228 VOLUME 114 | NUMBER 2 | February 2006 • Environmental Health Perspectives

Research

Recreational waters are monitored worldwide

to protect the health of bathers from infectious diseases caused by waterborne pathogens

that are associated with the pollution of natural recreational waters by human and animal

feces, and the results of monitoring are the

basis for corrective action where necessary.

Water quality standards are typically based on

measurements of the concentrations of fecal

indicator organisms. In 1998 a review carried

out on behalf of the World Health Organization (WHO) (Prüss 1998) assessed all available epidemiologic information on associations

between the risk of infection for bathers and

the concentration of fecal indicator organisms

in fresh and marine recreational waters. Most

studies reported an increase of health risk in

swimmers with an increase in the indicatorbacteria count in recreational water. Relative

risk (RR) values for swimming in polluted

water versus clean water or versus staying on a

beach without entering the water were often

significant and usually ranged between 1 and

3. Both Prüss (1998) and the current guidelines of the WHO (2003) have identified the

randomized controlled trial design as the

one that yields the most reliable results when

associations between the degree of fecal water

pollution and the risk of infection are investigated. This conclusion has been based primarily on the fact that the randomized controlled

trial design minimizes nondifferential misclassification bias resulting from the inaccurate

assignment of microbial concentrations in the

water to exposed participants. However, to this

day, results from only one study of this kind

are available for exposure to seawater. The

results presented here are the first ever collected in a randomized controlled trial in fresh

water. In addition, this randomized controlled

trial is the first to study a cohort that includes

children and teenagers, who usually make up a

considerable percentage of the visitors at public

bathing sites.

Materials and Methods

Study sites. We performed the present study at

five German freshwater bathing sites. All study

sites had been registered to the European

Commission’s atlas of European bathing beaches

(European Commission 2005). They had complied with current European standards (Council

of the European Communities 1976) for at least

the three previous bathing seasons. Bathing sites

in the European Union comply with the microbiological quality criteria when 95% of all sample results of a complete bathing season do not

exceed the “imperative values” (legally binding)

of 2,000 fecal coliforms and 10,000 total coliforms per 100 mL. In addition, it is recommended that 80% of the sample results comply

with a “guide value” of 100 fecal coliforms and

that 90% of the samples comply with a guide

value of 100 fecal streptococci per 100 mL.

Study locations were situated in the

north, northeast, southwest, and southeast of

Germany: four sites on lakes and one site on a

river. Sources of fecal contamination included

treated and untreated municipal sewage, agricultural runoff, and contamination from

water fowl [details in Supplemental material

Annex 1 (http://ehp.niehs.nih.gov/docs/

2005/8115/supplemental.pdf)].

Address correspondence to A. Wiedenmann,

Landratsamt Lörrach, Fachbereich Gesundheit,

Palmstr. 3, 79537 Lörrach, Germany. Telephone:

+49-7621-410-2140. Fax: +49-7621-410-2199.

E-mail: albrecht.wiedenmann@arcor.de

Supplemental material is available online (http://

ehp.niehs.nih.gov/docs/2005/8115/supplemental.pdf).

We thank our international consultants D. Kay,

J. Fleisher, and I. Leenen for their invaluable advice

on planning the study; S. Gommel, M. Eissler,

M. Hirlinger, A. Paul, K. Jüngst, U. Luther-Kliegl,

W. Langhammer, C. Hock, P. Kühner, and C. Führer

for their dedicated collaboration and their team spirit;

and our cooperation partners at the five study locations,

especially S. Brockmann, R. Waidmann, H. Gabler,

R. von Butler, B. Kiß, C. Höller, B. Majeran,

P. Schindler, and S. Schweitzer, for their efforts and

support. We are especially indebted to our 202 project

helpers and to the 2,196 volunteers who participated

in the trials. We thank D. Blaurock for the linguistic

revision of the manuscript.

The project was organized by the Institute for

General and Environmental Hygiene of the University

of Tübingen with the support of a grant from the

German Federal Environmental Agency (UFOPLAN

project 298 61 503).

The authors declare they have no competing

financial interests.

Received 13 March 2005; accepted 29 September

2005.

A Randomized Controlled Trial Assessing Infectious Disease Risks from

Bathing in Fresh Recreational Waters in Relation to the Concentration of

Escherichia coli, Intestinal Enterococci, Clostridium perfringens, and Somatic

Coliphages

Albrecht Wiedenmann,1 Petra Krüger,2 Klaus Dietz,3 Juan M. López-Pila,4 Regine Szewzyk,4 and 

Konrad Botzenhart5

1District Government Stuttgart, State Health Office, Stuttgart, Germany; 2District Government Tübingen, Tübingen, Germany; 3Department of Medical Biometry, University Hospital, Tübingen, Germany; 4 Federal Environmental Agency, Berlin, Germany; 5Institute

of General and Environmental Hygiene, University of Tübingen, Tübingen, Germany

We performed epidemiologic studies at public freshwater bathing sites in Germany to provide a

better scientific basis for the definition of recreational water quality standards. A total of 2,196

participants were recruited from the local population and randomized into bathers and nonbathers. Bathers were exposed for 10 min and had to immerse their head at least three times.

Water samples for microbiological analysis were collected at 20-min intervals. Unbiased concentration–response effects with no-observed-adverse-effect levels (NOAELs) were demonstrated for

three different definitions of gastroenteritis and four fecal indicator organisms. Relative risks for

bathing in waters with levels above NOAELs compared with nonbathing ranged from 1.8 (95%

CI, 1.2–2.6) to 4.6 (95% CI, 2.1–10.1), depending on the definition of gastroenteritis. The effect

of swallowing water provided additional evidence for true dose–response relationships. Based on

the NOAELs, the following guide values for water quality are suggested: 100 Escherichia coli,

25 intestinal enterococci, 10 somatic coliphages, or 10 Clostridium perfringens per 100 mL.

Recreational water quality standards are intended to protect the health of those consumers who

are not already immune or resistant to pathogens that may be associated with indicator organisms.

In contrast to current World Health Organization recommendations, we concluded that standards

should be based on rates of compliance with NOAELs rather than on attributable risks determined above NOAELs, because these risks depend mainly on the unpredictable susceptibility of

the cohorts. Although in theory there is no threshold in real concentration–response relationships,

we demonstrated that a NOAEL approach would be a more robust and practical solution to the

complex problem of setting standards. Key words: bathing, Clostridium perfringens, Escherichia

coli, fecal indicators, fecal water pollution, fresh recreational water, gastroenteritis, health risks,

intestinal enterococci, somatic coliphages. Environ Health Perspect 114:228–236 (2006).

doi:10.1289/ehp.8115 available via http://dx.doi.org/ [Online 29 September 2005]
Ethical clearance. The trial design was

approved by the ethics commission of the

medical faculty of the Eberhard Karls

University Tübingen.

Recruitment of the study cohort. A total of

2,196 participants were recruited from the

local population. In the 3 weeks before each

trial, the study design and the purpose of the

study were promoted with the help of the

local media, and information flyers were distributed at information desks and on the

beaches. People could register for participation either directly at the information desks or

by calling a telephone number and making an

appointment for an initial interview and medical examination. For the pilot trial at study

location 1, only volunteers > 18 years of age

were accepted for participation. After additional ethical clearance, the lower age limit

was set to 4 years for the remaining trials at

study locations 2–5. All adult participants and

teenagers between 14 and 18 years of age gave

their written informed consent before participating. Teenagers between 14 and 18 years of

age were accepted with the written, signed

consent of their parents or legal guardians.

Children < 14 years of age were accepted with

the written, signed consent of both parents or

legal guardians and had to be accompanied by

at least one parent or a legal guardian.

Children < 4 years of age were not accepted

because they were considered to be a potential

source of fecal accidents during the trials.

Enrollment. Two to three days before

exposure, the participants were interviewed in

person, and all participants underwent a short

medical examination. The medical checkups

were performed by professional physicians and

included monitoring the questionnaire data

with respect to the details on the participants’

health conditions; a brief discussion on specific

health problems such as chronic diseases, infections, or injuries in the participant’s medical

history if required; an inspection of the eyes,

ears, and throat; and an electronic temperature

measurement in the ear. Volunteers were

included if they appeared to be physically and

mentally fit for participation in the study trials.

Reasons for exclusion were serious acute infectious diseases or fever (> 38.0°C), acute nausea

or diarrhea, wounds that had not healed, or

health conditions where bathing might have

presented a serious or life-threatening risk.

Allocation. On the exposure day, participants were randomized into equal-sized groups

of bathers and nonbathers. Randomization

took place after registration at a registration

desk on the beach using a block randomization

procedure with blocks of 10. Parents of children < 14 years of age were asked whether their

children would accept a randomization result

independent of the result of their parents. If

the answer was yes, the children were independently randomized. If children preferred to be

accompanied by one of their parents, they had

to decide by which one. Parents were also

given the option of making this decision for

their children. Subsequently, the parents

received their randomization result.

Trial design. Each participant received a

standardized lunch package and a bottle of

mineral water; 20 of the lunch packages per

trial were microbiologically analyzed and

monitored for food pathogens in a state food

safety laboratory. All food items complied

with their specific quality standards. The purpose of serving this controlled lunch package

was to exclude the theoretical possibility of a

food-borne outbreak that might have occurred

if the participants had bought their lunch at

local fast-food stores on the beach.

The bathers were assigned to one of four

roped-off bathing areas of approximately 10 m

width and 20 m length that were distributed

across the beach, with each divided into a nonswimmers’ zone with shallow water and a

swimmers’ zone. The nonbathers were directed

to a roped-off area of the beach with lawn or

sand grounds where they could not come into

contact with the water.

When the participants arrived at their destination—either the nonbathers’ area or one of

the four bathing areas—they were interviewed

again by one of the project helpers. In this second interview, the questions focused on symptoms having occurred after the first interview

and on nutritional details of the preceding 2 or

3 days. Participants who reported any new

symptoms were sent to one of the physicians

on site, who decided whether these symptoms

were a reason for exclusion. After the interview,

the participants were free to have their lunch,

and nonbathers were free to stay in the nonbathers’ area for normal beach activities such as

sunbathing or playing, depending on the

weather conditions.

Participants who had been randomized for

bathing entered the water under the individual

supervision of their interviewers. Bathing duration was limited to exactly 10 min, and participants were asked to stay inside the roped-off

swimming zones and to stay or swim around

balloons that had been placed like buoys in the

center of the zones. They were also instructed

to completely immerse their heads at least three

times during the 10 min. The supervisors individually recorded the start and ending times of

the bathing period and the number of head

immersions performed in each of the 10-minperiods. After leaving the water, the participants were asked whether they had accidentally

swallowed water.

Microbiological analyses. For the entire

time the participants were being exposed, water

samples were collected every 20 min from the

centers of the swimmers’ and nonswimmers’

zones in all four areas. These samples were analyzed in a nearby mobile laboratory for six

microbiological parameters: Escherichia coli

[International Standardization Organization

(ISO) 1998b], intestinal enterococci (ISO

1998a), Clostridium perfringens (Council of the

European Union 1998), somatic coliphages

(ISO 2000), aeromonads (Schulze 1996), and

pyocyanine-positive Pseudomonas aeruginosa

(Deutches Institut für Normung, European

Normalization 2002). The values of the

method-specific lower detection limits were

assigned to all samples with analytical results

below the detection limit, and the results were

censored. Upper detection limits were not

reached in any of the samples. Method details

are given in Supplemental material Annex 2

(http://ehp.niehs.nih.gov/docs/2005/8115/

supplemental.pdf).

Microbiological quality control. Quality

control procedures included positive and negative media controls for all target organisms and

temperature control of all incubators with

continuously operating digital temperature displays and additional electronic devices recording minimum and maximum temperatures

throughout the incubation period. As a quality

control procedure for E. coli and intestinal

enterococci, external quantitative reference

materials were applied that had been evaluated

in earlier international round-robin trials

(“reference lenticules K,” donated by Institut

Pasteur de Lille, Lille, France; European

Community contract SMT4-CT95-1603/

DG12-RSMT; Contreras-Coll et al. 2002).

PhiX-174 coliphages were used as positive controls in the somatic coliphage assay.

Follow-up. One week after exposure, all

the participants were interviewed again in person and underwent a medical inspection of the

throat, eyes, and ears. Interviewers and doctors

were unaware of the exposure status of the

participants. Three weeks after exposure, the

participants received a last questionnaire by

mail. After receipt of this fourth questionnaire,

each participant received a compensation of

25 Euros to cover personal expenses.

Data entry, verification, and analysis.

All questionnaire data were entered into an

electronic database created with Epi Info (version 6.2; Centers for Disease Control and

Prevention, Atlanta, GA, USA) and verified

by an independent second entry. Statistical

analyses were performed using the JMP (version 5.0; SAS Institute Inc., Cary, NC, USA),

STATCALC in Epi Info, and MS Excel 97

(Microsoft Corporation, Redmond, WA,

USA).

Calculation of individual exposure concentrations. For each minute of trial duration

(between 220 and 240 min, depending on the

number of participants per site), microbial

concentrations in the water were calculated by

arithmetic interpolation between the results

obtained by analyzing the water samples. This

was done for all bathing areas and in both the

Health risks from freshwater bathing

Environmental Health Perspectives • VOLUME 114 | NUMBER 2 | February 2006 229
swimmers’ and the nonswimmers’ zones. The

microbial concentrations of each of the 10 min

of water contact were individually assigned to

each of the bathers in their individual exposure

area. The values from the nonswimmers’ or the

swimmers’ zones were used, depending on

where the participant was staying. Finally, individual exposure concentrations for all microbiological parameters were assigned to each

bather by calculation of the arithmetic mean

concentration of the 10 individual exposure

minutes. Participants with unacceptable exposure data were excluded from further analysis.

Exposure data were considered to be unacceptable if the participants did not comply with

their randomization status, if they entered the

water too early or too late, or if the total exposure time was less or more than 10 min.

Exposure definitions. We defined exposure in two different ways. The first definition

was “10 min bathing with at least three head

immersions,” which is equivalent to the

instructions that the participants had received

from the study organizers. For this definition

the arithmetic mean of the 10 concentrations

that had been calculated and assigned as

described above was used as the mean exposure concentration of every individual bather.

We used the second definition of exposure

to assess the influence of the number of head

immersions. It takes into account the fact that

the participants followed the instructions to

immerse their heads at least three times to a

varying extent. Because each immersion of the

head could be looked on as an equivalent for

the uptake of a certain small amount of water

via the eyes and the lacrimal duct, the nose

and throat, and the mouth, the minutespecific concentrations of all minutes during

which the head was immersed were multiplied

with the number of head immersions during

each of these minutes and added. The result

can be considered to be the theoretical equivalent of a single head immersion at that concentration. This second theoretical definition

of exposure was therefore called “single head

immersion.”

Calculation of incidence rates, RRs, and

attributable risks. We calculated incidence

rates as the number of cases with onset of disease in the week or the 3 weeks after exposure

divided by the number of individuals observed

during these periods in a defined exposure category [nonbathers, bathers, bathers exposed

below or above no-observed-adverse-effect levels (NOAELs), bathers exposed in quartile or

quintile categories of microbial concentrations], and used percentage as the unit of measurement. We calculated RRs as the incidence

rates among participants in a defined exposure

category (bathers or bathers exposed above

NOAELs) divided by the incidence rates

among unexposed participants (nonbathers).

We calculated attributable risks (aRs) as the

difference between the incidence rates (percent) observed in two different exposure categories (e.g., incidence rates of bathers above

NOAELs minus the incidence rates of nonbathers). Initially, we compared the crude incidence rates of disease among bathers and

nonbathers, the crude RRs, and the crude aRs

after the 1-week evaluation interval with those

after the 3-week interval. After it became obvious from this comparison that there was no

additional scientific benefit from using the

3-week interval, all subsequent analyses concentrated on the results obtained within a

period of 1 week.

Determination of NOAELs. To determine

potential NOAELs, we sorted participants by

their individual exposure concentration in

ascending order. We then performed a Pearson’s

chi-square test for every actually occurring

concentration to compare the incidence rates

among bathers exposed below and above this

concentration, and the resulting p-value was

recorded. The most probable estimate for a

potential NOAEL was considered to be the

concentration that revealed the lowest p-value,

provided that the following conditions were

met: a) the incidence rate among bathers

above this concentration was significantly

higher than the incidence rate among bathers

below this concentration (p < 0.05); b) the

incidence rate of bathers below this concentration was not significantly lower than the incidence rate of nonbathers (exclusion of a

coincidental imbalance in the distribution of

cases); and c) no expected cell value was less

than five (exclusion of unreliable test results).

The reliability of this procedure was tested

with sets of simulated data with defined dose–

response relationships.

Control for bias. We tested potential

NOAELs against a variety of possible confounding variables in the total cohort, including

age, sex, study location, weather conditions,

accidental swallowing of water during the trial,

previous or additional water contact or waterrelated activities, previous diseases, diseases in

household members, other household members participating in the study, consumption of

prescription drugs, various nutritional factors,

consumption of alcohol and tobacco, travel

history, socioeconomic status, leisure activities,

risk perception, membership in environmental

organizations, and background information on

recreational water monitoring. Initially, all

variables were univariately screened for possible effects in the total final cohort of bathers

and nonbathers (Pearson’s chi-square tests).

Variables showing significant univariate effects

(p < 0.05) were considered to be potential

confounders of the NOAELs and were further

analyzed using a multiple logistic regression

procedure (effect likelihood ratio test) in

which the disease was modeled as the response

variable, and the potential NOAEL and the

potential confounder as model effects. If

NOAELs remained significant effects in these

tests (p < 0.05), the NOAELs were considered

to be unbiased, and the potential confounder

was considered to be an independent predictor

of disease. In addition, all models were analyzed for possible interaction effects between

NOAELs and potential confounding variables

by crossing both effects in separate effect likelihood ratio tests. Significant interaction

effects (p < 0.05) were recorded but NOAELs

were not rejected. We testesd all potential

NOAELs and the possible confounding variables one by one to exclude effects caused by a

possible collinearity of the indicator organisms. Stratified analysis as a possible alternative

method to test for confounding was considered inappropriate because of the limited

number of cases and the loss of test power that

would have been associated with stratification.

In addition, we compared potential

NOAELs with the results of classifying bathers

into quartile and quintile categories of exposure concentrations. Potential NOAELs were

considered acceptable and unbiased estimates

for a true NOAEL if they remained significant

model effects (p < 0.05) in all effect likelihood

ratio tests and if none of the incidence rates in

quartile or quintile categories with upper

range limits below the potential NOAEL was

significantly higher than the incidence rate of

the nonbathers.

We evaluated the influence of the accidental swallowing of water and the plausibility of

the NOAELs by categorical analysis of the

incidence rates of disease in bathers exposed

below and above NOAELs.

Disease definitions. We analyzed data for

the following diseases as outcome variables:

acute febrile respiratory infections, common

cold, ear inflammation, eye inflammation,

skin infections or cutireactions, urinary tract

infections, and finally three more or less stringent definitions of gastroenteritis: a) definition

“GE_UK” according to Kay et al. (1994),

b) definition “GE_UK-wf” according to the

present study, and c) definition “GE_NL-2”

according to van Asperen et al. (1998).

We defined a case of gastroenteritis by

Boolean combination of symptom variables

from the questionnaire as follows (Boolean

operators in capital letters): GE_UK, (diarrhea

AND three or more bowel movements per

day) OR vomiting OR (nausea AND fever)

OR (indigestion AND fever) [Note: there

were no occurrences of (nausea AND fever) or

(indigestion AND fever)]; GE_UK-wf, that is,

GE_UK without consideration of stool frequency: diarrhea OR vomiting OR (nausea

AND fever) OR (indigestion AND fever)

[Note: there were no occurrences of (nausea

AND fever) or (indigestion AND fever)];

GE_NL-2, diarrhea OR nausea OR vomiting

OR stomach pains.

Wiedenmann et al.

230 VOLUME 114 | NUMBER 2 | February 2006 • Environmental Health Perspectives
Results

Participant flow. The participant flow

through each stage of the trial is displayed in

Figure 1 in CONSORT format (Moher et al.

2001). The follow-up rate was 91.9%; that is,

2,018 of initially 2,196 participants returned

the last questionnaire.

Cohort characteristics. The age distribution among bathers and nonbathers in the

final cohort of 1,981 participants was almost

identical. Minimum, 25th percentile, median,

75th percentile, and maximum in the group

of bathers were 4, 14, 23, 39, and 79 years

versus 4, 15, 25, 39, and 89 years in the

group of nonbathers. The ratios of male to

female participants in the group of bathers

were 50.8:49.2% and 46.5:53.5% in the

group of nonbathers; 1.7% of the participants

were preschool children, 33.4% were schoolchildren, 11.9% were students, 35.9% were

employed, 7.6% were homemakers or retired,

5.2% were unemployed, and 4.3% did not

disclose their employment status; 7.1% were

members of an environmental organization.

Exposure intensity. Only 9% of the participants immersed their heads less often than

required, whereas 53% voluntarily did it more

often (median, 4; mean, 5.5; range, 0–87).

High numbers of head immersions were

recorded for some well-trained swimmers who

immersed their head with almost every swimming movement.

Disease-specific cohorts. Disease-specific

cohorts were formed by exclusion of participants who reported disease-specific precursor

symptoms at the first or second interview or

whose disease status was undefined. Diseasespecific precursor symptoms used to evaluate

the risk of acquiring gastroenteritis were considered to be any of the following symptoms:

fever, loss of appetite, nausea, vomiting, stomach pains or cramps, indigestion, loose bowel

movements, or diarrhea. For the evaluation of

other disease risks, specific precursor symptoms

were chosen accordingly. The disease status

was considered to be undefined if any of the

symptom variables that were required to define

a case was missing or answered by “not sure.”

For the analysis of gastroenteritis, 188 participants were excluded because of typical precursor symptoms. In addition, the following

numbers of participants were excluded because

of their undefined disease status, depending on

the definition of gastroenteritis: 36 (GE_UK),

25 (GE_UK-wf), or 26 (GE_NL-2).

Parameter-specific cohorts. From the disease-specific cohorts, parameter-specific

cohorts were formed by exclusion of data sets

with missing microbiological data for each of

the microbiological parameters. For the analysis of gastroenteritis, the following number of

data sets had to be excluded depending on the

kind of fecal indicator organism: none (C. perfringens), 9 (E. coli and intestinal enterococci),

or 12 (somatic coliphages). Thus, the parameter-specific cohort sizes for the analysis of gastroenteritis ranged between 1,745 and 1,768.

Microbial concentrations in the water. The

total number of available sample results per

parameter, the median concentrations, and the

correlation coefficients between the microbiological parameters are listed in Supplemental

material Annexes 2 and 3 (http://ehp.niehs.nih.

gov/docs/2005/8115/supplemental.pdf). The

concentration ranges for the fecal indicators

were as follows: E. coli, 4.7–5,344/100 mL;

intestinal enterococci, 3.0–1,504/100 mL;

C. perfringens, 9–260/100 mL; somatic coliphages, 10–3,780/100 mL. The lower range

limits are equivalent to the lower detection limits. Upper detection limits were not reached in

any of the samples.

Microbiological quality control. The results

obtained from the quantitative quality control

tests for E. coli and intestinal enterococci using

“reference lenticules K” were well within the

range of results that could be calculated in a

round-robin trial performed by nine European

expert labs as described above. The means and

the standard deviations of the log10-transformed

results obtained during the study trials were

2.76 ± 0.08 E. coli/100 mL and 2.47 ± 0.09

Health risks from freshwater bathing

Environmental Health Perspectives • VOLUME 114 | NUMBER 2 | February 2006 231

Enrollment

Medical exclusions

Dropout

17

112

1st interview:

2–3 days before exposure,

n = 2,196 participants

Allocation

Medical exclusions

Dropout

15

6

Follow-up

Dropout 13

Analysis

Unacceptable 37

exposure dataa

Final cohort

2nd interview:

Exposure day,

2,067 participants

Block randomizationa

1,033 bathers 1,034 nonbathers

1,012 1,022

3rd interview:

1 week after exposure,

2,034 participants

999 1,019

962 1,019

1,981 participants

213

7

5

3

0

Postal questionnaire:

3 weeks after exposure,

2,018 participants

(follow-up rate: 91.9%)

Disease-specific cohort

Parameter-specific cohortd

1,759 participants

9

Disease-specific precursor

symptoms at 1st or 2nd

interview or undefined

disease statusb

Missing microbiological

data (incomplete analysis

analytical failures)c

Eligible:a

> 18 years (location 1)

> 4 years (location 2–5)

No medical contraindications

Figure 1. Participant flow through each stage of the trial in CONSORT format (Moher et al. 2001) for the

analysis of gastroenteritis (definition GE_UK-wf) and the concentration of E. coli and intestinal enterococci. aDetails are explained in “Materials and Methods.” bNumber depends on the disease definition. cNumber depends on the microbiological parameter. dNumber depends on the disease definition and the

microbiological parameter.
intestinal enterococci/100 mL (n = 38) compared with 2.77 ± 0.18 E. coli/100 mL and 2.49

± 0.30 intestinal enterococci/100 mL (n = 43)

obtained in the round-robin trial.

Compliance of the study sites with current

European Union standards. In 95.2% of the

samples (401 of 421) collected during the

5 trial days, E. coli concentrations were below

the imperative value of 2,000/100 mL. Thus,

in the time periods during which the trials

were performed, the total water quality of all

five study locations would have just passed

the current European standard (95% of sample results < 2,000/100 mL).

Crude incidence rates and RRs of bathers

versus nonbathers. Significant or borderline

significant differences between the crude incidence rates of bathers versus nonbathers could

be observed only for gastroenteritis 1 week

after exposure and for skin ailments. The incidence rates of gastroenteritis in the nonbathers

group (“baseline risks”) corresponded well with

the three different definitions of gastroenteritis,

with the most stringent definition (GE_UK)

revealing the lowest rate of 1.4% [95% confidence interval (CI), 0.8–2.4] and the less stringent definitions (GE_UK-wf and GE_NL-2)

revealing higher rates of 2.8% (95% CI,

1.9–4.1) and 5.2% (95% CI, 3.9–6.9). The

same effect could be observed in the group of

bathers. Crude aRs of gastroenteritis slightly

decreased after 3 weeks, indicating that virtually all episodes of bathing-associated gastroenteritis occurred within an incubation period of

1 week and that symptoms were less likely to

be remembered for a time period of 3 weeks

than for a period of 1 week. Further data evaluation therefore focused on the results

obtained after 1 week. Detailed figures are displayed in Supplemental material Annex 4

(http://ehp.niehs.nih.gov/docs/2005/8115/

supplemental.pdf).

NOAELs, incidence rates, RRs, and aRs.

NOAEL estimates that were unbiased by any

of the potential confounding variables could be

determined for all three definitions of gastroenteritis and all four fecal indicator organisms,

whereas the concentrations of aeromonads and

Pseudomonas aeruginosa were not associated

with gastroenteritis in any case. Detailed

descriptions of the 160 tested variables are

given in Supplemental material Annex 5

(http://ehp.niehs.nih.gov/docs/2005/8115/

supplemental.pdf). Details of the results of

the effect likelihood ratio tests are recorded

in Supplemental material, Annexes 6 and 7

(http://ehp.niehs.nih.gov/docs/2005/8115/

supplemental.pdf). NOAELs for nonenteric

diseases were either nonexistent or nondetectable with the available cohort size or within

the range of microbial concentrations encountered during the present study; in the case of

the common cold, they were confounded by

other risk factors. A possible partial association

between the concentration of aeromonads and

skin ailments (cutireactions) was detectable,

but it was overlaid by an unknown independent water-related effect; that is, there must

have been other causes for cutireactions among

bathers that were not associated with the

microorganisms monitored in the present

study. Because all NOAELs were above the

lower detection limits of the microbiological

methods, there was no need for application of

special techniques to account for censored data

below the detection limits.

The incidence rates, RRs, and aRs for gastroenteritis below and above the potential

NOAELs, the incidence rates and aRs depending on the accidental swallowing of water, and

the corresponding raw numbers are listed

in detail in Supplemental material Annex 8

(http://ehp.niehs.nih.gov/docs/2005/8115/

supplemental.pdf). Table 1 summarizes

NOAELs, RRs, and aRs for all combinations of

exposure definitions, definitions of gastroenteritis, and fecal indicator organisms. For exposure

definition 1 (“10 min bathing with at least

three head immersions”), NOAELs of the most

widely used fecal indicators E. coli and intestinal

enterococci, for example, ranged between 78

and 180 E. coli/100 mL and between 21 and

24 intestinal enterococci/100 mL. RRs for these

two indicators ranged from 1.9 (95% CI,

1.3–2.8) to 3.5 (1.8–7.0), and aRs for bathing

above NOAELs ranged from 3.1 to 5.0%

depending on the definition of gastroenteritis.

Swallowing water compared with not swallowing water resulted in significantly higher aRs

above NOAELs than below NOAELs [arithmetic mean aRs: 3.6% vs. 1.3%; p < 0.0001,

analysis of variance (ANOVA)]. In addition,

swallowing water below NOAELs never

resulted in any significant effect compared with

nonbathing, whereas swallowing water above

Wiedenmann et al.

232 VOLUME 114 | NUMBER 2 | February 2006 • Environmental Health Perspectives

Table 1. RRs and aRs of bathing above NOAELs versus nonbathing, and aRs of swallowing water versus not swallowing water below and above NOAELs by exposure

definition, fecal indicator, and definition of gastroenteritis 1 week after exposure.

Exposure Definition NOAEL RR, aR (%), aR (%), aR (%),

definitiona Fecal indicator of GE (MO/100 mL) bathing > NOAEL 95% CI bathing > NOAEL SW ≤ NOAEL SW > NOAEL

1 E. coli UK 180 3.55 1.79–7.02 3.6 1.3 3.9

UK-wf 78 2.51 1.55–4.05 4.3 1.6 2.9

NL-2 167 1.96 1.32–2.89 5.0 1.0 3.3

Intestinal enterococci UK 24 3.2 1.64–6.27 3.1 1.3 3.7

UK-wf 21 2.67 1.65–4.32 4.7 0.7 3.4

NL-2 24 1.9 1.30–2.77 4.7 2.8 2.3

C. perfringens UK 13 3.34 1.72–6.51 3.3 0.4 4.1

UK-wf 13 2.61 1.60–4.25 4.5 0.9 3.3

NL-2 13 1.86 1.27–2.73 4.5 0.8 3.5

Somatic coliphages UK 150b 4.61 2.1–10.11 5.1 1.3 6.7

UK-wf 10 2.47 1.51–4.04 4.2 1.6 2.8

NL-2 10 1.77 1.21–2.60 4.0 2.1 2.1

2 E. coli UK 1,453 4.41 2.17–8.96 5.2 1.6 5.3

UK-wf —c — — — ——

NL-2 2,163 2.33 1.48–3.67 8.6 0.5 8.6

Intestinal enterococci UK 123 3.41 1.74–6.68 3.7 1.1 4.0

UK-wf 123 2.78 1.71–4.51 5.7 1.9 2.8

NL-2 145 1.93 1.31–2.84 6.0 3.8 1.6

C. perfringens UK 38 3.12 1.62–6.03 3.2 –0.4 4.1

UK-wf 38 2.43 1.50–3.93 4.6 0.8 3.3

NL-2 36 1.71 1.18–2.47 4.6 0.0 3.2

Somatic coliphages UK 330 3.77 1.87–7.61 4.2 1.4 3.2

UK-wf 50 2.44 1.49–3.98 4.6 1.7 2.7

NL-2 119 1.76 1.18–2.62 4.9 0.7 3.1

Abbreviations: CI, confidence interval; GE, gastroenteritis; MO, microorganisms; SW, swallowing water.

a Exposure definitions 1 and 2 as explained in “Materials and Methods.” bValue is probably too high because of a second local minimum of the Pearson’s chi-squared p-values. cPotential NOAEL estimate does not fulfill all criteria of validity (incidence rate of bathers below NOAEL is significantly lower than incidence rate of nonbathers).
NOAELs always revealed significant effects (all

p-values < 0.003), thus providing evidence for

true dose–response relationships.

The results from classifying bathers into

quartiles and quintiles of the exposure concentrations generally corresponded well with the

determined NOAELs. The incidence rates of

gastroenteritis in the first quartile and in the

first quintile were below the upper limit of the

95% CI of the incidence rate of nonbathers for

all 24 combinations of exposure definition,

definition of gastroenteritis, and fecal indicator

organism. Pearson’s chi-square tests did not

reveal a significant difference between the incidence rates of nonbathers and the incidence

rates in the first quartile or quintile in any of

the 24 combinations. On the other hand, all

incidence rates within the fourth quartile and,

with only one exception, all incidence rates

within the fifth quintile were within the 95%

confidence intervals of the incidence rates of

bathers above NOAELs. For most quartiles

and quintiles with concentration ranges above

NOAELs, Pearson’s chi-square tests or Fisher’s

exact tests revealed significant differences

between the incidence rates within the quantiles and the incidence rates among nonbathers. Only the presumptive NOAEL of 150

somatic coliphages/100 mL for GE_UK and

exposure definition 1 was obviously too high

and seems to mark the level where the concentration–response curve reaches its maximum

rather than the true NOAEL. A complete list

with incidence rates of gastroenteritis in quartile and quintile categories of fecal indicator

concentrations for the three different definitions of gastroenteritis and the two exposure

definitions is given in Supplemental material

Annexes 9 and 10 (http://ehp.niehs.nih.gov/

docs/2005/8115/supplemental.pdf). An example with incidence rates in quartile and quintile

categories of microbial exposure concentrations

for GE_UK-wf is given in Table 2. The charts

in Figure 2 demonstrate a comparison of the

incidence rates of gastroenteritis in quartile and

quintile categories of E. coli concentrations and

the incidence rates below and above the

NOAEL depending on the accidental swallowing of water for GE_UK-wf.

Influence factors on NOAELs, RRs, and

aRs. The NOAELs depended mainly on the

definition of exposure, with significantly

higher values for exposure definition “single

head immersion” (p = 0.009, ANOVA for

log10-transformed data). They also depended

on the microbiological parameter (p = 0.006).

They did not, however, depend on the definition of gastroenteritis (p = 0.32).

Like the incidence rates of nonbathers and

the incidence rates of bathers exposed below

NOAELs, the incidence rates of bathers above

NOAELs mainly depended on the definition

of gastroenteritis (p < 0.001). There was no significant difference between the two exposure

definitions (p = 0.90) and between the four

fecal indicator parameters (p = 0.88).

The mean risk attributable to swallowing

water above NOAELs was significantly higher

than that attributable to swallowing water

below NOAELs [3.6 (range, 1.6–8.6) vs. 1.3

(range, –0.4 to 3.8); p < 0.001, ANOVA].

Severity of bathing-associated gastroenteritis. The RRs were higher for the more stringent

definitions of gastroenteritis. Mean RRs of

bathers above NOAELs versus nonbathers were

3.7 (range, 3.12–4.61) for GE_UK, 2.6 (range,

2.43–2.78) for GE_UK-wf, and 1.9 (range,

1.71–2.33) for GE_NL-2. Mean aRs above

NOAELs were 3.8% (range, 3.1–5.2), 4.4%

(range, 4.2–5.7), and 4.7% (range, 4.0–8.6),

respectively. This demonstrates that most gastroenteritis cases attributable to bathing were

cases meeting the most stringent definition of

gastroenteritis; that is, they were not simply

mild forms of gastroenteritis. Two of the

26 nonbathers (7.7%; 95% CI, 0.9–25.1) who

developed gastroenteritis (GE_UK-wf) within

1 week after the trial day consulted a doctor,

versus 3 of the 40 bathers (7.6%; 95% CI,

1.6–20.4) who were exposed above NOAEL.

Thus, there was no indication that waterborne

gastroenteritis was less severe than gastroenteritis acquired through other sources of infection.

Discussion and Conclusions

The rationale behind the use of indicator

organisms since their introduction into water

hygiene more than 100 years ago was simply

the empirical observation that below certain

concentrations—below certain extents of fecal

pollution—the disease risk from certain fecal–

orally transmittable pathogens was negligible.

It has never been assumed that certain concentrations of indicator organisms above these levels are inevitably associated with certain

predictable risks. Fecal indicator organisms

such as E. coli and intestinal enterococci are

part of the normal bacterial flora of the guts of

all warm-blooded animals, including human

beings. With only rare exceptions (e.g., enterohemorrhagic E. coli), they do not cause disease.

Health risks from freshwater bathing

Environmental Health Perspectives • VOLUME 114 | NUMBER 2 | February 2006 233

Table 2. Incidence rates of gastroenteritis (definition GE_UK-wf) in quartile and quintile categories of microbial exposure concentrations for exposure definition 1

(“10 min bathing with at least three head immersions”) 1 week after exposure.

Quartile UL Quintile UL

Parameter MO/100 mL Cases No. IR (%) p-Valuea MO/100 mL Cases No. IR (%) p-Value

Unexposed 26 921 2.8 26 921 2.8

E. coli 72 4 207 1.9 0.47 61 3 166 1.8 0.61

181 11 212 5.2 0.08 116 6 168 3.6 0.62

379 14 211 6.6 0.007** 245 10 170 5.9 0.04*

4,600 17 208 8.2 < 0.001# 445 12 166 7.2 0.004**

4,600 15 168 8.9 < 0.001#

Intestinal enterococci 14 5 208 2.4 0.74 12 3 167 1.8 0.61

53 9 212 4.2 0.28 27 5 167 3.0 0.80

101 14 210 6.7 0.007** 68 15 169 8.9 < 0.001#

1,190 18 208 8.7 < 0.001# 114 7 168 4.2 0.35

1,190 16 167 9.6 < 0.001#

C. perfringens 9 6 224 2.7 0.91 9 6 224 2.7 0.91

18 9 202 4.5 0.23 13 4 115 3.5 0.57

33 18 211 8.5 < 0.001# 22 12 172 7.0 0.006**

148 13 210 6.2 0.02* 36 14 166 8.4 < 0.001#

148 10 170 5.9 0.04*

Somatic coliphages 10 7 302 2.3 0.64 10 7 302 2.3 0.64

35 9 115 7.8 0.01* 11 3 31 9.7 0.06

142 12 212 5.7 0.04* 85 11 167 6.6 0.01*

3,598 16 206 7.8 < 0.001# 153 10 171 5.8 0.04*

3,598 13 164 7.9 0.001**

Abbreviations: IR, incidence rate; MO, microorganisms; UL, upper range limit.

aThe p-value was calculated by Pearson’s chi-square test or Fisher’s exact test that compared the number of cases among the bathers who were exposed in one of the given categories

of fecal indicator concentrations with the number of cases among the unexposed participants (nonbathers). Fisher’s exact test results were used when an expected cell value was < 5.

*p < 0.05; **p < 0.01; #

p < 0.001.
In food matrices where they can multiply,

microorganisms of fecal origin may be of only

little value as indicators of health risks.

According to federal law in Switzerland, cheese

may contain up to 10,000 E. coli per gram

(Eidgenössisches Volkswirtschaftsdepartement

1999). In recreational waters, fecal indicator

organisms can be associated with varying

amounts and changing kinds of pathogens,

depending on the spread of infection at the

source of the fecal pollution, and the susceptibility of exposed cohorts is highly variable and

hardly predictable. Therefore, the assumption

that the risk of acquiring gastrointestinal illness from exposure to certain concentrations

of fecal indicator organisms is predictable, as

implied by recent publications (Havelaar et al.

2001; Kay et al. 1994, 2004), may be true

only for a defined population under the condition of a constant degree of endemicity of

always the same kinds of pathogens. It would

not, however, be a sufficient basis for the

derivation of generally applicable microbiological standards for fecal indicator organisms

in water. According to Prüss (1998), maximum detectable aRs of gastroenteritis from

exposure to recreational water ranged between

0.4 and 27.7% in studies involving freshwater

exposure, and from 0.5 to 19.5% in studies

involving seawater exposure. A comparison of

the results from randomized exposure to fresh

water (present results) with the results from

randomized exposure to seawater (Kay et al.

1994) confirms that different aRs (4.5% vs.

19.5%) can occur in two different cohorts

(Germany vs. United Kingdom) above almost

identical NOAELs [24 intestinal enterococci/100 mL in the present study vs. 32 fecal

streptococci/100 mL reported by Kay et al.

(1994)] even if the type and intensity of exposure are nearly the same (10 min bathing, three

or more head immersions). If the pathogen:

indicator ratios in the German and U.K. studies had been completely different, this would

most likely have caused larger differences

between the NOAELs. The most probable

explanation for the differences in the baseline

risks (nonbathers) and the aRs (bathers above

NOAELs) observed in the German and the

U.K. trials are differences in the cohort susceptibilities (Dizer et al. 2005).

If the determination of NOAELs is based

on observable differences between groups—

nonbathers, bathers below NOAEL, and

bathers above NOAEL—then NOAEL is also

a function of statistical significance, which is a

function of sample size and of the susceptibility

status of the population. A possible explanation

for the slightly lower NOAELs in the German

trial may therefore be the relatively larger

cohort size (1,748 vs. 1,216) and the relatively

lower background rate of disease in the group

of nonbathers (4.8% vs. 9.7%; GE_UK, each

within 3 weeks after exposure) and in the

group of bathers below NOAEL (4.4% vs.

10.9% among bathers exposed at concentrations of up to 20 intestinal enterococci or fecal

streptococci per 100 mL). On the other hand,

the lower susceptibility of the German cohort

may have partially compensated this effect.

If recreational water standards are based on

predefined maximum “acceptable” disease risks

for bathers (or estimates of the “disease burden” from bathing) as identified in a certain

epidemiologic study, the resulting values for

“acceptable” concentrations of fecal indicator

organisms mainly depend on three variables:

a) the NOAEL detected in that study, b) the

susceptibility of the cohort in that study, and

c) the definition of the disease. NOAELs

mainly depend on the pathogen:indicator ratio

(the higher the ratio the lower the NOAEL)

and on the intensity of exposure (the more

intense the exposure the lower the NOAEL).

The measurable susceptibility of a cohort

depends on the degree of immunity—the percentage of individuals who can be infected—

and on the percentage of infected individuals

who develop symptoms, because only symptomatic disease will be reported. The definition

of gastroenteritis (more or less stringent) does

not affect the NOAEL; however, it significantly

affects the baseline level of gastroenteritis in

nonbathers and bathers below the NOAEL,

the RR, and the aR.

It has been proposed that imperative

(legally binding) values for fecal indicator concentrations in recreational water standards

be set at a level that limits the aR of acquiring

gastroenteritis to 5% and that guide (recommended) values should be set at a level that

limits the aR to 3% (Havelaar et al. 2001; Kay

et al. 2004). This concept has been applied in

the current revision of the European bathing

water directive [Commission of the European

Communities (CEC) 2002] using both the

NOAEL and the aR depending on the fecal

indicator concentration above the NOAEL as

determined in the Kay et al. (1994) studies at

British seawater bathing sites. These studies

revealed an overall aR of approximately 9.5%

above a concentration of 26 fecal streptococci/

100 mL. This is about three times more than

we have observed in the German trials above a

concentration of 24 intestinal enterocci/

100 mL using the same definition of gastroenteritis (GE_UK).

The reason why the concept of basing water

quality standards mainly on aRs is less robust

than basing them solely on NOAELs is demonstrated in Figure 3, which shows the effect of

different cohort susceptibilities (British vs.

German cohort) and different definitions of

gastroenteritis (GE_UK, GE_UK-wf, and

GE_NL-2) on the “acceptable” level of fecal

water pollution if standards are based on predefined “acceptable” aRs (5% or 3%) above an

identical NOAEL. The probability density

function (PDF) in Figure 3 was calculated

using a geometric mean value (μ) of 100 intestinal enterococci/100 mL, which is the current

European guide value for 90% of the sample

results (Council of the European Communities

1976) and also the proposed value for the 95th

percentile in the proposal for a revised guideline

(CEC 2002). The standard deviation was chosen according to similar calculations that have

been used in recommendations of the WHO

Wiedenmann et al.

234 VOLUME 114 | NUMBER 2 | February 2006 • Environmental Health Perspectives

Figure 2. Incidence rates of gastroenteritis (definition GE_UK-wf) within 1 week after exposure to fresh recreational water in quartile and quintile categories of

E. coli concentrations (A) and incidence rates below and above the presumed NOAEL of 78 E. coli /100 ml (B), depending on the accidental swallowing of water.

*p < 0.05 in a chi-square test or Fisher’s exact test comparing the incidence rate of gastroenteritis in one of the specified exposure categories with the incidence rate of gastroenteritis

in the group of unexposed participants (nonbathers). **p < 0.001.

10

9

8

7

6

5

4

3

2

1

0

1 10 100 1,000

E. coli/100mL

Incidence rate of gastroenteritis (%)

A B

10,000

10

9

8

7

6

5

4

3

2

1

0

Unexposed < NOAEL

– W

< NOAEL

+ W

> NOAEL

– W

> NOAEL

+ W

*

*

* *

*

Incidence rate of gastroenteritis (%)

2.8%

1.3%

2.9%

5.7%

*

8.6%**

– W = participants did not swallow water

+ W = participants swallowed water

Nonbathers and bathers above NOAEL

Bathers: quartile ranges of exposure

Bathers: quintile ranges of exposure

95% confidence limits

Quartile range means

Quintile range means

26/921 2/155 3/104 17/296 24/279
(Kay et al. 2004). The “disease burden” associated with exposure to recreational water with

fecal indicator concentrations that vary according to a PDF with a given μ and an SD of

100.75 (which can also be characterized by its

corresponding 95th percentile) was calculated

according to the following equation: disease

burden = [1 – p(cumulative PDF)] × (mean aR

above NOAEL). This formula represents a simplification of the WHO concept, which uses a

hypothetical square root function to describe a

successive increase in aR above the NOAEL

(32 fecal streptococci/100 mL) and a constant

risk above the upper range limit of experimental

data (158 fecal streptococci/100 mL) (Kay et al.

1994, 2004). The aRs (bathers’ excess risks)

that were used in the sample calculations in

Figure 3 were chosen on the basis of results

from studies with a randomized controlled

study design and an exposure intensity characterized by 10 min of bathing with three or

more head immersions: 9.5% is the aR that was

observed in the trials performed at British seawater bathing sites (Kay et al. 1994) in the

group of bathers exposed at fecal streptococcus

concentrations of 26/100 mL or higher (British

cohort); 3.35, 4.5, and 4.85% are the arithmetic means of the aRs associated with exposure above the presumptive NOAELs for

E. coli and intestinal enterococci that could be

observed at German freshwater bathing sites

(German cohort; present results) for three more

or less stringent definitions of gastroenteritis.

These calculations demonstrate that,

despite the clear effects that we have found, it

would be impossible to set imperative values

for German freshwater bathing sites based on

the results of the present study, and that guide

values for compliance at 95th percentile levels

of monitoring results for intestinal enterococci

would range from approximately 200 to

approximately 4,000/100 mL depending on

nothing else but different cohort susceptibilities in the German and the U.K. trials and on

different definitions of gastroenteritis. If the

concept of basing regulatory standards on a

maximal acceptable aR of 5% and 3% were

applied to studies other than those performed

by Kay et al. (1994), no imperative or guide

values would be necessary at all—for example,

for Egyptian beaches (Cabelli 1983) or Hong

Kong beaches (Cheung et al. 1990) or bathing

areas in any other part of the world where the

maximal aRs were < 5% or 3%.

Microbiological quality standards are

intended to protect the health of those consumers who are not already immune or for

some other reason resistant to the pathogens

from which they are to be protected. For example, they should provide a certain degree of protection not only for the local population under

endemic conditions but also for tourists who

may have a completely different susceptibility

status with respect to a typical pattern of pathogenic organisms to which local residents are

already adapted. In addition, standards should

also protect the consumers’ health in the situation of epidemics caused by newly introduced

pathogens, when even the local population may

be extremely susceptible. Therefore, the highly

variable cohort susceptibilities that can be

observed in epidemiologic studies investigating

the risk of acquiring gastroenteritis from recreational water contact should not play a determining role in the setting of standards. Instead,

the standards should be based solely on acceptable rates of compliance with guide values that

correspond to experimentally and empirically

verified NOAELs. In this case, the level of a

standard would depend on only one variable,

the NOAEL, instead of three: the NOAEL, the

cohort susceptibility, and the disease definition.

The advantages of choosing guide values in the

same order of magnitude of NOAELs are manifold: a) the levels of the guide values would be

easy to communicate to the public, because the

meaning of a NOAEL is easy to understand;

b) different definitions of gastroenteritis and

different cohort susceptibilities in certain epidemiologic studies would not automatically

result in different standards; and c) the results

of individual water samples could be directly

compared with the standard (i.e., values below

NOAEL vs. values above NOAEL or, e.g.,

10 times above NOAEL), whereas such a comparison is not informative if standards are

expressed as 95th percentiles of the distribution

of microbial concentrations as currently proposed by the European Commission (CEC

2002). We are fully aware that a NOAEL for

gastroenteritis is not a natural constant if it

is expressed as a concentration of indicator

organisms in the water. In an experimental

study design, the NOAEL is influenced by the

pathogen:indicator ratio, the virulence of the

pathogens, the amount of water that is ingested,

and, depending on the mathematical method

used to determine the NOAEL, possibly the

cohort size and the associated power of the

statistical analysis. Therefore, the intensity of

exposure in experimental studies should be

similar to an average bathing activity, and

NOAELs determined in experimental studies

such as the randomized trials in the United

Kingdom (Kay et al. 1994) and in Germany

(present results) should be compared with

empirical observations before they are used as a

basis for standards.

According to the results of the present

study and the considerations described above,

the following values are suggested as reasonable

estimates for NOAELs at an average bathing

intensity and as a practicable basis for setting

recreational water standards: 100 E. coli,

25 intestinal enterococci, 10 somatic coliphages, or 10 C. perfringens per 100 mL.

These values are well in accordance with

empirical observations for the concentration of

intestinal enterococci and E. coli as demonstrated by the meta-analysis performed by

Wade et al. (2003) for the U.S. Centers for

Disease Control and Prevention. They concluded that there was evidence that the risk of

gastrointestinal illness was considerably lower

in studies with indicator densities below the

Health risks from freshwater bathing

Environmental Health Perspectives • VOLUME 114 | NUMBER 2 | February 2006 235

0.5

0.4

0.3

0.2

0.1

0.0

Probablility density

A B

Intestinal enterococci/100 mL

NOAEL = 25 IE/100 mL

95th percentile = 1,713

PDF

of exposure to log-normal

distributed intestinal

enterococci concentrations

with μ = 102 and SD = 100.75

10.0

9.0

8.0

7.0

6.0

5.0

4.0

3.0

2.0

1.0

0.0

95th percentiles of lognormal distributions

of intestinal enterococci/100 mL

0 1 10 100 1,000 10,000 100,000 1 10 100 1,000 10,000 100,000 Disease burden (%)

5% risk level

3% risk level

“Acceptable”

levels of fecal

water pollution

aR above NOAEL:

9.5%

4.85%

4.5%

3.35%

Exposed below NOAEL

(not at risk)

Exposed above NOAEL

(at risk)

“Disease burden”:

symptomatic waterborne

infection among those

exposed above NOAEL

Figure 3. Calculation of the disease burden from exposure to log-normal distributed intestinal enterococci concentrations (A) and the influence of cohort susceptibility

and disease definition on the acceptable level of fecal water pollution if standards are based on predefined attributable risks (in this case, 3 and 5%) above an assumed

identical NOAEL of 25 intestinal enterococci/100 mL (B). Details on the calculation of the PDF, the disease burden, and the aRs are explained in “Discussion.”
Wiedenmann et al.

236 VOLUME 114 | NUMBER 2 | February 2006 • Environmental Health Perspectives

freshwater guidelines proposed by the U.S.

Environmental Protection Agency (EPA) for

both enterococci (33/100 mL) and E. coli

(126/100 mL). The results of the randomized

trials in German fresh waters are also in accordance with the ratio of roughly 1:4 between

the guideline values for enterococci and E. coli

recommended by the U.S. EPA and are in

clear contrast to the ratio of 1:2.5 that is

assumed by the European Commission in the

current draft directive (CEC 2002).

A novel and practicable monitoring system

based on compliance rates with NOAELs (calculation of “time-integrated quality scores”)

was recently proposed (Wiedenmann A and

Botzenhart K, unpublished data). Current

WHO recommendations and the concept

used to derive bathing water standards in the

European Union should be reconsidered in

light of the results and conclusions from this

randomized trial.

The results of the present study also

demonstrate that C. perfringens and somatic

coliphages may be alternative fecal indicators

that could be used to set standards for fresh

water just as well as E. coli and enterococci.

These results can be of interest in regions such

as those with tropical climates, where E. coli

and enterococci may be less reliable as indicator organisms, because there is some indication

that these organisms might multiply in such

environments (Fujioka and Byappanahalli

2003).

For the analysis of the trial results reported

in this article, we have applied the most conservative concentration–response model consisting of only a NOAEL, the incidence rate of

disease below the NOAEL, and the incidence

rate of disease above the NOAEL. The modeling of complex concentration–response functions to only few cases of disease may lead to

highly speculative results, especially when the

models are extrapolated (Kay et al. 2004). We

are, however, ready to test any of the various

possible modeling approaches (Kay et al. 2004;

Wymer and Dufour 2002) or to perform

meta-analyses in cooperation with other

research groups.

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and of the Council Concerning the Quality of Bathing Water.

Brussels:Commission of the European Communities.

Available: http://europa.eu.int/eur-lex/en/com/pdf/2002/

com2002_0581en01.pdf [accessed 1 August 2004].

Cheung WH, Chang KC, Hung RP, Kleevens JW. 1990. Health

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