the  beaker was very short, the losses of PAH due



to adsorption were  assumed to be insignificant.  Both foam columns  were changed



after a certain  volume of  water (maximum  25 liters)  passed through the columns




and the sampling was  continued with fresh foam columns.   In case of raw water,






                                       21

-------
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                                                                                         13  
-------
depending on  the  suspended solid  content,  this maximum volume  varied  from 10




liters to as high  as 20 liters.  After sampling the desired volume of water,  the



columns were  wrapped  in aluminum  foil,  cooled in ice, and  transported  to  the



laboratory.    In the laboratory, a major part  of  the foam eluate  obtained by




solvent extraction  was  used for PAH  analysis and a  fraction  of the remaining



extract was used for determination of mutagenic activity.



     In  addition  to  collection of  samples  by concentration on  polyurethane




foams, 2 gallons of unconcentrated water were also collected from each sampling




point for determination  of  mutagenic activity.   Collapsable polyethylene bottles



of one gallon capacity were rinsed three times with the water to  be collected




prior to  filling  the  bottles  with water.   A small air space was  left  in each




bottle for  expansion  of water  during  cooling.  The  bottles were  capped with



plastic screw  caps,  cooled in  ice,  and transported to the  laboratory  via  air



freight.




Sample Collection Points at Each Water Supply—



     At each of the sampling locations,  samples  were collected at four different



points.   These were raw water  and finished  water,  both  usually at  the water




treatment plant,  and  two samples  of  finished water.   The finished  water  was



sampled after  passage through  different lengths of distribution pipes.   Where



possible the two sampling points were chosen  at the  furthest distance from  the



treatment plant which would be consistent with obtaining water which had passed



through two  different kinds of distribution pipes.  These samples represented the



maximum exposure of the  finished water to the two different pipes.  In locations




where the distribution system consisted  of only  one kind of pipe,  one sample  was






                                      23

-------
taken at the most distant  practical  point  from the treatment plant and  repre-




sented maximum exposure  to the distribution pipe,  while the other  sample  was




obtained from  an intermediate  point in the system.   The sampling  points  are




diagrammatically shown in Figure 2.




     The two  finished  water samples representing  exposure to  the distribution




lines were usually collected  from water taps inside motels, private or public




buildings at approximately  the  desired  distances  from  the treatment plant.   To




avoid the collection  of contaminants which  resulted from leaching plumbing lines




in these buildings  (as opposed to contaminants leaching  from  the distribution




line), running water from well flushed lines was used to obtain samples.




     Table 3 describes the type of pipes the finished water passed through before




reaching the sampling points  (sample 3  and sample  4 in Figure  2),  the distance




the finished waters  travelled  through these pipes  and  the configuration of the




distribution system with respect  to  the treatment  plant.  It should be pointed




out  that  the  distances  given  in Table 3  refer  to the  approximate distances




travelled by the  waters through the pipes (the distribution lines were not always




straight and  could be  circuitous) and not  the  actual  distance  of the sampling




point from the treatment plant.




Elution of PAH from Foam Plugs




     The PAH were eluted from the foam columns  by washing  each column with 30 ml




acetone and 125 ml cyclohexane.  The details of the elution procedure are given




elsewhere (Saxena et  al.,  1977a).   Several  impurities  belonging  to foams were




also collected during the elution process.   With the exception of trace amounts




of fluoranthene and benzo(ghi)perylene,  other interfering  impurities originating






                                      24

-------
   RAW
 WATER
SOURCE


TREATMENT
PLANT
TYPE A DISTRIBUTION PIPES

            SAMPLE 1
          (RAW WATER
          AT INTAKE)
SAMPLE 2
(FINISHED
WATER)
   SAMPLE 3
  (MID-POINT
FINISHED WATER I
                                       1
 SAMPLE 4
 (FARTHEST POINT
 FINISHED WATER)
   RAW
 WATER
SOURCE
                                                SAMPLE 3
                                                (MID-POINT FINISHED WATER)
            SAMPLE 1
          (RAW WATER
          AT INTAKE)
SAMPLE 2
(FINISHED
WATER)
                    SAMPLE 4
                    (FARTHEST POINT
                    FINISHED WATER)
      WATER SUPPLY WITH ONE TYPE(A) OF DISTRIBUTION PIPES
   RAW
 WATER
SOURCE


TREATMENT
PLANT
                  f     l
            SAMPLE 1
          (RAW WATER
           AT INTAKE)
SAMPLE 2
(FINISHED
WATER)
                                              SAMPLE 3
                                              (FARTHEST POINT OF TYPE A PIPE)
                        SAMPLE 3
                        ( FARTHEST POINT OF TYPE B PIPE )
RAW \
SOURCE 1

TREATMENT
PLANT
TYPE A
DISTRIBUTION PIPE
TYPE B
DISTRIBUTION PIPE
1
             SAMPLE 1
          (RAW WATER
           AT INTAKE)
SAMPLE 2
(FINISHED
WATER)
   SAMPLE 3
(FARTHEST POINT
 OF TYPE A PIPE)
                                                              1
                                         SAMPLE 4
                                         (FARTHEST POINT OF
                                         TYPE B PIPE)
SAMPLE 3
(FARTHEST POINT
 OF TYPE A PIPE)
      WATER SUPPLY WITH MORE THAN ONE TYPE (A & B) DISTRIBUTION PIPES
      Figure  2.  Locations and  configurations of  sampling points
                  after  the passage of potable waters  through
                  distribution pipes
                                  25

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from the  foam  plugs could  be  eliminated by precleaning plugs  with  benzene or




cyclohexane as detailed by Saxena _et al. (1977a).




PAH Separation and Analysis



PAH Separation—



     During the  concentration  of PAH  from  water,  several  other  water contam-




inants were also concentrated on the foam plugs  and  some  of them coeluted with



PAH during the elution  step.   These impurities interfered with the analysis of




PAH and  a  cleanup procedure  was  found  necessary.    The  first  cleanup  step




consisted of the solvent partitioning procedure of Hoffman and Wynder (1960) as



modified by Acheson jet al. (1976). However,  this  procedure alone was inadequate,




and additional cleanup involving liquid chromatography through a short Florisil



column was found to be  necessary to further separate the PAH from impurities.



Following cleanup,  the  extract was  concentrated to  0.1 ml  by a combination of



evaporation in  a rotary evaporator  and  under a stream  of  nitrogen gas.   The




details of the cleanup  and  concentration  procedure are described elsewhere (Basu




and Saxena, 1978b; Saxena j§_t al_., 1977a).



Addition of Internal Standard for the Determination of Recovery Factor—




     The loss of PAH during the elution and  purification procedure was monitored


                                                              14
for each analysis by the addition of an internal standard of   C-benzo(a)pyrene



to the foam plugs prior to  initiating sample workup.   A  known amount of radio-




labeled material (quantity usually less than 10 ng) was added  to the foam plugs,



PAH eluted, purified and  concentrated  to 0.1 ml as  described in the preceding



section.   A  10  u.1  aliquot of  the concentrate  was  assayed for radioactivity by
                                       29

-------
liquid scintillation  counting.    The  recovery factor  was  determined  from  the

amount of radioactivity initially added to the foam plugs.

PAH Analysis

     The PAH quantified in the present study were fluoranthene, benzo(j)fluora-

nthene,   benzo(k)fluoranthene,    benzo(a)pyrene,   indeno(1,2,3-cd)pyrene   and

benzo(ghi)perylene.  The compounds selected can be regarded as representative of

the whole  polynuclear family,  and examination  of  their presence  in drinking

waters has  been  recommended by  -the World Health Organization  (1971).   PAH

analysis was performed using  two  techniques:   gas  chromatography-FID detection

and thin-layer chromatography-fluorescence detection.

     Our earlier studies (Saxena  et al.,  1977a)  demonstrated  that  a packed and

temperature-programmed Dexsil-300  GC  column failed  to  provide adequate resolu-

tion for some of the PAH, resulting in erroneously high values.  Therefore, with

the exception of the  samples from the  first six locations, all samples collected

after 8-1-80 were  analyzed  by SE-54  wall-coated glass capillary columns.   The

details of the operating conditions for  this packed column are given elsewhere

(Basu and Saxena, 1978b).  The conditions used for the capillary GC analaysis are

given below:


                    Injection mode:  Splitless
                      Vent delay:    40 seconds
                    Septum purge flow rate:   3 ml/min
                      Split vent flow rate:  60 ml/min
                    He carrier gas linear velocity:   25 cm/sec

                    Detector gases:
                      Np make-up gas  flow rate:  51  ml/min
                      N  gas flow rate:  39 ml/min
                      Air flow rate:   300 ml/min
                                       30

-------
                    Column Programming:
                      Initial temp.:  160°C
                      Program rate :  8°C/min
                      Final temp.  :  270°C
                      Final hold   :  65 mins for 30 m column and 45 mins for
                                      the 15 m column

                    Injection port temp.:  250°C
                    Detector temp.       :  350°C

     Nitrogen was used as the detector make-up gas to enhance the sensitivity of

the  detector.   The  sample injection technique  and  the  special nature  of  the

injection  port  septa  used have  been  previously described  (Basu  and  Saxena,

1978b).

     Thin-layer chromatography was performed using a slight modification of the

plate coatings of Borneff  and Kunte  (1969).   The plates were  developed  in  two

dimensions.   The  details  of the  preparation of TLC plates and  the method  for

solvent development have been described by Saxena et al.  (1977a).

     The identification of PAH spots on  the TLC  plates were made by two methods.

The first method, an approximate one, was  based  on the R_  values of the spots.
                                                         D

The  R_  values were defined  as  the ratio  of distance  travelled by  a specific
     D

compound to that of benzo(a)pyrene in the two solvent systems.   Since R~ values

were  found  to  be  somewhat variable  depending  on  the  thickness   of  coating

material, solvent composition and other undetermined factor(s), the  unequivocal

identification of a compound was made by the excitation and emission spectra of

the  individual spot  directly  on the plate  at  room  temperature  with an Aminco

thin-film scanner attached to an Aminco-Kiers spectrophotofluorimeter.

     For  quantification  of the  spots,   the signals  of  fluorescence  emission

intensities of the scanned spot  produced  by the photomultiplier were  transferred


                                      31

-------
(as input) to a strip  chart  recorder  (50  mv full scale deflection).  The peaks




were  manually  integrated  for  area  determination.    The  in situ  scanning



eliminated  the  losses  usually encountered  during  removal  of  PAH spots  for




fluorescence measurement  in  solvents,  and  the  interferences  arising due  to



solvent interactions.  The details of the TLC identification and quantification




of the  spots are reported  elsewhere  (Basu and  Saxena,  1978b;  Saxena jit  al.,




1977a).




Assay of Mutagenic Activity




     The bioassay employed in the  present  study utilizes the histidine-requiring



strains of the bacteria Salmonella  typhimurium  developed  by Ames and coworkers



(1975).   This  In  vitro bioassay is presently one  of the  most  widely known and



frequently  used   test  systems  to  detect the  mutagenic  effects   of  chemical



substances.   The  Ames test  has  the advantages  of speed,  ease  of  testing,  low




cost, and the generation of  relatively precise  results.   This  assay can detect




point mutations of  both  the frameshift and  base-pair  substitution types.   The



assay has the capacity  to approximate metabolic activation  of the test substances




by the incorporation of a microsome-containing subcellular preparation obtained




from rat liver (S-9  mix).  A  flow diagram  of the bioassay procedures used during



the present studies is presented in Figure 3.



     The assays were performed in both the  presence  and  absence of a mammalian




metabolic activation (S-9) system.  The following is a brief description of the



tester strains used in the present study:






                                       32

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                                         f^
     TA1535   -  Strain reverted by mutagnes which cause base-pair substitutions

     TA1537   -  Strain that is reverted by mutagens causing frame shift
                 suppression in G-C rich regions

     TA1538   -  Strain reverted by mutagens causing frame shift suppression as
                 small deletions in G-C base-pair regions

     TA98     -  Similar to TA1538 above, but possessing a pKM 101 plasmid
                 containing an ampicillin resistance gene and a "mutator" gene,
                 which increases sensitivity to mutagens

     TA100    -  Strain derived from TA1535 and like TA98, possesses a pKM 101
                 plasmid for ampicillin resistance, and a "mutator" gene

Methods for Routine Bioassays—

     Strains of S_.  typhimurium are routinely cultured in our laboratory, and are

stored as  frozen bacterial  concentrates at -80°C.   The  following procedures

describe the methods employed for the routine handling and storage of the tester

strains,  the preparation of the metabolic activation system, and the preparation

of minimal media/agar plates used in the assay.

     Preparation of Cell Suspension—Cell suspensions of the  Salmonella tester

strains for  the present study  were  prepared using a modification  of  the Ames

procedure.  Instead of growing fresh cultures each time an assay was performed,

large batches of subcultured bacteria were prepared and stored frozen at -80°C.

Aliquots of these frozen cells were used directly in the bioassay after thawing

and making the  appropriate dilutions.    In  this  manner  the  cells from a single

subculture could be used for a number of bioassays performed on different days.

The bacteria were grown, harvested, and stored by the following methods.

     A subculture  of  the  tester  strain was  prepared  by  innoculating  100 ml

sterile nutrient broth (8  gm DIFCO  Nutrient Broth,  15 ml  Q.2%  L-cysteine;  in

-------
1000 ml distilled water) with bacteria from a  frozen stock culture.  The innocu-




lated broth was gently shaken in a water bath at 37°C for 16 to 18 hours, followed



by  as^eptically  harvesting  the cells by  centrifugation  at  5500 rpm for  15




minutes.  The spent broth  was discarded  and the remaining bacteria pellet was



resuspended in sterile nutrient broth supplemented  with 2Q% sterile glycerine as




a cryo-protective agent. The cell density  of the suspension was in the range of




5 to 9 x 10  cell/ml.   The  cell suspension  was as^eptically transferred in 15 ml



aliquots into vials and stored  at -80°C  until used.   This  technique eliminated



the necessity for the  growing of bacterial  strains  for  each experiment, and also



eliminates problems of  sparse "lawns" which may arise  with  the  slower growing



Salmonella  strains.     This   technique   leads   to  an  improvement   in  the



reproducibility  of  the Ames'  assay,  and  saves  time.   This  procedure  yields




reproducible results even  when  aliquots  of cells  were used  after two months of



storage at -80°C.




     Preparation of the Mammalian Metabolic Activation System (S-9)—Metabolic



activation  in the  bioassay  was achieved by  incorporation  of the  9000 x g



supernatant of a liver homogenate from rats previously  injected  with a polychlor-




inated  biphenyl  (PCB)  in  order to  induce microsomal enzyme  activity.    The



procedure as reported by Ames et al.  (1975) for the preparation of the rat liver



homogenate, the  S-9  fraction,  and the S-9 cofactor  mix was  employed without



modification.   The agent used for induction of the  rat  liver enzyme system was a



mixture of polychlorinated biphenyls (PCB) under  the  trade  name Aroclor  1254.



Aroclor dissolved in corn  oil (100 mg in 0.5  ml  cornoil) was administered as a




single intraperitoneal injection at  a dosage of 5 mg/kg.  To minimize potential






                                       35

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differences  among  individual  animals,  at  least three  rats  were used  in the

preparation of the  S-9 mix.  The rats were allowed 5 days for induction of liver

raicrosomal enzymes.  Food and water were provided ad libitum for  4 days at which

time the rats were  starved 24  hours  prior to being sacraficed.  The animals were

decapitated and their livers surgically removed under asceptic conditions.  The

rat livers  were weighed and  minced in cold  0.15 M KC£  (KC2/g liver  =  3:1  by

weight).   The  minced  liver  tissue  was  homogenized  using a  Polytron  tissue
                                 •ğ  ti
homogenizer followed by centrifuge^- of the homogenate at  9000 x g for 15 minutes

at  4°C.   The  supernatant  (S-9)  was  asceptically removed  and  7-0 ml aliquots

placed into sterile screw-top  glass vials.   Aliquots of the  S-9 fraction were

frozen and stored at -80°C.

     The complete S-9 mix (liver supernatant plus cofactors) was  prepared by the

following procedure.   All  ingredients were  asceptically  combined to  a final

volumn of 1 ml:


               Rat liver supernatant (S-9)        100 ul
               0.4 M MgCS-?                         20 ul
               1.65 M KC&                          20 ul
               1.0 M glucose-6-phosphate            5 u-1
               1.0 M NADP"1"                         40 ul
               0.2 M sodium phosphate buffer,
                 pH = 7.4                         500 p,l
               Sterile distilled water            315 ul
                                                 1000 |il

To insure sterility of  the  final  solution,  the homogenate-cofactor mixture was

filter sterilized through a 0.2 urn membrane filter.  Fresh S-9 mix was prepared

each day, and kept in an ice bath.  Unused portions  of S-9 mix were discarded at

the end  of  the day.   Each  batch of  S-9  was checked for  metabolic activation


                                       36

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     potential  using  diagnostic mutagens  of known  activity,  and the S-9  was also
     checked for possible bacterial contamination.
          Preparation  of  Minimal   Media  Plates  for  Liquid  Suspension and  Plate
     Incorporation Assays—A concentrated (50x) minimal salts solution (Vogel-Bonner
     minimal salt  concentrate)  was  prepared by adding the  following ingredients in
     sequence and stirring with a magnetic stirring apparatus until dissolved:
                    670 ml  distilled water
                      5 gm  MgSOjj (anhydrous)
                    100 gm  citric acid (monohydrate)
                    500 gm  potassium phosphate (dihydrous, anhydrous)
                    170 gm  sodium ammonia phosphate Ğ4 M
     This solution was not autoclaved,  and could be stored  at  room temperature for
     long periods of time.
          Minimal glucose agar was prepared by adding in a 2 liter Florence flask:
                     20 ml  50x Minimal salts concentrate
                    980 ml  distilled water
                     15 gm  DIFCO Bacto-agar if plates were desired
                                                       r
I/  The contents of the  flask  were autoclaved at 250°JS  and 15  psi  for  15 minutes.
     After removal from the autoclave,  5  ml of sterile 40$ glucose was asceptically
     added to the flask.   If plates were desired, 25-28 ml of media were asceptically
     transferred  to  100  x 15  mm  plastic petri  dishes  (Lab  Tex,  VWR  Scientific
     Supplies).  Minimal  media plates were used for the liquid suspension assays, and
     for the "1 ml" dose in the plate incorporation assays.
          Preparation  of Media/Water  Plates—The  following  method was   used  for
     preparing Media/Water plates in order to enhance the  possibility of detecting low
     levels of mutagens in  unconcentrated water samples.   In  a 600 ml prescription

                                            37

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  bottle,   30  gm  DIFCO  Bacto-agar   was  added  to   550  ml   distilled  water.
  Simultaneously, 40 ml of 50x minimal salts concentrate was added to a. two liter
  Florence  flask.   For those flasks  for  which half-strength media was required,
  700 ml distilled water was  added to the appropriate flask.   Both the agar and the
/                                                    \,
  salts or  salts/water flasks were autoclaved  at 250°C  and  15 psi for 15 minutes.
  After autoclaving, 5 ml of sterile 40?  glucose was added  to each of the flasks.
  For those flasks  used to  prepare plates with the maximum amount of test water,
  1400 ml of filter-sterilized test water  was added.  For the half-strength flasks,
  700 ml  filter-sterilized  test water was  added to the  appropriate  flask.   The
  contents  of all flasks were brought to 50 °C in  a water bath.  The molten sterile
  agar was  added and mixed in each flask.  Into  15 x 100 mm plastic petri dishes,
  25 to 28 ml of the media/water was asceptically poured.  The final amount of test
  water  in  the full-strength plates was  approximately  25 to 28 ml per plate and
  12.5 ml per  plate in the   half-strength plates.  These  media/water  plates were
  used to assay  unconcentrated water samples in  the plate incorporation assays.
       Preparation  of  Minimal Top  Agar and Minimal-Nutrient Broth — A  minimal top
  agar  media was  made by  adding to  a   2  liter Florence  flask,  the  following
  ingredients:
                       20 ml  50x minimal salts concentrate
                        5 gm  NaCl
                       75 gm  DIFCO Bacto-Agar
/                      980 ml  distilled  water
                                                  5
  The contents of the flask were autoclaved at 250 °p and  15 psi for 15 minutes.  The
  top agar was  stored  in sterile prescription bottles,  in  volumes of 100 ml, at
  room  temperature  until  used.   Before using the agar was  remelted by heating in

                                         38

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boiling water, and 10 ml of a sterile 0.5 mM L-histidine and 0.5 ml biotin stock




solution was added to each  bottle.  The contents of the bottle were gently mixed



by swirling.  The trace amount of histidine incorporated into the top agar will



allow  the  bacteria,  to  undergo  several  divisions,  and  is  necessary  for




rautagenesis to be observed.



     A minimal-nutrient broth (Vogel-Bonner solution supplemented with glucose)




was used  as  a pre-incubation medium for the  liquid suspension assays.   These



assays required a one hour preincubation of bacteria,  test organic concentrate,




and S-9 mix where  appropriate,  before  plating onto minimal-media  plates.   The



broth was prepared by adding 20 ml of the 50x minimal salts concentrate to 980 ml



distilled water and sterilizing this solution in an autoclave at 250°F and 15 psi




for 15 minutes.  After sterilization, 5  ml  of  a sterile Wf> glucose solution was



asceptically added.  For use in  the  assay 1.0 ml of  the  sterile minimal-media




broth was asceptically dispensed  into each sterile culture tube before bacteria,




mutagen, or S-9 mix were added.



     Preparation of Water Concentrates for Ames Testing—Organic concentrates of



water were received  from the analytical  laboratory  for  mutagenicity testing in



20 ml of cyclohexane.  The  water concentrates represented 10 liters  of water from



the raw-water  source or 15 liters of water  from the treated  and  distribution



sources.  Aliquots of each cyclohexane  concentrate were transferred to separate



tubes.  The cyclohexane was  evaported with nitrogen until dry.   Upon complete



drying the residue was  resuspended in sterile DMSO  to provide a stock solution



from which subsequent dilutions were made.  The stock solutions provided the "H"




(high) dose solution for each sample location.  For the raw water samples the "H"






                                      39

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dose represented 150 ml water per 0.1 ml  of the final DMSO solution.  Subsequent
raw water dilutions were:  "X" (intermediate)  dose, 100 ml water per 0.1 ml DMSO,
"M" (medium) dose,  50 ml water per 0.1  ml DMSO,  and "L" (low)  dose, 25 ml water
per 0.1 ml DMSO.  For the treated and both of the distribution water samples the
"H" dose represented 300 ml per 0.1 ml  DMSO solution.  Subsequent dilutions for
each sample were "X"  dose, 200 ml  water,  "M" dose,   100 ml water,  or  "L" dose,
50 ml water per 0.1  ml DMSO solution.   For each concentration assayed by liquid
suspension 0.1 ml of the appropriate DMSO water concentrate solution was used.
     Quality Controls—The performance  of the mutagenicity assays was routinely
examined  using diagnostic mutagens as  positive  controls.    Diagnostic  test
substances  of known mutagenic  potential and  not  requiring prior  metabolic
activation, included:
                    TA98     4-nitro-o-phenylenediamine
                    TA100    methylmethane sulfonate
                    TA1535   methylmethane sulfonate
                    TA1537   9-aminoacridine
                    TA1538   4-nitro-o-phenylenediamine
The performance of  the mutagenicity assays involving metabolic  activation was
verified by the use of 2-aminoanthracene as a  diagnostic positive control mutagen
for all tester strains in the presence  of the S-9 metabolic mix.
     Confirming the  tester strains' genotypes for the  presence of the R-factor
conferring  ampicillin  resistance  in  strains  TA98 and  TA100  was  routinely
performed.    Amplicillin-containing filter-paper   discs  containing  10 p.g  of
ampicillin were placed in the center of nutrient agar petri dishes overlaid with
each of the desired tester strains.  Zones of bacterial growth inhibition after

                                      40

-------
24 hours  incubation at  37°C  were observed,  as expected, with  tester  strains




TA1535, TA1537, and TA1538, but not with strains TA98 or TA100.



     To confirm the presence of  the  tester  strains'  deep rough mutation (fra),




which permits large molecules to enter the bacteria and inhibit growth, a crystal



violet test was carried out in conjunction with the ampicillin resistance test.



A sterile filter paper disc containing 10 ng crystal violet (10 ul of a 1 mg/ml



solution) is placed onto nutrient-agar plates containing bacteria.  After 12 to




24 hours incubation at 37°C a  clear  zone of growth  inhibition  around  the disc



indicates the presence of the deep rough mutation.




Experimental Procedures—




     Two variations of the  standard plate incorporation assay were used to assess



the mutagenic potential of drinking waters.  One  such  procedure was designated as



the "Media water assay,"  and employed unconcentrated, filter-sterilized drinking



water  samples.   The  second procedure  was  designated  the "liquid  suspension




assay," and employed organic concentrates of the drinking water  samples.  Details



of the two experimental procedures are provided in the following sections.



     Media Water Plate Incorporation Assay—To  facilitate the  detection of low




concentrations  of  mutagens  in  unconcentrated drinking  water  samples,  the



bioassay procedure  was  modified by  incorporating  filter-sterilized  test water



into the  base agar.  Two  different   amounts  of test water  in the  media were



obtained by dilution of  the test water  with an appropriate volume of distilled



water prior to the preparation of the media.




     For the actual plating procedure sterile  100  x  13  mm glass tissue culture




tubes containing 3  ml of the top agar  and  0.5  mM  histidine/0.5 mM biotin solution






                                      41

-------
were supplemented with 0.1  ml  of  the appropriate bacterial suspension and 0.5 ml



of the S-9 mix (where appropriate). The contents of each tube were overlaid onto



the base agar layer.   Suspect mutagens would  therefore be  present  in the base



layer, and test organisms and the S-9  mix would be  present in the top agar.  This



procedure allowed for  incorporation of approximately  25 or  12.5  ml  of drinking



water per plate  (70% of the total volume for the highest concentration).



     A third level of exposure was included in the protocol  by adding 1.0 ml of




filter-sterilized  drinking  water  into  the  agar-histidine/biotin  top  agar




solution, before  supplementation with  the appropriate  bacterial  tester strain




and S-9  mix  (where  required).  This  "1 ml"  exposure  was plated  onto standard



minimal-media plates in a manner  similar to a  plate incorporation assay.  Assays



on all the water samples collected from one location were performed  at the same



time  along  with distilled  water controls for  spontaneous  reversion  rate and




positive mutagenesis controls.  Therefore, three concentrations of 25, 12.5 and



1 ml per assay-plate were used to obtain dose response information.



     All assays  were performed in quintuplicate.   Plates were incubated at 37°C



in a  Sherer  Lab  incubator for 96 hours before enumerating  the colonies on the



Biotran  II automatic  colony counter.   The results  were recorded as  number of



revertants counted per plate, and expressed  as means  (+ standard deviation) of



the quintuplicate determinations. Also, the  ratio of  experimental ^control was



calculated.




     In previous laboratory studies conducted  at  SRC  (Neal  et al.,  1980) using



known mutagens,  it was demonstrated that  the  same mutagens  added  to the bottom




agar were detected with lower efficiency than those added to the top agar.  For






                                       42

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example, benzo(a)pyrene,  an extremely hydrophobia molecule,  was detected in the




media water assay with 10  times  less sensitivity compared to  the  procedure  in




which the mutagen was added  to the  top  agar;  however,  mutagens which were more



hydrophilic were detected with high  efficiency.  Thus, although some sensitivity



is  lost in detecting very hydrophobia  mutagens, the  ability to add  a larger




quanity of sample justifies the use of this procedure.



     Liquid Suspension Assay of Drinking Water Concentrates—This assay involved




incubation of an organic  concentrate of water and bacterial  tester  strains  in




liquid suspension prior to plating  on minimal-media.   Liquid  suspension assays




are  particularly  valuable  with  assays  that involve  complex  uncharacterized



mixtures, and where  the possibility  exists that some of  the compounds present,  or



a metabolically activated form of the compounds  present, may  be inactivated  by



the  agar.   All of  the positive  features  of the plate incorporation  assay are




retained by the liquid  suspension assay, therefore  this  assay can  detect any




compound  which is  positive  in  the conventional  plate  incorporation  assay.



DeSerres and Shelby  (1979)  have  recommended the  use of the  liquid  suspension



assay where  the  results  of  the  standard  plate incorporation  assay  are incon-



clusive .



     The liquid suspension assay was  used in assessing the mutagenic potential of



drinking water concentrates.   The pre-incubation medium for this assay contained




1 ml  of minimal media  (minus agar)  broth, 0.5 ml  rat-liver  homogenate  plus



cofactors  (S-9  mix) where  required for metabolic  activation, 0.1  ml of the



appropriate  bacterial  tester  strain,  4  u-g histidine,  6 ug  biotin,  and  the




designated amounts  of the  test  drinking water concentrate.   Identical vehicle






                                      43

-------
control  tubes  were made  with 0.1 ml  of DMSO  added.   Following a  60  minute




incubation of the mixture at 37°C in a water bath, 3 ml of soft agar containing



24 |ig histidine and 36 ug biotin per ml was  added to each tube.  The contents of



each tube were mixed and overlaid onto minimal glucose agar plates.  The plates



were inverted and incubated at 37°C for 96 hours.  The colonies were counted and




the results  expressed as the  ratio  of the number  of revertants  colonies  per



plates  receiving  exposure   to test  waters  over  the  number  of  spontaneous




revertants on control plates.




     Intrepretation of  the  Results—In the plate incorporation  assays  and the



liquid suspension  assays with test  water  concentrates,  a ratio  of revertant



colonies (experimental/spontaneous control)  of 1.5 to 2.0 was considered to be a



weak mutagenic  response,  and  a  ratio  of  > 2.0 was  considered  as  a positive



mutagenic response.  Particularly in  samples with low activity,  the presence of a



positive dose-response relationship was  used  to assist  in the  determination of




whether the sample was clearly mutagenic.

-------
                                   SECTION 6



                             RESULTS AND DISCUSSION



     The leaching of  PAH from coal tar-coated potable  water  storage tanks and




water distribution pipes has been demonstrated by a few investigators (Borneff,



1976; McClanahan, 1979; Roebeck,  1979;  Alben,  1980a).  However, only six percent



of the total installed potable water distribution pipes in  the U.S.  consist of



steel  pipes  that are typically  lined with  coal  tar-based  coating materials




(Goldfarb, 1979).  Instead,  the majority  (75%) of water distribution pipes are



made of  cast/ductible iron (Goldfarb,  1979).  Unlined  and  cement mortar-lined




cast/ductile iron  pipes are  typically coated with  petroleum  asphalt  paints,



instead  of coal  tar,  for corrosion inhibiton and as a  curing aid for the pipe



linings  (Goldfarb, 1979).   That  these  asphalt-based paints contain  PAH  can be




inferred from  the investigation  of Radding et al.,  1975 (see Section 4).  The




leachability of  PAH   in  potable  waters travelling  through distribution  pipes




coated with  asphalt-based  paints, however,  is  not  well  studied.   The primary



objective of the present investigation was to examine  both the  change  in PAH




concentration and the pattern of mutagenic activity in treated  waters  as a result



of their passage through commonly used distribution/transmission pipes.



     The sampling locations  selected for  the  study  represent  the largest water




supplies within  24 selected counties  in the  U.S.   These water  supplies  serve



areas  that   represent both  high   and  low  overall  cancer  incidence  rates  as




established by U.S. EPA epidomiological study  (Kopfler,  1977).  To avoid bias in




the  analysis,  however,  the  relative cancer mortality   rates  among  these  areas






                                       45

-------
remained unknown to us.  As  shown  in  Table  3,  the samples collected from these




water supplies  represent  contact with  a  variety of  distribution  pipes.   The



majority of  the distribution  lines  in these  locations  consist of  unlined or



cement-lined cast/ductile iron pipes.   A small number of samples were collected



from concrete,  asbestos/cement, cement-lined steel, plastic and copper pipes, or



a combination of these.  The exact sampling points  for each of the water supplies




tested  are  shown in  Table 3 and  Figure  2.   The  rationale  for selecting  the




sampling points is given in Section 5.




     The presence  of a wide variety of PAH, including several animal carcinogens,




in environmental samples is  not uncommon.   For  example,  in one study  (Kunte,



1979),  more  than 200  different PAH  were detected in  an ambient  air  sample.



Because  it  is  difficult and generally unnecessary  to  quantify all PAH in  a



complex environmental sample,  it seems practical to choose a  few  specific  and




representative compounds for water  monitoring purposes.  The six PAH analyzed in




this  study  included  fluoranthene  (PL); benzo(j)fluoranthene  (BjF);  benzo(k)-



fluoranthene  (BkF);   benzo(a)pyrene   (BaP);  indeno(1,2,3-Gd)pyrene  (IP);  and




benzo(ghi)perylene  (BghiP).   The  World Health Organization  (WHO)  recommends,




with the exception of benzo(j)fluoranthene, the analysis of these PAH in drinking



water.   Benzo(b)fluoranthene,  which  is recommended  for  analysis  by WHO,  was



replaced with its isomer benzo(j)fluoranthene in our  analysis  because benzo(b)-



fluoranthene was not available to us  as a  reference standard.  This group of six



PAH is regarded by WHO as a  reliable  indicator of the entire  polycyclic family



(WHO, 1971).  Except  for the ease  of  fluorescence detection  and their frequent




occurrence  in   environmental  samples,  the   reason  for  selecting  these  six






                                       46

-------
compounds by WHO is further explained (Borneff and Kunte,  1979).  In the absence




of other guidelines, we decided to analyze the above  six  PAH for the following



additional  reasons:    the  six  PAH  represent  compounds  varying from  strongly



carcinogenic  (benzo(a)pyrene)  to  non-carcinogenic  (fluoranthene)  (NAS,  1972),



and  include compounds that  are hard-to-separate;  i.e.,  the  isomers benzo(j)-



fluoranthene and benzo(k)fluoranthene.  The ability of the analytical method to




separate  carcinogenic  PAH  (for example, benzo(a)pyrene)  from its  nonmutagenic



isomer  (for  example,  benzo(e)pyrene)  can be  tested  by including  the  last two




isomers, which are difficult to separate.



     PAH represent only one class of biologically active compounds which may be



either deposited in raw water or leached from distribution  pipes.   In order to



detect the presence of other biologically active compounds  of importance to water



quality,  an in vitro mutageniciity  assay  was performed  on samples  from each



collection site.  This assay was performed on the same organic concentrate used



for PAH analyses, and  also on  the  unconcentrated water.  The  use  of unconcen-




trated water allows for detection of biologically active compounds that are not



absorbed by polyurethane foam plugs,  as well as compounds which are lost during



the  extraction and  preliminary chemical  clean  up  phases.    The  experimental



bioassay data, expressed as  revertants per  plate,  for each  sampling  site are



tabulated in Appendix A.




     The PAH in water samples taken from the different locations were collected



by a polyurethane foam retention method.  In a previous study (Basu and Saxena,



1978b), the  retention  efficiency of  polyurethane foams for  six PAH in finished




waters was  reported to be  nearly  90$.  Using  the  foam concentrative technique

-------
described in Section 5, the investigators collected water samples from 24 potable




supplies.   Water samples  from  5 of  the 24  supplies  were  resampled for  the




following reasons:   the water samples collected during the first trip to Fremont,




OH; Elkhart, IN; and Fairborn,  OH were not analyzed for mutagenic activity,  and




the original  samples collected from Champaign,  IL,  and Ft.  Myers,  FL  showed




elevated levels of mutagenic activity.  Therefore,  water  from these five supplies




were resampled  primarily  for the analysis  or confirmation of  their  mutagenic




activity.




RESULTS OF CHEMICAL ANALYSIS FOR PAH




     The results of PAH analyses are  given in Table 4.  The values  given  for




fluoranthene and benzo(ghi)perylene  in  Table 4  have been corrected for the trace




amounts of these PAH contributed by  the foam plugs from each lot.  The details of




these corrections  are given elsewhere (Saxena et al.,   1977).   Benzo(a)pyrene




values have been corrected for the amount of radiolabelled compound added as an




internal standard.   Correction has not been made for the PAH retention efficiency




of  the  foams,  which was  assumed to  be  quantitative.   Because the  retention




efficiency of PAH  from  foam plugs will  always be lower than 100?, the  actual




amount of PAH in waters should  be slightly  higher than the values  shown in the




table.   The  loss of PAH during solvent extraction of the  foams and the subsequent




chemical clean up was determined by  the addition of radiolabelled benzo(a)pyrene




as an internal  standard.   The  amount of  radiolabelled  benzo(a)pyrene  added to




each of the samples  was such that it  provided statistically significant counts




over the background and  yet did not interfere with  the analysis of  low level




benzo(a)pyrene.






                                       48

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     The values  of the  six  PAH determined  were corrected using  the  recovery




factor from radiolabelled benzo(a)pyrene.  It was assumed  that  the recovery of



all six  PAH was the same as  the  recovery of the benzo(a)pyrene.   The average




recovery of PAH  for all finished water was slightly higher (68?) than the average



recovery for the raw waters (63?).  This  lower recovery in the  case  of raw waters



is understandable because in some instances  the  impurities in raw  waters,  pro-



duced emulsions during the solvent extraction steps.



     The final  concentrations  of the  six PAH presented  in Table H are  based




primarily  on  TLC-spectrofluorometric values.   The  GC-FID  method  in  the  vast



majority of  cases  failed to  detect  PAH  because less than 5 u& of the  final




concentrate (less  than  556  of the total)  could be used for quantification,  and



because the method  has  a lower sensitivity  than the  fluorometric method.   In



cases where both values could be obtained, the GC results served as a crosscheck




for the TLC values.  In a limited number of cases, the TLC method did not provide




concentration values  for one  or  two PAH  compounds  in the samples  because of




incomplete  separation  from  impurity spot(s).    Some  of  these   compounds  were



separated and quantified by the capillary GC-FID method.   The separability and




sensitivity of the  capillary  GC-FID  method was  superior for  the early eluting



peaks, such as fluoranthene,  benzo(j)fluoranthene and benzo(k)fluoranthene.   As



found by  other  authors  (Bjrirseth,  1979), benzo(j)fluoranthene  and  benzo(k)f-



luoranthene were separated by an SE-5^ capillary column.



     The detection  limit for  the  six PAH  by both TLC-fluorometric  detection



(Saxena et al.,  1977) and capillary  GC-FID detection  is  given in Table 5.   The




capillary GC-FID detection limit is  based  on a minimum output response of four






                                       59

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            Table 5.  Detection Limit for Six PAH by Thin Layer and
                      Capillary Gas Chromatographic  Methods
TLC-fluorometric
PAH
FL
BjF
BkF
BaP
IP
BghiP
Absolute
limit, ng
14.0
7.5
5.0
10.0
10.0
20.0
Limit in 60 1
water (ppt)
2.3
0.1
0.2
0.2
0.2
0.3
Capillary GC-FID
Absolute
limit, ng
6.2
8.3a
10.3
	
	
Limit in
water
3.5
6.9a
8.6
	
	
60 1
(ppt)





compound not separated by GC
                                           60

-------
times the background  values  obtained at an output attenuation of  1 x  6H  and a



maximum sample loading volume of 2 u& from a total of  TOO  uA  concentrate.   The



detection  limits  for  indeno(1,2,3-cd)pyrene   and  benzo(ghi)perylene  by  the




GC-method are  not shown  in  Table 5  because  neither  of  these compounds  were



detected by the capillary GC-FID method  in  any of the samples.  Nevertheless the




detection limits for these two compounds by the capillary GC method were higher



than  those  determined by  the  packed column method.   With the slower eluting




peaks, similar results were obtained with other capillary columns  by Lao et al.




(1973).



     If PAH contaminants are leached from the  pipes or their linings,  one would




expect enhancement  of PAH levels  as the  treated  waters  travel through  these



pipes.  Also,  in a distribution system served by one kind of  pipe, an enhancement



of PAH level in waters at an intermediate point of the distribution system should




signal a further enhancement of PAH concentration  in water at the most distant



point of  the  distribution system.   In  general, an  increase  in PAH level was



observed  in waters after their  passage through cast/ductile iron  and cement-




coated cast/ductile iron  pipes.  Of the 24 sites monitored for the possibility of



enhancement of PAH concentation  by  the distribution system, 67% showed increases



in PAH concentrations  in waters travelling through at least one sample collection



point in  the  distribution lines.   The  remainder of the water  supplies showed



reductions in PAH concentrations.



     A correlation between the PAH level in water and the distances these waters



passed through  the distribution lines   was  also found.    An  increase in the




distance usually enhanced  the PAH  levels in water.   Because petroleum asphalt-






                                      61

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based  paints  and  sealants  are  employed  with  cement-coated  and  uncoated




oast/ductile iron pipes, this enhancement of PAH levels may  be  attributable  to



the increased leaching of the pipe linings associated with increased contact time



inside the pipes.  The amount of PAH leached into water,  however,  was small and



found to produce incremental increases in the part per trillion range.



     The amount of PAH  leached was  also  found  to be related to the  age  of the



installed pipes.   The maximum incremental difference in PAH concentrations  of




53.4 ppt, 61.2  ppt,  and 42.2 ppt  were  detected  in waters  from Fremont,  OH;




Elkhart,  IN;  and  Tascaloosa,  AL   respectively.    The  Fremont,  OH  water  was



collected from a recently installed cement-coated  cast/ductile  iron  section  of




the distribution  system.   The cast/ductile  iron pipes in Tascaloosa,  AL were



coated with  asphalt-based paints  and probably were installed recently.  Although



the age of  the  pipes  in Elkhart, IN was  not known, the substantive increase in



PAH concentration in water may indicate that a major new section of pipe in the




distribution system had  been recently installed.  It is interesting to note that




in all  three  locations, the  increase  in the concentration  of  the  more  water




soluble PAH  (Santodonato et  al.,  1979) accounted exclusively for the substantial




increase in  the  total  PAH concentration in the water.  Distribution pipes coated



with PAH-containing materials tend  to leach out the  more water soluble PAH, such



as flouranthene, at the  earlier stages of their use.  As the pipes  become older,



their surfaces become depleted of the more water soluble PAH and they leach out



less  soluble  PAH,  such as  benzo(a)pyrene  and  benzo(ghi)perylene,  in  higher




proportions.  This age  dependence  of  PAH leaching was also  observed  by Alben




(1980a) in studies on  a  coal tar-coated storage tank.  Therefore, an increase in






                                      62

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the concentrations  of  the more water  soluble PAH in waters  from  distribution




lines provided a further indication that they were recently installed.



     The  small amount  of  PAH detected  in  the  water  after passage  through



cast/ductile iron pipes is  not surprising.   In a  recent  investigation,  Miller



et al. (1981) measured the concentration of the six PAH in water by circulating



PAH-free water up  to 232 hours  through 34' x 6" petroleum asphalt-coated ductile



iron pipes.  The total PAH concentration in the water after passage through the



pipes did not exceed 10 ppt.  The concentrations of the six PAH measured in the




leached waters from the present investigation were of the same  order of magnitude



as those determined by Miller et al.  (1981).




     In certain instances, particularly in waters from Fond Du Lac, WI; Austin,



TX; and Wheeling,  WV, marked reductions of as much as  98$ were observed in PAH



concentrations in the waters after distribution.  This reduction in PAH concen-



tration cannot be  correlated with  any specific kind of distribution pipe.  While




the distribution pipes  in  Wheeling, WV consisted of a  combination of copper, cast



iron, cement-coated ductile iron and asbestos/cement, the distribution pipes in



Fond Du Lac,  WI  and Austin, TX consisted of  cast  iron  and cement  mortar lined



steel pipes,  respectively.   A  possible explanation for  the  reduction  in PAH



concentration lies in the age of the distribution pipes.  Cast iron and cement-



coated cast/ductile iron and steel pipes may be coated with asphalt-based paints



and sealants.  Up to a certain age,  the  asphalt coating might continuously leach



slowly decreasing amounts of PAH in the travelling waters.  When the surface of



the pipes becomes depleted of PAH  leachables,  the bare metal and cement surfaces




may act as a sorbent of PAH as water containing PAH travels through them.






                                      63

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     The quality  of water as  a  result of  its  passage through two  other  non-




metallic pipes,  for example,  asbestos/cement  (Wichita Falls, TX; Hutchinson, KS;




and Fayetteville, NC) and reinforced  concrete  (Olympia, WA) ,  was  also examined




during this investigation.  As expected, with pipes not  coated with PAH leachable




materials the water passing through these  distribution  lines showed no change in




PAH concentrations  other  than fluctuation in the concentration of  benzo(ghi)-




perylene.




     Water quality  parameters, such as  the  pH,  hardness,  and  alkalinity of the




water may also  affect the leachability of PAH  from the pipe surfaces.   It was




shown by McFarren et al. (1977) that the aggressive index of travelling water is




important  in  determining  the  leachability  of    asbestos   fibers  from  the




asbestos/cement pipes.  The  laboratory studies of a few  investigators  (Alben,




1980b; Oyler et  al.,  1978)  suggest the possibility of interaction  of residual




chlorine in  potable waters  with the  compounds  leached from  the  pipe surfaces.




Chlorinated  and  other oxidized products  were  identified  as evidence  of these




interactions.   Because no  dramatic  leachings  of PAH were  observed from the




distribution pipes in the present investigation, no attempt was  made to study the




effect of water quality parameters on the  leachability  of PAH from the distribu-




tion pipes.




     No data regarding  the  precision and accuracy of  the  results can be given




because a single analysis for each sample was performed.  The data presented in




Table 4, however,  were  obtained  under the  following  quality  assurance condi-



tions.






                                       64

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     (1)  The retention  efficiencies  of the 6  PAH by polyurethane  foams  were



determined with PAH-spiked water every time a new batch of foams was purchased.




The retention efficiencies were found to exceed 80%.



     (2)  The recovery of PAH in each sample resulting from foam desorption and




chemical cleanup was  determined from  the  recovery of radiolabelled benzo(a)-



pyrene that was added as an internal standard.




     (3)  We analyzed external quality control  samples received from U.S.  EPA,



and the results of  these  analyses indicated that our quantification methods were




satisfactory.



     (4)  Whenever there  was  more than  a  10$  variation  in  the sensitivity of



fluorometric detection (due to change in lamp intensity or  photocell response) as



determined  by  a  standard  quinine  sulfate  spot,  fresh  fluorometric  standard



curves were made and used for the determination of unknown sample concentrations.




     (5)  For samples  quantified  by the GC-FID  method,  at  least  one  standard




mixture was  injected  into the GC following  sample injection  to  ascertain any




changes in relative retention times and sensitivity of detection.



     (6)  A fresh solvent and reagent blank was run with the samples whenever a




new lot of solvent and/or chemicals was used.



     It is  evident  from  Table 4 that  with  the  exception  of treated water from



Austin, TX,  more than  90% removal of PAH was  accomplished by  the treatment



processes in the water supplies when the concentration of  total  PAH in raw water



exceeded 10 ppt.   The efficiency of PAH  removal  by the  treatment processes was



less significant when the level of total PAH in raw water was less than 10 ppt.




In fact, in certain cases as in treated water from Augusta; GA, Ft.  Myers, FL;






                                       65

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Eau Claire,  WI;  Fond Du  Lac,  WI;  Hutchinson,  KS; Olyrapia,  WA;  and San  Luis




Obispo; CA, the PAH level might  have  increased  as  a result of treatment.   The




increase may be due to the presence of PAH impurities  in treatment chemicals or




due to leaching of the pipes employed to transport  the treated water to  the tap




(where they were sampled), or both.




     We studied the possibility  of variations in PAH  concentration  of  surface




waters in samples  collected from Fremont, OH and Ft. Myers,  FL at  two different




times.  The  raw surface water collected from Fremont,  OH  during  the spring of




1981  showed  a  large  increase  of  PAH  concentration  compared to  the  sample




collected from the same location  in the  summer  of  1977.  The raw  surface water




collected from Ft. Myers, FL,  however,  showed no change in  PAH  concentration.




The observed concentration variation  in  the  Fremont,  OH raw  water sample might




have been due to seasonal variation of PAH  concentration,  but this  possibility




was not  further  explored.   In the absence  of  home heating  during  the  winter




months, which represents the largest source of PAH contamination in the environ-




ment (NAS, 1972),  the surface water from Ft.  Myers would not be expected to show




any seasonal variation in PAH concentration.




     In addition to the  six PAH identified and quantified by the TLC-fluorometric




and GC-FID methods, a number of  unidentified  compounds were detected  by  both




methods.   For  example,  in Wheeling,  WV  raw  water   samples,  as  many as  13




luminescent  spots  remained unidentified  on  the   TLC plate.    Some  of  the




unidentified  spots on  the  TLC  plates  showed  characteristic absorption  and




emission spectra.   One such  compound with a characteristic emission spectra that




recurred in many water  samples  is shown  in  Figure  4.   Similarly,  the capillary






                                      66

-------
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                                         67

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GC-FID  method  showed  the  presence  of  several unidentified  compounds  in  the



chromatograms of many of the water  samples.   The  capillary GC-FID chromatogram



obtained from Elkhark, IN water samples collected from the four sampling points




illustrate the  complexity  of these chromatograms (Figure 5).   These chromato-



grams also qualitatively demonstrate the changes in  the water quality as the raw



water is treated to finished water and is finally passed through the distribution



pipes.  No attempt was made, however, to identify the unidentified compounds.



RESULTS OF MUTAGENICITY ASSAYS




     In most cases,  the raw water and finished water  (before and after distribu-



tion) tested  in this investigation  demonstrated  no significant  or  consistent




mutagenic activity in the  Ames test system.   Thus  it  is  not  clear whether the



leachate from certain distribution pipes actually  contributes to the presence of



rautagens or, on the other hand, whether some pipes adsorb mutagens which had not




previously been removed during  treatment.  Of the 2U water supplies tested, three




showed no detectable  mutagenic activity in  either the  raw water or water taken



from any  of  the collection points  (Table 6).   Six  other  water supplies  had a



number  of sampling  sites  in  which an  individual  assay  point demonstrated a




mutation ratio  (number of revertants in experimental plate divided by number of



revertants in control plates) of between 1.5 and 2.0.  The random appearance of



these weak  mutagenic  responses  and  the concomitant  lack  of a  dose-related



increase  in  mutation rate indicates  that  these values fall within  the normal



variation of the Ames assay.  This interpretation  is more difficult, however, in




the cases of Fayetteville,  NC and Tascaloosa, AL a ratio of greater than 1.5 was






                                       68

-------



                                              131

Figure 5.  Gas chromatograms of Elkhart,  IN samples  collected
           at the four sampling points
                             69

-------











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observed at the highest concentration tested, since  this  could  indicate that a




higher dose level would produce a clearly positive response.



     The lack of  observed  rautagenic response in most  samples probably results




from the absence of sufficient quantities of mutagens  in  the sample.   There is



also the possibility that some of these samples  contain mutagenic compounds and




that the biological activity of  these  compounds was masked by  the  presence of



toxic substances  in  the  sample.   The interference  by  toxic  materials with the



performance of the Ames assay has been reported  by Heartlein et  al.  (1981) in a




study  of the  mutagenicity of  drinking water  in  agricultural  areas.   Since




toxicity tests were not performed on any of  the water  samples or their concen-



trates,  the  only  detectable evidence of  severe toxicity in our investigation



would  be the absence  of  a bacterial  lawn   (the  limited  growth  of  histidine-



dependent bacteria allowed  by the trace amount of histidine supplied by the media




in the minimal plate).  The absence of a bacterial lawn did  not  result from the




testing  of  any  samples in  this  study,  however, slight bacterial toxicity was




occasionally encountered as indicated by a dose-dependent  decrease in the number



of revertants with some samples.



     An example of this toxic  effect was  observed with  the raw water sample from



Fremont, Ohio where the average number of revertants for strain  TA1535 declined



from 79.5 for control  to 62.0, 50.0, 56.5, and 8.4 as the dose increased.  In the



study of Hearthein et al.  (1981)  the addition of S-9 mix decreased the toxicity



of the sample to the -feafe- bacteria,  however, in the present study the presence of




S-9 mix  decreased toxicity  in  certain  cases  while in  other  cases it increased




toxicity.  It is  apparent  from the  effect  of metabolic activation on bacterial






                                      71

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toxicity that  the  chemical composition and/or  amounts of the  toxic  agents  in



individual samples varied extensively among the different collection locations.



In most samples,  the  lowest dose tested appeared to be nontoxic to the  bacteria.



     The  types of  pipe associated  with  water samples  that  demonstrated  no




mutagenic activity are shown in Table 6.   The pipes listed in Table 6 represent



all the various types encountered in this  study, ranging  from the cast iron to



asbestos/cement.   Although  many  variables  are unknown about  the  pipes such as




age, condition, and the  physical properties (pH, types of solutes) of the water,




it is apparent that this study shows  no consistent relationship between the type



of pipe and the absence  of observed  mutagenic activity in the  water  which has



been transported by these pipes.



     Fifteen of the  water  supplies  provided at least  one sampling point which



either produced a weak mutagenic response or a mutagenic response in at least one




of the four strains of bacteria tested.   For a response to be considered weakly




mutagenic in this study  we  require that the reversion ratio fall between 1.5 and



2.0 and  be  accompanied  by  a  positive  dose-response trend.   A  clear  mutagenic




response in this  study was  considered to occur when the reversion ratio exceeded



2.0.  It should be noted  that  it is not  unexpected to observe mutagenic responses



with environmental samples where the high dose point has a lower ratio than the



intermediate and  low dose  point.   Such data  are  generally  indicative  of the



toxicity to  the  tester  strain of  bacteria used in  the  Ames  assay.    Even  in



samples demonstrating clear mutagenic potential in this study, the response was



not great, with most ratios being only slightly greater than 2.0.






                                      72

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     As a  result  of the low  level  of activity,  even in  samples  designated as




showing a  positive mutagenic  response,  it was  not possible  to have  precise



guidelines to interpret the effects of water treatment  and type of distribution




system on  mutagenic  activity.   Since the  interpretation  of  these mutagenicity



data required a higher degree of subjective evaluation than for most Ames assay



data,  three  individuals in our group independently interpreted the data,  as



presented in Appendix A.  Where differences in interpretation exist a consensus




was obtained and this consensus is presented in Table 7.




     The interpretation of these data was based on weight of evidence approach.




A positive mutagenic  response was considered more  credible  if a dose-response



relationship could be  demonstrated  even  when  individual values which comprised



this  dose-response relationship  could  be demonstrated  even  when  individual




values which  comprised  this  dose-response were  below  a ratio  of  1.5.   The



metabolic activation (S-9 mix) was considered when comparing mutagenic response




among different points in the sampling scheme;   e.g., mutagenicity was considered



to  be  enhanced if  distribution point I  showed  activity  in the presence  and



absence of S-9 mix, while the  finished water showed  activity only in the absence



of S-9 mix.  It was necessary to do this in order to account for the appearance of



different  classes  of  mutagens  in the raw,  finished,  and distribution system



water.  In the same manner,  the information from the two types of assays (liquid



suspension and plate incorporation)  and all bacterial tester strains was used to



arrive at a final  interpretation with  the realization that each tester strain is



sensitive to different types of compounds and  that each  of the two assay systems




might  actually  be evaluating  both  qualitatively and quantitatively different






                                       73

-------



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chemical mixtures from the  same sampling point.  This information was combined to



indicate  the  change  in  mutagenic activity  in the raw water  and water  from



distribution  points  I and  II  as compared with the mutagenic activity  of  the




finished water.



     The  summary  of  the data in  Tables  6  and 7 indicates in general  that  the




distribution system neither adds  nor  substracts  from the mutagenicity present in



the finished water, in any consistent manner.  The types of pipes used in these




water systems included cast/ductile iron, cement-lined steel and asbestos/cement



pipes.   The water systems which indicated  either  an increase or  decrease in



mutagenic activity  during distribution  represented all the  various  types of



pipes.   Thus,  the  data suggest  that,  although  in  some  cases  the  mutagenic



activity  of  finished water may  be  affected  by the distribution  system,  this



effect cannot be simply be attributed solely to the type of pipe used to trans-



port the water.




     A further  indication  of the  complexity  of  the  interaction  of compounds in



drinking  water  with  the distribution  pipes  is demonstrated  in the  two water



supplies  which  were  tested twice.  The  sample  obtained  from  the  first trip to



Champaign was only tested in a limited manner  for mutagenic activity, with only a



single concentration of the organic concentrate tested.   This  limited assay



produced over a five  fold increase in  the  reversion rate for TA98 in the presence



of metabolic activation at  the two distribution points.   This result contrasts



with the  finding  of  no mutagenic activity in this  strain with samples  of  raw



water and finished water.  Upon collecting and assaying a new water sample from




Champaign the total mutagenic response was considerably lower, and this low level






                                       75

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of activity was observed in samples from all collection points.  In testing two



independent samples from Ft.  Myers similar  results  were obtained.   The  first



samples  showed  a  decrease  in mutagenic  activity in  water obtained  from  the



distribution system while the second indicated that the distribution system had



no effect on mutagenic  activity.   Since  the distribution lines had not changed



during the interval between the sampling trips, other variables must account for



the observed differences.



     Some of the variables  which may  affect the  mutagenic activity in drinking




water, and the interaction of this activity with the distribution pipes include




variation in raw water quality, treatment processes in the prepartion of finished



water, and differences in the microbiological population in the  pipes.  The first



two variables can cause qualitative changes in the trace organic content of the



finished water.  If these changes generate different mutagens or promutagens it




might not be unexpected  that different types of interaction with the distribution



system may occur.  Also, changes in the raw water quality  and treatment processes




may alter the pH of the water, the chlorine content,  and the amount of disolved



and suspended solids.  Any of these changes  could  affect the ability of the water



to  leach material  from the  distribution  pipe,  or  may affect  the  biological



activity  of leached  compounds.    Finally,  Alexander  (197*0  has  reported  the



synthesis of mutagens by microbiological  action,  and  if this is occurring in the



distribution lines  then changes in the  microbiological population  will  cause



changes in the patterns of mutagenicity.



     It  is  difficult from  the  results  of  the present  study to  speculate  on




whether  any of the above  processes  could  account  for  the  different  results






                                      76

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obtained  from the  two  water  samples  taken  from  Champaign  and  Fort  Myers.



However, a recent  report  by Clark and  Johnston  (1981) describes an  extensive



analysis of the mutagenic activity in the drinking water of Champaign.  In this



study not only was marked  seasonal  variation was observed.  A  correlation was



made between mutagenic activity and the alternating use of choramine and break-



point chlorination in the treatment processes.  In a similar manner those changes



in the treatment  processes  could account for both  the quantitative differences in



mutagenic activity and the differences  in  the patterns of  mutagenic activity




observed by us in the  two sampling trips to Champaign.  There is no  information



on what caused the differences in mutagenicity in  the Ft.  Myers samples except




that during the second trip there was a severe drought condition which resulted



in poor water quality as measured by total organic carbon levels.




     PAH, many of which are mutagenic (Santodonato et al.,  1979), were detected




in most of the water samples.   Although mutagenic PAH were present,  the concen-



tration of PAH did not  correlate with the level of mutagenic activity.  The range



of mutagenic  response included ratios of 1.51  to 4.13 for samples which contained




no  detectable levels  of  the six  representative PAH.   The highest  mutagenic



activity was  observed in Champaign (first trip)  even though the total PAH concen-



tration was  only  6.2 ng,  whereas  the sample from  Wheeling had the  highest PAH



level, 1586 ng/&, but produced no demonstrable mutagenic activity.   It is also



important to note that PAH require metabolic activation before mutagenicity can



be observed in the Ames  assay, and  a number of  water systems  showed mutagenic




activity in the absence of  metabolic activation.  Although the PAH either present




in the raw water or leached from distribution  pipes may contribute to some of the






                                      77

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mutagenic  response  observed,  the cannot  be  the  major  source  of  biological




activity in the sample.  Also, it is clear that the  levels  of PAH would be a poor



indicator of the overall mutagenic potential  of drinking waters examined in this




study.



     The results of this study indicate that the mutagenic compounds present in



drinking water represent a  diverse group  of contaminants when the water supplies



are compared with each  other.   As can be determined from an examination of the



mutagenicity data in Appendix A most of the positive results were obtained with




the plate incorporation assay, however, at least some of the positive responses



were observed in  the  liquid suspension assay.   The starting material  for the




adsorbed and  eluted from polyurethane foam  plugs.  The  compounds  adsorbed to



polyurethane foam  plugs probably  represent  relatively hydrophobic  compounds,



whereas the unconcentrated  water  containing  both polar and nonpolar compounds,




was  assayed only with  the  plate incorporation technique.    The presence  of




mutagenic  activity  in  only  one assay system  is suggestive evidence  that the



mutagens are predominantly  polar (plate incorporation assay) or nonpolar (liquid




suspension assay) compounds.  Some of the water supplies appear to have mutagens



which do not adsorb to polyurethane foam plugs while others have mutagens which



could be concentrated by this technique.



     The second  indication  that  the  mutagens in  drinking  water  represent  a



diverse group of compounds  is  the varying requirement  for metabolic activation



for expression of mutagenicity.  Mammalian metabolic activation (i.e., S-9 mix)




is needed to convert  pro-mutagens to ultimate mutagens in the Ames  assay.   As




shown in Table 7  some water supplies  displayed  mutagenic  activity  only in the






                                       78

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presence of S-9 mix, indicating that only pro-mutagens were present while other



supplies revealed the presence of ultimate mutagens exclusively, as indicated by




activity only in the absence of S-9.  The absence in these cases of activity in



the presence of S-9 indicates that mammalian enzymes present in the S-9 mix are



capable of metabolizing the ultimate mutagens  to  inactive compounds.  In samples



where activity  is  present  both with and  without metabolic  activation  it  is




impossible to say whether both ultimate  and  pro-mutagens are present or that the



ultimate mutagens are  refractory  to mammalian metabolism.  The above  evidence




suggests  that  a  number  of  classes  of  compounds  may be  responsible  for  the



mutagenic activity observed in the water supplies tested in this study.




     Along with the  effect  of  the  distribution  system on  mutagenic activity of



drinking  waters,   this  study  also  provides  additional  information  on  the



efficiency of  removal  of mutagens  by  water treatment, and on  the presence  of



mutagens in raw waters.  One  of the  earliest  reports which described mutagenic




activity in raw water was the  study of Pelon et al. (1977).   The  water tested was



from the  Mississippi River which  receives  wastewater from  a  large number  of



industries.  Pelon  et  al. (1977)  used  unconcentrated  raw  water in their study




while in  a more recent  investigation,  Kool  et  al.  (198la)  observed mutagenic



activity in organic concentrates from surface  waters  in the Netherlands.  In the



present study  10  of the  24 raw waters  tested  displayed  detectable mutagenic



activity (Table 8).   It is of  interest to note that most of  the locations showing



mutagenic activity  received  their  raw water from surface  water sources,  while



most of the ground waters tested in this study showed no mutagenic activity.  The




presence of pollutants in surface waters is well documented, with the pollution






                                      79

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occurring primarily from intentional discharge,  atmospheric fallout,  and ground




run off.  On the other hand,  filtration through sand has long been used to purify



water, and this type of  filtration along with biological degradation which would



naturally occur prior to water  entering  the aquifer probably accounts  for  the



general lack of activity shown in ground  water.



     The presence of mutagens in drinking water has also  been  well  documented



(Kool et al.,  198lb,  198lc;  Loper et al., 1978; Schwartz  et al.,  1979;  Simmon




et al., 1977).   As  can be seen in Table 7 we found in many cases  the water after




treatment was more mutagenic than the raw water.  In three of the water systems



we tested the raw water  was nonmutagenic while the finished water contained some



mutagenic activity.  A  number of studies  have linked the mutagenic activity in



drinking water  to  the  formation of low molecular weight  chlorinated compounds



produced by  chlorination  (Simmon et al.,  1977;  Rapson et  al.,  1980;  Brett  and




Calverly, 1979; Dolara  et al.,  1981;  Anonymous, 1981).   Although  chlorination




may be the source of the increased mutagenicity  observed in finished water,  the



activity  probably  did  not  result  from  chlorinated  low  molecular  weight




compounds, since neither  of the assay procedures  used in this  study are very



effective at the detection of volatile compounds.  In addition to chlorination,



mutagens  may also  be  added to  finished  water   through  the  presence  of  trace



contaminants in the chemicals used  during purification  (e.g.,  lime  addition).



Without more extensive  investigation  of each process employed in the purifica-



tion of drinking waters we studied the specific  source of the increase in muta-



genic activity cannot be determined.   Table 7 also  shows that  in two cases  the






                                       81

-------
treatment process was effective in the  removal  of  mutagenic  activity,  while in


six cases the treatment had no effect on the level  of mutagens.


     Many of the samples tested, including both finished water and distribution

      c" '
water,'low levels of mutagenic activity (Appendix A).  The implication to human


health of these low levels of mutagenic activity are  not fully understood at the


present.  This is particularly  true  in  the  assessment of  mutagenic activity in


complex environmental mixtures in which a large number of interactions may occur.


Supression  of mutagenic  activity  has  been  observed in other  environmental


samples  as  a  result of  interactions  (Shahn  and Fournier,  1978).   Although


enhancement of  activity has not been reported  there have been  no systematic


investigations of the performance of the  Ames  assay  with  complex environmental


mixtures.  The interpretation of mutagenicity is further complicated by the low


levels of activity  commonly observed in water  samples and the toxicity to the


bacteria of the organic concentrates which prevent the use of high dose levels.


Overall the results  obtained with the Ames assay in this study indicate that some


drinking  water  suplies  contain  compounds  that  have  potential  biological


activity.   The  presence  of  biologically  active  compounds  in  drinking  water


indicate that these water supplies should be first studied in greater detail to


determine the source of  this contamination before attempting to  assess any public


health; arising from the consumption of this water.
                                       82

-------
                                   SECTION 7

                                   REFERENCES


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Kunte,  H.   1979.  Separation, detection and identification of polycyclic aromatic
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Shahin, M.M., and  F.  Fournier.   1978.  Suppression of  mutation induction and
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                                      86

-------
             APPENDIX A




Mutagenic Activity in Drinking Waters

-------
                             Key to Tables  1-131:
 Metabolic Activation = rat liver S-9 homogenate/extract  for  enzymatic  activa-
 tion is present ( + )  or absent (-)

2
 Dose:  C = control;  L = low;  M = medium; X = intermediate;  H = high


 Dose:  C = control; 1 ml =  1 ml of filter sterilized water per plate, 1/2 = equal
 volumes of media water and distilled water used in preparing agar; Full = only
 filter sterilized media water used to prepare agar

 *Weak mutagenic response indicated; **Mutagenic response indicated
 NA = not applicable;  NC =  Not calculated; NT = not tested; 3D = standard devia-
tion

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