Prepared in cooperation with the Ohio Environmental Protection Agency 

Use of DNA Markers for Investigating Sources of 

Bacteria in Contaminated Ground Water: Wooster 

Township, Wayne County, Ohio 

By Denise H. Dumouchelle 

Open-File Report 2006–1382 

U.S. Department of the Interior 

U.S. Geological Survey 
U.S. Department of the Interior 

DIRK KEMPTHORNE, Secretary 

U.S. Geological Survey 

Mark D. Myers, Director 

U.S. Geological Survey, Reston, Virginia 2006 

For product and ordering information: 

World Wide Web: http://www.usgs.gov/pubprod 

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its natural and living resources, natural hazards, and the environment: 

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Suggested citation: 

Dumouchelle, D.H., 2006, Selected ground-water-quality data from wells in the Scenic Heights-Batdorf 

Road area, Wooster Township, Wayne County, Ohio: U.S. Geological Survey Open-File Report 2006-1382, 

13 p. 

Any use of trade, product, or firm names is for descriptive purposes only and does not imply 

endorsement by the U.S. Government. 

ii 

Although this report is in the public domain, permission must be secured from the individual 

copyright owners to reproduce any copyrighted material contained within this report. 
Contents 

Abstract................................................................................................................................................................................1 

Introduction .........................................................................................................................................................................1 

Purpose and Scope ........................................................................................................................................................2 

Description of Study Area.............................................................................................................................................2

Sampling and Analytical Methods ..................................................................................................................................4 

Ground-Water-Quality Data..............................................................................................................................................7 

Summary ............................................................................................................................................................................11 

References Cited ..............................................................................................................................................................12

Figure 

1. Map showing location of study area and wells sampled in the Scenic Heights Drive-Batdorf Road 

area of Wayne County, Ohio. ..............................................................................................................................3 

Tables 

1. Wastewater-method compound names, USGS National Water-Quality Laboratory reporting limits, 

and possible compound uses .............................................................................................................................5 

2. Well data and water-quality field data for ground-water samples from wells in the Scenic Heights 

Drive-Batdorf Road area of Wayne County, Ohio, June 2006.......................................................................8 

3. Results of bromide, chloride, and nitrate analyses for ground-water samples from wells in the 

Scenic Heights Drive-Batdorf Road area of Wayne County, Ohio, June 2006 ..........................................9 

4. Results of analyses of bacterial concentrations and bacterial DNA in ground-water samples from 

wells in the Scenic Heights Drive-Batdorf Road area of Wayne County, Ohio, June 2006...................10 

iii 
Conversion Factors 

Multiply By To obtain 

Length 

inch (in.) 2.54 centimeter (cm) 

inch (in.) 25.4 millimeter (mm) 

foot (ft) 0.3048 meter (m) 

Volume 

gallon (gal) 3.785 liter (L) 

Temperature in degrees Celsius (°C) may be converted to degrees Fahrenheit (°F) as follows: 

°F=(1.8×°C)+32 

Small sample volumes are reported in milliliters (mL) and microliters (μL). Concentrations of 

chemical constituents in water are given either in milligrams per liter (mg/L), micrograms per liter 

(μg/L), or nanograms per microliter (ng/μL). 

Bacteria concentrations are given in colony-forming units per 100 milliliters (CFU/100 mL).

iv 
Use of DNA Markers for Investigating Sources of 

Bacteria in Contaminated Ground Water: Wooster 

Township, Wayne County, Ohio 

By Denise H. Dumouchelle 

Abstract 

In 2004, a public-health nuisance was declared by the Wayne County Board of Health in the 

Scenic Heights Drive-Batdorf Road area of Wooster Township, Wayne County, Ohio, because of concerns 

about the safety of water from local wells. Repeated sampling had detected the presence of fecal-indicator 

bacteria and elevated nitrate concentrations. In June 2006, the U.S. Geological Survey (USGS), in 

cooperation with the Ohio Environmental Protection Agency (Ohio EPA), collected and analyzed samples 

from some of the affected wells to help investigate the possibility of human-origin bacterial 

contamination. Water samples from 12 wells and 5 home sewage-treatment systems (HSTS) were 

collected. Bromide concentrations were determined in samples from the 12 wells. Samples from 5 of the 

12 wells were analyzed for wastewater compounds. Total coliform, enterococci and Escherichia coli (E. 

coli) bacteria concentrations were determined for samples from 8 of the 12 wells. In addition, two 

microbial source-tracking tools that employ DNA markers were used on samples from several wells and a 

composite sample of water from five septic tanks. The DNA markers from the Enterococcus faecium

species and the order Bacteroidales are associated with specific sources, either human or ruminant 

sources. 

Bromide concentrations ranged from 0.04 to 0.18 milligrams per liter (mg/L). No wastewater 

compounds were detected at concentrations above the reporting limits. Samples from the 12 wells also 

were collected by Ohio EPA and analyzed for chloride and nitrate. Chloride concentrations ranged from 

12.6 to 61.6 mg/L and nitrate concentrations ranged from 2.34 to 11.9 mg/L (as N). 

Total coliforms and enterococci were detected in samples from 8 wells, at concentrations from 2 to 

200 colony-forming units per 100 milliliters (CFU/100 mL) and 0.5 to 17 CFU/100 mL, respectively. E. 

coli were detected in samples from three of the eight wells, at concentrations of 1 or 2 CFU/100 mL. Tests 

for the human-specific marker of enterococci, the esp gene, were negative in the seven samples tested, 

including the composite sample of HSTS water. DNA with the general Bacteroidales marker was detected 

in samples from four wells, but the tests for both the human- and ruminant-associated markers were 

negative. The presence of the PCR (polymerase chain reaction) -detectable DNA for the general fecal 

Bacteroidales marker is indicative of fecal contamination and recently recharged water. 

Introduction 

In October 2004, the Wayne County Board of Health declared a public-health nuisance in the 

Scenic Heights Drive-Batdorf Road area of Wooster Township, Ohio (fig. 1) because of concerns about 

the safety of the local water supply. In January 2006, at the request of Wayne County officials, the Ohio 

Environmental Protection Agency (Ohio EPA) began an investigation. Repeated sampling of the private 

water wells in the area found elevated nitrate concentrations and total coliform and Escherichia coli (E. 

1
coli) bacteria on multiple occasions (Ohio Environmental Protection Agency, 2006). One possible source 

of contamination is nearby home sewage-treatment systems (HSTS); other possible sources include 

domestic pets and wildlife (bacteria) and fertilizers (nitrate). 

Several water-quality and microbial source tracking (MST) methods can be used to indicate 

sewage contamination. Unfortunately, other potential sources can complicate interpretation of the data 

from many of these methods. Therefore, studies have found that use of multiple methods, including 

chemical and microbiological indicators, is the best approach for evaluating potential sewage 

contamination (K. E. Hyer, U.S. Geological Survey, written commun., 2006; Dumouchelle, 2006; Noble 

and others, 2006). To help identify whether the observed bacterial contamination originated from a human 

source, the U.S. Geological Survey (USGS), in cooperation with the Ohio EPA, collected and analyzed 

samples from wells in the Scenic Heights Drive-Batdorf Road area. In particular, the USGS used two 

MST tools that employ human-associated DNA markers found in bacterial strains of the order 

Bacteroidales and the species Enterococcus faecium (Bernhard and Field, 2000; Dick and others, 2005; 

Scott and others, 2005). Additionally, the presence of the general Bactroidales marker was used as another 

indicator of fecal contamination. The USGS Ohio Water Science Center is testing MST methods for their 

utility in combination with more traditional tools for contaminant-source identification. The present study 

is one of several case studies in Ohio. 

Purpose and Scope 

The purpose of this report is to present data from ground-water samples collected from selected 

residential wells in the Scenic Heights Drive-Batdorf Road area and to describe the use of DNA markers 

for investigating the sources of bacteria in contaminated ground water. In June 2006, the USGS collected 

and analyzed water samples from 12 wells and 5 HSTS. Bromide concentrations were determined at all 12 

wells. Wastewater compounds were analyzed in samples from 5 of the 12 wells. Total coliform, 

enterococci, and E. coli bacteria concentrations were determined for samples from 8 of the 12 wells. 

Analysis of DNA markers was done on water samples from seven wells. A composite sample of HSTS 

water from 5 of the 12 properties where wells were sampled was analyzed for DNA markers. 

Ohio EPA concurrently collected water from the same 12 wells and analyzed the samples for 

chloride and nitrate concentrations. Data from samples collected and analyzed by the Ohio EPA also are 

presented. 

Description of the Study Area 

The study area is in Wooster Township, southwest of the city of Wooster in northeastern Ohio, 

near the intersection of Batdorf and Tolbert Roads (fig. 1). The land use is rural residential, with most 

homes on lots ranging from half an acre to more than an acre. The homes are served by residential water 

wells and leach-line HSTSs. The residential wells in the area are from 70 to 185 ft deep and are completed 

as open holes in fractured sandstone, siltstone, and shale. The Ohio Department of Health used a 

downhole camera in July 2003 to investigate a well in the vicinity of WN-36 (fig. 1), and numerous 

fractures and openings were observed both above and below the water level (Craig Smith, Ohio 

Environmental Protection Agency, written commun., 2006). In addition, during the sampling for the 

current study, cascading water was heard in one well (WN-40). 

2
WN-46

WN-38

WN-39

WN-45

WN-40

WN-35

WN-44

WN-42

WN-43

WN-36

WN-41

WN-37

County location

OHIO

WN-36 Sampled well and number

EXPLANATION

Figure 1

Wooster Township

WAYNE COUNTY

Study area

Figure 1. L. 

ocation of study area and wells sampled in Scenic Heights Drive - Batdorf Road area, ayne County, Ohio. Location of study area and wells sampled in Scenic Heights Drive-Batford Road W

area, Wayne County, Ohio.

3
Sampling and Analytical Methods 

Selection of wells was based on results of previous sampling by various agencies. Five wells with 

frequent detections of fecal-indicator bacteria were selected for analysis of wastewater compounds, 

bacteria, and bromide. Three additional wells were selected for analysis of bacteria and bromide. Another 

three wells were selected for the analysis of bromide only. A twelfth well, just north of study area, was 

sampled and analyzed for bromide to obtain background concentration data. 

Homeowners of the selected wells gave permission for sampling and identified household spigots 

from which untreated water could be drawn. All samples were collected from these spigots as supplied by 

existing plumbing and well pumps. In most cases, an outside spigot was used as the sampling point. Sterile 

tubing and fittings were connected to the spigot for purging and sample collection. Although the sampled 

wells were in regular use, all wells were purged before sample collection. During purging, field 

measurements of pH, specific conductance, dissolved oxygen concentration, and temperature were made. 

Water samples were collected after the field measurements had stabilized. 

The wastewater compounds and bromide analyses were done by the USGS National Water-Quality 

Laboratory in Denver, Colo. Bromide analyses were done by ion chromatograpy, with a reporting limit of 

0.02 mg/L. The wastewater compounds and detection limits are given in table 1; analytical methods used 

are documented in Zaugg and others (2002). A field blank sample also was collected and analyzed for 

wastewater compounds to determine potential contamination from field and transport procedures. 

Bacterial analyses were done at the USGS Ohio Water Microbiology Laboratory in Columbus, 

Ohio. Samples were analyzed within 26 hours of collection for total coliforms and E. coli by use of the MI 

membrane-filtration method (U.S. Environmental Protection Agency, 2002a) and for enterococci using the 

mEI membrane-filtration method (U.S. Environmental Protection Agency, 2002b). Because of the need to 

collect enterococci colonies for DNA analysis, up to 1 L of sample water was filtered, which is more than 

the standard 100 mL filtered for the method. The MST methods that can detect the presence of genetic 

markers within the order Bacteroidales and the species Enterococcus faecium were used to try to 

determine whether fecal contamination included a human source. Water samples were tested for the 

presence of a general fecal, human-associated, and ruminate-associated marker in the order Bacteroidales,

as described in Dick and others (2005), according to the protocol originally described by Bernhard and 

Field (2000). Water samples were tested for the presence of Enterococcus faecium human-specific esp 

marker (enterococci marker) according to the protocol originally described by Scott and others (2005). 

About 500 mL of wastewater was collected from 5 septic tanks and combined into a single sample that 

also was analyzed for bacterial DNA markers. 

Procedures for the microbiology quality-assurance/quality-control (QA/QC) laboratory practices 

are described in Francy and others (2005). In the laboratory, filter blanks were included for at least every 

third E. coli sample (and every sample for Bacteroidales or enterococci markers) to document that 

filtration equipment and buffered water were not contaminated. 

Control DNA for human contamination sources (Delaware, Ohio, wastewater influent sample) and 

ruminant contaminant sources (Delaware, Ohio, cattle feces) were similarly processed for the presence of 

the Bacteroidales and enterococci markers. The presence of the general Bacteroidales marker (Bac32) 

was used as evidence that the polymerase chain reaction (PCR) was successful; in the absence of a 

positive response for Bac32, a matrix spike was included. For the enterococci marker, matrix spikes were 

included for each environmental sample, including the composite HSTS sample, to identify any possible 

matrix inhibition. PCR reagent blanks were included to test for contamination of PCR reagents with 

amplifiable target DNA. 

4
Table 1. Wastewater-method compound names, USGS National Water-Quality Laboratory reporting limits, 

and possible compound uses (modified from Zaugg and others, 2002). 

[Laboratory reporting limits are in micrograms per liter; F, fungicide; H, herbicide; I, insecticide; GUP, general-use pesticide; 

FR, flame retardant; WW, wastewater; Manuf, manufacturing; %, percent; >, greater than; CP, combustion product; UV, 

ultraviolet] 

Compound name 

Laboratory 

reporting 

limit 

Possible compound uses or sources 

1,4-Dichlorobenzene 0.5 Moth repellant, fumigant, deodorant 

17beta-Estradiol 5 Estrogen replacement therapy, estrogen metabolite 

1-Methylnaphthalene 0.5 2-5% of gasoline, diesel fuel, or crude oil 

2,6-Dimethylnaphthalene 0.5 Present in diesel/kerosene (trace in gasoline) 

2-Methylnaphthalene 0.5 2-5% of gasoline, diesel fuel, or crude oil 

3beta-Coprostanol 2 Carnivore fecal indicator 

3-Methyl-1H-indole (skatol) 1 Fragrance, stench in feces and coal tar 

3-tert-Butyl-4-hydroxyanisole (BHA) 5 Antioxidant, general preservative 

4-Cumylphenol 1 Nonionic detergent metabolite 

4-n-Octylphenol 1 Nonionic detergent metabolite 

4-tert-Octylphenol 1 Nonionic detergent metabolite 

5-Methyl-1H-benzotriazole 2 Antioxidant in antifreeze and deicers 

Acetophenone 0.5 Fragrance in detergent and tobacco, flavor in beverages 

Acetyl-hexamethyl-tetrahydro-naphthalene 

(AHTN) 

0.5 Musk fragrance (widespread usage) persistent in ground water 

Anthracene 0.5 Wood preservative, component of tar, diesel, or crude oil, CP 

Anthraquinone 0.5 Manuf dye/textiles, seed treatment, bird repellant 

Benzo[a]pyrene 0.5 Regulated PAH, used in cancer research, CP 

Benzophenone 0.5 Fixative for perfumes and soaps 

beta-Sitosterol 2 Plant sterol 

beta-Stigmastanol 2 Plant sterol 

Bisphenol A 1 Manuf polycarbonate resins, antioxidant, FR 

Bromacil 0.5 H (GUP), >80% noncrop usage on grass/brush 

Bromoform 0.5 WW ozonation byproduct, military/explosives 

Caffeine 0.5 Beverages, diuretic, very mobile/biodegradable 

Camphor 0.5 Flavor, odorant, ointments 

Carbaryl 1 I, crop and garden uses, low persistence 

Carbazole 0.5 I, Manuf dyes, explosives, and lubricants 

Chlorpyrifos 0.5 I, domestic pest and termite control (domestic use restricted as of 

2001) 

Cholesterol 2 Often a fecal indicator, also a plant sterol 

Cotinine 1 Primary nicotine metabolite 

Diazinon 0.5 I, > 40% nonagricultural usage, ants, flies 

Dichlorvos 1 I, pet collars, flies, also a degradate of naled or trichlofon 

d-Limonene 0.5 F, antimicrobial, antiviral, fragrance in aerosols 

Equilenin 5 Hormone replacement therapy drug 

Estrone 5 Biogenic hormone 

5
Table 1. Wastewater method compound names, USGS National Water-Quality Laboratory reporting limits, 

and possible compound uses.— Continued

 

Compound name 

Laboratory 

reporting 

limit 

Possible compound uses or sources 

Ethynyl estradiol 5 Oral contraceptive 

Fluoranthene 0.5 Component of coal tar and asphalt (only traces in gasoline or diesel 

fuel), CP 

Hexahydrohexamethylcyclopentabenzopyran (HHCB) 0.5 Musk fragrance (widespread usage) persistent in ground water 

Indole 0.5 Pesticide inert ingredient, fragrance in coffee 

Isoborneol 0.5 Fragrance in perfumery, in disinfectants 

Isophorone 0.5 Solvent for lacquer, plastic, oil, silicon, resin 

Isopropylbenzene (cumene) 0.5 Manuf phenol/acetone, fuels and paint thinner 

Isoquinoline 0.5 Flavors and fragrances 

Menthol 0.5 Cigarettes, cough drops, liniment, mouthwash 

Metalaxyl 0.5 H, F (GUP), mildew, blight, pathogens, golf/turf 

Methyl salicylate 0.5 Liniment, food, beverage, UV-absorbing lotion 

Metolachlor 0.5 H (GUP), indicator of agricultural drainage 

N,N-diethyl-meta-toluamide (Deet) 0.5 I, urban uses, mosquito repellent 

Naphthalene 0.5 Fumigant, moth repellent, major component (about 10%) of 

gasoline 

Nonylphenol, diethoxy- (total, NPEO2) 5 Nonionic detergent metabolite 

Octylphenol, diethoxy- (OPEO2) 1 Nonionic detergent metabolite 

Octylphenol, monoethoxy- (OPEO1) 1 Nonionic detergent metabolite 

para-Cresol 1 Wood preservative 

para-Nonylphenol (total) 5 Nonionic detergent metabolite 

Pentachlorophenol 2 H, F, wood preservative, termite control 

Phenanthrene 0.5 Manuf explosives, component of tar, diesel fuel, or crude oil, CP 

Phenol 0.5 Disinfectant, manuf several products, leachate 

Prometon 0.5 H (noncrop only), applied prior to blacktop 

Pyrene 0.5 Component of coal tar and asphalt (only traces in gasoline or diesel 

fuel), CP 

Tetrachloroethylene 0.5 Solvent, degreaser, veterinary anthelmintic 

Tri(2-chloroethyl) phosphate 0.5 Plasticizer, flame retardant 

Tri(dichloroisopropyl) phosphate 0.5 Flame retardant 

Tributyl phosphate 0.5 Antifoaming agent, flame retardant 

Triclosan 1 Disinfectant, antimicrobial (concern for acquired microbial 

resistance) 

Triethyl citrate (ethyl citrate) 0.5 Cosmetics, pharmaceuticals 

Triphenyl phosphate 0.5 Plasticizer, resin, wax, finish, roofing paper, FR 

Tri(2-butoxyethyl) phosphate 0.5 Flame retardant 

6
Ground-Water-Quality Data 

Data on the wells and the onsite measurements (pH, specific conductance, temperature, and 

dissolved oxygen (DO)) are listed in table 2. The pH, specific conductance, and temperature values are 

typical for ground water from bedrock in northeastern Ohio (Eberts and others, 1990; Jagucki and Darner, 

2001); however, the DO concentrations are unusually high for ground water. Although the use of the 

residential well pumps in the sample collection could introduce some oxygen into the water, the DO 

concentrations are still unusually high. In general, DO concentrations in ground water are expected to be 

less than 0.1 mg/L because buried organic matter and oxidizable minerals deplete available oxygen 

quickly; however, ground water in recharge areas can have relatively high DO concentrations, similar to 

concentrations in surface water (Hem, 1989; Freeze and Cherry, 1979). The DO concentrations measured 

in the wells in this study are consistent with recently recharged water. 

Bromide concentrations from 12 ground-water samples ranged from 0.04 to 0.18 mg/L (table 3). 

Samples were collected concurrently by Ohio EPA and analyzed by their laboratory for chloride and 

nitrate1

 concentrations (table 3). Chloride concentrations ranged from 12.6 to 61.6 mg/L. Nitrate 

concentrations ranged from 2.34 to 11.9 mg/L (as N). Water samples from three of the wells had nitrate 

concentrations that exceeded the Maximum Contaminant Level of 10 mg/L (U.S. Environmental 

Protection Agency, 2004). Samples from 10 of the wells had nitrate concentrations greater than 3.1 mg/L; 

in Ohio, this may indicate effects of human activity (Baker and others, 1989). High nitrate concentrations 

are not common in ground water because aquifers are normally low in oxygen, forcing soil bacteria to 

strip oxygen from nitrate. These nitrate concentrations are consistent with the high DO concentrations 

described earlier. 

Samples from eight wells were collected and analyzed for bacteria concentrations (table 4). 

Enterococci bacteria were found in samples from every well, although the concentrations in samples from 

three wells were less than 0.5 CFU/100 mL (volumes filtered were greater than 100 mL). Total coliforms 

were found in samples from every well at concentrations from 2 to 200 CFU/100 mL. E. coli were found 

in samples from three wells at concentrations greater than 1 CFU/100 mL. 

Concentrations of enterococci from wells WN-37, WN-39, WN-40, WN-42, and WN-43 did not 

allow for the collection of more than 100 colonies for the DNA-marker analysis. This is significant 

because Scott and others (2005) reported consistent detection of the esp gene in human-derived 

enterococci when at least 100 colonies were cultivated. Regardless, the colonies were tested for the 

presence of the esp gene. The presence of this gene indicates fecal contamination from a human source; 

the absence of the gene indicates either that fecal contamination is from nonhuman sources, or that the 

enterococci concentrations from human sources were below the detection limit, or that the marker was not 

present in the fecal source. The esp gene was not found in any of the samples, including the composite 

sample from five septic tanks (table 4). In another study, the esp gene was reported to be present in 8 of 10 

septic-system samples that had enterococci concentrations of 58 ± 24 CFU/100 mL (Scott and others, 

2005), so the failure to detect the marker in the composite sample with 51,000 CFU/100 mL is a strong 

indication that the gene would not have been detected in the ground-water samples even if the enterococci 

concentrations were greater. Therefore, the fecal contamination in the wells is likely from either human 

sources that did not have the marker or from nonhuman sources. 

 

1

 Actual analysis was for nitrite plus nitrate; however, “nitrate” is used in this report for simplicity because the proportion of 

nitrite to nitrate is negligible in most ground water. 

7
Table 2. Well data and water-quality field data for ground-water samples from wells in the 

Scenic Heights Drive-Batdorf Road area of Wayne County, Ohio, June 2006

[μS/cm, microsiemens per centimeter at 25 degrees Celsius; deg. C, degrees Celsius; mg/L, milligrams per liter; U, 

unknown; <, less than; --, no data] 

Site 

name 

Date 

sampled 

Approximate

depth of 

well/length 

of casing 

 (feet) 

Depth to 

water, below 

ground 

surface 

(feet) 

pH 

(standard 

units) 

Specific 

conductance 

(μS/cm) 

Temperature,

water 

(deg. C) 

Dissolved 

oxygen 

(mg/L) 

WN-35 6-21-2006 150*/U 102 7.0 485 13.0 9.45 

WN-36 6-21-2006 118 / 70 59.7** 6.9 430 12.2 1.26 

WN-37 6-19-2006 80*/U 29.4 6.3 417 13.1 8.56 

WN-38 6-21-2006 140 / 116 94.8 6.5 298 11.3 6.77 

WN-39 6-20-2006 140*/U 82.4 6.3 273 13.0 7.65 

WN-40 6-19-2006 116 / 52 51.6 6.6 465 13.8 6.51 

WN-41 6-20-2006 135 / 79 55.0 7.0 466 13.5 2.25 

WN-42 6-20-2006 140*/U 65.8 6.5 361 13.2 6.73 

WN-43 6-20-2006 100 / 79 63.0 6.5 355 12.6 4.04 

WN-44 6-21-2006 140*/U 104.3 7.0 554 12.8 4.96 

WN-45 6-19-2006 150 / 120 98.0 6.8 422 12.9 4.59 

WN-46 6-19-2006 -- -- 7.0 618 12.7 7.15 

* Reported by the Ohio Environmental Protection Agency. 

** Depth to water is below the top of casing. 

8
Table 3. Results of bromide, chloride, and nitrate analyses for groundwater samples from wells in the Scenic Heights Drive-Batdorf Road area 

of Wayne County, Ohio, June 2006. 

[Samples for chloride and nitrate analyses were collected and analyzed by the Ohio 

Environmental Protection Agency; mg/L, milligrams per liter] 

Site 

name 

Bromide, 

dissolved 

Chloride, 

dissolved 

(mg/L) 

Nitrite + nitrate, 

 (mg/L 

(mg/L) as N) 

WN-35 0.19 43.6 11.9 

WN-36 .07 39.7 2.34 

WN-37 .14 59.2 9.87 

WN-38 .09 13.0 10.9 

WN-39 .16 12.6 11.3 

WN-40 .18 61.6 9.15 

WN-41 .06 37.1 2.42 

WN-42 .12 37.2 8.66 

WN-43 .08 46.8 5.88 

WN-44 .05 28.0 6.50 

WN-45 .05 44.5 5.08 

WN-46 .04 53.2 6.38 

9
Table 4. Results of analyses of bacteria concentrations and bacterial DNA in ground-water 

samples from wells in the Scenic Heights Drive-Batdorf Road area of Wayne County, Ohio, June 

2006. 

[CFU/100 mL, colony-forming units per 100 milliliters; <, less than; E, results based on estimated colony count; k, 

results based on colony count outside of the ideal range, potentially reducing accuracy of estimates; g, present at 

concentrations less than 0.5 colonies per 100 milliliters; --, not determined; Pos., positive for detection of bacterial 

DNA marker; Neg., negative for detection of bacterial DNA marker] 

Bacteroidales c

markers present Site name 

Enterococcia 

(CFU/100mL) 

Total 

Coliform a

(CFU/100ml) 

E. coli a

(CFU/100mL) 

Enterococcus 

esp markerb

present Fecal Human Ruminant 

WN-36 g

d

14 E, k < 1 -- Neg. -- -- 

WN-37 17 E, k 200 E, k 1 E, k Neg. Pos. Neg. Neg. 

WN-39 2 E, k 28 E 2 E, k Neg. Neg. -- -- 

WN-39 replicate 2 E, k 9 E, k < 1 Neg. Neg. -- -- 

WN-40 2 E, k 32 E 1 E, k Neg. Pos. Neg. Neg. 

WN-41 g

d

2 E, k < 1 -- -- -- -- 

WN-42 2 E, k 26 E < 1 Neg. Pos. Neg. Neg. 

WN-43 2 E, k 16 E, k < 1 Neg. Pos. Neg. Neg. 

WN-45 gd 4 E, k < 1 -- Neg. -- -- 

Septic 

composite 51,000 E -- -- Neg. -- Pos. Neg. 

a Samples were processed within 26 hours of collection. 

b The esp marker is on a gene in the Enterococcus faecium DNA that is specific to bacteria from a human source. Presence of 

the marker indicates fecal contamination of human origin but does not indicate humans as a dominant source. Absence of the 

marker indicates either no human-origin fecal contamination or concentrations less than the minimum detection limit.

c

 The presence of the general fecal Bacteroidales marker indicates fecal contamination. The presence of the human-associated 

or ruminant-associated marker indicates human or ruminant sources contributed to the fecal contamination. The absence of a 

marker indicates either no associated host-source for the fecal contamination or that the concentrations were less than the 

minimum detection limit. 

d

 Concentrations less than 1 CFU/100 mL were determined by filtering up to 1 liter of water. 

10
Samples from seven wells were analyzed for the detection of the general fecal Bacteroidales marker; four 

were positive (table 4). DNA samples from these four wells were then tested for the host-specific, humanand ruminant-associated markers; all tests for host-specific markers were negative. The presence of the 

general fecal marker indicates fecal contamination. Although the presence of the human- or ruminantassociated marker indicates human or ruminant sources contributed to the contamination, the absence of 

the marker may indicate one or more of the following situations: (1) there was no fecal contamination 

from the host-associated sources, (2) the amount of contamination from the host-associated sources was 

below the detection limit of the method, or (3) the marker was not present in the fecal sources or was not 

present at expected concentrations. 

Fecal bacteria of the order Bacteroidales are anaerobes and are expected to survive for only a 

relatively short time in oxygenated waters. A study by Kreader (1998) found that in river water at 4°C, 

PCR-detectable DNA from Bacteroidales was detectable for at least 2 weeks; at 14° C, the DNA was 

detectable for only 4 to 5 days. Given the temperatures in the sampled water in this study (11-13°C), the 

presence of detectable DNA from Bacteroidales in samples from four wells —WN37, WN-40, WN-42, 

and WN-43 — is indicative of recently recharged water. 

Water from five wells — WN-37, WN-39, WN-40, WN-42 and WN-43 — was sampled and 

analyzed for wastewater compounds (table 1). Metolachlor, an herbicide and general-use pesticide and an 

indicator of agricultural drainage (table 1), was detected in all five samples but at concentrations below the 

laboratory reporting limit of 0.5 μg/L (data on file at the USGS Ohio Water Science Center). The 

estimated concentrations of metolachlor ranged from 0.0102 to 0.1160 μg/L. The only other compound 

detected was caffeine, in the sample from WN-40; the concentration was estimated at 0.120 μg/L, below 

the reporting limit of 0.5 μg/L. 

Summary 

In 2004, a public-health nuisance was declared by the Wayne County Board of Health in the 

Scenic Heights Drive-Batdorf Road area of Wooster Township, Wayne County, Ohio, because of concerns 

about the safety of water from local wells. Repeated sampling of the private water wells had found 

elevated nitrate concentrations and the fecal-indicators total coliform and Escherichia coli (E. coli). To 

help identify whether the bacterial contamination was from a human source, the U.S. Geological Survey 

(USGS), in cooperation with the Ohio Environmental Protection Agency (Ohio EPA), collected and 

analyzed samples from some of the affected residential water wells. The wells in the area are completed as 

open holes in fractured bedrock. In June 2006, the USGS collected water samples from 12 wells and 5 

septic tanks. Bromide concentrations were analyzed in samples from all 12 wells. Samples from 5 of the 

12 wells were analyzed for wastewater compounds. Total coliform, enterococci, and E. coli bacteria 

concentrations were determined in samples from 8 of the 12 wells. In addition, two microbial source 

tracking (MST) tools that employ DNA markers were tested on several samples from wells and a 

composite sample of water from 5 septic tanks. The DNA markers from the Enterococcus faecium species 

and the order Bacteroidales are associated with specific sources, either human or ruminant. The general 

Bacteriodales marker is also an indicator of fecal contamination from warmblooded animals. 

Onsite measurements of pH, temperature, specific conductance, and dissolved oxygen were made 

during sampling. The unexpectedly high concentrations of dissolved oxygen, ranging from 1.26 to 9.45 

mg/L, are indicative of recently recharged waters. Bromide concentrations in samples from 12 wells 

ranged from 0.04 to 0.18 mg/L. No wastewater compounds were detected at concentrations above the 

reporting limits in five wells. Metolachlor was detected in the five samples but at concentrations below the 

laboratory reporting limit of 0.5 μg/L. Caffeine was detected in one sample but also at a concentration 

below the reporting limit of 0.5 μg/L. Samples from the 12 wells were collected by Ohio EPA and 

analyzed for chloride and nitrate; chloride concentrations ranged from 12.6 to 61.6 mg/L and nitrate 

concentrations ranged from 2.34 to 11.9 mg/L. 

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Total coliforms were detected concentrations from 2 to 200 CFU/100 mL in eight wells. Similarly, 

enterococci bacteria were detected in all eight wells sampled, ranging in concentration from 0.5 to 17 

CFU/100 mL. E. coli were detected in samples from only three of the eight wells, at concentrations of 1 or 

2 CFU/100 mL. The test for the human-specific marker, the esp gene, of enterococci was negative in 

seven samples tested, including the composite sample of HSTS water. The general fecal Bacteriodales 

marker was detected in samples from four wells, but the tests for both the human- and ruminant-associated 

markers were negative. The presence of the PCR-detectable DNA for the general fecal Bacteroidales 

marker is indicative of fecal contamination and recently recharged water. 

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