Identification of an Assemblage of Indicator Organisms to Assess Timing and Source of Bacterial...

IDENTIFICATION OF AN ASSEMBLAGE OF INDICATORORGANISMS TO ASSESS TIMING AND SOURCE OF BACTERIAL

CONTAMINATION IN GROUNDWATER

M. J. CONBOY∗ and M. J. GOSSDepartment of Land Resource Science, University of Guelph, Guelph, Ontario, Canada

(∗ author for correspondence, Ontario Federation of Agriculture, 40 Eglinton Ave. East, 5th Floor,Toronto, ON, M4P 3A2, Canada)

(Received 4 August 1999; accepted 11 July 2000)

Abstract. Bacterial contamination of drinking water wells is a common problem in many ruralareas. Some of this contamination may be related to manure spreading or housing of livestock;another source is on-site septic systems. Current indicator organisms are able to detect the presenceof fecal contamination, but where there may be more than one potential source of fecal material, thecurrent indicators are unable to ascertain the origin. This laboratory investigation was undertaken todetermine the longevity and reliability of a selected suite of indicator organisms. Total coliform, fecalcoliform, fecal streptococci and Clostridium perfringens were monitored in a simulated contaminatedgroundwater environment for 6 months. All four indicator organisms were present at the end of 6months. The number of fecal streptococi bacteria decreased most noticeably, allowing assessment ofrelative age of contamination. C. perfringens was found to be a reliable indicator of contaminationfrom animal manure. Fecal material from 28 different animals and three septic systems were assessedfor the presence of the indicator organisms. Total coliform, fecal coliform and fecal streptococci werepresent in the fecal material of all animals tested including reptiles. C. perfringens was detected infeces from all but two of the animals assessed. Using an assemblage of indicator organisms providesmore information regarding source and timing of contamination than just testing for total coliformand fecal coliform bacteria.

Keywords: bacterial contamination, Clostridium perfringens, coliforms, fecal streptococci, indicatororganisms, source of pollution, well water

1. Introduction

Contamination of rural drinking water by bacteria is commonly more prevalentthan contamination due to excessive nitrate or pesticides (Goss et al., 1998). TheAmerican Academy of Microbiology indicated that the safety of drinking wateris declining throughout the world because of microbiological contamination (Hart,1996). This decline is not only in Third World countries, but in developed countriesas well. Eric Minz of the U.S. Centre for disease control estimated that there aremore than 3 billion cases of diarrhea world wide each year resulting in 10 milliondeaths, many caused by waterborne microorganisms (Hart, 1996). Ontario’s cur-rent Best Management Practices provide guidance to farmers to ensure that theirfarming operation is efficient and minimizes any contribution of contaminants to

Water, Air, and Soil Pollution 129: 101–118, 2001.© 2001 Kluwer Academic Publishers. Printed in the Netherlands.

102 M. J. CONBOY AND M. J. GOSS

TABLE I

Recommendations for minimum separation distance between wells andpotential contaminant source (OMAFRA, 1991)

Item Recommendation for

minimum distance

(m)

Septic tank 15

Weeping Bed 30

Manure 30

Feedlot 30

Crop 30

Unused well that has not been properly plugged 30

Lagoon and landfills 300

the environment. The minimum separation distance from any potential source ofcontaminant to a well has been established for Ontario (Table I). Nonetheless,many farming operations still have problems with chronic bacterial contaminationof their drinking water wells even though current guidelines have been adoptedfor the housing of livestock, application of manure, and separation of all sourcesof contamination. Some of the guidelines may therefore be too broad for safeapplication on all soil types and topographies. Animal manure and on-site humanwaste-disposal systems, such as septic units, represent the main sources of ruralwell water contamination by bacteria of fecal origin. Such contamination is a sig-nificant cause of gastro-intestinal illness. Any immunity to gastro-intestinal illnessdue to chronic exposure to contaminated drinking water is not complete (Raina etal., 1998).

The aim of this study was to identify different indicator organisms and to es-tablish their reliability in determining the source, and proximity or relative age ofcontamination.

2. Bacterial Survival

The first requirement was to determine the ability of the components of a possiblesuite of indicator organisms to survive long enough in a groundwater environ-ment to be detected. If the numbers of an indicator organism decreased extremelyrapidly, there would be little chance of obtaining groundwater samples with theorganism present. It is generally assumed that bacteria have a very short life cycle,and when outside of their ideal environmental conditions, the stresses placed uponthe organisms are too great for reproduction or prolonged survival. This is likely

INDICATOR ORGANISMS AND GROUNDWATER CONTAMINATION 103

the case for organisms that are introduced into the environment without a carbonsource, but the presence of organic matter provided by manure allows the organismto live in a somewhat isolated environment from the general soil.

The fecal coliform population while still in a bovine system is constantly in theexponential growth phase. This quasi steady-state results from the continuous inputof substrate and output of bacteria with the frequent defecation (average of 12 perday) of cattle. Once voided from the body, the fecal coliform population is con-sidered to immediately pass successively through retardation, stationary, and deathphases. The retardation phase lasts about 1 day, the stationary population phaseabout 2–6 days and the death phase continues beyond 30 days for an unknownperiod (Lynch and Poole, 1979; Thelin and Gifford, 1983; Sinton, 1980; Martin andNoonan, 1977). Die-off times in groundwater for some displaced bacteria appearto be slow and a reduction in abundance by one order of magnitude can involveseveral weeks or longer (Crane and Moore, 1984; Bitton et al., 1983). Results fromlaboratory studies show that E. coli can remain alive for up to 5.5 months (Gerbaand Keswick, 1981). This survival occurred despite the reduction of 99.9998% ofE. coli in 20 days. In most cases 2–3 months is sufficient to reduce pathogenicbacteria to negligible numbers once they have been applied to the soil. However,survival as long as 5 yr has been reported (Rudolfs et al., 1950).

3. Indicator Organisms

The second objective of this study was to develop a suite of indicator organisms thatcould be assessed to provide information regarding the likely source of bacterialcontamination. Drinking water standards used in this study were the same as thoseestablished for municipal supplies. However, for such supplies, testing ensures thatwater treatment is effective and seeks to identify whether there are any leaks inthe delivery system that would permit bacteria from sewage pipes to enter thetreated water. In this case, a very simple bacterial test is usually adequate. Thepresence of bacteria would signal a need to assess the treatment facility or identifya pipe fracture, and thereby allow the problem to be located. However, in ruralareas, the problem is different. Rural wells sample local groundwater resources.The groundwater is untreated, and there are usually a number of possible sourcesof bacteria. The most likely sources of contamination are feces from livestock andseptic systems, but other possible sources include wild animals, organic materialfalling in to the well and soil-borne bacteria.

Historically, coliform bacteria have been used as water quality indicators due totheir association with the intestinal tract and with pathogenic bacteria. Total coli-form includes those belonging to the genera Escherichia, Citrobacter, Enterobacterand Klebsiella. These coliforms are discharged in high numbers in human andanimal feces, but not all of them are of fecal origin. They are less sensitive thanviruses or protozoan cysts to environmental factors and to disinfecting. Some mem-


bers of this group (Klebsiella) may grow under some environmental conditions inindustrial and agricultural wastes (Bitton, 1994).

The absence of total coliforms is a good indication that the water is clean. Anepidemiology study, conducted among farm families in Ontario, Canada showedthat human health was not affected by the presence of total coliforms in drinkingwater (Raina et al., 1998). A more specific test is also required that can relateto affects on human health. The presence of fecal coliform bacteria in drinkingwater is a better indication that human health may be compromised. Fecal coli-forms include all coliforms that can ferment lactose at 44.5 ◦C. The fecal coliformgroup comprises bacteria such as Escherichia coli and Klebsiella pneumonae. Thepresence of these organisms in water samples is thought to indicate the presence offecal material from warm-blooded animals in the water supply (APHA, 1989).

Currently, E. coli is used as a standard indicator organism to determine thepresence of fecal contamination. Although this may be adequate for water qualitytesting, it could be misleading for studies where the source of fecal pollution is tobe determined. Commonly used indicators of fecal pollution such as coliform bac-teria will not distinguish between fecal pollution of human and animal origin. Thispresents a serious limitation in determining the source of contamination, which canhinder remedial action. Fecal coliforms also do not meet many of the criteria forindicator organisms. They are less resistant to disinfection than associated patho-gens, they have been detected in pristine sites (Hazen, 1988), they may re-growin water and wastewater under appropriate conditions (Bitton, 1994) and they maysurvive for extended periods. Despite these problems, the presence or absence ofE. coli is relied on as a drinking-water quality indicator in many areas.

If the indicator organism of choice is E. coli, there is a significant limitation indetermining the source of bacterial contamination, as E. coli is present in human,domestic animal and wild animal feces. The presence of E. coli in a water samplegives no clues as to whether the source is due to a malfunctioning septic system,inadequate protection of the groundwater from the manure management system, orwhether local wildlife has had access to the water source.

Fecal coliforms and fecal streptococci are considered to originate in the digest-ive tract of humans and warm-blooded animals. The presence of these bacteria inwell water indicates that there is a source of fecal matter entering the water supply.Fecal streptococci die-off more rapidly in the environment than other bacterialindicators (Crane and Moore, 1986), therefore when there are no new additions ofbacteria, the proportion of streptococci to coliforms will decrease rapidly. The pres-ence of fecal streptococci indicates that a source has been contaminated by fecalmaterial relatively recently, or that a very high level of contamination, possiblyassociated with organic matter, was present. The ratio of fecal coliform to fecalstreptococci serves as an indicator of the origin of pollution of surface waters. Theratio of fecal coliform to fecal streptococci is different in the feces of humans, wildand domestic animals. A ratio of 4 or more indicates a contamination of humanorigin whereas a ratio below 0.7 is indicative of animal pollution (Geldrich, 1976).


However, these ratios are only valid for recent pollution (24 hr) (Thelin and Gifford,1983). Studies by Doran and Linn (1979) and Doran et al., (1981) used the fecalcoliform to fecal streptococci ratio to determine if the source of bacterial contam-ination was domestic or wild animals. They found that a ratios below 0.05 wereindicative of wildlife sources and ratios above 0.1 were characteristic of grazingcattle.

Coliform organisms provide information that water is contaminated. An as-semblage of indicator organisms that also includes fecal streptococci can providean assessment of relative age or proximity of contamination. The ratio of fecalcoliforms to fecal streptococci may provide some information on source of bacteriabut can not be considered reliable for more than 24 hrs and is not used for thispurpose in this investigation.

The presence of an organism that is unique to animals or to humans is animportant component of an assemblage of indicator organisms. Recent studieshave suggested that Clostridium perfringens, which shows resistance to antibiot-ics, could be a specific indicator of pollution originating from domestic livestockmanure (Huysman et al., 1993). C. perfringens is a gram-positive, spore-formingrod-shaped bacterium. The spores are quite resistant to environmental stresses andto disinfecting which make it a poor indicator of water quality when consideredindependently. It does not provide any information about timing of the contamin-ation entering the well. However, the presence of C. perfringens indicates that atsome time in the past animal manure entered the water supply.

There are many limitations associated with adopting a single indicator organismto establish the presence of fecal contamination and determine the likely source ofcontamination. To overcome some of these problems, an assemblage of micro-organisms was considered for this study. We have assessed the effectiveness oftotal and fecal coliforms as indicators of a contamination event, and consideredwhat new information could be obtained by enumerating fecal streptococci and C.perfringens that is resistant to two antibiotics. This survival of fecal-origin bacteriawas monitored in a simulated groundwater environment. The presence of indicatororganisms in feces from twenty-eight different animals and three septage sampleswas also assessed.

4. Methodology

The experiment was confined to the laboratory.

5. Samples

In early summer, fresh fecal deposits from at least five different animals of a givenspecies were located and each mixed to provide a uniform sample with as little


bedding material as possible. These samples were transported to the laboratory insterile containers, inside coolers with icepacks. Groundwater was collected from awell at a local farm, sterilized by autoclave and cooled to 7 ◦C. Immediately uponreturn to the laboratory (less than 3 hr from collection of first sample) manure fromchicken, dairy and beef cattle, and pigs were mixed with the sterile groundwater(10 g wet weight manure per litre water) and aged. The poultry manure samplescontained a significant amount of bedding material, and as much of this as possiblewas removed prior to mixing with groundwater. A further treatment that was in-tended to simulate a mixed farm, was constructed by taking 12.5 g of wet manurefrom chickens, dairy and beef cattle and pigs respectively, and combining the totalof 50 g with 5 L of groundwater. The ratio of wet manure to groundwater waschosen to simulate a contamination event, which introduced organic matter andbacteria, as might occur in vulnerable locations during or shortly after the spreadof manure. Samples of stored manure were also collected immediately prior tomanure spreading in the fall and represented manure applied to fields. The samebasic approach was used to transport and mix the samples with groundwater. Theduration of the incubation of the stored manure was shorter than that of the othertreatments because the samples were collected later in the year.

The mixtures of manure and groundwater were stored at 7 ◦C to represent a typ-ical groundwater environment in Ontario. When bacterial assessments were made,a sub-sample was taken and the remainder of the solution was maintained at 7 ◦C,so that the long-term storage temperature was not altered. The presence of the suiteof indicator organisms in the groundwater samples was investigated throughout thestorage period. Indicator organisms were assessed by monitoring their survival andrelative abundance.

To assess organisms in animal fecal material other than in domestic livestock,samples were obtained from the Ontario Veterinarian College, the Biology Depart-ment at the University of Guelph, and local pet shops. Samples were collected inplastic sample bags, weighed and suspended in nanopure water (18 M�). Mixtureswere assessed for indicator organisms the day the sample was collected and after 2weeks. Samples of avian feces were collected as a mix of droppings of three birdspecies including, pigeon, lovebird and canary. Samples from three different septictanks were assessed.

6. Assessment of Indicator Organisms

Standard membrane filtration techniques were employed for all filtration enumera-tions (APHA, 1989). Water (100 mL) was filtered through a Magna M-PAC Nylonsupported grid (0.45 µm) (MSI) and the filters were then placed on the appropriatemedia in a 55 × 15 mm plastic petri plate. All filtrations were performed in du-plicate. All results were reported as colony forming units. Counts were obtainedby a series of dilutions in Nanopure water, direct, 1:10, 1:100, 1:1000, 1:10 000


and 1:100 000. Samples that were overgrown at this lowest dilution had more than200 CFU/100 mL−1 and were considered to contain a minimum of 10 000 000 CFU100 mL−1. Dilution at 1:100 000 was the lowest dilution used to assess survival. Asthe purpose of the investigation was to determine whether bacteria could survive inlarge enough numbers to be detectable, it was not considered necessary to includemore dilutions so that actual numbers in the initial mixture could be determined.

To enumerate total coliforms, filters were incubated for 24 hr at 37 ◦C onM-ENDO agar (Becton-Dickinson, Detroit, Michigan, U.S.A.) with 95% ethanol.Organisms enumerated as total coliforms produced a colony with a green metallicsheen on M-ENDO agar. Confirmation of the presence of coliforms was indicatedby gas production in inverted fermentation tubes, after incubation for 48 hr at 37 ◦C,in Lauryl Tryptose Broth (Difco, Detroit, Michigan, U.S.A.) and Brilliant GreenBile Broth (Difco, Detroit, Michigan, U.S.A.).

Fecal coliforms were incubated on M-FC agar (Becton Dickinson, Detroit,Michigan, U.S.A.) with 1% rosalic acid in 0.2 M NaOH for 24 hr at 44 ◦C. Coloniesenumerated as fecal coliforms were blue.

Fecal streptococci were incubated for 48 hr at 37 ◦C on KF streptococcus agar(Becton Dickinson, Detroit, Michigan, U.S.A.) containing 1% triphenyltetrazoliumchloride (TTC) (Becton-Dickinson, Detroit, Michigan, U.S.A.). Organisms enu-merated as fecal streptococci were red.

Two hundred colony forming units (CFU) were used as a standard cut-off pointfor bacterial enumeration. More than this number of CFU present on a plate caninhibit the expression of characteristic features of the colonies due to overcrowding,and the actual count can not be considered reliable (APHA, 1989). If there weremore than 200 colonies present on the plate, the plate was considered overgrown(o/g).

C. perfringens is an anaerobic organism, and an anaerobic chamber would havebeen required for enumeration. Instead, presence or absence was monitored usingscrew-cap test tubes to create an anaerobic environment. SFP agar (Difco, Detroit,Michigan, U.S.A.) was sterilized then Bacto-egg yolk additive (Difco, Detroit,Michigan, U.S.A.), polymyxin B sulphate (Difco, Detroit, Michigan, U.S.A.) andkanamycin sulphate (Difco, Detroit, Michigan, U.S.A.) were mixed into the agar.The entire mixture was poured into screw cap test tubes. The agar was warmed toliquefy, prior to inoculation. Approximately 1 mL of agar was removed from thetest tube, replaced with 1 mL of sample and thoroughly mixed. The tubes werefilled completely to create an anaerobic environment inside the test tube. Sampleswere incubated at 37 ◦C. Only direct inoculation was used, in case the dilutionprocess affected the ability of anaerobic C. perfringens’ to reproduce reliably.The presence of black colonies suggested the presence of C. perfringens. Tubeswere checked for black colonies (positive reaction) after 24 hr and again after 1week. Samples that were positive after 24 hr were removed from further incubationand sterilized. Those that did not have any black colonies present after one week


Figure 1. Survival curves for indicator organisms in stored dairy manure.

Figure 2. Survival curves for indicator organisms in fresh dairy cattle manure.

were considered to be devoid of C. perfringens. Two replicates of each test wereperformed.

One ‘nanopure’ water sample was incubated on each medium for a maximumof every 20 fecal samples assessed.

Petri plates and test tubes with characteristic growth indicative of each indi-vidual organism were submitted to the University of Guelph, Determinative Bac-


Figure 3. Survival curves for indicator organisms in beef manure.

Figure 4. Survival curves for indicator organisms in pig manure.


Figure 5. Survival curves for indicator organisms in mixed manure.

Figure 6. Survival curves for indicator organisms in chicken manure.

teriology Laboratory. Tests performed by this lab confirmed the identification of in-dicator organisms. Some plates with non-characteristic growth were also submittedto determine what other organisms were growing on the plates.

At the completion of monitoring bacterial die-off, the volume and wet and dryweight of the manure samples were measured. Samples were dried at 40 ◦C untilall liquid had evaporated and solid had dried.


7. Results

Total coliform bacteria were good indicators of contamination in the control samples.However, samples from the manure experiments yielded little useful informationas the total coliform plates were overgrown for almost every treatment, throughoutthe entire experiment (Figures 1–6, squares). The M-ENDO agar is an indicatormedium, but it is not selective; it allows counts of total coliforms based uponorganisms present with characteristic colonial morphology, in this case colonieswith a metallic green sheen. Where there was significant growth on the plates,from coliforms and other organisms, there was no way to determine how manyof the organisms on the plate were coliforms unable to express the characteristiccolony due to overcrowding of the plate, or if coliforms were present, but only insmall numbers. The confirmation tests using Lauryl Tryptose Broth and BrilliantGreen Bile Broth allowed assessment of the presence of coliforms, but these testsdid not give any indication of number of organisms present.

Enumeration of fecal coliforms was also problematic where there were verylarge concentrations of bacteria of manure origin. This medium was not selective;and relied on a reaction between fecal coliforms and the medium for the formationof the characteristic blue colony.

Incubating the plates at 44.5 ◦C is considered to be selective for fecal coliforms,and indeed this was the case. On a few occasions plates were incubated at 37 ◦C,as well as at 44.5 ◦C. This cooler incubation temperature lead to overgrowth ofcontaminants on fecal coliform plates.

In many cases, there were other colonies present on the plates incubated at44.5 ◦C that were usually distinguishable by colours other than blue (specific tofecal coliforms). Klebsiella and Staphylococcus were identified on one contamin-ated plate that was assessed by the Determinative Bacteriology laboratory.

There were many samples that were extremely overgrown with competitor bac-teria. On overgrown plates blue colonies were masked. These same samples wouldreveal the presence of blue fecal coliform colonies when the samples were diluted.This same phenomenon occurred in well water samples (Conboy and Goss, 1998).Fecal coliforms were more useful indicators as the concentration of bacteria de-creased. Fecal coliforms lived for several months in all treatments (Figures 1–6,closed diamonds).

Fecal streptococci were able to survive for over 140 days, in some treatments(Figures 1–6, closed triangles). Fecal streptococci tended to show a reduction innumbers earlier than total coliform bacteria. The stored manure, beef and dairycattle treatments contained little straw or bedding, had a definite reduction in num-bers within 60 days. The straw or bedding would act as a carbon source, whichbreaks down more slowly than manure itself.

On all figures, a value of 100 000 CFU mL−1 represents an overgrown plate atthe lowest dilution and should be considered a minimum concentration value.

Treatments in which the number of CFU of fecal streptococci declined also


TABLE II

Dry matter remaining after 6 month of incubation

Type of manure Dry matter Fecal streptococci count

(g mL−1) (end, CFU mL−1)

Stored dairy cattle 0.0001 27

Dairy cattle 0.0056 32

Beef cattle 0.0069 31

Pig 0.0168 6000

Mix 0.0272 10000

Chicken 0.0554 10000

TABLE III

Comparison of colony forming units in fresh and storeddairy cattle manure for selected days

Day Fresh dairy cattle Stored manure

Fecal Total Fecal Total

1 100000 100000 100000 3800

28 100000 100000 10000 10000

63 100000 100000 1000 1400

119 10000 10000 5.4 30

showed a decrease in the dry weight of solids per unit volume of manure (Table II).The main components of the manure are carbon compounds that would serve as anutrient source for the bacteria.

Comparison of fresh and stored dairy cattle manure showed that stored manurehad lower number of total coliform and fecal coliform bacteria present throughoutmuch of the experiment (Table III).

Monitoring presence or absence of C. perfringens was satisfactory, but whenserial dilutions were used in an attempt to predict the order of magnitude of CFUpresent, the results for comparable treatments were variable (results not shown).Control samples were always free of C. perfringens, and none of the samples fromthe three septic systems were found to test positive for the organism. However,C. perfringens was present in all treatments of animal manure for the entire 6-month duration of the experiment. C. perfringens, while not a very useful indicatorfor determining the age of the contamination, could be very useful in determiningsources of contamination.


Manure from 28 different animals and 3 septic systems were assessed for thepresence of the four indicator organisms. These results showed that total coli-form, fecal coliform and fecal streptococci bacteria were not unique to warm-blooded animals, as several reptiles contained these organisms in their fecal ma-terial (Table IV). The results also showed that most animals tested positive for C.perfringens, not just domestic livestock. Human waste did not test positively.

8. Discussion

This study highlighted several common misconceptions in the literature regardingbacterial indicator organisms. Total and fecal coliforms, as well as, fecal strepto-cocci have been reported as originating in the intestinal tract of warm-blooded an-imals and that once voided, these organisms start to cease activity and die (AHPA,1989). All of the indicator organisms tested in our study were found in the manureof frogs, snakes, and other cold-blooded animals and thus, are not only repres-entative of warm-blooded animals. C. perfringens was reported by Huysman etal. (1993) as a unique domestic livestock indicator but was found in almost everyanimal tested.

Reviews by Reddy et al. (1981) and Crane and Moore (1984, 1986) reportedresults of more than 50 die-off studies which have indicated that more than 90% ofenteric bacteria can die-off within 30 days. Outside of the intestine, the ideal en-vironment for enteric bacteria is in a cool, dark location with plenty of water. Oncebacteria reach this type of environment, provided there are adequate nutrients, thebacterial populations will continue to live. Although ours was a laboratory study, itshowed that after 6 months of incubation in a simulated groundwater environment,bacteria still thrived. This extended survival may negatively impact water qualityfor longer time periods or over larger areas of the aquifer than would be predictedbased on the literature.

Our results are consistent with those of Verstraete and Voets (1976), who foundthat enteric bacteria introduced without organic material died-off rapidly, but whennutrients were introduced with the microorganisms, the die-off of enteric organismswas delayed for a period of time in which they survived or even grew. In our study,populations of fecal streptococci bacteria declined with decreases in added drymatter. In stored dairy manure, the treatment that showed the greatest reduction inbacterial populations, the dry matter remaining after 6 months was also the smal-lest. In chicken manure, a treatment with very little decline in bacterial numbers,there was a great deal of bedding material which acted as a carbon source andcontributed to the greater dry weight. Storage of manure may also reduce bacterialconcentrations.


TAB

LE

IV

Sum

mar

yof

indi

cato

ras

sess

men

tfor

diff

eren

tani

mal

s

Ani

mal

Man

ure

Tota

lB

rilli

ant

Lau

ryl

Fec

alF

ecal

C.p

erfr

inge

ns

conc

entr

atio

nco

lifo

rmgr

een

Try

ptos

eco

lifo

rmS

trep

toco

cci

Hor

se12

.90

gL

−119

3po

sitiv

epo

sitiv

e21

0>

400

posi

tive

She

ep10

.00

gL

−111

5po

sitiv

epo

sitiv

e12

219

nega

tive

Cow

11.2

0g

L−1

105

posi

tive

posi

tive

122

posi

tive

Rab

bit

1.70

g20

0m

L−1

o/ga

nega

tive

posi

tive

01

posi

tive

Ferr

et2.

09g

200

mL

−1o/

gpo

sitiv

epo

sitiv

eo/

g20

0po

sitiv

e

Ham

ster

0.22

g20

0m

L−1

1po

sitiv

epo

sitiv

e1

80po

sitiv

e

Dog

2.30

g20

0m

L−1

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posi

tive

posi

tive

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>40

0po

sitiv

e

Mou

se0.

20g

200

mL

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gpo

sitiv

epo

sitiv

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go/

gpo

sitiv

e

Cat

3.05

g20

0m

L−1

o/g

posi

tive

posi

tive

o/g

o/g

posi

tive

Chi

nchi

lla

0.28

g20

0m

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o/g

posi

tive

posi

tive

280

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13g

200

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sitiv

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Rat

1.60

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posi

tive

posi

tive

>40

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400

posi

tive

Bla

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0.20

g20

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TAB

LE

IV

(con

tinu

ed)

Ani

mal

Man

ure

Tota

lB

rilli

ant

Lau

ryl

Fec

alF

ecal

C.p

erfr

inge

ns

conc

entr

atio

nco

lifo

rmgr

een

Try

ptos

eco

lifo

rmS

trep

toco

cci

Igua

nave

rysm

allu

nkno

wn

wei

ght

2po

sitiv

epo

sitiv

e2

54po

sitiv

e

Sal

aman

der

0.16

g20

0m

L−1

>20

0po

sitiv

epo

sitiv

e3

20po

sitiv

e

Bir

d2.

20g

200

mL

−1o/

gpo

sitiv

epo

sitiv

eo/

go/

gpo

sitiv

e

Mil

ksn

ake

1.50

g20

0m

L−1

o/g

posi

tive

posi

tive

o/g

o/g

posi

tive

Gui

nea

pig

1.00

g20

0m

L−1

o/g

posi

tive

posi

tive

o/g

>80

posi

tive

Woo

dfr

og0.

22g

200

mL

−1o/

gpo

sitiv

epo

sitiv

eo/

g>

400

posi

tive

Dai

ryca

ttle

10.0

0g

L−1

o/g

posi

tive

posi

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9. Choosing Appropriate Indicators

In Ontario, Canada, the Ministry of Health will test well water for total coliformand fecal coliform bacteria as a complimentary service. This is also common inother areas. The use of these two indicators in any well survey can be benefi-cial, as there can be direct comparison of the well’s history of contamination andcurrent results. This also would allow farmers to have well water tested betweensampling dates and the results could be incorporated into information obtained. Inany well survey, assessing rural well water quality, total and fecal coliforms shouldbe included as part of an assemblage of bacteria. This allows a direct comparisonbetween results obtained and the results from previous tests on the same wells, andwith other studies.

Current indicators do not reveal any information with regards to timing of con-tamination or source. This study investigated other organisms to include in anassemblage of indicator bacteria that would be able to provide more information.Fecal streptococci commonly inhabit the intestinal tract of humans and warm-blooded animals. Their presence indicates fecal contamination in water. Membersof this group persist well but do not reproduce in the environment (Bitton, 1994).

The presence of fecal streptococci was a useful indicator in samples, with enu-meration giving an idea of the relative age of contamination. Where fecal strep-tococci were present in large numbers, the contamination must be of relativelyrecent origin, or the contamination must also introduce nutrients or organic matteron which the organisms could live. In a well water sample, if there was no possiblemeans for bacterial infiltration to have taken place for an extended period, but fecalstreptococci numbers remained high, it would be reasonable to conclude that sig-nificant amounts of organic material also entered the well water. Fecal streptococciwere present in smaller numbers than were total coliforms, and the absence of theformer would indicate that the contamination event was much earlier than an eventassociated with both indicators being present in the water sample.

C. perfringens, with antibiotic resistance, was found to be an indicator of fecalmaterial of animal origin. On the farm there can be several animals; however, whenlooking at the history of contamination of the well or by doing a visual inspectionaround the well, the sources can be limited. As livestock operations will generallyhave a concentrated area of manure, which would house a very large number ofbacteria, this would be the most likely source of animal origin bacteria on a farm.Other possibilities include: rodents or other animals falling into and dying in thewell; or an incorrectly sealed well which allows rodents or frogs, or other animalsto basically live and defecate within the well. Visual inspection should reveal thepresence of anything inside the well. If a well water sample tests high for bacteria,but has never had any bacteria prior to this occasion, this would be a clue tothe homeowner that a visual inspection of the well is necessary. The two mainpotential sources of fecal contamination in rural areas are manure from livestockoperations and septic tank systems. This assemblage of indicator organisms will


provide information as to which source is more likely to be entering the well watersupply.

10. Conclusions

Animal manure is present on many farms and its use as a soil amendment can bevery beneficial to soil and crops. However, current manure management practicesmay not be sufficient to ensure safe drinking water.

Indicator organisms are commonly considered to originate in the intestines ofwarm-blooded animals, but we also found them in the fecal material of severalcold-blooded animals.

Fecal origin bacteria can survive for extended periods in a groundwater envir-onment. This extended survival of enteric bacteria means that groundwater has thepotential to be unsafe for consumption for an extended period of time after con-tamination, and that contamination may affect a significant portion of an aquifer.

Results from laboratory testing showed that the total coliform test was of lim-ited use. Enumeration of fecal coliforms was also problematic when there werevery large numbers of bacteria of manure origin. Nevertheless, total coliforms andfecal coliforms are important indicator organisms to include in any water qualitystudy. These organisms can be assessed frequently by the homeowner and allowcomparison with previous test results on the same well and comparison with otherwater quality studies.

The number of fecal streptococci decreased more than total coliform numbers inthe laboratory experiments. Thus this organism can be used to provide informationon the relative timing of a contamination event.

C. perfringens was found to be a good indicator for animal origin pollution, butwas not found to be unique to domestic livestock. This organism was isolated fromall but one of the animals assessed. This included cold-blooded animals. This orother specific organisms should also be included in tests of rural drinking water toaid in locating the source of contamination.

Fecal pollution of human and animal origin can be reliably distinguished bycombinations of appropriate indicators. The results from this investigation showthat it would be beneficial to use an assemblage of bacterial indicators to assesswater quality.

There are added benefits to testing for an assemblage of bacteria. First, there aremore indicators of fecal contamination, and the additional indicators give confirm-ation of any contamination. In addition, information regarding source and age ofcontamination can be used to identify appropriate remedial action. Public Healthlaboratories should consider regular testing for fecal streptococci and C. perfrin-gens in addition to total and fecal coliforms to assist rural residents in identifyingthe source of contamination entering their wells.


Acknowledgements

This research was supported in part by research grants issued by the Ontario Min-istry of Agriculture, Food and Rural Affairs/University of Guelph Enhanced Part-nership and The Environmental Capacity Enhancement Project. We thank CarrolMallory of Ontario Ministry of Health, Palmerston Laboratory for carrying outconfirmatory tests on our samples, and Sara Mclellan for laboratory assistance withsome aspects of this research.

References

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Division, Ministry of Work and Development, New Zealand, p. 25.Raina, P., Pollari, F. Teare, G. Goss, M. J., Barry, D. A. J. and Wilson, J. B.: 1998, Can. J. Public

Health 90, 172.Reddy, K. R., Khaleel, R. and OverCash, M. R.: 1981, J. Environ. Qual. 10, 255.Rudolfs, W., Franks, L. L. and Ragotzkie, R. A.: 1950, Sewage Indust. Wastes. 22, 1261.Sinton, L. W.: 1980, J. Hydrol. (N. Z.) 19, 119.Thelin, R. and Gifford, G. F.: 1983, J. Environ. Qual. 12, 57.Verstraete, W. and Voets, J. P.: 1976, Water Research 10, 129.

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