iii

Contents

Abstract .......................................................................................................................................................... 1Introduction ................................................................................................................................................... 2

Purpose and Scope ............................................................................................................................. 3Enteric Viruses and Microbiological Indicators of Fecal Contamination ................................... 3Previous Studies .................................................................................................................................. 4

Enteric Viruses in Ground-Water Supplies ............................................................................ 4Factors that Affect the Presence of Enteric Viruses in Ground-Water Supplies ............ 6Indicators of Fecal Contamination in the Subsurface .......................................................... 7

Environmental setting ......................................................................................................................... 7Methods of Study .......................................................................................................................................... 9

Sampling procedures .......................................................................................................................... 9Microbiological analyses ................................................................................................................. 10

Enteric Virus Analysis by RT-PCR .......................................................................................... 10Enteric Virus Analysis by Cell Culture ................................................................................... 11Indicator Bacteria ..................................................................................................................... 11Coliphage ................................................................................................................................... 11

Ancillary Environmental and Water-Quality Information ............................................................ 11Quality Assurance and Quality Control .......................................................................................... 12

Enteric Viruses and Microbiological Indicators of Fecal Contamination in Small Public Ground-Water Supplies .............................................................................................................. 12

Quality-Control Considerations for Enteric Viruses by RT-PCR ................................................. 14Occurrence of Enteric Viruses and Microbiological Indicators of Fecal Contamination ...... 15Microbiological Indicators of Fecal Contamination as Predictors of the Presence of Enteric Viruses ...................................................................................................................... 16

Environmental Factors and Chemical-Quality Constituents Related to the Presence of Indicators and Enteric Viruses ................................................................................................... 17Summary, Conclusions, and Suggestions for Future Study ................................................................. 23Acknowledgments ...................................................................................................................................... 25References Cited ........................................................................................................................................ 25

Figures 1. Map showing well location and surficial geology of the study area ................................... 8 2. Example of hybridization results from reverse transcriptase-polymerase chain reaction (RT-PCR) analysis for enteric viruses ............................................................. 13 3. Graph showing chloride concentrations in relation to chloride to bromide (Cl:Br) ratios for small-public-supply wells sampled July 1999 through July 2001: Distribution of wells by map number, and at least one detection of total coliforms, enterococci, Escherichia coli, and (or) coliphage, and enteric viruses .................................................................................................................................. 21

iv

Tables 1. Summary of literature on the occurrence of enteric viruses, bacterial indicators, and coliphage in ground-water-supply wells ......................................................................... 5 2. Results of enteric virus analysis by reverse transcriptase-polymerase chain reaction (RT-PCR) of regular and replicate spike samples collected from small- public-supply wells in southeastern Michigan, July 1999 through July 2001 .................. 14 3. Detections of enteric virus by reverse transcriptase-polymerase chain reaction (RT-PCR), culturable viruses, and microbiological indicators of fecal contamination from small-public-supply wells in southeastern Michigan, July 1999 through July 2001 ..................................................................................................... 15 4. Small-public-supply wells in southeastern Michigan with detections of enteric viruses, July 1999 through July 2001, and detections of microbiological indicators from the same well .................................................................... 16 5. Predictive accuracy of microbiological indicators for virus occurrence in the same small-public-supply well in southeastern Michigan, July 1999 through July 2001 ...................................................................................................................................... 17 6. Rankings of environmental and chemical-quality categorical variables by risk of fecal contamination and relations to detections of microorganisms in small- public-supply wells in southeastern Michigan, July 1999 through July 2001 .................. 18 7. Relations between detections of microorganisms and continuous environmental and chemical-quality variables of small-public-supply wells in southeastern Michigan, July 1999 through July 2001 .................................................................................. 20 8. Characteristics of small-public-supply wells in southeastern Michigan sampled from July 1999 through July 2001 ............................................................................ 29 9. Oligonucleotide primers and probes used for virus detection in the reverse transcriptase-polymerase chain reaction (RT-PCR) method ............................................. 35 10. Quality-control samples for reverse transcriptase-polymerase chain reaction (RT-PCR) analysis ...................................................................................................................... 36 11. Field water-quality measurements and microbiological data for small-public- supply wells sampled July 1999 through July 2001 .............................................................. 37 12. Chemical-quality data for small-public-supply wells in southeastern Michigan sampled from July 1999 through July 2001 ............................................................................ 51

Enteric Viruses and Microbiological Indicators 13

Figure 2. Example of hybridization results from reverse transcriptase-polymerase chain reaction (RT-PCR) analysis for enteric viruses.Columns 1 and 2 show results for enterovirus; 3 and 4 for reovirus; 5 and 6 for rotavirus; 7 and 8 for hepatitis A virus; 9 and 10 for Norwalk virus.)

Environmental Factors and Chemical-Quality Contituents 21

BMaximum total coliform concentration81

No detection of total coliformsCl:Br = 400

23

5

18

35

32

22

24

1

1620

29

1417

25

34

12

1928

11 837

36

7

33 2130

3

9

13

10

638

Sewage + halite

100

1,000

10,000

Background GW

GW + sewage

GW + halite

GW+brine upper limit

GW+brine lower limit

Sewage

10

1 10 100 1,000

100

1,000

10,000

Background GW

GW + sewage

Sewage + halite

CHLORIDE CONCENTRATION, IN MILLIGRAMS PER LITER

GW + halite

Sewage

10

GW+brine upper limit

GW+brine lower limit

CH

LOR

IDE

TO

BR

OM

IDE

RAT

IO

18

5

17

1

250

4

1

81

1

CH

LOR

IDE

TO

BR

OM

IDE

RAT

IO

1Map identifier

Figure 3. Chloride concentrations in relation to chloride to bromide (Cl:Br) ratios for small-public- supply wells sampled July 1999 through July 2001: A, Distribution of wells by map number, and at least one detection of B, total coliforms, C, enterococci, Escherichia coli, and (or) coliphage, and D, enteric viruses. (Halite end-member from Knuth and others, 1990; domestic sewage end-member from Davis and others, 1998, and Peavy, 1978; sewage has a Cl:Br ratio of 30–600 and chloride concentration of 37–101 mg/L (Peavy, 1978, as cited in Thomas, 2000); brine end-members from Eberts and others, 1990; a Cl:Br ratio of 400 is the theoretical maximum for natural water (Thomas, 2000).)

A

Cl:Br = 400

22 Enteric Viruses in Ground Water from Small Public Water Supplies in Southeastern Michigan

1 10 100 1,000

Sewage + halite

100

1,000

10,000

Background GW

GW + sewage

GW + halite

GW+brine upperlimit

GW+brine lower limit

Sewage

10

Enteric virus detection

D

C

100

1,000

10,000

Background GW

GW + sewage

Sewage + halite

GW + halite

10

GW+brine upper limit

GW+brine lower limit21

688

1

1

9

Sewage

Enterococci concentrationEscherichia coli concentration1

1

No detection of Escherichia coli, enterococci or coliphage

Coliphage detected

CHLORIDE CONCENTRATION, IN MILLIGRAMS PER LITER

CH

LOR

IDE

TO

BR

OM

IDE

RAT

IO

Cl:Br = 400

Cl:Br = 400

CH

LOR

IDE

TO

BR

OM

IDE

RAT

IO

Figure 3. Chloride concentrations in relation to chloride to bromide (Cl:Br) ratios for small-public- supply wells sampled July 1999 through July 2001: A, Distribution of wells by map number, and at least one detection of B, total coliforms, C, enterococci, Escherichia coli, and (or) coliphage, and D, enteric viruses. (Halite end-member from Knuth and others, 1990; domestic sewage end-member from Davis and others, 1998, and Peavy, 1978; sewage has a Cl:Br ratio of 30–600 and chloride concentration of 37–101 mg/L (Peavy, 1978, as cited in Thomas, 2000); brine end-members from Eberts and others, 1990; a Cl:Br ratio of 400 is the theoretical maximum for natural water (Thomas, 2000).) – Continued.