The influence of sewage cleaning plants and rainfall on ...

Master thesis in Infection Biology and Epidemiology

The influence of sewage cleaning plants and rainfall on

the microbiological surface water quality

and

the backflow into groundwater

by Pascal Scheidegger

2007

Swiss Tropical Institute, Basel

Cantonal Laboratory Basel-Landschaft

Bureau for Environment and Energy, Basel-Landschaft

Supervision

Prof. Dr. phil. II Marcel Tanner (Director of the Swiss Tropical Institute)

&

Dr. phil. II Paul Svoboda (Department Head of the Division Microbiology, Cantonal Laboratory

Basel-Landschaft)

&

Dr. phil. II Adrian Auckenthaler (Bureau for Environment and Energy, Basel-Landschaft)

Table of contents__________________________________________________________________ 1

Table of contents

ACKNOWLEDGEMENTS ............................................................................................................................................3

ABBREVIATIONS .........................................................................................................................................................4

SUMMARY .....................................................................................................................................................................5

1. INTRODUCTION .......................................................................................................................................................7

1.1 BACTERIOLOGY ....................................................................................................................... 7

1.1.1 ENTEROBACTERIACEAE.......................................................................................................... 7

1.1.2 CAMPYLOBACTER................................................................................................................. 18

1.1.3 PSEUDOMONAS..................................................................................................................... 21

1.2. VIROLOGY ............................................................................................................................. 23

1.2.1 NOROVIRUS.......................................................................................................................... 23

1.2.2 ROTAVIRUS .......................................................................................................................... 24

1.3 PROTOZOOLOGY .................................................................................................................... 25

1.3.1 GARDIA LAMBLIA ................................................................................................................. 26

1.3.2 CRYPTOSPORIDIUM PARVUM................................................................................................ 27

1.4 M ICRO-ORGANISMS IN WATER : INITIAL POSITION ............................................................... 28

PART A: SURFACE WATER ........................................................................................................... 29

DRAINAGE OF SETTLEMENT AREAS................................................................................................ 30

SCP OPERATING MODE.................................................................................................................. 31

PART B: GROUNDWATER AND DRINKING WATER ....................................................................... 32

RAW WATER AND RAW WATER PROCESSING.................................................................................. 33

DRINKING WATER.......................................................................................................................... 34

WATER SUPPLY IN SWITZERLAND ................................................................................................. 35

PROBLEMS OF WATER SUPPLY....................................................................................................... 35

PROBLEMS WITH CONTROL SYSTEMS............................................................................................. 36

1.5 SIGNIFICANCE , AIMS , OBJECTIVES ........................................................................................ 37

2. MATERIAL AND METHODS ................................................................................................................................39

2.1 SAMPLING AND SAMPLING AREAS .......................................................................................... 39

2.1.1. PART A: SURFACE WATER................................................................................................... 39

2.1.2. PART B: GROUNDWATER..................................................................................................... 41

FURTHER PARAMETERS................................................................................................................. 42

2.2 ANALYTICAL PROCEDURES .................................................................................................... 42

Table of contents__________________________________________________________________ 2

2.2.1 BACTERIOLOGY.................................................................................................................... 42

2.2.2 VIROLOGY ............................................................................................................................ 46

3. RESULTS..................................................................................................................................................................48

3.1. PART A: SURFACE WATER .................................................................................................... 48

3.1.1. INDICATOR ORGANISMS....................................................................................................... 48

3.1.2. PATHOGENS......................................................................................................................... 52

3.1.3 WATER QUALITIES................................................................................................................ 62

3.2 PART B: GROUNDWATER ....................................................................................................... 66

4. DISCUSSION............................................................................................................................................................70

4.1. PART A: SURFACE WATER .................................................................................................... 70

4.1.1. INDICATOR ORGANISMS: ...................................................................................................... 70

4.1.2. PATHOGENS AND THE INFLUENCE OF SCPS ......................................................................... 73

4.1.3. WATER QUALITY IN THE RIVERS.......................................................................................... 78

4.2. PART B: GROUNDWATER ...................................................................................................... 80

4.3. SUMMARY OF THE MOST IMPORTANT POINTS ...................................................................... 82

5. PROSPECT...............................................................................................................................................................83

6. REFERENCES.......................................................................................................................................................... 84

APPENDIX ....................................................................................................................................................................88

APPENDIX I. DATA OF SURFACE WATER SAMPLES ...................................................................... 88

APPENDIX II. DATA OF GROUNDWATER SAMPLES ...................................................................... 98

APPENDIX III. SAMPLE SITE DIAGRAMS .................................................................................... 102

APPENDIX IV. SAMPLE EVENT DIAGRAMS ................................................................................ 104

APPENDIX V. TABLES ‚ INFLUENCE OF SCPS’ ........................................................................... 111

Acknowledgements________________________________________________________________ 3

Acknowledgements

The present thesis based on a framework of cooperation between the Swiss Tropical Institute (STI)

in Basel and the Cantonal Laboratory Basel-Landschaft in Liestal (KLBL). I want to thank Prof.

Dr. Marcel Tanner (Director of the STI) and Dr. Paul Svoboda who made this collaboration

possible and gave me the opportunity for doing this thesis. Moreover I thank the KLBL for

financial support of this MSc thesis.

I thank Prof. Dr. Marcel Tanner, my supervisor from the STI, for his support and advices in the

beginning of the study.

I thank Dr. Paul Svoboda, my supervisor from the KLBL, for the guidance and support through my

thesis, and for the creative discussions.

I thank Dr. Adrian Auckenthaler from the Bureau for Environment and Energy Basel-Landschaft

for his support concerning groundwater and for the creative discussions.

I want to thank the stuff of the laboratory for the great support and the excellent atmosphere. A

special thank goes to Jürg Grimbichler for introducing me to the lab techniques and for helping out

in difficult situations. I thank Elisabeth Thommen and Ursula Löffel for their support in the

laboratory.

A special thank goes to Anne Blomstein for proof-reading the MSc thesis and the critical remarks.

I thank Jan Hattendorf from the STI for helping me in statistical questions.

Finally I thank my family for their support and confidence during the whole time.

Abbreviations_____________________________________________________________________ 4

Abbreviations AIDS Acquired immunodeficiency syndrome

AMK Aerobic mesophyl germs

BAG Bundesamt für Gesundheit (Federal bureau for health)

E. coli Escherichia coli

EHEC Enterohaemorrhagic E.coli

GW Groundwater

HIV Human immunodeficiency virus

HUS Haemolytic uraemic syndrome

KLBL Cantonal laboratory Basel-Landschaft

PCR Polymerase chain reaction

SCP Sewage cleaning plant

SD Standard deviation

SLBL Schweizerisches Lebensmittelbuch

STI Swiss tropical institute

SW Surface water

UV Ultra-violet

VT1, VT2 Verocytotoxin 1 and 2

WHO World health organisation

WWCP Waste water cleaning plant

Summary________________________________________________________________________ 5

Summary According to the World Health Organization (WHO), gastrointestinal infectious diseases are

among the most important infectious and parasitic diseases worldwide. They follow directly on

respiratory infectious diseases and HIV/AIDS and cause greater mortality than malaria and

tuberculosis.

There are an increasing number of food-related and waterborne diseases during the summer.

Recreational activities pose a risk factor in acquiring such diseases, leading to infection with

waterborne micro-organisms.

SCPs are likely to have a particularly large effect on the microbiological surface water quality.

Following rainfall, environmental micro-organisms may be washed into surface waters on different

routes.

Surface water infiltrates into groundwater which is pumped up and processed for drinking water.

Problems with micro-organisms in raw water and drinking water have been increasing in the last

couple of decades, leading to small epidemics.

In this survey the influence of SCP’s and rainy events on the microbiological surface water quality

in bathing areas and the dilution downstream of the effluents was tested. Two SCP’s in the canton

Basel-Land were chosen, one at the Birs (SCP Zwingen) and one at the Ergolz (SCP Frenkendorf

2). Sample sites above the effluent were compared with effluents and sample sites downstream.

Samples were analysed for E. coli, Salmonella, EHEC, Shigella, Yersinia, Campylobacter,

Pseudomonas, Rotavirus and Norovirus.

If a river is contaminated microbiologically, micro-organisms can infiltrate into groundwater. This

water is pumped up and processed for drinking water. Several outbreaks due to contaminated wells

have been described in the last decade. To validate the infiltration of surface water into ground

water, samples of surface water and groundwater from well at the Birs were taken parallel and

tested for presence of E. coli.

In all tested surface water cases, the number of E. coli/100 ml never fell below 100. Salmonella,

EHEC and Pseudomonas were very abundant in all sample sites. The other pathogens were

detectable sporadically (viruses, Campylobacter) or were absent (Shigella, Yersinia).

The influence of an SCP on the microbiological river water quality and the quality downstream

depends on the river's discharge. The quality downstream can decrease, possibly due to a laminar

flow. Additional inflows (mixed-water overflows, feeding rivers) also seem to play a role.

Summary________________________________________________________________________ 6

During poor weather, contamination of rivers is higher than during good weather. However, the

river water quality is very dynamic and there are great differences for both good and poor weather.

The river water quality improved again two to three days after a rain event.

For Pseudomonas and EHEC, the influence of SCPs on river water quality is largely negative. The

same applies for Salmonella in the Ergolz. For all other pathogens, there was a negative influence

in 50% or less of cases.

The river water quality is very dynamic. Sites downstream can have different qualities within one

day, or the quality of one site can change within a few days, even when the weather conditions

remain stable.

The filtering of surface water performed by the unsaturated zone in the aquifer reduced the number

of E. coli/100 ml groundwater by about a factor of 1000 (log 3). By simple observation of surface

water (discharge, turbidity) one can estimate if E. coli are likely to be found in groundwater. For

each well influenced by river water, a critical value regarding discharge and turbidity should be

defined. This value gives the point from which E. coli are likely to be found in groundwater. If the

river exceeds this value, tests should be mandatory.

Introduction______________________________________________________________________ 7

1. Introduction

The following section provides insights into important properties of surface water, groundwater

and micro-organisms. Systematic, pathogenesis and epidemiological aspects together with

virulence factors of waterborne micro-organisms will be described. Problems related to the

presence of micro-organisms in surface water and groundwater and how the human environment

influences these natural cycles will be discussed.

1.1 Bacteriology

1.1.1 Enterobacteriaceae

The family of Enterobacteriaceae includes more than 100 species and is the most important

bacterial family for human medicine. Their natural environment is the gut of human beings and

animals. Some of them are harmless commensals, whereas others are slightly to strongly

pathogenic.

Enterobacteriaceae are the most important micro-organisms causing intestinal infections in

humans. Some species cause diseases with typical clinical symptoms such as diarrhoea, typhoid

fever, dysentery or pest. Other species are opportunists that cause nosocomial infections such as

urinary tract infection, pneumonia, wound infections and sepsis.

Enterobacteriaceae are Gram-negative, motile, aerobic and facultative anaerobic rods with rounded

ends. They are about 0.5–1.5 µm thick and 2–4 µm long and often peritrichously flagellated.

Typical and common species are Escherichia coli (including VTEC, EPEC, ETEC, EIEC, EaggEC

— see Table 1.1), Salmonella, Shigella, Yersinia, Proteus, Citrobacter, Klebsiella, Enterobacter,

Serratia, Providencia, Morganella.

Introduction______________________________________________________________________ 8

A. Escherichia

E. coli is a natural habitant of the human and animal gut. In healthy people it is a harmless

commensal. The presence of E. coli in drinking water, surface water bathwater and food is often

attributed to faecal contamination from agricultural and urban/residential areas. For surface and

bathwater there are four classes of quality in Switzerland (see chapter 1.4, Part A: Surface water).

Most types of E. coli are harmless. However, some strains can cause severe diseases, such as

bloody diarrhoea, and occasionally kidney failure.

General pathogenesis

E. coli possesses a range of pathogenic factors.

O- and K-antigen protect the organism from complement and phagocytosis in the absence

of antibodies.

Many strains express haemolysin(s). These enzymes are important for the release of

essential ferric ions bound to haemoglobin.

Some strains express siderophores (e.g. enterobactin) which remove ferric ions from

mammalian iron transport proteins such as transferrin and lactoferrin.

Pathogenesis and epidemiology of extraintestinal infections

E. coli can generally cause both intestinal infections such as diarrhoea and extraintestinal infections

such as urinary tract infections. Extraintestinal infections occur when E. coli colonizes places other

than the gut. Typical manifestations are wound infections, infections of the gall bladder,

appendicitis, peritonitis, neonatal meningitis, sepsis and urinary tract infections.

About 15% of all nosocomial sepsis is caused by E. coli, usually by strains of septic E. coli

(SEPEC). SEPEC are serum resistant.

E. coli is also the most common cause of urinary tract infection outside the hospital. This infection

manifests in the lower part of the urinary tract (urethritis, cystitis, urethrocystitis) or it can affect

the renal pelvis and the kidney (cystopyelitis, pyelonephritis).

Introduction______________________________________________________________________ 9

Most E. coli infections are thought to be caused by organisms originating from the patient's own

flora.

Infections occurring in the absence of mechanical anomalies are thought to be caused by the

pathovar UPEC (uropathogenic E. coli).

Pathogenesis and epidemiology of intestinal infections

Table 1.1 provides an overview of enteropathogenic strains of E. coli.

Table 1.1: Pathogenic strains of E. coli

Abbreviation Name Description EPEC Enteropathogenic E. coli Infantile enteritis, especially in tropical countries ETEC Enterotoxigenic E. coli Community-acquired diarrhoeal disease in areas

of poor sanitation, most common cause of travellers' diarrhoea (up to 50%)

EIEC Enteroinvasive E. coli Shigella dysentery-like disease in patients of all age groups

EAggEC Enteroaggregative E. coli Chronic diarrhoeal disease in certain developing countries

EHEC / VTEC

Enterohaemorrhagic / verocytotoxin producing E. coli

Mild, watery diarrhoea to haemorrhagic colitis and haemolytic uraemic syndrome (HUS)

Taken from Greenwood et al. 2007

EHEC

Infections with EHEC can be associated with symptoms ranging from mild, watery diarrhoea to

bloody haemorrhagic colitis, mostly without fever, and potentially fatal haemolytic uraemic

syndrome (HUS). The incubation time is 12 hours to maximally 3–10 days. Illness duration is 7–

10 days. HUS is characterized by acute renal failure accompanied by thrombocytopenia and

anaemia. From 2 to 7% of all infections with EHEC develop HUS, predominately in children under

5 years and elderly people. The case fatality rate is 3–5%.

The main virulence factor is the production of two types of cytotoxin. These toxins are toxic to

Vero cells and are hence named verocytotoxins 1 and 2 (VT1, VT2 or Stx1, Stx2). A second factor

is found in strains of serovar O157, which express an 'attaching and effacing' genotype.

Introduction______________________________________________________________________ 10

VT 1 and 2 are located on a bacteriophage plasmid and are very similar to the Shiga toxin

produced by Shigella dysenteriae. Through infestation with such a toxin-coding phage any strain of

E. coli could change to EHEC (Burnens, 2001).

VT1 and 2 bind to the surface protein Gb3 (globotriosylceramide) of several eukaryotic cells.

Binding to Gb3 receptors located in the kidney leads to HUS. There are several VT2 variants with

similar effects but immunological differences that bind to Gb4 (globotetraosylceramide). After

binding to eukaryotic cells, the toxins are internalized and remain active within the endosomes.

One subunit of the toxin prevents protein synthesis and results in cell death.

Strains of serovar EHEC O157 carry a gene for 'attaching and effacing' (eae). As for EPEC, this

gene is located on a 'pathogenicity island' in the bacterial chromosome. In areas of attachment of

these genotypes to target cells the brush border microvilli are lost (effaced).

The three genes (vt1, vt2, eae) are not associated with each other. Each gene can occur alone or in

combination with the others within one EHEC, depending on strain.

EHEC is an emerging pathogen that is gaining in importance worldwide. The number of infections

and outbreaks is rising. In the EU and EEA/EFTA countries, the incidence rate has more than

doubled during the last 10 years (Figure 1.1).

Source: The first European communicable disease report

Figure 1.1: Incidence rate of VTEC cases in EU and EEA/EFTA countries by year reported, 1995–2004

The yearly distribution of EHEC cases shows a clear seasonality (Figure 1.2).

Introduction______________________________________________________________________ 11

In 2005 data from 11 EU countries, Iceland and Norway showed the highest incidence rate in

children ≤ 4 years of age (9.04 per 100 000), falling rapidly with increasing age. There is no real

difference between the incidence rates of males and females (European Epidemiological Report,

2007).

Outbreaks often occurred within communities, in nursing homes for elderly people or in day care

centres for young children. In May 2000 an outbreak in the community Walkerton (Ontario,

Canada) led to 7 deaths and more than 2300 illnesses (Canada Communicable Disease Report,

2000). The drinking water supply was contaminated by rainwater runoff containing cattle faeces.


Figure 1.2: Distribution of VTEC cases by month, for selected European countries, 2005 (n = 2031)

The infection dose is estimated to be 10–100 germs. The main source of infection is the

consumption of raw minced meat. Unpasteurized milk, apple juice, cooked meat, sprouts and

contaminated water are important sources as well. Humans are the main reservoir. Livestock such

as cattle, sheep and to a lesser extend goats, pigs and chickens are also important. Infection can be

transmitted by the faecal-oral route due to unhygienic behaviour.

B. Salmonella

In the last 20 years, Salmonella enterica has become one of the most common causes of food

poisoning in various countries. Nowadays it is accepted that there is only one species, S. enterica,

Introduction______________________________________________________________________ 12

with seven subspecies (enterica, arizonae, diarizonae, houtenae, indica, salamae, bongori) (Le

Minor und Popoff, 1987). Reeves et al. (1989) suggested that S. enterica ssp. bongori should be

described as a separate species. A clearly defined classification system is still lacking.

Most clinical important serotypes belong to the species S. enterica ssp. enterica. As the name

suggests, S. enterica primarily causes diseases of the intestines. There are more than 2000

serotypes that are classified due to their somatic (O) and flagellar (H) antigens.

Pathogenesis

There are four clinical manifestations:

• gastroenteritis

• bacteraemia / septicaemia

• typhoid fever / enteric fever

• asymptomatic carrier state

With respect to the enteric illness they elicit, Salmonella spp. can be divided into two fairly distinct

groups: typhoidal serovars and non-typhoidal/enteric serovars. Typhoid salmonellosis is caused by

serovars typhi and paratyphi A, B, C. Important serovars for enteric salmonellosis are enteritidis

and typhimurium.

In enteric salmonellosis the bacteria are taken up orally in contaminated food of animal origin

(eggs, meat) or water. They adhere to cells of the ileum and colon and invade the mucosa. Germs

persist in epithelial cells and sometimes in macrophages, producing an enterotoxin, giving rise to

local inflammation (Figure 1.3). After an incubation time of 6–72 hours after ingestion, the disease

usually begins with acute diarrhoea and sickness. Diarrhoea lasts for 3–5 days, accompanied by

fever and abdominal pain. The disease is usually self-limiting. In cases of strong loss of water and

electrolytes, hospitalization is necessary.

Introduction______________________________________________________________________ 13

Source: www.gsbs.utmb.edu/microbook/ch021.htm

Figure 1.3: Infection cycle of enteric salmonellosis

In typhoid salmonellosis, bacteria are taken up orally in contaminated food or water or via smear

infection. They adhere to cells of the jejunum and enter the lymphatic tissue by transcytosis

through cells of the mucosa and phagocytosis by macrophages. Germs proliferate in mesenterial

lymph nodes and spread in the lymph and blood. Secondary sites of infection include the spleen,

liver and bone marrow Subsequently, after 1–14 days incubation, a generalized septic disease

pattern develops, beginning with gradually increasing fever, followed by headache, leucopoenia,

splenic swelling, abdominal roseola and occasionally bloody diarrhoea after 3 weeks. Typhoid

fever is a more severe illness and can be fatal.

Virulence factors

Salmonella possess a wide range of effector proteins, which are injected directly into host cells

with help of two type III secretion systems. These proteins disturb several cellular metabolic

processes, with the result that water and electrolyte regulation are disturbed or Salmonella is taken

up into host cells. Other factors are invasins or adhesins on the bacterial surface.

Introduction______________________________________________________________________ 14

Epidemiology

Typhoid salmonellosis is not a problem in countries with good sanitation. In northern and central

Europe, typhoid salmonellosis is imported by travellers and epidemic outbreaks only following the

rare coincidence of several risk factors. However, in countries with poor sanitation, typhoid

salmonellosis is still prevalent with many millions of cases each year. S. typhi and S. paratyphi are

restricted to humans, although livestock can occasionally be a source of S. paratyphi.

Enteric salmonellosis is endemic and epidemic worldwide. Although it has been gaining in

importance since the end of the Second World War (Tauxe, 1997), cases reported in 25 EU

member states, Norway and Iceland have been declining since 1995 (Figure 1.4).


Figure 1.4: Incidence rate of salmonellosis cases in EU and EEA/EFTA countries by year reported, 1995–2004

The highest incidence rate was reported in children younger than 4 years of age. Cases of

salmonellosis show a clear seasonality (Figure 1.5).

Introduction______________________________________________________________________ 15


Figure 1.5: Distribution of salmonellosis cases by month, for selected European countries, 2005 (n = 89317)

C. Shigella

Shigellae are the causative organism of bacterial dysentery. The genus comprises four species:

Shigella dysenteriae, S. flexneri, S. boydii and S. sonnei. Shigellae are non-motile and rod-like.

They have complex antigenic patterns and classification is based on somatic O-antigens.

Pathogenesis

Shigellae are predominately transmitted by the faecal-oral route through direct person-to-person

contact or via contaminated food and water. Flies have been identified as a transmission vector

from contaminated faecal waste. More recently, sexual transmission among men having sex with

men has become a more common cause of outbreaks in several countries. The infective dose is

very low (10 to several hundred germs). The incubation time for disease is 2–5 days. Bacteria

remain localized in the intestinal epithelial cells of the terminal ileum and colon. They are taken up

by M-cells and phagocytosed by macrophages. Inside macrophages they lyse the phagosome and

induce apoptosis. After cell death, bacteria are taken up by enterocytes on the basolateral side.

Inside enterocytes bacteria proliferate, leading to cell destruction. Neighbouring enterocytes are

invaded by lateral transfer from infected cells. Depending on species, the clinical picture can vary

from a mild, self-limiting diarrhoea to very serious presentations such as high fever, dysentery

Introduction______________________________________________________________________ 16

(bloody and pyic diarrhoea, enterospasms and tenesmus, massive intestinal bleeding), megacolon

or HUS. Reactive arthritis can follow the enteric symptoms. Severe illnesses are usually caused by

S. dysenteriae.

Virulence factors

Shigella possesses a 180- to 240-kb plasmid encoding invasins. S. dysenteriae produces the

chromosome-coded Shiga toxin, which inhibits eukaryotic protein synthesis.

Epidemiology

Bacterial dysentery is present worldwide. In industrialized countries with good sanitation it occurs

only sporadically, and shigellosis is a typical travellers' disease. In developing countries with poor

sanitation and hygiene, the disease is endemic and probably epidemic. Worldwide there are over 2

million infections each year, resulting in 600 000 deaths. In developing countries, shigellosis is a

leading cause of childhood death.

In 25 EU member states, Iceland and Norway, the incidence has been declining over the last 10

years. The highest incidence was in children younger than 4 years of age (3.5 per 100 000),

representing 10% of all cases. The incidence rises again at age group 25–44. There is a clear

seasonality, with an increase from winter to summer and a peak in August and September

(European disease epidemiological report, 2007).

Waterborne outbreaks of shigellosis have been recorded. As the organisms are not particularly

stable in water environments, their presence in drinking water indicates recent human faecal

pollution.

D. Yersinia

The genus Yersinia includes 11 species of which 3 are of medical importance for humans: Yersinia

pestis (plague), Y. enterocolitica and Y. pseudotuberculosis (both enteric diseases). Y.

enterocolitica is a pleomorphic, peritrichous flagellated, short rod. All types of yersiniosis are

zoonoses.

Introduction______________________________________________________________________ 17

Pathogenesis

Germs of Y. enterocolitica are taken up orally in raw/undercooked meat and water. Direct

transmission from animals or humans by smear infection is also possible, but seldom. Germs reach

the lower intestinal tract, penetrate the mucosa and are transported to mesenterial lymph nodes by

macrophages. Incubation time is 3–7 days. There are two different clinical patterns.

• Intestinal yersiniosis: fever, diarrhoea, abdominal pain and lymph adenitis are the main

symptoms. Mimicking appendicitis in adolescents and colitis in adults is possible.

• Extraintestinal yersiniosis: manifestations such as arthritis, lymphadenopathy, erythaema

nodosum (inflammation of fat cells under the skin) can appear.

Virulence factors

All pathogenic strains isolated from humans have a 70-kb virulence plasmid containing several vir

determinants. These plasmids code for polypeptides for cell adherence, phagocytosis resistance,

serum resistance and cytotoxicity. In addition there are chromosomal virulence genes for invasins,

enterotoxins and iron uptake systems.

Epidemiology

Y. enterocolitica is present worldwide in domestic and wild animals. The main reservoirs are pigs.

Pathogenic Y. enterocolitica has been detected in sewage and polluted surface waters. Y.

enterocolitica in drinking water are more commonly non-pathogenic strains, probably of

environmental origin.

In 2005, 9660 cases were recorded in EU countries with an overall incidence rate of 2.23 per

100 000. From 1995 to 2004, the incidence rates were relatively stable or slightly rising with two

clear peaks in 1998 and 2002. The main affected age group are children younger than 4 years of

age. There is no clear seasonality, but a slightly higher number of cases in the second half of the

year (European disease epidemiological report, 2007).

Although most Yersinia spp. detected in water are probably non-pathogenic, circumstantial

evidence has been presented to support transmission of Y. enterocolitica to humans from untreated

drinking water. But since Yersinia is sensitive to disinfection processes, protection of raw water

Introduction______________________________________________________________________ 18

supplies from human and animal waste and adequate disinfection minimizes the presence of

Yersinia in water supplies.

E. Opportunistic Enterobacteriaceae

Many Enterobacteriaceae have only marginal pathogenicity. They are classical opportunists.

Usually harmless for healthy people, they can occasionally cause fatal infections in patients with

underlying diseases, predominately in hospitals. Infections caused by such species are urinary and

respiratory tract infection, wound infections, infections of the skin and subcutis and sepsis. In

germs associated with hospitals, resistance against antibiotics is common, and multidrug resistance

has often been observed. Typical species are Citrobacter, Klebsiella, Enterobacter, Serratia,

Proteus, Morganella, Providencia.

1.1.2 Campylobacter

The genus Campylobacter includes many species of which Campylobacter jejuni, C. fetus and to a

lesser extend C. coli and C. lari are of medical importance.

Campylobacter species are small, spiral, Gram-negative rods. They have one flagellum at one or

both poles which enable their rapid, darting motility. Campylobacter grows in microaerophilic

conditions (5% oxygen, 10% CO2). It is the most common cause of bacterial enteritis in

industrialized countries and the most frequently reported zoonosis. Amongst all Campylobacter

species, C. jejuni is the most important (90–95% of all Campylobacter infections). About 1000

organisms can cause infection. Campylobacter species readily take up naked DNA from their

surroundings and as a consequence are genetically diverse.

Pathogenesis

Germs are taken up orally. Incubation time is 2–5 days. Typical disease patterns are watery or

sometimes bloody diarrhoea, accompanied by abdominal pain and fever, nausea and sometimes

vomiting. The infection is usually self-limiting after a few days and hospitalization is only

Introduction______________________________________________________________________ 19

necessary in very severe cases. However, 1 or 2 weeks after the onset of the disease, two different

relapses may occur in the form of reactive (aseptic) arthritis or, rarely, the potentially fatal

Guillain-Barré syndrome, a post-infective polyneuropathy (acute demyelinating disease of the

peripheral nerves).

It is primarily the jejunum and ileum that are colonized, with occasional extension into the colon

and rectum. The bacteria produce a cytotoxin, translocate directly across the cell layer or invade

host cells (Figure 1.6). Subsequent cell damage stimulates an inflammatory response. Bacteria are

still detectable in stool samples weeks after recovery.

Figure 1.6: Campylobacter pathogenesis

Virulence factors

Campylobacter species produce an enterotoxin and several cytotoxins. They have a capsular

polysaccharide which acts as an epitope for antibody production. This epitope is thought to initiate

Guillain-Barré syndrome. Antibodies cross-react with myelin of peripheral nerves.

Epidemiology

Campylobacter is transmitted from animals to humans by oral uptake of contaminated food and

water. Other routes of transmission are direct contact with infected animals and smear infection

between humans. Swimming in natural surface waters is also a risk factor. The main reservoirs are

animals, especially wild birds, poultry and cattle. The main sources of infection are meat,

Introduction______________________________________________________________________ 20

unpasteurized milk and contaminated water. Campylobacter does not multiply in food. Occurrence

of germs in the environment is strongly dependent on rainfall and waterfowl. Contaminated

drinking water supplies have been identified as a significant source of outbreaks of

campylobacteriosis.

Campylobacteriosis shows a clear seasonality, with a peak in August (Figure 1.7). The incidence in

14 EU countries, Norway and Iceland increased between 1995 (85 000 cases) to 2002–2004

(180 000–190 000 cases) (Figure 1.8).


Figure 1.7: Incidence rate of campylobacteriosis cases in EU and EEA/EFTA countries by year reported, 1995–2004


Figure 1.8: Distribution of salmonellosis cases by month, for selected European countries, 2005 (n = 28145)

Introduction______________________________________________________________________ 21

The highest incidence is in children younger than 4 years of age (European disease epidemiological

report, 2007). In developing countries the disease is hyperendemic.

1.1.3 Pseudomonas

Pseudomonads are Gram-negative, aerobic, non-fermenting rods. They are motile and have one or

two polar flagella. Pseudomonads are opportunistic human pathogens and usually cause

nosocomial diseases and diseases in people with predisposing factors, in immunocompromised

patients (AIDS, leukaemia, neutropenia) and in patients consuming immunosuppressive medicine.

In healthy people, infections are very rare and only mild.

Pseudomonads are ubiquitous in wetlands (soil, surface water, sewage, oceans, on plants), in faeces

and in small amounts in the gut of humans and animals. The clinically most important species is

Pseudomonas aeruginosa. This is due to several reasons:

• good adaptability

• innate resistance to many antibiotics and disinfectants

• many virulence factors

Pathogenesis

The pathogenesis of P. aeruginosa is very complex. It usually enters the body through damaged

sites. The germ adheres to host cells via adhesive pili. From the point of entry, the germ invades

the body. P. aeruginosa can infect almost any external site or organ.

There are several disease patterns caused by an infection with P. aeruginosa, depending on site of

entry and the host's immunocompetence. These patterns are colonization of wounds and burns,

post-operative wound infections, infection of the urinary tract, ears (otitis externa) and eyes, sepsis,

endocarditis in drug addicts, pneumonia in patients with cystic fibrosis, and septicaemia and

meningitis in infants.

Virulence factors

P. aeruginosa has many virulence factors:

Introduction______________________________________________________________________ 22

• exotoxin A (ADP-ribosyltransferase): blocks translation by inactivation of elongation factor

EF2

• exoenyme S (ribosyltransferase): inactivates cytoskeleton proteins of eukaryotic cells

• cytotoxin: damages cells by making pores in the cell membrane

• several metal proteases: hydrolysing elastin, collagen, laminin

• two phospholipases C: membrane active

Epidemiology

P. aeruginosa is able to proliferate in wet environments with poor life conditions. Thus there are

many sources of infection in hospitals, bathrooms and kitchens — washbasins, toilets, cosmetics,

air conditioners, inhalers, respirators, anaesthetic machines, dialysis machines — but also in

swimming pools and spas. Primary sources may also be infected patients and germ carriers.

P. aeruginosa is resistant to many disinfectants and antiseptics commonly used in hospitals. It may

even multiply in such liquids. Healthy carriers usually harbour strains in the gastrointestinal tract.

But in the community, the carriage rate seldom exceeds 10%.

Transmission may occur directly via medical staff or indirectly via contaminated apparatus. The

presence of germs in dust and eschar shed from burns suggests that infection can be airborne.

Severely burned patients and those with chest injuries that require artificial ventilation are very

susceptible. Pulmonary infection frequently precedes septicaemia, which is often fatal. Eye

infections may occur via contaminated lenses or by use of contaminated medicine during

ophthalmic procedures. Ear infections or folliculitis (jacuzzi rash) may occur during use of poorly

maintained whirlpools, where the warm and aerated conditions are perfect for P. aeruginosa.

Epidemics of gastrointestinal infection can occur in newborn and young infants in paediatric wards

as a result of contaminated milk feeds. Infections in infants include septicaemia and meningitis.

It is easier to prevent than to cure infections with P. aeruginosa. Preventive measures include very

good disinfection of surgical materials and surfaces, avoiding admittance of high-risk patients to

wards where P. aeruginosa infections have recently occurred, no use of multidoses of creams or

drops for several patients. Once P. aeruginosa has gained access to the hospital environment it is

difficult to eradicate.

Introduction______________________________________________________________________ 23

1.2. Virology

1.2.1 Norovirus

Noroviruses (genus Norovirus, family Caliciviridae) are a group of related, single-stranded RNA,

non-enveloped viruses that cause acute gastroenteritis in humans. Norovirus was recently approved

as the official genus name for the group of viruses provisionally described as 'Norwalk-like viruses'

(NLVs). They are 35–40 nm in diameter und their surface shows typical cup-like cavities (calyx =

cup) which can be seen under electron microscopy.

Until recently, norovirus could only be detected by electron microscopy, but the diagnosis of

norovirus as a cause of outbreaks of acute gastroenteritis has improved with increasing use of the

reverse transcriptase-polymerase chain reaction (RT-PCR). This method detects the norovirus

RNA and can be used to test stool as well as environmental samples. Norovirus cannot be

propagated in cell culture systems.

The genus Norovirus has two different clades: genogroup 1 and genogroup 2 (Vinjé et al., 1997).

Pathogenesis

The viruses are excreted by faeces and in vomit. Routes of transmission are person-to-person

contact, inhalation of contaminated aerosols, dust particles or airborne particles of vomit, ingestion

of contaminated water and food (often shellfish harvested from contaminated waters). Replication

takes place in the jejunum. Villi become broadened and blunted, enterocytes become cuboidal and

vacuolated. Incubation time is 12–72 hours. The disease is self-limiting and practically no

mortality has been reported (Dijuretic and al., 1996). Symptoms are nausea, vomiting and

abdominal cramps. Diarrhoea occurs in 40–100% of cases depending on the isolate. Some patients

have fever, chills, headache and muscular pain. Diarrhoea is more frequent in adults, while

vomiting is more often described in children and infants (Lodder et al. 1998). Symptoms are

generally mild, and hospitalization is seldom required. The disease can be asymptomatic.

Introduction______________________________________________________________________ 24

Epidemiology

The disease occurs worldwide in all age groups, but predominately in infants, schoolchildren and

elderly people. Together with rota- and adenoviruses, noroviruses are the most common viral

enteritis-causing agents. The disease is suggested to be underreported due to mild symptoms and

the asymptomatic course of the disease.

Humans are considered to be the only source of human-infectious species, although subclinical

infection and excretion of contaminated faeces of primates have been described.

Human caliciviridae often cause small epidemics, usually in winter. Since vomiting is often a main

symptom, the disease is also called 'winter-vomiting disease'.

Many outbreaks have been linked to contaminated drinking water supplies.

Studies indicate that excreted viruses can remain viable for several years and that the infectious

dose is 10–100 particles. Due to these low infection doses, minor contamination of hands, work

surfaces, taps, carpets etc. can be a major source of infection.

Asymptomatic excretion of noroviruses is not uncommon. Such individuals may serve as an

important reservoir of infection, particularly in hospitals or catering services.

1.2.2 Rotavirus

Rotaviruses belong to the family Reoviridae, together with coltiviruses, orthoreoviruses and some

genera infectious for plants and animals. Reovirus is an abbreviation for 'respiratory enteric orphan

virus', meaning that no disease could be associated with the virus after its discovery.

Members of the genus Rotavirus consist of 11 segmented, double-stranded RNA strands, each

strand coding for one protein. They have a non-enveloped, icosahedral capsid with a diameter of

50–65 nm. The capsid is surrounded by a double-layered shell, giving the virus the appearance of a

wheel — hence the name rotavirus. The diameter of the entire particle is about 80 nm.

Rotaviruses are serologically divided into seven groups, A–G, each with several subgroups.

Groups A–C are infectious for humans, group A being the most important. Groups D–G infect a

wide spectrum of animals.

Introduction______________________________________________________________________ 25

Pathogenesis

Human rotaviruses (HRVs) are transmitted by the faecal-oral route. One patient excretes up to 1011

particles in only 1 g of faeces. Excretion periods last approximately 8 days. The most important

transmission routes are person-to-person contact and inhalation of airborne HRVs or aerosols

containing the virus. Ingestion of contaminated food and water plays a less important role, but

outbreaks due to these sources have been described. The virus infects cells in the villi of the small

intestine where it disrupts the transport of sodium and glucose. Acute infection has an abrupt onset

of severe watery diarrhoea with fever, abdominal pain and vomiting. Dehydration and metabolic

acidosis may develop. The disease may be fatal if not treated appropriately.

Epidemiology

Due to the high number of excreted particles in stools, domestic sewage and environments polluted

by human faeces are likely to contain large numbers of HVRs. Viruses have been detected in

sewage, rivers, lakes and treated drinking water.

The presence of HRVs in drinking water constitutes a public health risk, even if transmission by

ingestion is not the most common route. There is some evidence that rotaviruses are more resistant

to disinfection than other enteric viruses.

HRVs are the most frequent pathogen for diarrhoea in children from 6 months to 2 years of age.

However, they also play a role in elderly people.

In developing countries, diarrhoeal diseases are the most common cause of death in children, and

20% of all these cases are due to rotaviruses. In industrialized countries, HRVs play a role in

hospitals and in children's homes, primarily in winter. They can persist for a long time on surfaces

and skin (hands) and are therefore very easily transmitted. The best prophylaxis is meticulous

hygiene.

1.3 Protozoology

The information here for Gardia spp. has been taken from Melanie Wiki, 2006 (A new method for

detection of Gardia lamblia and its application in monitoring surface water), that concerning

Introduction______________________________________________________________________ 26

Cryptosporidium spp. from Sandra Ruchti, 1999 (Zur Epidemiologie von Cryptosporidium sp.:

Oocysten-Dichten in Oberflächen-, Roh-, und Trinkwasser im Lützeltal (Bl/So)).

Protozoans are eukaryotic, unicellular, heterotrophic organisms which often have a parasitic life

cycle. Typical for many protozoans is the ability to build cysts or oocysts, which enable them to

survive for a long time in the environment, withstanding dryness, high temperatures and other

inhospitable conditions.

1.3.1 Gardia lamblia

Gardia lamblia is a flagellated parasite of the small intestine causing lamblia dysentery. The

parasite is found worldwide with different prevalence rates. Parasites are excreted as a cyst in high

numbers in human or animal faeces. These cysts can survive for several months in cold water and

are relatively resistant to environmental dehydration.

Together with Cryptosporidium and E. coli, Gardia is the most common pathogen identified as

causing human infections through contaminated drinking and surface water.

Pathogenesis and disease pattern

Parasites are taken up orally. Less than 10 cysts can cause disease. The incubation time is usually

1–2 weeks, but can vary from 1 to 45 days. The parasites manifest in the small intestine, where

they cause inflammation and disturb resorption. The pathogenesis is unclear, but they probably

produce toxin-like proteins. Symptoms are diarrhoea, abdominal pain, vomiting, signs of

malabsorption and loss of weight. The disease can be acute, chronic or asymptomatic. The parasite

is often eliminated spontaneously after a few weeks. In asymptomatic and chronic cases, the

parasite can persist for many years inside the intestine. Chronic cases may endure for many years.

Epidemiology

Routes of transmission are faecal-oral by direct contact of humans with other humans or animals,

or indirectly via contaminated food or water.

Introduction______________________________________________________________________ 27

Most frequent risks are swimming in surface water, uptake of contaminated water or travelling to

endemic areas.

Parasites are found worldwide. In Europe the average prevalence rate is 3–4%. In developing

countries, it is up to 50%. In both cases, rates can vary regionally.

Humans are the main host but there are several animal reservoirs, predominately mammals (cattle,

sheep, dog).

Males have a significantly higher risk for acquisition than females. The disease is very common in

children in developing countries and in day care centres in industrialized countries.

Reports about seasonality vary, and increases have been recorded in both summer and winter.

In chronic and asymptomatic cases, cysts are excreted in faeces for a long time in high numbers.

Due to the parasite’s resistance in the environment, infected patients contribute to a high

environmental contamination and a problem for public health.

1.3.2 Cryptosporidium parvum

Cryptosporidiosis is a zoonosis with a large animal reservoir. Parasites have been identified in

more than 70 mammals, including pets (dog, cat), livestock (cattle, sheep, goat, pig) and wild

animals (deer, badger, fox). Parasites form oocysts which are excreted in human or animal faeces.

These oocysts can survive in cool water for months and are resistant to normal concentrations of

chloride and ozone in water supply systems. They are, however, immobilized by UV and destroyed

by warm water (70°C for a few minutes).

Pathogenesis and disease pattern

Parasites are taken up orally. Thirty to 100 oocysts can cause disease. The incubation time is 5–28

days. They manifest in the small intestine where they destroy microvilli and cause infiltration of

mucosa into cells. Symptoms are watery, mucous, seldom bloody diarrhoea, rapid weight loss,

dehydration, nausea and headache. The disease is usually latent or self-limiting, lasting 1–26 days

with mild symptoms. In HIV and immunocompromised patients, the disease can be very strong,

chronic and occasionally fatal, with serious cholera-like diarrhoea, nausea, vomiting, fever and

abdominal pain. Parasites are excreted in faeces for a long time.

Introduction______________________________________________________________________ 28

Epidemiology

Route of transmission is faecal-oral by direct contact of humans with humans or animals, or

indirectly through drinking water and food.

The disease is found worldwide and is considered as one of the 'emerging diseases'. In

industrialized and developing countries, the general prevalence rate is 2% and 6%, respectively, for

HIV patients it is 14% and 24–50%, respectively. Young calves have a prevalence rate of 20–

100%.

Outbreaks have been associated with contaminated water (drinking water, surface water,

swimming pools and public supply systems).

There is no chemoprophylaxis. The best prophylaxis is good hygiene for excreters.

1.4 Micro-organisms in water: Initial position

A wide range of micro-organisms (viruses, bacteria, protozoa) and macro-organisms (helminths)

are found in watery environments such as surface water (Tougianidou et al. 1998), groundwater

and soil. Most of them are unidentified and not infectious. However, some of them are infectious

and are transmitted between humans via contaminated water or food. These food- and waterborne

pathogens can cause various diseases in humans, e.g. gastrointestinal or respiratory diseases or

infections of the skin and damaged sites.

According to the World Health Organization (WHO), gastrointestinal infectious diseases are

among the most important infectious and parasitic diseases worldwide. With an estimated 1.8

million deaths a year and 62 million disability-adjusted life years (DALYs), they follow directly on

respiratory infectious diseases and HIV/AIDS and cause greater mortality than malaria and

tuberculosis.

These germs are often distributed in human excretions, e.g. faeces or urine, and released into water.

Many micro-organisms can survive for a long time in surface water and groundwater without

proliferation. If their numbers exceed a critical value, they can become dangerous for the health of

people using these waters. Infections can be contracted after ingestion of contaminated water

during swimming and other activities.

In developing countries without a system for the distribution of clean drinking water, such

pathogens are a severe public health problem, because people use surface waters directly as

Introduction______________________________________________________________________ 29

drinking water, for doing their laundry, for spending free time, to wash and as a toilet. Waterborne

pathogens can therefore spread easily between humans. In addition, such populations usually do

not have facilities to clean sewage, and the dirty water flows directly into rivers and lakes, thus

contaminating it with microbes from faeces or household waste.

In industrialized countries with an officially organized water supply, drinking water is processed

before it is distributed. Nevertheless, problems with surface water remain, although their

dimensions differ to those in developing countries. Since we do not drink from surface waters

directly, recreational activities pose the major risk factor. Swimming and diving with accidental

ingestion of water or pathogen contact with damaged body sites can lead to infection with

waterborne micro-organisms (Schijven et al. 2006). A number of outbreaks due to surface water

have been described in North America and Europe (Lee et al. 2002; C. Furtado et al. 1998; Bocca

et al. 2002), including Switzerland (Maurer A.D., Sturchler D., 2000).

According to the BAG report, there are an increasing number of food-related und waterborne

diseases during the summer. Most of these infections are caused by barbeques e.g. due to

insufficiently grilled meat or unhygienic handling of food, but little is known about the impact of

surface water.

The human environment (cities and villages, farms, camping places, sewage cleaning plants) has a

great influence on the microbiological quality of water.

Part A: Surface water

With a high population density in Switzerland, the country's surface waters are greatly influenced

by the human environment. In general, intense water use and garbage, infected livestock such as

calves and lambs, dunging with manure and sewage cleaning plant (SCP) drains can lead to

contamination with zoonotic and anthropzoonotic micro-organisms (Lodder et al 2005, Rose et al.

1998). SCPs have a particularly large effect. Sewage from households and industry, as well as

water from the environment, flows into these plants, accumulating huge masses of waste and

micro-organisms. Since SCPs release cleaned water into rivers and lakes but do not filter out

micro-organisms from sewage, these waters may have a higher contamination with micro-

organisms than waters without SCP input. Thus swimming areas in surface waters close to SCPs

are possibly more burdened with pathogens and may be hazardous to human health.

Following rainfall, environmental micro-organisms may be washed into surface waters. Soil,

animal faeces (livestock and wild animals) or garbage are possible sources. Another possible

Introduction______________________________________________________________________ 30

source are SCPs. Since many plants do not have the capacity to clean all the additional rainwater,

the plants overflow and sewage drains off directly into surface waters uncleaned, contaminating

them with garbage and micro-organisms (Balayan M S. 1997).

Effluents from SCPs are believed to have the strongest influence on microbiological water quality,

and water quality improves as one moves further downstream from an SCP: after several hundred

meters, the downstream quality should again be good enough for recreational activities.

In Switzerland the microbiological qualities of surface waters and bathwaters are classified in four

ranges, A–D. Each range is defined by specific amounts of E. coli per 100 ml of water as an

indication of faecal pollution. The presence or absence of Salmonella is additionally indicated. For

each classification, there are recommendations concerning usage of described waters (see table

3.11, page 62).

Drainage of settlement areas

Sewage from households and industry flows into SCPs via drain systems (see Figure 1.9).

Source: Ferdinandy et al. 1996

Figure 1.9: Present urban water cycle

In addition clean water from fountains and rivers flows into the same drains. Groundwater may

penetrate through leaky drains. During rainfall events, additional water from roofs and streets

flows into the drain system. All this additional water reduces the cleaning capacity of SCPs. To

Introduction______________________________________________________________________ 31

protect drains and plants from these masses of water, overflows are integrated into the drain

systems (mixed water drains). If the water amount exceeds the drain’s capacity, the additional

water flows over into secondary drains which lead directly into surface waters.

These discharging systems pollute surface waters strongly. There are several options to reduce this

pollution. In new buildings, rainwater and sewage can be separated into different drains. These

rainwater drains lead directly into surface waters. The same drains can be used for water from the

streets.

In existing drain systems and SCPs, mixed water pools can be installed. Additional water flows

over into these pools instead of surface waters, and gets pumped back when the water amount

reduces.

However, planning and installation of such systems needs time and money, and there are not yet a

sufficient number of such systems to disburden surface waters from sewage.

SCP operating mode

Common SCPs in Switzerland use three different methods for cleaning waste water: mechanical

cleaning, biological cleaning and chemical cleaning.

Mechanical cleaning

In a first step, raking systems of different sizes remove solid and large materials such as leftovers,

toilet articles and other wastes.

In a sand trap, heavy material and sand settle down and are removed. In the following pre-cleaning

pool, the flow rate slows down. Small materials settle down and are removed. Materials floating on

the surface are removed by a surface broach.

Biological cleaning:

In an aerated pool, bacteria clean the water in two steps.

First step: organic material is decomposed and ammonia is metabolized to nitrate (nitrification).

The pool is aerated so that the bacteria receive enough oxygen.

Second step: Bacteria metabolize nitrate to nitrogen. For this step no aeration is needed.

Introduction______________________________________________________________________ 32

Chemical cleaning:

Phosphate must be removed from sewage. Metal salts (iron sulphate or aluminium chloride) are

added to which phosphates bind. The bound phosphate precipitates into the sewage sludge.

In the post-cleaning pool, bacteria settle down with sludge. Floating sludge is removed with a

surface broach. The clean water flows over the pool rim and is drained into local rivers or lakes.

Part B: Groundwater and drinking water

Switzerland is the moated castle in Europe. Every year there is an average rainfall of 60.1 m3 of

water, 13.1 billion m3 accrue from foreign countries and 20 billion m3 evaporate back to the

atmosphere. All in all, 262 000 million m3 of water reserves are stored in Switzerland (lakes,

glaciers, groundwater, dams and rivers), of which 1 billion m3 is processed for drinking water

every year. Twenty percent comes from surface waters and to 40% each from source waters and

groundwater (www.trinkwasser.ch). The so-called Mittelland of Switzerland is very rich in

groundwater which must be pumped up before it is processed. Figure 1.10 shows a scheme of a

typical groundwater pumping station.

Source: www.trinkwasser.ch

Figure 1.10: Groundwater pumping station

Introduction______________________________________________________________________ 33

Raw water and raw water processing

Water from rainfall, rivers, lakes and snow water flows into surface waters (Figure 1.11).

The longer surface water is under the influence of civilization, the higher the likelihood that it will

become contaminated. Depending on the filtration performance of the unsaturated zone in the

aquifer, surface water infiltrates into groundwater at different rates. In our region (Jura massif)

there is a karst geology. Water infiltrates quickly and is poorly filtrated; it descends into the ground

down to an impermeable layer, where it accumulates. This water is pumped up and processed for

drinking water. It is therefore important to keep raw water clean from faecal contamination from

households and agriculture by creating groundwater protection zones.

Source: www.trinkwasser.ch

Figure 1.11: From surface water to ground water

Thirty-eight percent of all groundwater and source water in Switzerland is naturally of drinking

water quality. The remaining 62% must be processed before it can be distributed (Michel &

Schweizer, 1998). For disinfection of raw water, several physical, chemical or mechanical

disinfection methods are in use (table 1.2).

Introduction______________________________________________________________________ 34

Table 1.2: Disinfection methods

Type Method Effect Description

Physical Heat; UV radiation

Protein denaturation; destruction of DNA

Radiation at 400 J/m2

Chemical Chlorination; ozonation

Toxic cell effects Chlorine, sodium hypochlorite, chlorine dioxide, ozone (O3)

Mechanical Filtration Nanopore (10–9 m) and micropore

(10–6 m) filters, filtration after flocculation

Taken from Roeske 2003

Thermal disinfection is the only technique which has a reliable effect for all types of pathogen. It

does not matter if the micro-organisms are suspended freely or if they are present in aggregates

(see below). However, thermal disinfection is not suitable for central distribution. It can be used

individually if contamination is present. For all chemical methods and UV radiation, there are

naturally occurring pathogens resistant to the permitted concentrations and intensities, respectively.

For filter techniques, efficiency is dependent on surface charge (Roeske 2003).

Drinking water

Drinking water is usually poor in but seldom free of micro-organisms (Roeske 2003). A low

number of non-pathogenic micro-organisms are allowed, but the water must be free of pathogenic

germs. In Switzerland three types of micro-organism are used as indicators to assess the

microbiological quality of drinking water: aerobic, mesophilic germs (bacteria, fungi and algae that

grow at 37°C), E. coli and enterococci (Auckenthaler, Huggenberger, 2003).

E. coli and enterococci must be absent in 100 ml of drinking water. Aerobic, mesophilic germs are

accepted in the following amounts:

• 300/ml in drinking water supply systems

• 100/ml in untreated raw water

• 20/ml in treated raw water

(The higher amount in drinking water supply systems may occur due to bacterial regrowth after

treatment, the formation of biofilms and long-standing water).

Tables 1.3 and 1.4 list some pathogenic micro-organisms, in addition to those described in sections

1.1–1.3, that can be found in raw water and supply systems.

Introduction______________________________________________________________________ 35

Table 1.3: Further globally important waterborne pathogens

Protozoa Bacteria Viruses Entamoeba histolytica (causative agent of amoebic dysentery)

Vibrio cholerae

Hepatitis A + E Coxsackieviruses Enteroviruses Adenoviruses


Table 1.4: Pathogens able to multiply in supply systems

Protozoa Bacteria Acanthameoba Naegleria

Legionella P. aeruginosa Acinetobacter Atypical mycobacteria Aeromonas hydrophila Yersinia


Legionella represent a health risk for all humans, independent of their health status. All others are

typical pathogens for nosocomial infections and can be a severe problem in hospitals and health

care centres.

Water supply in Switzerland

The Swiss water supply is administered by the cantons and the communities. This federal system

results in more than 3000 independent, mostly small suppliers (www.trinkwasser.ch).

In north-west Switzerland, 70% of all suppliers use a UV radiation plant, followed by chlorination,

filtering plants and ozonation (Auckenthaler, Huggenberger 2003).

Problems of water supply

Problems with raw water have been increasing in the last couple of decades. An expanding and

more mobile population has lead to rurally congested areas with rising livestock, agriculture and

Introduction______________________________________________________________________ 36

water consumption. Groundwater intakes are often close to agricultural areas, protection zones are

often not well located (Auckenthaler, Huggenberger 2003) and sewage sludge is often used as

manure, introducing viruses into farmland (Baumgartner 2001). From 1973 to 1997, 21 new

pathogenic germs were discovered (Sonntag, 1997).

Processing plants have often not adjusted to this new situation and small suppliers do not have the

financial potential to maintain processing plants in state-of-the-art condition. Highly technical

processes have not been adapted to suit small suppliers.

Despite good processing of raw water, pathogenic micro-organisms sometimes reach distribution

systems, leading to small epidemics (Swerdlov et al. 1992, Beller et al. 1997, Maurer & Stürchler

1998, Häfliger et al. 2000). There may be several sources and causes of such outbreaks.

Germs in water may be freely suspended or form aggregates (bound to particles, surrounded with

slime or mucus). Most freely suspended micro-organisms are easily eliminated by singe-stage

processing. Elimination of aggregated micro-organisms is more difficult. Chemical disinfection

and UV radiation are not strong enough to kill germs beneath the surface. For them, filtration is the

best method.

Some micro-organisms build a biofilm after they have bound to a surface and started to multiply.

This biofilm is a protective layer of extracellular substances, anorganic substances and dead

organic material. Micro-organisms can spread out of such biofilms. They are a problem if present

in supply systems.

Single-stage processing (UV, chlorination, ozonation) are usually sufficient for clear groundwater.

The situation becomes more complicated if germs are present that can only be eliminated by

combined processing (filtration, activated carbon), or if fluctuations in turbidity occur (which is

typical for the karst geology in jura massif). Chlorination and ozonation cannot disinfect

aggregates and UV is only suitable when turbidity is less than 1 FNU (see chapter 2.1.2., part

‘further parameters’).

Problems with control systems

The microbial quality of raw water and drinking water is determined by the presence or absence of

indicator organisms. In raw water there is a correlation between pathogenic germs and the density

of indicator organisms. The more indicators are present, the higher the likelihood that pathogens

will be present. If there is a high density of indicators in raw water, disinfection can be

Introduction______________________________________________________________________ 37

strengthened by use of more chemicals, or the supply can be halted until the quality has improved

naturally.

For drinking water, the correlation no longer holds. Indicator organisms are often eliminated easily

by single-state processing. The absence of indicator organisms in drinking water does not prove

that there are no pathogens present. Viruses and protozoa are more resistant to disinfection

methods and can reach the distribution system, leading to epidemics (MacKenzie et al., 1994;

Häfliger et al., 2000; Gornik et al., 2000).

1.5 Significance, aims, objectives

Surface waters are widely used for recreational activities. Quality controls occasionally indicate

that these places are contaminated with Salmonella. This bacterium can originate from inflows,

SCPs or humans themselves. According to the BAG report, there are an increasing number of

food-related and waterborne diseases during the summer. It is believed that some of these cases are

caused by contact and ingestion of contaminated surface water from bathing places.

Little is known about the influence of SCPs and rainy events on the microbiological quality of

surface waters. Two hypotheses were tested in this survey:

1) An SCP always has a great influence on the microbiological quality of surface water.

2) The quality after SCP effluent will be very poor for about 300 m. The pollution will decline with

growing distance from the effluent.

These two hypotheses are based on assumptions that have not yet been proven. This knowledge is

important for estimations of risks and dangers in spending time in pathogen-affected areas. If the

dilution downstream of a drain is known, one can appoint places that are safe and identify those

where it is not advisable to swim and spend time.

River water infiltrates into groundwater, which is used for drinking water processing. If the river

water is contaminated microbiologically, micro-organisms will also infiltrate into groundwater.

Several outbreaks due to contaminated wells have been described in the last decade (Swerdlov et

al. 1992, Beller et al. 1997, Maurer & Stürchler 1998, Häfliger et al. 2000). A better understanding

Introduction______________________________________________________________________ 38

of the ‘surface water – groundwater’ system will help to minimize the presence of micro-organisms

in distribution systems and to keeping the food product ‘drinking water’ safe.

Objectives

This work consists of five objectives:

1. Application of common analysis methods for the detection of different pathogens in surface

waters.

2. Validation of methods under field conditions and during different environmental situations

(water level, rainy events) and definition of sampling sites located at the Birs and the

Ergolz (frequency of sampling points).

3. Monitoring the initial microbiological water quality close to water-cleaning plants (before

and after the effluent) and downstream of effluents.

4. Monitoring the same places during bad weather events.

5. Monitoring the backflow of micro-organisms from surface water into groundwater.

Material and Methods______________________________________________________________ 39

2. Material and Methods

2.1 Sampling and sampling areas

2.1.1. Part A: Surface water

To test the surface water qualities and influences of SCPs, two cleaning plants were chosen, one

located at the Ergolz (SCP Ergolz2) and one at the Birs (SCP Zwingen) (see Map 1 and 2). Sample

sites above the effluent were compared with effluents and sample sites downstream.

Sample sites are indicated by letters and numbers. The first letter stands for the river (E and B).

Sample sites where the E or B is followed by a number were analysed for E. coli and all pathogens.

Sample sites where the E or B is followed by letters were only analysed for E. coli.

E0/B0 → Sample site directly above SCP effluent E1/B1 → Effluent of SCP E2/B2 → Sample site after SCP effluent (10 – 20 m downstream of effluent) E zw → Sample site between E2 and E3, about 350 m downstream of effluent E3 → About 450 m downstream of effluent E4 → Before waterfall, about 850 m downstream of effluent Ebb → Bicycle bridge, about 1300 m downstream of effluent Emb → Motorway bridge, about 1600 m downstream of effluent B EI → End of island, about 290 m downstream of effluent B3 → Before weir, about 500 m downstream of effluent B V → Vis-à-vis Verdyol, about 640 m downstream of effluent B T → Level with the Eggfluh tunnel, about 1600 m downstream of effluent B4 → Level with the bridge in Grellingen, about 4000 m downstream of effluent During the period from 12 March 2007 to 20 August 2007 samples were collected on 23 different

days, resulting in 137 samples for the Ergolz (22 sampling days) and 147 samples for the Birs.

During sampling days, the weather situation was recorded. Information about water levels (runoff

in m3/s) was provided by the Federal Bureau for Hydrology.

When a test for all micro-organisms was to be made, 5 litres of water were taken in 5-litre plastic

canisters. If samples were taken only for analysis of E. coli, 0.5 litre of water was taken in 500-ml

glass bottles. Samples were taken at the river border of rivers, just below the surface.


Source: GeoView, GIS-BL

Map1: Sampling Area Frenkendorf


Map2: Sampling Area Zwingen-Grellingen


2.1.2. Part B: Groundwater To validate the infiltration of surface water into ground water, samples of surface water and

groundwater were taken parallel. Theses samples were tested for E. coli. Map 3 shows the sample

area.


Map3: Sampling Area Duggingen The sample site was 65 m from the well. The blue areas are the groundwater protection zones 1

and 2. The distance from the pump station to beginning of protection zones 1 and 2 is about 340

and 610 m, respectively.

Prior investigations in this sampling area have defined the composition of the groundwater as

follows: 20% water from the Birs, 6% water from Lockergestein (water from the valley), 74%

water from Hauptrogenstein (water from the karstified hills).

Surface water was taken by an automatic sampler (Probenehmer ISCO Model 6700 and Model

3700) every 2 hours. One litre of water was taken and stored in 1-litre plastic bottles. Groundwater

samples were taken every 4–8 hours by the pump station attendant. Samples were placed in 1-litre

glass bottles and stored in a cool bag.


During three weather events, samples were taken: (1) from 18 to 19 July 2007, during which period

the weather was good and data were used as negative control; (2) from 6 to August 2007, during a

strong rainy event, at the end of which the sampler was swept away by a flood and the sampler

model was changed; (3) from 24 to 28 September, during a week with regular but weak rain

events.

Further parameters

For both surface water and groundwater, turbidity, conductance and UV light absorption were

measured.

Turbidity: the water was X-rayed by a light beam of 880 nm. Scattered light was measured at an

angle of 90°. Turbidity is a parameter for the quantity of particles in water.

UV absorption: UV absorption is a parameter for organic substances. It measures C=C double

bonds of organic material in water that are stimulated by UV light (254 nm). An apparatus

measures the light absorbance.

Conductance: conductance is a parameter for dissolved conducting particles. Low values in

groundwater predict a high amount of young water, since old water has more dissolved lime.

2.2 Analytical procedures

2.2.1 Bacteriology

E. coli: quantitative

The analysis was carried out according to Swiss food code law, chapter 56, E3 (2004). For the

sample taken at locations E1, E2, B1, B2, 0.1, 1 and 10 ml were analysed. For the other water

samples, volumes of 1, 10 and 100 ml were analysed. Samples were filtered with a 0.45-µm

cellulose nitrate membrane filter. Filters were placed onto Tryptone Bile Glucuronid Agar (TBX)


and incubated at 44°C for 18–24 hours. After incubation all blue colonies were classified as E. coli.

Confirmation is not necessary. E. coli/100 ml was calculated.

EHEC: qualitative

The analysis was carried out according to the field manual of the Basel-Land cantonal, B-P-14,

based on V.K. Sharma et al. 2002. For the sample taken at locations E1, E2, B1, B2, 0.1, 1 and 10

ml were analysed. For the other water samples, volumes of 1, 10 and 100 ml were analysed.

Filtration was done with a 0.45-µm cellulose nitrate membrane filter.

The filters were incubated in 10 ml of Modified Tryptone Soy Broth (MTSB) for 6 hours at 42°C.

After incubation the tubes were cooled down to 4°C until further processing. From each tube, 100

µl of each tube was placed on TBX and spread. Plates were incubated for 18–24 hours at 37°C.

All grown colonies were dissolved with a wet swab in 2 ml of molecular biology grade water.

DNA extraction was done by QIAGEN BioRobot EZ1 (magnetic particle technology) and the EZ1

DNA Tissue Kit.

Analysis was by real-time PCR using a LightCycler (Roche) and QuantiTect Probe PCR Kit

(Qiagen). This kit detects the three genes stx1, stx2, eae.

Primers and probes:

System stx1:

Ecoli stx1 F 5'- GAC TGC AAA GAC GTA TGT AGA TTC G -3'

Ecoli stx1 R 5'- ATC TAT CCC TCT GAC ATC AAC TGC -3'

Ecoli stx1 Probe 5’-FAM- TGA ATG TCA TTC GCT CTG CAA TAG GTA CTC -DQ-

System stx2:

Ecoli stx2 F 5'- ATT AAC CAC ACC CCA CCG -3'

Ecoli stx2 R 5'- GTC ATG GAA ACC GTT GTC AC -3'

Ecoli stx2 Probe 5’-FAM- CAG TTA TTT TGC TGT GGA TAT ACG AGG GCT TG -

System eae:

Ecoli eae F 5'- GTA AGT TAC ACT ATA AAA GCA CCG TCG-3'

Ecoli eae R 5'- TCT GTG TGG ATG GTA ATA AAT TTT TG -3'

Ecoli eae Probe 5’-FAM- AAA TGG ACA TAG CAT CAG CAT AAT AGG CTT GCT -


Salmonella/Shigella: qualitative

The analysis was carried out according to Swiss food law, chapter 56, E20 (2004). One litre of

sample water was filtered through a 0.45-µl cellulose nitrate membrane filter and a 12-µl cellulose

nitrate pre-filter. The filter was incubated in buffered peptone water, a non-selective pre-

enrichment broth, for 16–20 hours at 37°C.

- Salmonella

A 0.1-ml sample of the pre-enrichment broth was added to 10 ml Rappaport-Vasiliadis Soya Broth

and 1 ml was added to Tetrathionate Broth. Broths were incubated aerobically for 18–24 hours at

42°C.

Hektoen Enteric Agar and XLD medium (both Oxoid) were inoculated with each enrichment broth

and incubated aerobically at 37°C for 18–24 hours.

Presumptive colonies with a deep-black centre and transparent edge (fish eye) on a dark-green

(Hektoen) or red (XLD) surface were confirmed by API 20 E (Biomérieux).

-Shigella

A 10-ml sample of pre-enrichment broth was added to 100 ml of both MOSSEL-Bouillon and GN-

Enrichment bouillon HAJNA, and incubated aerobically for 18–24 hours at 37°C. Hektoen Enteric

Agar and XLD medium were inoculated with each enrichment bouillon and incubated aerobically

for 18–24 hours at 37°C.

Presumptive colonies that were green, moist, flat and transparent with a corded edge on Hektoen or

translucent with a corded edge and same colour as the medium on XLD medium were confirmed

by API 20 E (Biomérieux).

Campylobacter: qualitative

The analysis was carried out according to Swiss food code law, chapter 56, E22 (2004). A water

sample of 100 ml was filtered through a 0.2-µm mixed cellulose ester membrane filter. The filter


was incubated in a modified Campylobacter-selective enrichment broth [nutrient broth No 2

(Oxoid) + agar + lysed horse blood + antibiotic solution) for 18–24 hours at 42°C.

Gélose Campylosel (CAM, Biomeriéux)) and Karmali plates (Oxoid) were inoculated with liquid

from the microaerophile part of the jar (1–1.5 cm beneath the surface) and incubated for 18–24

hours at 42°C in a microaerophile chamber.

Presumptive colonies from both plates were inoculated on two Columbia plates (Columbia agar

with sheep blood plus; Oxoid). One plate was incubated microaerophilically, the other aerobically,

for 18–24 hours at 42°C.

Colonies that grew microaerophilically but not aerobically were confirmed with an Accuprobe

Culture Identification Reagent Kit.

Pseudomonas aeruginosa: quantitative

The analysis was carried out according to Swiss food code law, chapter 56, E4 (2004). Three

volumes of 100, 10 and 1 ml were filtered through a 0.45-µm cellulose nitrate membrane filter.

The filters were placed onto Cetrimid selective Agar and incubated aerobically for at least 24 hours

at 42°C.

All colonies pigmented green, blue, red and/or colonies fluorescing under UV light (360 nm) were

confirmed with an oxidase test kit.

Pseudomonas/100 ml was calculated.

Yersinia enterolitica:

The analysis was carried out according to Swiss food code law, chapter 56, E 7.08 (5th Edition,

volume 2). One litre of sample water was filtered through a 0.45-µl cellulose nitrate membrane

filter and a 12-µl cellulose nitrate pre-filter. The filter was incubated aerobically in Yersinia-

selective enrichment broth for 18–24 hours at 37°C.

Yersinia selective agar Nr. A13 (CIN) plates were inoculated with the enrichment broth and

incubated for 18–24 hours at 37°C. Presumptive colonies were confirmed with API E 20.


2.2.2 Virology

Quantitative real-time, one-step RT-PCR with sequence-specific probes

The analysis was carried out according to the field manual of the Basel-Land cantonal laboratory,

B-P-07 and B-P-15.

For RNA extraction, the QIAamp Viral RNA Mini Kit was used. The composition of the AVL

buffer differed from that in the handbook: 560 µl AVL buffer with carrier DNA mixed with 2440

µl AVL buffer without carrier DNA.

For rotavirus and norovirus, all steps were the same and the viruses were analysed together in 1

litre of filtered water.

Lysis of viral particles and RNA extraction

One litre of sample water was filtered through a charged 0.45-µm zetapor membrane filter and an

8-µl cellulose nitrate membrane pre-filter. The zetabor filter was placed into 3 ml of prepared AVL

buffer mixture, vortexed and incubated at room temperature for 15 min. The filter had to be in

constant contact with the buffer. Then, 3 ml ethanol (96–100%) was added and vortexed.

For extraction, a combination of the spin and vacuum protocols (Qiagen handbook, page 18 and

20) was performed: extraction was performed by using the vacuum protocol (QIAamp spin column

and a vacuum pump); washing and collection were performed using the spin protocol. The

collected RNA was frozen immediately.


Real-time, one step RT-PCR

PCR was performed using the QuantiTect Probe RT-PCR Kit.

Virus Primer - probe designation Sequence : 5'–3'

Norovirus ggI COG1F CGY TGG ATG CGN TTY CAT GA COG1R CTT AGA CGC CAT CAT CAT TYA C RING1(a)-TP FAM-AGA TYG CGA TCY CCT GTC CA- RING1(b)-TP FAM-AGA TCG CGG TCT CCT GTC CA- Norovirus gg II COG2F CAR GAR BCN ATG TTY AGR TGG ATG AG COG2R TCG ACG CCA TCT TCA TTC ACA RING2-TP FAM-TGG GAG GGC GAT CGC AAT CT- Rotavirus RVp3F, sense ACC ATC TAC ACA TGA CCC TC RVp3R, antisense GGT CAC ATA ACG CCC C

RV-Probe FAM-ATG AGC ACA ATA GTT AAA AGC

TAA CAC TGT CAA

Y = C/T, R = A/G, B ≠ A und N = A/T/G/C

Results__________________________________________________________________________ 48

3. Results

3.1. Part A: Surface water

To test the influence of SCPs on the microbiological quality of surface waters, two SCPs were

chosen. Sample sites above the effluent were compared with effluents and sample sites

downstream.

3.1.1. Indicator organisms:

Changes of E. coli density of within 0.5 log were considered as equal due to the naturally

inhomogeneous distribution of the bacterium in water or to counting mistakes on plates.

Figure 3.1 and 3.2 show box plots for E. coli density of at each sample site. Dots outside the plots

indicate outliers. Description of sample sites is in chapter 2.1.1., page 39.

Figure 3.1 Box Plot of density of E. coli at the Ergolz

Below the effluent of SCP Ergolz 2 at sample site E2, the number of E .coli was about 1 log higher

than above the effluent. The difference is significant (Mann-Whitney test, ntotal= 44, p< 0.001).The

river does not dilute the effluent water directly. For dilution it takes about 450 m (sample site E3),

where the number of E. coli was not significantly different to that at sample site E0 (Mann-

Results__________________________________________________________________________ 49

Whitney test, ntotal=40, p= 0.338) anymore. Further downstream the mean number of E. coli/100 ml

increased again and had increased by more than 0.5 log at 1200m from the SCP.

The number of E. coli in the effluent was usually higher than 10 000 E. coli/100 ml, with a

maximum of 101 000 E. coli/100 ml. Only once did it fall below 10 000 (2110 E. coli/100 ml,

20.03.2007).

Figure 3.2 Box plot of density of E. coli at the Birs

After the effluent of SCP Zwingen at sample site B2, the number of E .coli/100 ml was the same

again as above the effluent (Mann-Whitney test, ntotal= 43, p= 0.489). The river dilutes the

influence of the SCP directly. At B EI the mean number increased again, but the sample size was

very small (n= 7). Further downstream, the mean was almost equal for a distance of about 4000 m

(B4). However, the standard deviation increased, meaning that the numbers of E. coli/100 ml of

single samples were scattered over a larger range. The density range in the effluent was very broad:

from 400 to 84 000 E. coli/100 ml.

Not all sample events showed the same pattern. On several occasions, the sample sites 0 and 1 had

approximately the same number of E. coli. Twice, the number of E. coli/100 ml in B1 was smaller

than in B0 (30.4.2007, good weather, 5600 and 400 E. coli/100 ml; 30.5.2007, poor weather, 30

000 and 1600 E. coli/100ml). Table 3.1 gives an overview of the relationship between sample sites

0 and 1, increasing unit and the weather. Weather events are described as poor, if it was raining

during sampling, or if it had rained within the last 48 hours before sampling.

Results__________________________________________________________________________ 50

Table 3.1 Sample side relations of sample sides 0 and 1

Sample site relation Frequency Increase unit (log) Weather good Weather poor 1 7 4 E1 > E0 15 2 4 1

E1 = E0 6 - 1 5

1 5 3 B1 > B0 11 2 3 0

B1 = B0 9 - 3 7 B1 < B0 2 1 1 1

Sample site relation refers to number of E. coli. Weather events are described as good, if there was no rain for more than 48 hours before sampling.

Sample sites were compared for different weather events. Table AAC shows factors comparing

sample sites E0 with E1 and B0 with B1 (for good and bad weather) and factors comparing each

sample site during different weather events.

Table 3.2 Comparison of E. coli numbers between sample sites and weather events

Weather good Weather poor Factor

E0 778 (642.4 ) 5331 (6021.3) 6.9 E1 31 909 (21 767.7493) 37 874 (28 298.7) 1.2

Factor 41 7.1 B0 1335 (1668.79) 11 211 (11 230.8) 8.4 B1 26 372 (26 953.1) 19 150 (22 839.2) 0.73

Factor 19.8 1.7 Data: Mean E. coli/100ml (standard deviation). Outliers are included. Factors give the x-fold of compared sample sites.

Factors comparing sample site 0 with sample site 1 show a much higher difference for good than

for poor weather events (41 and 7.1 for the Ergolz, 19.8 and 1.7 for the Birs). In general, during

good weather, an SCP influences a river more strongly than during poor weather. Nonetheless

during poor weather, the densities for both sample sites are higher than during good weather.

Regarding single sample events during poor weather, there can be huge differences:

Birs 20.03.2007: B0 = 3900, B1 = 51 900

Birs 04.06.2007: B0 = 810 =, B1 21 000

Ergolz 05.05.2007: E0 = 3000, E1 = 61 000

Ergolz 30.05.2007: E0 = 1700, E1 = 21 000

Results__________________________________________________________________________ 51

The results suggest that the concentration of E. coli is approximately eightfold more during poor

than during good weather (6.9 for the Ergolz and 8.4 for the Birs). SCPs show almost no difference

between different weather events. The mean of SCP Ergolz 2 (E1) was a little higher for poor

weather than for good weather. SCP Zwingen had a slightly better water quality during poor than

during good weather.

Influence of weather

Each sample event was analysed with respect to weather conditions. Data from the Federal Bureau

of Hydrology were used. These data indicate discharge in m3/sec. An increasing discharge was

regarded as a poor weather event.

Figures 3.3 and 3.4 show E .coli density at sample sites E0 and B0, respectively, compared with

the discharge. These sample sites were chosen since they should be under the smallest influence of

an SCP.

Log (E. coli/100 ml) correlated with discharge except on a few dates (e.g. Ergolz 25.6.2007 –

2.7.2007, Birs 27.3.2007 – 3.4.2007).

100

1000

10000

100000

27.0

3.20

07

29.0

3.20

07

03.0

4.20

07

11.0

4.20

07

16.0

4.20

07

23.0

4.20

07

02.0

5.20

07

05.0

5.20

07

06.0

5.20

07

07.0

5.20

07

14.0

5.20

07

21.0

5.20

07

30.0

5.20

07

04.0

6.20

07

18.0

6.20

07

25.0

6.20

07

02.0

7.20

07

09.0

7.20

07

16.0

7.20

07

30.0

7.20

07

log

(E.c

oli/1

00m

l)

0.000

2.000

4.000

6.000

8.000

10.000

12.000

14.000

Dis

char

ge (

cubi

c m

eter

/sec

ond)

log (E.coli/100ml)

Discharge (cubic meter/second)

Figure 3.3 Comparison E. coli and flow (cubic meter/sec) in the Ergolz, sample site E0

Results__________________________________________________________________________ 52

100

1000

10000

100000

27.0

3.20

07

03.0

4.20

07

10.0

4.20

07

17.0

4.20

07

24.0

4.20

07

01.0

5.20

07

08.0

5.20

07

15.0

5.20

07

22.0

5.20

07

29.0

5.20

07

05.0

6.20

07

12.0

6.20

07

19.0

6.20

07

26.0

6.20

07

03.0

7.20

07

10.0

7.20

07

17.0

7.20

07

24.0

7.20

07

log

(E.c

oli/1

00m

l)

0.000

5.000

10.000

15.000

20.000

25.000

30.000

35.000

40.000

45.000

50.000

Dis

char

ge (

cubi

c m

eter

/sec

ond)

log (E.coli/100ml)Discharge (cubic meter/second)

Figure 3.4 Comparison E. coli and flow (cubic meter/sec) in the Birs, sample site B0

3.1.2. Pathogens

Analyses of surface water samples and SCP water for pathogens showed many differences between

different pathogens for both their presence/absence and the influence of the SCP.

Sample site prevalence

For the Ergolz, 137 samples were analysed during 22 sampling days. For the Birs, 147 samples

were analysed during 23 sampling days.

Results__________________________________________________________________________ 53

A. EHEC

105 samples were taken and analysed for EHEC (Table 3.3).

Table 3.3 Overview analysis for EHEC

Sample side Number of analysed samples

Number of positives

Months of sampling

E0 13 6 (46.2%) March - July E1 13 12 (92.3%) March - July E2 11 11 (100%) March - July E zw 1 1 (100%) March - April E3 8 8 (100%) March - July E4 3 3 (100%) July Total Ergolz 49 41 (83.7%) Total Ergolz (without E1) 36 29 (80.5%)

B0 14 12 (85.7%) March - July B1 14 13 (92.9%) March - July B2 13 12 (92.3%) March - July B EI 2 2 (100%) March - April B3 10 7 (70.0%) March - July B4 3 3 (100%) July Total Birs 56 48 (87.5%) Total Birs (without B1)

42 36 (85.7%)

Results__________________________________________________________________________ 54

B. Salmonella

119 samples were taken and analysed for Salmonella (Table 3.4).

Table 3.4 Overview analysis for Salmonella

Sample site Number of analysed samples

Number of positives

Months of sampling

E0 16 8 (50.0%) March - August E1 16 13 (81.3%) March - August E2 13 10 (76.9%) March - August E zw 2 0 March - April E3 10 7 (70.0%) March - July E4 4 4 (100%) July Total Ergolz 61 42 (68.8%) Total Ergolz (without E1)

45 29 (64.4%)

B0 15 4 (26.7%) March - August B1 15 6 (40.0%) March - August B2 12 4 (33.3%) March - August B EI 1 0 March - May B3 11 3 (27.3%) March - July B4 4 3 (75.0%) July Total Birs 58 20 (34.5%) Total Birs (without B1)

43 14 (32.6%)

Three isolates were sent to the National Centre for Enteropathogenic Bacteria to analyse

serovariety. They were identified as S. enterica ssp. enterica Enteritidis, S. enterica ssp. enterica

Kentucky and S. enterica ssp. enterica Brandenburg.

D. Shigella, Yersinia

A total of 83 samples each were examined between March and July. Yersinia and Shigella could

not be found in any samples.

Results__________________________________________________________________________ 55

C. Campylobacter

127 samples were taken and analysed for Campylobacter (Table 3.5).

Table 3.5 Overview analysis for Campylobacter


Number of positives

Months of sampling

E0 16 3 (18.75%) March - August E1 16 1 (6.25%) March - August E2 13 1 (7.7%) March - August E zw 2 0 March – April E3 10 0 March – July E4 4 0 July Total Ergolz 61 5 (8.2%) Total Ergolz (without E1)

45 4 (8.9%)

B0 17 1 (5.8%) March - August B1 17 0 March - August B2 14 3 (21.4%) March - August B EI 2 0 March - April B3 12 1 (8.3%) March – July B4 4 2 (50%) July Total Birs 66 7 (10.6%) Total Birs (without B1)

49 7 (14.3%)

One isolate was sent to the National Centre for Enteropathogenic Bacteria for analysis of

serovariety. It was identified as C. jejuni.

Results__________________________________________________________________________ 56

B. Pseudomonads:

95 samples were taken and analysed for Pseudomonas (Table 3.6).

Table 3.6 Overview analysis for Pseudomonas


Number of positives

Month of sampling

E0 13 11 (84.6%) March - July E1 13 12 (92.3%) March - July E2 10 10 (100%) March - July E3 7 7 (100%) March - July E4 3 3 (100%) July Total Ergolz 46 43 (93.4%) Total Ergolz (without E1)

33 31 (93.9%)

B0 13 13 (100%) March - July B1 13 12 (92.3%) March - July B2 10 10 (100%) March - July B3 10 10 (100%) March - July B4 3 3 (100%) July Total Birs 49 48 (97.9%) Total Birs (without B1)

36 36 (100%)

The concentration of bacteria in 100 ml of water ranged between 1 and 1200 cells for the Ergolz

and between 1 and 681 cells for the Birs.

Table 3.7 Quantities and statistical values for Pseudomonas in the Ergolz

Minimum 0 Range 1200 Median 170 Maximum 1200 Mean 300 SD 329.65 Amount of bacteria in 100 ml of water

Table 3.8 Quantities and statistical values Pseudomonas in the Birs

Minimum 0 Range 681 Median 22 Maximum 681 Mean 95.8 SD 161.22 Amount of bacteria in 100 ml of water

Results__________________________________________________________________________ 57

CFU = Colony forming unit

Figure 3.3: Box plot of density of Pseudomonas at the Ergolz

The effluent had a strong impact on the river Ergolz. No decrease of Pseudomonas could be

observed. The density of Pseudomonas at sample site E4 was still higher than at E0. For good

weather, E0 showed values of 0–38 CFU/100 ml (n = 5), E3 of 2 and 4 CFU/100 ml. There were

no data for E4. For poor weather there was only one event. E0 had 48 CFU/100 ml, values for E2

– E4 ranged between 160 – 460 CFU/100 ml. Up to 48 hours after poor weather, E0 showed

values of 8-480 CFU/100 ml (n = 5), E3 of 6–1200 CFU/100 ml (n = 5) and E4 130-870 CFU/100

ml (n = 3).

CFU = Colony forming unit

Figure 3.4: Box plot of density of Pseudomonas at the Birs

Results__________________________________________________________________________ 58

The river Birs diluted the influence of the SCP directly. Downstream, however, Pseudomonas/100

ml rose again (at a distance of 4000 m). For good weather, B0 showed values of 2–10 CFU/100

ml (n = 5), B3 of 2 - >30 000 CFU/100 ml (too dense to be counted, not shown). There are no data

for B4. For poor weather there was only one event. E0 had 30 CFU/100 ml, values for B2 – B4

ranged between 40 – 72 CFU/100 ml. Up to 48 hours after poor weather, B0 showed values of

5–600 CFU/100 ml (n = 5), B3 of 2–540 CFU/100 ml (n = 5) and B4 of 12–680 CFU/100 ml (n =

3).

F. Norovirus:

105 samples were taken and analysed for norovirus (Table 3.9).

Table 3.9 Overview analysis for norovirus


Number of positives

ggI ggII Months of sampling

E0 13 4 (30.8%) 2 2 March - July E1 13 4 (30.8%) 3 1 March - July E2 11 5 (45.5%) 2 3 March - July E zw 1 0 0 0 March - April E3 8 2 (25.0%) 0 2 March - July E4 3 1 (33.3%) 0 1 July Total Ergolz 49 16 (32.7%) 7 9 Total Ergolz (without E1)

36 12 (33.3%) 4 8

B0 14 2 (14.3%) 0 2 March - July B1 14 6 (42.9%) 2 4 March - July B2 13 2 (15.4%) 0 2 March - July B EI 2 0 0 0 March - Mai B3 10 2 (20.0%) 0 2 March - July B4 3 1 (33.3%) 0 1 July Total Birs 56 13 (23.2%) 2 11 Total Birs (without B1)

42 7(16.7%) 0 8

Downstream of a contamination the genogroup never changed. On 20 August, sample site B1 was

contaminated with both ggI and ggII.

Results__________________________________________________________________________ 59

E. Rotavirus

105 samples were taken and analysed for rotavirus (Table 3.10).

Table 3.10 Overview analysis for rotavirus


Number of positives

Months of sampling

E0 13 6 (46.2%) March - July E1 13 3 (23.1%) March - July E2 11 4 (36.4%) March - July E zw 1 1 (100%) March - April E3 8 1 (12.5%) March - July E4 3 0 July Total Ergolz 49 15 (30.6%) Total Ergolz (without E1) 36 12 (33.3%)

B0 14 5 (35.7%) March - July B1 14 7 (50.0%) March - July B2 13 7 (53.8%) March - July B EI 2 2 (100%) March - May B3 10 3 (30.0%) March - July B4 3 0 July Total Birs 56 24 (42.9%) Total Birs (without B1)

42 17 (40.4%)

Results__________________________________________________________________________ 60

Figures 3.5 and 3.6 summarize the findings for each pathogen at each sample site.

3

8

1

11

1313

13

13

38

1

11

41041316

16

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13

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38111

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Ezw E

3E

4

E0

E1

E2

Ezw E

3E

4

E0

E1

E2

E3

E4

E0

E1

E2

E z

w E3

E4

E0

E1

E2

E z

w E3

E4

E0

E1

E2

E z

w E3

E4

Sample side

% p

ositi

ve f

indi

ngs

Salmonella EHEC Pseudomonads

Campylobacter

Rotavirus

Norovirus

Numbers in bars = sample size

Figure 3.5 Percent of positive findings at all sample sites along the Ergolz

3

10

2

13

14

14

14

14

3

10

2

14

1717

4

128

4

31010

13

133

10

2

1314

14

4

11

1

12

15

15

0

10

20

30

40

50

60

70

80

90

100

B0

B1

B2

B E

IB

3B

4

B0

B1

B2

B E

IB

3B

4

B0

B1

B2

B3

B4

B0

B1

B2

B E

IB

3B

4

B0

B1

B2

B E

IB

3B

4

B0

B1

B2

B E

IB

3B

4

Sample side

% p

ositi

ve s

ampl

es

Salmonella EHEC Pseudomonads

Camplyobacter

Rotavirus

Norovirus

Numbers in bars = sample size

Figure 3.6 Percent positive findings at all sample sites along the Birs

Results__________________________________________________________________________ 61

Influence of SCPs

To assess the impact of SCPs, samples upstream were compared with samples of effluents for the

presence of pathogens. Rivers were negatively influenced if a) both sides (0 and 1) were positive,

or b) effluent (1) was positive and sample site above effluent (0) was negative.

Figures 3.7 and 3.8 show the percentage of negative influence.

In more than 90% of cases, both effluents (Ergolz and Zwingen) had a negative influence for

EHEC and Pseudomonas. Regarding Salmonella, the negative influence was 81.25% for the Ergolz

and 40% for the Birs, respectively. Regarding Noro- and Rotavirus, on only a few occasions was a

negative influence observed. Campylobacter was not analysed because there were few positive

samples. Figures are derived from tables in Appendix V.

0102030405060708090

100

Salmon

ella

EHEC

Pseudo

mon

ads

Rotavir

us

Norovir

us

%negative influence

no influence

Figure 3.7: Percentages of negative influence of SCP Ergolz 2

0102030405060708090

100

Salmon

ella

EHEC

Pseudo

mon

ads

Rotavir

us

Norovir

us

%negative influence

no influence

Figure 3.8: Percentages of negative influence of SCP Zwingen

Results__________________________________________________________________________ 62

3.1.3 Water qualities:

The following diagrams show water qualities in all sample sides, according to the Swiss water

quality classification system (see Table 3.9). These diagrams were used to provide an overview of

sample places over time and of different sample places during a sample event.

Figures 3.9, 3.10 and 3.11 show the water qualities of one sample site each. Figures 3.12 and 3.13

show water qualities of one sampling event each. Further figures are listed in the Appendix III &

IV.

The bars have been indicated by colours, depending on weather:

Weather good for at least the last 48 hours Rainy during sampling Rainy within the last 24 hours Rainy within the last 48 hours

Bars with a minus have no quality information since samples were not analysed for Salmonella.

Bars with a minus and 100-1000 E. coli/100 ml have quality B or C*, depending on

presence/absence of Salmonella. Bars with a minus and 1000-10 000 E. coli/100ml have quality C

or D*, depending on presence/absence of Salmonella.

Table 3.11 Classes of water quality for surface waters and bathwaters in Switzerland

Class Values Description Class A E. coli < 100 / Salmonella

not detectable Good water quality. No impairment of health expected. No recommendations

Class B E. coli 100-1000 / Salmonella not detectable

Good water quality. No impairment of health expected. No recommendations

Class C E. coli > 1000 / Salmonella not detectable E. coli up to 1000 / Salmonella detectable

Acceptable water quality. Impairment of health cannot be excluded. No diving; good shower after swimming recommended.

Class D E. coli > 1000 /Salmonella detectable

Poor water quality. Impairment of health possible. Health risk after swimming. Swimming discouraged.

E.coli: number per 100ml water. Salmonella: presence/absence in 1L of water

* = Salmonella present

Results__________________________________________________________________________ 63

E0

D*

D*

C*

D*D*

B

C

B

-

C*

C*

BB

CC*-

C- -

-

C

-

1

10

100

1000

10000

100000

20.0

3.

27.0

3.

29.0

3.

03.0

4.

11.0

4.

16.0

4.

23.0

4.

02.0

5.

05.0

5.

06.0

5.

07.0

5.

14.0

5.

21.0

5.

30.0

5.

04.0

6.

18.0

6.

25.0

6.

02.0

7.

09.0

7.

16.0

7.

30.0

7.

20.0

8.

log

(E.c

oli/1

00m

l)

Figure 3.9: Bar diagram of sampling place E0

E2

D*

C

D*D*

D*D*D*

C- -

- - CD*

D*-

- D*- D*

-

1

10

100

1000

10000

100000

20.0

3.

27.0

3.

29.0

3.

03.0

4.

11.0

4.

16.0

4.

23.0

4.

02.0

5.

05.0

5.

06.0

5.

07.0

5.

14.0

5.

21.0

5.

30.0

5.

04.0

6.

18.0

6.

25.0

6.

02.0

7.

09.0

7.

16.0

7.

30.0

7.

log

(E.c

oli/1

00m

l)

Figure 3.10: Bar diagram of sampling place E2

E3

-- -

C B - - --

- -C*

-C*

D*

B

D*D*

C*

D*

1

10

100

1000

10000

100000

20

.03

.

27

.03

.

03

.04

.

11

.04

.

16

.04

.

23

.04

.

02

.05

.

05

.05

.

06

.05

.

07

.05

.

14

.05

.

21

.05

.

30

.05

.

04

.06

.

18

.06

.

25

.06

.

02

.07

.

09

.07

.

16

.07

.

30

.07

.

log

(E.c

oli/1

00m

l)

09.07.2007: Density of E. coli too high to be counted (at least 30000/100ml). Figure 3.11: Bar diagram of sampling place E3

Results__________________________________________________________________________ 64

16.07.

--C*C*

D*D*

C*

1

10

100

1000

10000

100000

E0 E1 E2 E3 E4 E bb E mb

log

(E.c

oli/1

00m

l)

Figure 3.12: Sampling event 16.7.2007, Ergolz

16.07.

B

D*

C C* - - D*

1

10

100

1000

10000

100000

B0 B1 B2 B3 B V B T B4

log

(E.c

oli/1

00m

l)

Figure 3.13: Sampling event 16.07.2007, Birs

Table 3.12 provides an overview of all qualities of each sample site that was analysed for

Salmonella.

Table 3.12 Summary of all water qualities

B0 B2 B3 B4 E0 E2 E3 E4 A - - - - - - - - B 4 1 2 - 4 - 2 - C 7 7 6 - 4 3 1 - C* 2 1 1 1 4 - 3 1 D* 2 3 2 3 4 10 4 3

Results__________________________________________________________________________ 65

Sample places E0, B0:

Comparing sample sites above the effluents, the Birs was found to have a better quality than the

Ergolz. For both rivers, it is difficult to recognise a pattern of water qualities. The qualities differed

strongly from sample event to sample event, ranging from B up to D*. Qualities changed

frequently, e.g. at the Ergolz between the two samplings from 25 June and 2 July (B→D*), or

between 23 April and 2 May (B→C*, with only one small thunderstorm on 26 of April).

There is a slight tendency for water quality to diminish during or after rainy events. But even when

the weather remains stable, the water quality can change.


The water qualities of surface waters after an effluent differ between the Ergolz and Birs. As

already seen in part ‘3.1.2 A. Salmonella’, SCP Ergolz 2 has a stronger and more burdening

influence on the river (in 81.25% of all sample events) than SCP Zwingen (40%).


In both rivers, water qualities were similar again to those above the effluents, with qualities from B

to D*.


Only four samples each were analysed for Salmonella. All these samples were taken in July. There

was a poor water quality for all these samples. E4 tested positive for Salmonella in all four

samples. B4 tested positive three times.

The density of E. coli corresponds to the situation discussed in chapter 3.1.1. The density of E.

coli in the effluents was 1 log higher than above the effluents. In the Ergolz, the density fell again

between sample sites E2 and E3 (450 m). In the Birs, the density had already fallen by sample site

B2. Salmonella still might have been present at these sites and would have reduced the quality

further. In a few sample events, the quality of the effluent is similar to the quality above the

effluent (see figures in Appendix IV). At the Birs, on two occasions, the quality of the effluent

was better than the quality on the sample side above the SCP (density of E. coli more than 1 log

higher).

Results__________________________________________________________________________ 66

3.2 Part B: Groundwater

To validate the infiltration of surface water into groundwater, samples of surface water and

groundwater were taken in parallel. Samples were taken during three weather events: (1) from 18

to 19 July 2007, during which period the weather was good and data were used as a negative

control; (2) from 6 to 9 August 2007, during a strong rainy event (flood water); (3) from 24 to 28

September, during a week with regular but weak rainfall.

There are gaps in the results concerning E. coli in surface water, due to problems with the sampler.

During sample event 3, E. coli was found in groundwater four times (Figure 3.15). The number of

E. coli/100 ml groundwater was 1, 1, 2 and 2. The number of E. coli/100 ml surface water ranged

between 1000 and 3000. Negative groundwater samples are not shown.

During the flood water, E. coli was found in groundwater on four occasions (see Figure 3.14). The

number of E. coli/100 ml was 9, 15, 5, and 21. The number of E. coli/100 ml surface water ranged

around 10 000. Negative groundwater samples are not shown.

These data indicate that the filtering performed by the unsaturated zone in the aquifer reduced the

number of E. coli/100 ml by about a factor of 1000 (log 3).

During sample event 3, high numbers of other Enterobacteriaceae/100 ml were found (19 and 198;

Table 3.15). They were identified as Burkholderia cepacia, Enterobacter cloacae and

Acinetobacter baumanii. Under the assumption that these bacteria are reduced in the groundwater

by a factor of 1000, the number of germs/100 ml surface water must have been very high.

1

10

100

1000

10000

100000

12:3

0

16:3

0

20:3

0

00:3

0

04:3

0

08:3

0

12:3

0

16:3

0

20:3

0

00:3

0

04:3

0

08:3

0

12:3

0

16:3

0

20:3

0

00:3

0

04:3

0

08:3

0

12:3

0

log

(E. c

oli/1

00m

l)

0.000

80.000

160.000

240.000

320.000

400.000

Dis

char

ge(c

ubic

met

er/s

econ

d)

E.coli/100ml SW

E.coli/100ml GW

Water level SW

Figure 3.14: Sample event 2, 6.8. – 9.8.2007

Results__________________________________________________________________________ 67

1

10

100

1000

10000

100000

18:0

0

00:0

0

06:0

0

12:0

0

18:0

0

00:0

0

06:0

0

11:4

0

17:4

0

23:4

0

05:4

0

11:4

0

17:4

0

23:4

0

05:4

0

log

(E.c

oli/1

00m

l)

0.000

3.000

6.000

9.000

12.000

15.000

Dis

char

ge (

cubi

c m

eter

/sec

ond)

E.coli/100ml SW

E.coli/100ml GW

Other Bacteria/100ml GW

Water level SW

Figure 3.15: Sample event 3, 24.9 – 28.9.2007

A comparison of surface water turbidity with number of E. coli showed an increasing number of E.

coli with increasing turbidity (Figures 3.16 and 3.17). On the other hand, turbidity of surface water

seems to correlate with the discharge (Figures 3.18 and 3.19). Routine data for E. coli in

groundwater from the years 2000 to 2007 were compared with the discharge. These data showed

an increasing number of E. coli in groundwater with increasing discharge (Figure 3.20).

On September 10th there were 720 E. coli/100 ml ground water. The discharge was 123 m3/s. This

date was excluded in the tables.

0

2000

4000

6000

8000

10000

12000

14000

16000

0.00 1.00 2.00 3.00 4.00 5.00 6.00

Turbidity surface w ater (FNU)

E. c

oli/1

00m

l sur

face

wat

er

18.7.2007

24.09.2007

Trend line

y = 2162.3x - 73.329

Figure 3.16: Comparison of E. coli and turbidity in surface water for sample events (1) and (3)

Results__________________________________________________________________________ 68

y = 189.16x + 2216.5

0

10000

20000

30000

40000

50000

60000

0.00 50.00 100.00 150.00 200.00 250.00 300.00

Turbidity surface w ater (FNU)

E. c

oli/1

00 m

l sur

face

wat

er

06.08.2007

Trend line

Figure 3.17: Comparison of E. coli and turbidity in surface water for sample event (2)

0.000

10.000

20.000

30.000

40.000

50.000

60.000

70.000

12:30

18:30

00:30

06:30

12:30

18:30

00:30

06:30

12:30

18:30

00:30

06:30

12:30

Dis

char

ge (

cubi

c m

eter

/sec

ond)

0.00

45.00

90.00

135.00

180.00

225.00

270.00

315.00

Tur

bidi

ty s

urfa

ce w

ater

Discharge surface waterTurbidity

Figure 3.18: Comparison of discharge and turbidity for sample event (2)

0.000

2.000

4.000

6.000

8.000

10.000

12.000

14.000

16.000

18:0

0

00:0

0

06:0

0

12:0

0

18:0

0

00:0

0

06:0

0

11:4

0

17:4

0

23:4

0

05:4

0

11:4

0

17:4

0

23:4

0

05:4

0

Dis

char

ge (

cubi

c m

eter

/sec

ond)

0.00

0.80

1.60

2.40

3.20

4.00

4.80

5.60

6.40

Tur

bidi

ty s

urfa

ce w

ater

(F

NU

)

Discharge surface water

Turbidity

Figure 3.19: Comparison of discharge and turbidity for sample event (3)

Results__________________________________________________________________________ 69

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160

Discharge Birs

E. c

oli/1

00 m

l gro

und

wat

er

Figure 3.20: Routine data for E. coli in groundwater compared with discharge

To assess ground water quality, one can observe the surface water to estimate the likelihood of

finding E. coli. With increasing turbidity and discharge in the surface water, the likelihood of

finding E. coli in groundwater also increases.

There were 52 floodwaters with a discharge > 30 m3/s from 2000 – 2006 (excluding 2001; no

groundwater samples were taken the whole year). Twenty-nine of these 52 floodwaters had a

discharge greater than 50 m3/s. In 2006, there were 2 floodwaters with a discharge of 191 and 161

m3/s, respectively (according to the statistics, such floodwaters occur every 2 – 5 years). On only 3

of these 29 events was the groundwater tested for E. coli. The likelihood of E. coli infiltrating in

groundwater increases during such events (Figure 3.20).

Discussion_______________________________________________________________________ 70

4. Discussion

In this survey, the microbiological quality of surface waters bathing areas was tested. A

comparison was made between good and poor weather conditions. Two hypotheses were tested:

1) An SCP always has a great influence on the microbiological quality of surface water.

2) The quality after SCP effluent will be very poor for about 300 m. The pollution will decline with

growing distance from the effluent.

4.1. Part A: Surface water

In all tested cases, the number of E. coli/100 ml never fell below 100, hence the quality was always

classification B or worse (see table 3.10). At least one pathogen was usually detected during each

sampling event. Salmonella, EHEC and Pseudomonas were very abundant in all sample sites (see

tables 3.5 & 3.6). The other pathogens were detectable sporadically (viruses, Campylobacter) or

were absent (Shigella, Yersinia). With respect to the classification system, the quality of the Ergolz

was poorer than that of the Birs.

4.1.1. Indicator organisms:

The first hypothesis seems to be incorrect. The data showed that there can be no significant

influence (Birs). In the Ergolz, however, there was a significant influence.

These differences are probably ascribable to the different sizes of the rivers and the SCPs. The

Ergolz has a lower annual discharge than the Birs. In 2006 the mean discharge was 5.36 m3/s for

the Ergolz and 22.5 m3/s for the Birs. From 1934 to 2006, the Ergolz had a mean discharge of 3.73

m3/s with a maximal mean of 6.7 m3/s and a minimal mean of 1.59 m3/s. From 1917 to 2006, the

Birs had a mean discharge of 15.4 m3/s with a maximal mean of 28.2 m3/s and a minimal mean of

6.12 m3/s. These data indicate that the water flow through the Birs is fivefold that of the Ergolz.

The box plots on pages 48 & 49 include all data for E. coli. Splitting up data for different weather

events shows that the influence is smaller during rainy weather or the day after (see tables in

appendix IV).

Discussion_______________________________________________________________________ 71

The assumption that surface water quality improves with growing distance from the effluent also

seems to be false (Description of sample sites is in chapter 2.1.1., page 39).

The quality in the Birs was identical from sample sites B2 to B4. The increasing density at B EI is

probably due to a small sample size (n = 7): the number of E .coli/100 ml at B EI was always in the

range of sample sites B2 and B3 within one sampling event. However, there are two outliers at

sample site B4 which show a much higher number of E .coli than all sample sites upstream, the

values being even higher than in the effluent. These two events were observed during poor weather

(6.5.2007 and 18.6.2007). Somewhere between B V and B4 there must be at least one inflow

(feeding river or mixed-water overflow) that is reducing the water quality. Since the Birs has a

discharge which dilutes the influence of the SCP directly, there may well be several inflows which

are also diluted directly. Thus the observed outliers at B4 indicate a huge amount of pollution from

an inflow.

The quality in the Ergolz improved to the level from E0 somewhere between E2 and E3 (450 m

after the effluent). After this point, the quality showed continuous deterioration.

There might be several reasons for this. There is possibly a laminar flow for the water from the

effluent, which then takes a long time to mix well with the river water. In the Rhine basin, a tracer

experiment showed a general persistence of the temporal skewness of concentration distribution

(van Mazijk & Veling 2004). Two cross-analyses in the Ergolz showed higher concentrations on

the right river bank with differences of 1700 – 3600 E. coli/100 ml at sample site E3 and 300 – 500

at sample sites E bb and E mb. The effluent is located on the right river bank. Sample sites E0 and

E2 were chosen on the right bank, all others were on the left river bank. The microbiologically

burdened water from the effluent is mixing slowly with the river water on the left side, with the

result that the E. coli concentration downstream slowly increases on the left river bank. E3 showed

approximately the same concentration as E0, suggesting that at this point, effluent was still very

poorly mixed with the river water on the left side.

However, below sample site E4 there is a waterfall which mixes the water well. Sample sites E bb

and E mb downstream of the waterfall still showed slightly increasing numbers of E. coli. This fact

weakens the assumption that a slow mix of water is the only reason for worsening quality

downstream. There are probably additional inflows which burden the quality. Such an inflow was

found in the Birs which occasionally showed very high numbers of E. coli. (89/100 ml to more

than 300 000/100 ml). Several inflows were identified in the Ergolz, but only one was tested. This

inflow had very small numbers of E. coli/100 ml. The other inflows were either not water-bearing

during good weather or were not accessible. Probably the dynamics of the waterflow and the

Discussion_______________________________________________________________________ 72

consequential unequal water quantities in the cross-profile play a role in the unequal distribution of

the bacterial concentrations.

On two occasions, sample site B0 showed a higher number of E. coli/100 ml than B1, once during

good weather and once during bad weather. On these occasions, the number of E. coli/100 ml of all

sample sites downstream were more than 0.5 log lower. A ‘polluted wave’ was possibly collected

during sampling. These waves might have originated from a mixed-water overflow during poor

weather or from a polluted feeder river.

Rivers are more polluted during poor weather. Uncleaned sewage flows into rivers via mixed-water

overflows in drainage systems. SCPs have a limited capacity to clean additional rainwater. Much

of it overflows into the river. The SCP Ergolz 2 has the capacity to clean three times the maximal

inflow during good weather, while SCP Zwingen has the capacity to clean the 1.5 times the mean

inflow during good weather. During rainfall the flow into SCPs increases about fivefold, and

sewage flows into the rivers via mixed-water overflows. Hence the rivers are more polluted. Two

to three days after rainfall, the quality of the rivers improved again.

The difference in the number of E. coli/100 ml between sample site 0 (above the effluent) and 1

(effluent) is much higher during good weather than during poor weather (see table 3.2). Effluents

have only a small difference in the number of E. coli/100 ml during good weather versus poor

weather.

However, the standard deviations are very high for different weather types, rendering it difficult to

identify weather-specific patterns. For both good and poor weather, the system is very dynamic and

irregular. In general, the contamination is higher during poor than during good weather. However,

for specific weather types there are great differences.

Tables 3.3 and 3.4 show correlations for E. coli/100 ml surface water and discharge of the river.

However, there are occasions when there is no correlation. After rainfall of approximately 2 mm,

the water overflows via mixed-water overflows. Depending on river size, it can take longer for the

discharge to increase than for sewage to overflow via mixed-water overflows. This might affect the

correlation. Hence a comparison between E. coli/100 ml with amount of rainfall could have shown

better correlation.

Discussion_______________________________________________________________________ 73

4.1.2. Pathogens and the influence of SCPs:

EHEC

EHEC originates from both human and animal sources, although it is usually difficult to appraise

the precise origin. EHEC is very abundant in water. For all 78 surface water samples, 65 were

positive. For each river, more than 80% of the samples were positive, at all sample sites. In the

effluents, 25 of 27 samples (> 92%) were positive. In summer detection rate rose. EHEC was even

found in samples of 1 ml, both in surface water and effluents. No association with weather could

be observed.

The abundant presence of this bacterium in surface water is a problem for groundwater quality.

One can assume that they infiltrate into ground water with same efficiency as E. coli. If

groundwater has a high turbidity, e.g. after a long period of poor weather, EHEC might enter water

catchments and reach distribution systems. Consumption of untreated or insufficiently treated

water can lead to outbreaks, such as those observed in Montbovon (Switzerland) in 2005 (NZZ

2005; La Liberté 2005), in Swaziland in 1992 (Effler et al. 2001) and in Missouri in 1990

(Swerdlof et at. 1992). For the latter two outbreaks, serotype 0157 was the causal agent of

infection.

Samples for EHEC were considered positive if one gene was detected. Each serotype has a specific

pattern of one, two or all of the three genes stx1, stx2, stx3. To detect serotypes with combined

genes (Sharma 2002), a hybridization technique is needed, which was not available for the current

study. The results show the presence or absence of EHEC, but cannot provide information about

serotypes and their pathogenicity.

Salmonella

Salmonella is largely discussed in the section ‘Water quality in the rivers’ on page 78.

64.4% of surface water samples were positive for Salmonella in the Ergolz and 32.6% in the Birs.

E1 (effluent of SCP Ergolz 2) had a high number of positive samples, influencing the river

negatively in 81.25% of samples. At B1 (effluent of SCP Zwingen), the number of positive

samples was lower, and in 60% there was no negative influence. However, in both rivers,

Salmonella was frequently present at all sample sites.

Discussion_______________________________________________________________________ 74

Shigella, Yersinia

Shigella and Yersinia were never detected in water samples. Shigellosis and yersiniosis are very

rare in Switzerland and are usually acquired during travels to developing countries. For that reason,

the probability of detecting these germs in surface water are very small. However, according to a

staff member's statement, there was one case of shigellosis 2 years ago in which a canoeist was

infected with Shigella. This person had not travelled in the weeks prior to the infection. Hence the

inclusion of Shigella and Yersinia in this survey.

Campylobacter

Campylobacter was found in a few samples. Given that Campylobacter is the most common cause

of bacterial enteritis in industrialized countries and that it is the most commonly reported zoonosis,

high densities of Campylobacter are to be expected in surface water. A former investigation, which

evaluated an optimized method for detection of C. jejuni in surface water samples, showed very

high densities (Wiesli, 1996).

There might be several reasons for the current findings. Campylobacter can be viable, but not

culturable if present in unfavourable conditions (Rollins & Collwell, 1986), and it is possible that

the conditions may have been unfavourable during parts of the survey period.

At the beginning of survey, the volume of blood broth was too small (10 ml) and the volume of

analysed water too high (1 litre) to detect the pathogen. This was changed from the sample date

09.07.2007 onwards, after which the number of positive findings rose slightly, but still remained

low.

Pseudomonas

Pseudomonas is very abundant in surface waters. From all 67 surface water samples (Birs and

Ergolz), only two tested negative (97% positives, both negatives at E0). Up to 48 hours after poor

weather, the number of Pseudomonas/100 ml is much higher than during good weather. For

Pseudomonas, the SCPs have a negative influence on the river.

Pseudomonas contributes to non-faecal microbial pollution of waters. Bathing water standards do

not include guidelines for non-faecal pathogens, but it is these autochthonous pathogens that may

Discussion_______________________________________________________________________ 75

flourish in surface waters with high water temperatures in summer months. In Achterhoek,

Netherlands, an outbreak of otitis media was strongly associated with swimming in lakes in 1994.

These lakes met the Dutch bathing water standards and those set by the European Commission for

faecal pollution (van Asperen, 1995). Samples of lakes were analysed and showed 1–17 CFU/100

ml (median = 2). However, samples were taken after the infections had occurred, and suggested

that the number had decreased in the interim. A review of whirlpool-associated folliculitis in the

USA reported values of 1 CFU/100 ml – 107 CFU/ml (Ratnam et al. 1985). Russin et al. (1997) set

the oral infectious dose for healthy people at 108 – 109 germs, but with much lower numbers for

immunocompromised people or patients undergoing antibiotic treatment.

The levels found in the Ergolz and Birs samples are much lower (maximum of 1200 and 681,

respectively) than those for oral dose. However, the infectious dose for otitis media is likely to be

lower than for oral uptake, since the bacteria do not have to pass through the digestive system. The

surface waters examined could be a source for an infection with Pseudomonas .

Noro- and Rotavirus

Viruses were detected sporadically in both rivers. In the Ergolz, 33.3% of surface water samples

were positive for both Norovirus and Rotavirus. The effluent was positive in 30.8% samples for

Norovirus and in 23.1% for Rotavirus.

In the Birs, surface water samples were positive in 16.7% of cases for Norovirus and in 40.4% for

Rotavirus. The effluent was positive in 42.9% of cases for Norovirus and in 50% for Rotavirus.

There was usually no negative influence from the effluents. Only SCP Zwingen showed a negative

influence for Rotavirus in 50% of all samples. All other negative influences ranged between 23–

36%.

Norovirus was found four times at sample site E0 and twice at sample site B0 during good weather,

but never during poor weather.

Rotavirus was found five times at sample site E0 and three times at sample site B0 during good

weather, and once and twice, respectively, during poor weather. Taken together, 14 samples from

E0 and B0 were positive for viruses during good (n = 11 for each river) weather and 3 samples

during poor weather (n = 3 for each river).

Rota- and Norovirus are thought to be anthroponoses, and thus one can assume that virus particles

reach surface waters only via human faecal contamination. The 14 findings at sample sites E0 and

B0 do not fit well with this hypothesis. Particles did not reach the rivers via mixed-water overflows

Discussion_______________________________________________________________________ 76

since many samples were positive during good weather. Their origin is also unlikely to have been

game or cattle. Manuring with sewage sludge has been prohibited since 2005. Negative influences

of SCPs were analysed as being small. It seems likely, therefore, that the viruses originated from

effluents upstream, indicating that the particles are present in detectable amounts for a long

distance.

A recent study of Norovirus in Switzerland showed person-to-person contact being the most

important transmission route for community-acquired, sporadic Norovirus infection. There was no

association between the risk of viral gastroenteritis and consumption of foodstuffs or bottled

mineral water (Fretz et al. 2005). Other studies found virus particles in bottled mineral water

(Beuret et al. 2002) or showed association between an outbreak and contaminated wells (Beller et

al. 1997; Häfliger et al. 2000).

Specialists are aware of the problem with viruses in drinking-water processing. The

Umweltbundesamt Berlin has discussed possibilities about removal of viruses during drinking-

water processing (Szewzyk et al. 2006). Since virus particles seem to be present in detectable

amounts in surface water for a long distance, contamination of surface water might be a problem

for groundwater processing stations further downstream.

Gardia lamblia

In a survey from 2006 (Wicki 2006), an influence of SCPs on cysts of G. lamblia was established.

Two SCPs were chosen, one on the Birs (Reinach) and one on the Ergolz (Frenkendorf, ARA

Ergolz 2). Sample sites were chosen above and after the effluent and 40 samples were taken and

analysed. Of these, 39 (97.5%) tested positive. The median number of cysts found in the Birs was

16.5 (0–145) per 20 litres. More cysts were found in the Ergolz with a median of 32.5 cysts per 20

litres (2–261).

An increased cyst concentration was found after both treatment plants, with the highest

concentration found in the Ergolz. In the Ergolz, the difference before and after the effluent was

statistically significant. In the Birs, the difference was lower and not statistically significant.

However, these data and former studies (Regli 1999) indicate a wide distribution of G. lamblia in

Swiss waters. Given a low infectious dose of 1 to 10 cysts and an extrapolated initial concentration

of 0.2 – 53 cysts/litre, acquiring of an infection is possible. There could also be an infiltration into

groundwater, which serves as a supply for drinking water.

Discussion_______________________________________________________________________ 77

Cryptosporidium parvum

In a survey from 1998/1999 (Ruchti 1998/1999), a pump station fed by two groundwater sources

was investigated for C. parvum. Water from the river Lützel infiltrates into the groundwater. One

kilometre above the pump station an SCP flows into the river. Close to the pump station, there are

farms with cattle. Sample sites were chosen above and downstream of the SCP.

Forty surface water samples were taken. All of them were positive, with no significant differences

in number between sample sites. After rainy events, the number of oocysts/20 litres was

significantly higher than during good weather. Four analyses were performed for SCP water. All of

them were positive. The two groundwater sources were contaminated in 46.2 and 84.6% of cases.

Thirteen drinking water samples were taken. Two of them were positive (1 oocyst and 2 oocysts in

20 litres). An additional three samples were taken during high turbidity in groundwater. One of

these samples was positive (5 oocysts in 20 litres).

Cryptosporidium oocysts are widely distributed in the Lützel. Various studies have shown similar

results to those described above for surface waters in Switzerland (Regli, 1994), Germany (Gronik

et al. 1991; Wagner-Wiening et al. 1998) and the USA (Ongerth & Stibbs, 1987).

The SCP has no influence on the number of oocysts in the river. The two positive findings in

drinking water are not surprising given that the two sources were contaminated in 46.2% and

84.6% of samples. The situation is more difficult if turbidity is present in groundwater.

In the same area, a risk analysis was performed in 1998/1999 to assess risk factors for

contamination of surface water and groundwater with C. parvum (Hardegger, 1998/1999). Both

valley sides were investigated for potential risk factors, and showed similar risk values for trickling

and flooding. Thus the identified risk differences for both sources must have been due to different

farming methods.

Cattle farming represented the greatest animal-related risk factor. Use of sewage sludge as manure

showed no risk differences. However, very small amounts were used and sewage sludge cannot be

excluded as a potential risk factor. Analysis of field factors (dung, manure, sewage sludge and

grazing time) with respect to rainfall showed higher risks during spring and autumn. There is a

potentially higher risk for oocysts in water during spring and autumn; the oocysts may remain in

the soil for several weeks before reaching groundwater.

The only observed clearly significant difference at the human level was disposal of sewage.

Discussion_______________________________________________________________________ 78

4.1.3. Water quality in the rivers

During rainy days up to 24 hours after a rainy event, qualities ranged from C to D* (for description

of the quality classification see table 3.11, page 62). Twenty-four to 48 hours after a rainy event,

qualities ranged from B to C*. During good weather, qualities ranged from B to D*. Occasionally,

qualities changed within short time periods from B to C* or D*, even without a rainy event. During

the second half of the survey, Salmonella was detected more frequently in the Ergolz. In the Birs,

the detection rate increased only slightly. It is unclear if this increase was due to seasonality or to

poorer weather in the second survey half. However, Salmonella was also detected during good

weather. Hence their presence is not tied to weather events. In general, Salmonella are present in

both rivers frequently, with no predictable pattern.

It is difficult to predict when it is safe to use surface waters for recreational activities. Occasionally

one river had positive and negative sample sites during one sampling event. There might be several

reasons for this. A ‘contaminated or clean wave’ may have been collected during sampling.

Contaminated waves might have flowed into the rivers from effluents, mixed-water overflows or

feeding rivers. These germs could be of human or animal origin, since salmonellosis is a zoonosis.

Cattle farming is likely to have a great influence.

These results raise questions about the current inspection system (every second week if the weather

is good, no inspection during poor weather). Good results might suggest that a bathing place is

safe, but the water quality can change quickly. On the other hand, poor results may originate from

a short-term pollution event which is soon over.

According to the unequal concentration distribution described for the Ergolz, it is possible that a

bathing place which is close to an SCP, but on the opposite river bank, has a better quality than a

bathing place further downstream. Figure 5.1 shows a schematic representation of this situation.

Dark blue: contaminated water

Figure 5.1: Schematic representation of the distribution of contaminated water from the effluent in

the river

effluent

Discussion_______________________________________________________________________ 79

Rivers with similar conditions to those in the Ergolz (low annual mean discharge, non-turbulent

flow, straight streambed) should be investigated individually, to define specific sites for bathing

and to make recommendations for their use under different circumstances.

Authorities recommend not swimming for 2 – 3 days after rainfall. The only micro-organisms for

which this recommendation is relevant are E. coli, C. parvum and P. aeruginosa. E. coli and C.

parvum are detected in higher numbers during poor weather. P. aeruginosa has the highest

concentrations during the three days following rainfall. Salmonella concentrations are not clearly

tied to weather events and differences might be due to seasonality. EHEC has no association with

weather, but there is a clear seasonality. Viruses are found frequently during good weather.

The inspection system should be reconsidered and broadened. The ‘surface water’ system is too

dynamic to be validated by spot checks. Consideration must be given to not only the weather and

SCP location, but a better understanding of the entire system is also needed. Perhaps it will never

be possible to state with certainly that one specific place will be safe on a specific day.

Nevertheless, investigations should be performed which can be used to estimate risks for specific

locations during specific time periods.

Two factors should be taken into account.

1) With the help of case-control studies, risks of acquiring infections should be performed for

different pathogens. Non-faecal microbes (e.g. Pseudomonas) and viruses should be

included. [Chronic diseases have been associated with viral infections, e.g. coxsackievirus

with myocarditis. This could be significant, given that 41% of all deaths in the elderly are

associated with diseases of the heart (Rose 1996)]. If there is an increased risk for a

pathogen, further investigations should be performed regarding its behaviour in the ‘surface

water’ system.

2) Pathogens with an increased risk must be observed with a long-term survey to define their

behaviour regarding seasonality, yearly differences, weather differences, inflow by feeding

rivers and mixed-water overflows, and for small rivers, also with regard to the influence of

SCPs. Additional influences such as cattle or game animals should not be neglected for

zoonoses.

Discussion_______________________________________________________________________ 80

Such investigations are very complex and time-consuming, but they would be meaningful.

Investigations in specific bathing places could be carried out more rapidly. However, it should

never be forgotten that there are always short-term fluctuations (e.g. polluted waves), and

swimming in surface waters is never completely safe.

4.2. Part B: Groundwater

Former groundwater sample analyses showed that there is infiltration of E. coli into the

groundwater catchment of the pump station Gillmatten. Due to missing data in this study, however,

it was not possible to define the time span during which E. coli infiltrates into the groundwater.

Unfortunately (in the current study) the sampler was swept away by a flood on the day on which

the highest discharge ever was measured in the Birs (Figure 5.2). It then took some time before a

new sampler could be installed. During sample event 3, there was a problem with the batteries

leading to a gap in surface water results. However, E. coli were found in groundwater between 18 –

24 hours after the discharge of the river started to rise.

In figures 3.14 and 3.15, only positive groundwater samples are shown. During sample event 2

(floodwater), no groundwater samples were taken for 12 hours after the second positive sample

(Figure 3.14). It is not certain if E. coli were present in between. For future surveys, a sampler

should be installed in the well to take groundwater samples more regularly.

The Enterobacteriaceae that were found during sample event 3 grew on the same plates as E. coli.

No other analysis methods were carried out for other micro-organisms. Additional bacteria may be

detectable in groundwater by different methods.

http://oldie70.ch/Fotoalbum.htm picasaweb.google.com/.../GCNOd6JpBEQRVsUsRK9_cw

Figure 5.2: Floodwater in the Birs (8.8.2007)

Discussion_______________________________________________________________________ 81

The parameters turbidity, UV absorption and conductance for groundwater were very constant and

E. coli was found while these parameters did not change. It is for that reason that the parameters

for surface water were primarily analysed.

By simple observation of surface water, one can estimate if E. coli is likely to be found in

groundwater (rising turbidity and discharge). There are several floodwaters every year during

which this likelihood rises. Most of the routine data were obtained during discharges lower than 30

m3/s, and there are probably no or only a few indicator organisms detectable in groundwater during

these periods. Only during 7 of 52 floodwaters (discharge > 30 m3/s) were groundwater samples

analysed. Of these, 5 tested positive. It is highly likely, therefore, that E. coli would have often

been present in groundwater during the other 45 floodwaters.

For each well influenced by river water, a critical value regarding discharge and turbidity should be

defined. This value gives the point from which E. coli are likely to be found in groundwater. If the

river exceeds this value, tests should be mandatory.

For definition of this value, the interaction ‘river-groundwater’ must be investigated (how much

water from the river is infiltrating into the catchment). The filter capacity of the aquifer must be

defined. Additional aspects should be taken into account. After a rainfall of approximately 2 mm,

the water in drains overflows via mixed-water overflows. Contamination can vary depending on

the amount and duration of rainfall. The number of E. coli is likely to decrease during a long

rainfall due to the fact that contaminants from the environment are diluted. During heavy rainfall

the dilution probably outweighs the additional contamination. A comparison of the amount of

rainfall with the number of E. coli in surface water over time would be useful for estimation of the

point with highest contamination.

Other factors that need to be considered include the influence on the river of feeding rivers, mixed-

water overflows, cattle farms and SCPs. All of them are important regarding pathogens, which can

infiltrate into groundwater.

Improved understanding of the ‘surface water’ system will assist in the control of the

‘groundwater’ system. Quality management of groundwater is important for food safety. If one

knows the quality of the groundwater (raw product) under specific circumstances (in this case

discharge and turbidity of the river), it is easier to control drinking water quality (end product).

By defining a critical value, one can react quickly to critical situations and turn off the pump until

groundwater samples are tested. If they are found to be positive, the pump should remain switched

Discussion_______________________________________________________________________ 82

off until the contamination has decreased naturally. In this way, contamination of the end product

and the supply systems can be minimized.

4.3. Summary of the most important points

• River water qualities ranged between classification B – D*. Quality A was never detected.

• The influence of an SCP on microbiological river water quality depends on the river's

discharge.

• The surface water quality downstream of an effluent seems to depend on the discharge of

the river. Quality can decrease, possibly due to a laminar flow. Additional inflows (mixed-

water overflows, feeding rivers) also seem to play a role.

• During poor weather, contamination of rivers is higher than during good weather.

However, the river water quality is very dynamic and there are great differences for both

good and poor weather. Nevertheless, in general, river water quality improved again two to

three days after a rain event.

• For Pseudomonas and EHEC, the influence of SCPs on river water quality is largely

negative. The same applies for Salmonella in the Ergolz. For all other pathogens, there was

a negative influence in 50% or less of cases.

• River water quality is very dynamic. Sites downstream can have different qualities within

one day, or the quality of one site can change within a few days, even when the weather

conditions remain stable.

• By simple observation of surface water (discharge, turbidity) one can estimate if E. coli are

likely to be found in groundwater.

• Groundwater should be tested depending on weather events and the discharge and turbidity

of the rivers.

Prospect_________________________________________________________________________ 83

5. Prospect

This survey provides information about the presence of E. coli and various pathogens in river

water. These data lead to the suggestion that bathing in river water could have an impact on the

bather’s health, but do not provide direct evidence for this. To investigate if bathing in river water

can lead to specific diseases, quantitative water analyses and case-control studies should be

performed in parallel. This could be important for viruses, since they are associated with chronic

diseases (e.g. coxsackievirus with myocarditis). Specific bathing places should be investigated and

recommendations published.

Further studies leading to a remodelling of the control system should be performed, as described in

the discussion.

A comparison of E. coli with the amount of rainfall instead of discharge could provide more exact

curves of contamination. This could be important for further studies with groundwater. The

definition of critical values (see discussion) could affect the control system in future, leading away

from the current spot check procedure to situation-based controlling. Further studies need to be

performed in this direction. More regular sampling of groundwater is very important. In addition,

different wells should be analysed to compare different influences such as geology and the impact

of a river.

References_______________________________________________________________________ 84

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Appendix___________________________________________________________________________________________________________________________ 88

Appendix Appendix I. Data of surface water samples Weather ID: 0 Weather good since more than 48 hours

1 Rainy during sampling 2 Rainy within last 24 hours before sampling 3 Rainy within last 48 hours before sampling

A: Birs

Nr Date Sample site Weather ID E Coli Salmonella Quality EHEC Pseudomonads Campylobacter ggI ggII Rotavirus 1 12-Mrz-07 B0 1 350 neg B pos - neg neg neg pos 2 12-Mrz-07 B1 1 9900 neg C pos - neg neg neg pos 3 12-Mrz-07 B2 1 650 neg B neg - neg neg neg pos 4 12-Mrz-07 B EI 1 470 neg B pos - neg neg neg pos 5 20-Mrz-07 B0 1 3900 - - pos - neg neg neg pos 6 20-Mrz-07 B1 1 51900 - - pos - neg neg neg pos 7 20-Mrz-07 B2 1 30300 - - pos - neg neg neg pos 8 20-Mrz-07 B AI 1 32800 - - pos - neg neg neg pos 9 20-Mrz-07 B EI 1 28400 - - pos - neg neg neg pos

10 27-Mrz-07 B0 0 3100 - - - - - - - - 11 27-Mrz-07 B1 0 2400 - - - - - - - - 12 27-Mrz-07 B2 0 2500 - - - - - - - - 13 27-Mrz-07 B3 0 3700 - - - - - - - - 14 29-Mrz-07 B0 0 1300 - - - - - - - - 15 29-Mrz-07 B1 0 1100 - - - - - - - - 16 29-Mrz-07 B2 0 1500 - - - - - - - - 17 29-Mrz-07 B3 0 1700 - - - - - - - - 18 29-Mrz-07 B T 0 1500 - - - - - - - - 19 03-Apr-07 B0 0 5600 neg C - 3 neg - - - 20 03-Apr-07 B1 0 400 neg B - neg neg - - -

Appendix___________________________________________________________________________________________________________________________ 89

Nr Date Sample site Weather ID E Coli Salmonella Quality EHEC Pseudomonads Campylobacter ggI ggII Rotavirus 21 03-Apr-07 B2 0 490 - - - - - - - - 22 03-Apr-07 B3 0 290 - - - - - - - - 23 03-Apr-07 B V 0 330 - - - - - - - - 24 11-Apr-07 B0 0 190 pos C pos pos neg neg neg pos 25 11-Apr-07 B1 0 29700 neg C pos pos neg pos neg pos 26 11-Apr-07 B2 0 580 - - - - - - - - 27 11-Apr-07 B3 0 210 - - - - - - - - 28 11-Apr-07 B V 0 200 - - - - - - - - 29 16-Apr-07 B0 0 250 - - - - neg - - - 30 16-Apr-07 B1 0 56000 - - - - neg - - - 31 16-Apr-07 B2 0 1200 - - - - neg - - - 32 16-Apr-07 B3 0 350 - - - - neg - - - 33 16-Apr-07 B aw 0 770 - - - - neg - - - 34 16-Jul-07 B V 0 590 - - - - neg - - - 35 23-Apr-07 B0 0 350 pos C neg 10 neg neg neg pos 36 23-Apr-07 B1 0 7300 neg C pos 90 neg neg neg pos 37 23-Apr-07 B2 0 460 pos C pos 13 neg neg neg pos 38 23-Apr-07 B EI 0 670 - - - - - - - - 39 23-Apr-07 B3 0 250 neg B neg dense neg neg neg pos 40 23-Apr-07 B V 0 290 - - - - - - - - 41 23-Apr-07 B T 0 330 - - - - - - - - 42 23-Apr-07 B4 0 151 - - - - - - - - 43 02-Mai-07 B0 0 190 neg B pos 2 neg neg neg neg 44 02-Mai-07 B1 0 8200 neg C pos 98 neg neg neg pos 45 02-Mai-07 B2 0 8500 neg C pos 9 neg neg neg pos 46 02-Mai-07 B EI 0 1500 - - - - - - - - 47 02-Mai-07 B3 0 200 neg B neg 2 neg neg neg pos 48 02-Mai-07 B V 0 330 - - - - - - - - 49 02-Mai-07 B T 0 510 - - - - - - - - 50 02-Mai-07 B4 0 404 - - - - - - - - 51 05-Mai-07 B0 1 25000 - - - - - - - - 52 05-Mai-07 B1 1 4000 - - - - - - - - 53 05-Mai-07 B2 1 7500 - - - - - - - -

Appendix___________________________________________________________________________________________________________________________ 90

Nr Date Sample site Weather ID E Coli Salmonella Quality EHEC Pseudomonads Campylobacter ggI ggII Rotavirus 54 05-Mai-07 B EI 1 7800 - - - - - - - - 55 05-Mai-07 B3 1 6600 - - - - - - - - 56 05-Mai-07 B V 1 6200 - - - - - - - - 57 05-Mai-07 B T 1 6800 - - - - - - - - 58 05-Mai-07 B4 1 1800 - - - - - - - - 59 06-Mai-07 B0 2 9100 - - - - - - - - 60 06-Mai-07 B1 2 7400 - - - - - - - - 61 06-Mai-07 B2 2 8200 - - - - - - - - 62 06-Mai-07 B EI 2 8200 - - - - - - - - 63 06-Mai-07 B3 2 8800 - - - - - - - - 64 06-Mai-07 B V 2 6200 - - - - - - - - 65 06-Mai-07 B T 2 7600 - - - - - - - - 66 06-Mai-07 B4 2 15700 - - - - - - - - 67 07-Mai-07 B0 3 1500 neg C neg 5 neg neg pos pos 68 07-Mai-07 B1 3 2100 neg C neg 25 neg neg pos pos 69 07-Mai-07 B2 3 8400 neg C pos 10 neg neg pos pos 70 07-Mai-07 B EI 3 8800 - - - - - - - - 71 07-Mai-07 B3 3 1100 neg C neg 7 neg neg pos pos 72 07-Mai-07 B V 3 1100 - - - - - - - - 73 07-Mai-07 B T 3 900 - - - - - - - - 74 07-Mai-07 B4 3 650 - - - - - - - - 75 14-Mai-07 B0 0 480 - - - - - - - 76 14-Mai-07 B1 0 6300 - - - - - - - - 77 14-Mai-07 B2 0 1500 - - - - - - - - 78 14-Mai-07 B3 0 560 - - - - - - - - 79 14-Mai-07 B V 0 960 - - - - - - - - 80 14-Mai-07 B T 0 760 - - - - - - - - 81 14-Mai-07 B4 0 1200 - - - - - - - - 82 21-Mai-07 B0 0 1500 neg C pos 3 neg neg neg neg 83 21-Mai-07 B1 0 5000 neg C pos 35 neg neg pos neg 84 21-Mai-07 B2 0 2900 neg C pos 19 neg neg neg neg 85 21-Mai-07 B EI 0 2700 - - - - - - - - 86 21-Mai-07 B3 0 1600 neg C pos 18 neg neg neg neg

Appendix___________________________________________________________________________________________________________________________ 91

Nr Date Sample site Weather ID E Coli Salmonella Quality EHEC Pseudomonads Campylobacter ggI ggII Rotavirus 87 21-Mai-07 B V 0 1700 - - - - - - - - 88 21-Mai-07 B T 0 2100 - - - - - - - - 89 21-Mai-07 B4 0 4700 - - - - - - - - 90 30-Mai-07 B0 2 30000 - - - - - - - - 91 30-Mai-07 B1 2 1600 - - - - - - - - 92 30-Mai-07 B2 2 2000 - - - - - - - - 93 30-Mai-07 B3 2 1700 - - - - - - - - 94 30-Mai-07 B V 2 1700 - - - - - - - - 95 30-Mai-07 B T 2 2400 - - - - - - - - 96 30-Mai-07 B4 2 2500 - - - - - - - - 97 04-Jun-07 B0 3 810 neg B pos 6 neg neg neg neg 98 04-Jun-07 B1 3 21000 pos D pos 23 neg neg neg pos 99 04-Jun-07 B2 3 1200 neg C pos 15 neg neg neg neg

100 04-Jun-07 B3 3 850 neg D pos 2 neg neg neg neg 101 04-Jun-07 B V 3 1200 - - - - - - - - 102 04-Jun-07 B T 3 1200 - - - - - - - - 103 04-Jun-07 B4 3 950 - - - - - - - - 104 18-Jun-07 B0 2 4700 pos D pos 230 neg neg neg neg 105 18-Jun-07 B1 2 9000 pos D pos 180 neg neg neg neg 106 18-Jun-07 B2 2 7100 neg C pos 230 neg neg neg pos 107 18-Jun-07 B3 2 4900 pos D pos 38 neg neg neg neg 108 18-Jun-07 B V 2 3300 - - - - - - - - 109 18-Jun-07 B T 2 3400 - - - - - - - - 110 18-Jun-07 B4 2 14400 - - - - - - - - 111 25-Jun-07 B0 0 1400 neg C pos 6 pos neg neg neg 112 25-Jun-07 B1 0 24000 pos D pos 110 neg neg neg neg 113 25-Jun-07 B2 0 1400 neg C pos 18 neg neg neg neg 114 25-Jun-07 B3 0 1200 neg C pos 9 neg neg neg neg 115 25-Jun-07 B V 0 1500 - - - - - - - - 116 25-Jun-07 B T 0 1500 - - - - - - - - 117 25-Jun-07 B4 0 1500 - - - - - - - - 118 02-Jul-07 B0 2 2800 neg C pos 15 neg neg neg neg 119 02-Jul-07 B1 2 78000 pos D pos 170 neg neg pos neg

Appendix___________________________________________________________________________________________________________________________ 92

Nr Date Sample site Weather ID E Coli Salmonella Quality EHEC Pseudomonads Campylobacter ggI ggII Rotavirus 120 02-Jul-07 B2 2 6000 pos D pos 62 pos neg neg neg 121 02-Jul-07 B3 2 3000 neg C pos 14 neg neg neg neg 122 02-Jul-07 B V 2 4900 - - - - - - - - 123 02-Jul-07 B T 2 6300 - - - - - - - - 124 02-Jul-07 B4 2 1700 pos D pos 12 neg neg neg neg 125 09-Jul-07 B0 1 6800 neg C pos 30 neg neg neg neg 126 09-Jul-07 B1 1 30400 pos D pos 210 neg neg neg neg 127 09-Jul-07 B2 1 7700 pos D pos 40 pos neg neg neg 128 09-Jul-07 B3 1 7000 neg C pos 72 pos neg neg neg 129 09-Jul-07 B V 1 8100 - - - - - - - - 130 09-Jul-07 B T 1 8000 - - - - - - - - 131 09-Jul-07 B4 1 10600 pos D pos 45 pos neg neg neg 132 16-Jul-07 B0 0 330 neg B - - neg - - - 133 16-Jul-07 B1 0 84000 pos D - - neg - - - 134 16-Jul-07 B2 0 1040 neg C - - pos - - - 135 16-Jul-07 B3 0 770 pos C - - neg - - - 136 16-Jul-07 B V 0 680 - - - - - - - - 137 16-Jul-07 B T 0 1200 - - - - - - - - 138 16-Jul-07 B4 0 1300 pos D - - neg - - - 139 30-Jul-07 B0 2 26600 pos D pos 600 neg neg pos neg 140 30-Jul-07 B1 2 15000 neg C pos 230 neg neg neg neg 141 30-Jul-07 B2 2 27000 pos D pos 400 neg neg pos neg 142 30-Jul-07 B3 2 18300 pos D pos 540 neg neg pos neg 143 30-Jul-07 B V 2 22600 - - - - - - - - 144 30-Jul-07 B T 2 23400 - - - - - - - - 145 30-Jul-07 B4 2 26000 neg C pos 681 pos neg pos neg 146 20-Aug-07 B0 2 5400 neg C pos 21 neg neg neg neg 147 20-Aug-07 B1 2 23000 neg C pos 50 neg pos pos neg

Appendix___________________________________________________________________________________________________________________________ 93

B: Ergolz

Nr Date sample site Weather ID E Coli Salmonella Quality EHEC Pseudomands Campylobacter ggI ggII Rotavirus1 20-Mrz-07 E0 1 1300 neg C neg - neg neg neg pos 2 20-Mrz-07 E1 1 2110 neg C neg - neg neg neg pos 3 20-Mrz-07 E2 1 1500 neg C pos - neg neg neg pos 4 20-Mrz-07 E zw 1 1300 neg C pos - neg neg neg pos 5 20-Mrz-07 E3 1 1500 neg C pos - neg neg neg pos 6 27-Mrz-07 E0 0 2500 - - - - - - - - 7 27-Mrz-07 E1 0 15000 - - - - - - - - 8 27-Mrz-07 E2 0 3900 - - - - - - - - 9 27-Mrz-07 E3 0 1500 - - - - - - - -

10 27-Mrz-07 E nF 0 1100 - - - - - - - - 11 29-Mrz-07 E0 0 440 - - - - - - - - 12 29-Mrz-07 E1 0 15000 - - - - - - - - 13 29-Mrz-07 E2 0 2700 - - - - - - - - 14 29-Mrz-07 E4 0 620 - - - - - - - - 15 29-Mrz-07 E nF 0 700 - - - - - - - - 16 03-Apr-07 E0 0 1000 pos C - neg neg - - - 17 03-Apr-07 E1 0 33600 neg C - neg neg- - - - 18 03-Apr-07 E2 0 13000 - - - - - - - - 19 03-Apr-07 E zw 0 11500 - - - - - - - - 20 03-Apr-07 E3 0 860 - - - - - - - - 21 11-Apr-07 E0 0 1070 neg C neg pos neg pos neg neg 22 11-Apr-07 E1 0 41200 pos D pos pos neg pos neg neg 23 11-Apr-07 E2 0 18700 - - - - - - - - 24 11-Apr-07 E3 0 1100 - - - - - - - - 25 11-Apr-07 E4 0 2000 - - - - - - - - 26 11-Apr-07 E nF 0 -2000 - - - - - - - - 27 16-Apr-07 E0 0 360 neg B - - neg - - - 28 16-Apr-07 E1 0 62000 neg C - - neg - - - 29 16-Apr-07 E2 0 15400 neg C - - neg - - - 30 16-Apr-07 E zw 0 690 neg B - - neg - - - 31 16-Apr-07 E3 0 780 neg B - - neg - - -

Appendix___________________________________________________________________________________________________________________________ 94

Nr Date sample site Weather ID E Coli Salmonella Quality EHEC Pseudomands Campylobacter ggI ggII Rotavirus 32 23-Apr-07 E0 0 280 neg B neg neg neg neg neg pos 33 23-Apr-07 E1 0 81000 pos D pos 680 neg neg neg pos 34 23-Apr-07 E2 0 35000 pos D pos 200 neg neg neg pos 35 23-Apr-07 E zw 0 2000 - - - - - - - - 36 23-Apr-07 E3 0 2700 - - - - - - - - 37 23-Apr-07 E zw2 0 4200 - - - - - - - - 38 23-Apr-07 E4 0 4300 - - - - - - - - 39 02-Mai-07 E0 0 850 pos C neg 38 neg neg neg neg 40 02-Mai-07 E1 0 28000 pos D pos 500 neg pos neg neg 41 02-Mai-07 E2 0 21000 pos D pos 90 neg pos neg neg 42 02-Mai-07 E3 0 3300 - - - - - - - - 43 02-Mai-07 E nF 0 2800 - - - - - - - - 44 02-Mai-07 E bb 0 5700 - - - - - - - - 45 02-Mai-07 E mb 0 5800 - - - - - - - - 46 05-Mai-07 E0 1 3000 - - - - - - - - 47 05-Mai-07 E1 1 61000 - - - - - - - - 48 05-Mai-07 E2 1 56000 - - - - - - - - 49 05-Mai-07 E3 1 3700 - - - - - - - - 50 05-Mai-07 E4 1 7900 - - - - - - - - 51 05-Mai-07 E bb 1 9600 - - - - - - - - 52 05-Mai-07 E mb 1 13700 - - - - - - - - 53 06-Mai-07 E0 2 6800 - - - - - - - - 54 06-Mai-07 E1 2 30500 - - - - - - - - 55 06-Mai-07 E2 2 27000 - - - - - - - - 56 06-Mai-07 E3 2 6700 - - - - - - - - 57 06-Mai-07 E4 2 6800 - - - - - - - - 58 06-Mai-07 E bb 2 6200 - - - - - - - - 59 06-Mai-07 E mb 2 9300 - - - - - - - - 60 07-Mai-07 E0 3 1600 neg C neg 20 neg neg pos pos 61 07-Mai-07 E1 3 26000 pos D pos 200 neg neg pos neg 62 07-Mai-07 E2 3 22000 pos D pos 127 neg neg pos neg 63 07-Mai-07 E3 3 1400 - - - - - - - - 64 07-Mai-07 E4 3 2100 - - - - - - - -

Appendix___________________________________________________________________________________________________________________________ 95

Nr Date sample site Weather ID E Coli Salmonella Quality EHEC Pseudomands Campylobacter ggI ggII Rotavirus 65 07-Mai-07 E bb 3 4800 - - - - - - - - 66 07-Mai-07 E mb 3 4900 - - - - - - - - 67 14-Mai-07 E0 0 305 - - - - - - - - 68 14-Mai-07 E1 0 15000 - - - - - - - - 69 14-Mai-07 E2 0 13500 - - - - - - - - 70 14-Mai-07 E3 0 1400 - - - - - - - - 71 14-Mai-07 E4 0 2300 - - - - - - - - 72 14-Mai-07 E bb 0 2300 - - - - - - - - 73 14-Mai-07 E mb 0 2100 - - - - - - - - 74 21-Mai-07 E0 0 270 pos C neg 1 pos neg neg pos 75 21-Mai-07 E1 0 21000 pos D pos 32 neg neg neg pos 76 21-Mai-07 E2 0 14000 pos D pos 27 neg neg pos pos 77 21-Mai-07 E3 0 450 pos C pos 4 neg neg pos neg 78 21-Mai-07 E4 0 1100 - - - - - - - - 79 21-Mai-07 E mb 0 1600 - - - - - - - - 80 30-Mai-07 E0 2 1700 - - - - - - - - 81 30-Mai-07 E1 2 21000 - - - - - - - - 82 30-Mai-07 E2 2 7800 - - - - - - - - 83 30-Mai-07 E3 2 1600 - - - - - - - - 84 30-Mai-07 E4 2 2400 - - - - - - - - 85 30-Mai-07 E bb 2 2500 - - - - - - - - 86 30-Mai-07 E mb 2 2900 - - - - - - - - 87 04-Jun-07 E0 3 550 neg B neg 8 neg neg nge pos 88 04-Jun-07 E1 3 71000 pos D pos 550 neg neg neg neg 89 04-Jun-07 E2 3 12000 pos D pos 58 neg neg neg neg 90 04-Jun-07 E3 3 860 pos C pos 6 neg neg neg neg 91 04-Jun-07 E4 3 1700 - - - - - - - - 92 04-Jun-07 E bb 3 2500 - - - - - - - - 93 04-Jun-07 E mb 3 2500 - - - - - - - - 94 18-Jun-07 E0 2 2300 neg C pos 48 neg neg neg neg 95 18-Jun-07 E1 2 31000 pos D pos 750 neg neg neg neg 96 18-Jun-07 E2 2 7300 pos D pos 250 neg neg neg neg 97 18-Jun-07 E3 2 2700 pos D pos 180 meg neg neg neg

Appendix___________________________________________________________________________________________________________________________ 96

Nr Date sample site Weather ID E Coli Salmonella Quality EHEC Pseudomands Campylobacter ggI ggII Rotavirus 98 18-Jun-07 E4 2 3200 - - - - - - - - 99 18-Jun-07 E bb 2 5000 - - - - - - - -

100 18-Jun-07 E mb 2 5600 - - - - - - - - 101 25-Jun-07 E0 0 750 neg B pos 6 neg neg neg neg 102 25-Jun-07 E1 0 24000 pos D pos 910 neg neg neg neg 103 25-Jun-07 E2 0 6400 pos D pos 360 neg neg neg neg 104 25-Jun-07 E3 0 700 neg B pos 2 neg neg neg neg 105 25-Jun-07 E4 0 1600 - - - - - - - - 106 25-Jun-07 E bb 0 2300 - - - - - - - - 107 25-Jun-07 E mb 0 1800 - - - - - - - - 108 02-Jul-07 E0 2 10700 pos D pos 70 pos neg neg neg 109 02-Jul-07 E1 2 29000 pos D pos 660 neg neg neg neg 110 02-Jul-07 E2 2 22000 pos D pos 450 neg neg neg pos 111 02-Jul-07 E3 2 29700 pos D pos 1200 neg neg neg neg 112 02-Jul-07 E4 2 30000 pos D pos 870 neg neg neg neg 113 02-Jul-07 E bb 2 27000 - - - - - - - - 114 02-Jul-07 E mb 2 26000 - - - - - - - - 115 09-Jul-07 E0 1 20200 pos D pos 48 pos neg neg neg 116 09-Jul-07 E1 1 21000 pos D pos 1000 neg pos neg neg 117 09-Jul-07 E2 1 18000 pos D pos 350 pos pos neg neg 118 09-Jul-07 E3 1 99999 pos D pos 460 neg neg neg neg 119 09-Jul-07 E4 1 20900 pos D pos 160 neg neg neg neg 120 09-Jul-07 E bb 1 13400 - - - - - - - - 121 09-Jul-07 E mb 1 12700 - - - - - - - - 122 16-Jul-07 E0 0 730 pos C - - neg - - - 123 16-Jul-07 E1 0 15200 pos D - - neg 124 16-Jul-07 E2 0 5800 neg C - - neg - - - 125 16-Jul-07 E3 0 630 pos C - - neg - - - 126 16-Jul-07 E4 0 630 pos C - - neg - - - 127 16-Jul-07 E bb 0 1600 - - - - - - - - 128 16-Jul-07 E mb 0 1300 - - - - - - - - 129 30-Jul-07 E0 2 9100 pos D pos 480 neg neg pos pos 130 30-Jul-07 E1 2 23000 pos D pos 350 neg neg neg neg

Appendix___________________________________________________________________________________________________________________________ 97

Nr Date sample site Weather ID E Coli Salmonella Quality EHEC Pseudomands Campylobacter ggI ggII Rotavirus 131 30-Jul-07 E2 2 13000 pos D pos 560 neg neg pos neg 132 30-Jul-07 E3 2 6400 pos D pos 120 neg neg pos neg 133 30-Jul-07 E4 2 8800 pos D pos 130 neg neg pos neg 134 30-Jul-07 E bb 2 7500 - - - - - - - - 135 30-Jul-07 E mb 2 7900 - - - - - - - - 136 20-Aug-07 E0 2 1400 pos D pos 950 neg pos neg neg 137 20-Aug-07 E1 2 101000 pos D pos 300 pos neg neg neg

Appendix___________________________________________________________________________________________________________________________ 98

Appendix II. Data of groundwater samples

Date: 18.07.2007

Surface water Groundwater

Time 1ml 10ml 100ml Result*

discharge turbidity (FNU)

UV-Absorption/m

conductance (µs/cm)

Time 1ml 10ml 100ml Result* Water level (m)

turbidity (FNU)

UV-Absorption/m

conducta(µs/cm

1 08:45 4 80 d 760 11.766 1.19 4.23 424 1 08:45 0 0 0 0 300.02 - - -

2 10:45 14 90 d 950 12.496 0.86 4.19 431 10:45 300.02 3 12:45 7 82 d 800 11.341 1.28 4.08 429 12:45 300.023

4 14:45 4 68 d 650 11.200 0.68 3.99 428 2 15:00 0 0 0 0 299.96 0.1 2.08 461

5 16:45 6 69 d 680 12.244 0.62 4.12 417 16:45 299.945

6 18:45 5 63 d 620 12.103 0.66 4.00 417 18:45 299.988

7 20:45 9 48 d 520 11.620 0.70 4.53 416 20:45 299.959

8 22:45 7 60 d 610 11.583 0.90 3.98 424 3 22:00 0 0 0 0 299.93 0.1 2.06 460

9 00:45 4 41 d 410 11.382 0.84 4.01 423 00:45 300.007

10 02:45 4 53 d 520 11.177 1.03 4.01 424 02:45 299.991

11 04:45 6 103 d 990 10.817 1.20 4.05 423 04:45 300.006

12 06:45 11 79 d 820 11.698 0.78 4.22 422 06:45 299.965

13 08:45 13 127 d 1300 11.738 1.17 4.00 422 4 08:45 0 0 0 0 299.98 0.09 1.75 452

Groundwater

* E.coli/100ml

Date: 6.-9.08.2007

Surface water

Ground water

Appendix___________________________________________________________________________________________________________________________ 99

Time Date 1ml 10ml 100ml Result*

discharge turbidity (FNU)

UV-Absorption/m


Time Date 1ml 10ml 100ml Result* Water level (m)

turbidity (FNU)

UV-Absorption/m


1 12:30 6.8. 2 33 255 290 8.172 1.20 4.22 413

2 14:30 6.8. 3 34 297 300 8.116 0.90 4.10 412 1 14:00 6.8. 0 0 0 0 299.861 1.08 2.32 438

3 16:30 6.8. 2 32 212 220 8.051 0.93 4.10 411 16:30 6.8. 299.85

4 18:30 6.8. 0 27 223 250 8.296 0.91 4.10 416 2 17:00 6.8. 0 0 0 0 299.858 1.53 2.53 440 5 20:30 6.8. 1 12 127 126 8.343 0.90 4.10 412 20:30 6.8. 299.85

6* 22:30 6.8. - - - 8.210 22:30 6.8. 299.815

7* 00:30 7.8. - - - 8.343 00:30 7.8. 299.873 8* 02:30 7.8. - - - 15.291 02:30 7.8. 299.919 9* 04:30 7.8. - - - 12.124 04:30 7.8. 299.876

10* 06:30 7.8. - - - 24.180 3 07:00 7.8. 0 0 0 0 299.94 0.18 1.72 448 11* 08:30 7.8. - - - 23.096 08:30 7.8. 299.919 12* 10:30 7.8. - - - 22.814 10:30 7.8. 300.006

13* 12:30 7.8. - - - 27.670 4 12:00 7.8. 0 0 0 0 300.049 0.13 1.66 446

14 14:30 7.8. d d d 50000 41.419 268.00 9.83 371 14:30 7.8. 300.122

15 16:30 7.8. 372 d d 37000 40.317 164.00 8.73 344 5 17:00 7.8. 0 0 0 0 300.124 0.12 1.71 447

16 18:30 7.8. 324 d d 32000 35.617 130.00 11.04 327 18:30 7.8. 300.191

17 20:30 7.8. 281 d d 28000 30.452 125.00 12.93 329 20:30 7.8. 300.162

18 22:30 7.8. 248 d d 25000 28.235 123.00 15.10 340 22:30 7.8. 300.205

19 00:30 8.8. 220 d d 22000 27.295 102.00 15.47 355 00:30 8.8. 300.214

20 02:30 8.8. 176 d d 18000 25.306 68.00 14.90 371 02:30 8.8. 300.197

21 04:30 8.8. 138 d d 14000 30.114 58.00 13.20 390 04:30 8.8. 300.211

22 06:30 8.8. 209 d d 21000 32.759 55.00 11.80 391 6 07:00 8.8. 0 0 9 9 300.21 0.15 1.94 448

23 08:30 8.8. 106 d d 11000 39.083 122.00 11.10 388 08:30 8.8. 300.263

24 10:30 8.8. 175 d d 18000 43.466 58.00 10.81 399 7 10:30 8.8. 0 4 12 15 300.234 0.12 1.94 448

25 12:30 8.8. - - - 44.974 12:30 8.8. 300.324

26 14:30 8.8. - - - 39.915 14:30 8.8. 300.281

27 16:30 8.8. - - - 37.252 16:30 8.8. 300.339

28 18:30 8.8. - - - 40.235 18:30 8.8. 300.286

Appendix___________________________________________________________________________________________________________________________ 100

29 20:30 8.8. - - - 66.361 20:30 8.8. 300.396

30 22:30 8.8. - - - 134.533 22:30 8.8. 300.66

31 00:30 9.8. - - - 219.462 00:30 9.8. 301.458

32 02:30 9.8. - - - 239.844 02:30 9.8. 302.121

33 04:30 9.8. - - - 282.694 04:30 9.8. 303.032

34 06:30 9.8. - - - 328.556 06:30 9.8. 303.623

35 08:30 9.8. - - - 344.771 08:30 9.8. 304.002

36 10:30 9.8. - - - 366.831 8 11:00 9.8. 0 1 4 5 304.236 0.16 1.88 447

37 12:30 9.8. - - - 369.934 12:30 9.8. 304.216

38 14:30 9.8. - - - 356.289 9 14:00 9.8. 0 3 20 21 304.134 0.18 1.91 449

E.coli / 100 ml water

* Sampling error, no samples 22:30-12:30

Date: 24.-28.9.2007 Surface water Groundwater

Time Date 1ml 10ml 100ml Result* discharge turbidity

(FNU) UV-

Absorption/m

Conduc Tance

(µs/cm) Time Date 1ml 10ml 100ml Result* discharge turbidity

(FNU)

UV-Absorption/

m


1 18:00 24.9. 6 61 d 600 7.184 0.42 3.4 417 1 16:30 24.9. 0 0 0 0 299.852 1.4 2.2 415 2 20:00 24.9. 8 46 d 490 7.492 0.59 3.5 421 20:00 299.852

Appendix___________________________________________________________________________________________________________________________ 101

3 22:00 24.9. 5 34 d 350 7.102 0.56 3.5 420 22:00 299.85

4 00:00 25.9. 7 59 d 600 7.415 0.63 3.6 417 00:00 299.85 5 02:00 25.9. 12 73 d 770 7.393 1.07 4.0 422 02:00 299.85 6 04:00 25.9. 12 87 d 900 7.431 1.19 3.8 425 04:00 299.852 7 06:00 25.9. 13 106 d 1080 7.215 0.86 3.7 427 06:00 299.852 8 08:00 25.9. 16 100 d 1050 7.436 0.75 3.6 427 2 07:30 25.9. 0 0 0 0 299.85 5.18 3.1 431 9 10:00 25.9. 21 170 d 1700 8.454 0.82 3.8 432 10:00 299.8

10 12:00 25.9. 33 218 d 2300 7.535 0.76 4.1 432 12:00 299.858 11 14:00 25.9. 27 237 d 2400 7.036 0.80 4.3 431 14:00 299.858 12 16:00 25.9. 27 214 d 2200 7.305 0.78 4.0 429 3 16:30 25.9. 0 0 0 0 299.847 0.5 2 418 13 18:00 25.9. - - - 7.668 18:00 299.85 14 20:00 25.9. - - - 7.168 4 19:30 25.9. 0 0 0 0 299.792 0.41 1.9 423 15 22:00 25.9. - - - 7.082 22:00 299.844

16 00:00 26.9. - - - 6.868 00:00 299.844 17 02:00 26.9. - - - 7.062 02:00 299.829 18 04:00 26.9. - - - 6.879 04:00 299.848 19 06:00 26.9. - - - 7.046 06:00 299.844 20 08:00 26.9. - - - 6.918 5 07:30 26.9. 0 0 1 1 299.795 0.21 1.8 416 14 09:40 26.9. 25 225 d 2500 7.285 1.01 4.0 425 6 10:30 26.9. 0 0 0 1 299.829 0.4 1.9 419 15 11:40 26.9. 13 123 d 1300 6.744 1.66 4.0 424 11:40 299.832 16 13:40 26.9. 16 101 d 1100 6.617 0.49 4.0 429 7 13:00 26.9. 0 0 0 0 19 299.78 1.21 2.1 420 17 15:40 26.9. 20 106 d 1200 7.179 0.87 4.4 415 8 16:00 26.9. 0 0 2 2 198 299.835 0.5 1.9 416 18 17:40 26.9. 8 116 d 1100 7.272 0.48 4.0 417 17:40 299.835 19 19:40 26.9. 15 75 d 820 6.839 0.75 3.9 416 19:40 299.821 20 21:40 26.9. 11 107 d 1100 7.190 0.32 3.7 415 21:40 299.835 21 23:40 26.9. 9 144 d 1400 7.029 0.75 3.9 423 23:40 299.809

22 01:40 27.9. 8 66 d 670 7.518 0.53 4.0 428 01:40 299.838 23 03:40 27.9. 16 166 d 1600 7.993 0.49 4.0 418 03:40 299.85 24 05:40 27.9. 27 258 d 2600 9.035 1.70 4.1 413 05:40 299.864 25 07:40 27.9. 27 239 d 2400 9.187 1.74 4.0 417 9 07:15 27.9. 0 0 0 0 299.821 0.18 1.8 412 26 09:40 27.9. 76 d d 7600 10.675 4.60 4.4 425 09:40 299.902 27 11:40 27.9. 120 d d 12000 10.993 0.53 6.1 438 11:40 299.878 28 13:40 27.9. 141 d d 14100 11.748 2.09 5.0 425 10 13:15 27.9. 0 0 0 0 299.933 0.45 2.2 415 29 15:40 27.9. 73 d d 7300 12.063 2.05 5.0 411 15:40 299.962 30 17:40 27.9. 93 d d 9300 13.083 3.10 5.3 402 11 18:00 27.9. 0 1 1 2 299.95 0.2 2.1 0.419 31 19:40 27.9. 119 d d 11900 12.476 4.71 6.0 402 19:40 299.977 32 21:40 27.9. 97 d d 9700 12.080 5.69 6.0 402 21:40 299.919 33 23:40 27.9. 88 d d 8800 13.442 4.12 5.7 407 23:40 299.977

34 01:40 28.9. 65 d d 6500 12.569 2.23 5.6 408 01:40 299.983 35 03:40 28.9. 92 d d 9200 12.016 3.80 5.5 406 03:40 299.977 36 05:40 28.9. 48 d d 4800 11.523 2.94 5.6 407 12 07:15 28.9. 0 0 0 0 299.89 0.51 1.9 416

Appendix___________________________________________________________________________________________________________________________ 102

37 10:00 28.9. - - - 11.147 - - - 13 10:00 28.9. 0 0 0 0 299.89 0.19 2.1 410 * E.coli/100ml

Tuesday and Wednesday raining sporadically 7 13:00 26.9. 0 6 15 19 → Burkholderia cepacia, Enterobacter cloacae, * loss of energy, 8 samples missing 8 16:00 26.9. 0 28 190 198 Acinetobacter baumanii

Appendix III. Sample site diagrams A: Birs

B0

B

- --

C

C*

-C*

B

--

C

-

C

-

B

D*

CC

C

B

D*C

1000

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log

(E.c

oli/1

00m

l wat

er)

B2

B-

---

-

-

C*

C - - C

-C -

C

C

C

D* D*

C

D*

100

1000

10000

100000

Appendix___________________________________________________________________________________________________________________________ 103

B: Ergolz

B3

C

D*

C-C

-C

--

BB

--

- --

CC

C*

D*

1

10

100

1000

10000

100000

27.0

3.

29.0

3.

03.0

4.

11.0

4.

16.0

4.

23.0

4.

02.0

5.

05.0

5.

06.0

5.

07.0

5.

14.0

5.

21.0

5.

30.0

5.

04.0

6.

18.0

6.

25.0

6.

02.0

7.

09.0

7.

16.0

7.

30.0

7.

log

(E.c

oli/1

00m

l wat

er)

B4

C

D*

D*

D*-

-

---

--

-

--

-

1

10

100

1000

10000

100000

23.0

4.

02.0

5.

05.0

5.

06.0

5.

07.0

5.

14.0

5.

21.0

5.

30.0

5.

04.0

6.

18.0

6.

25.0

6.

02.0

7.

09.0

7.

16.0

7.

30.0

7.

log

(E.c

oli/1

00m

l)

E0

-

C

---

C

-C* C

B B

C*

C*

-

B

C

B

D*D*

C*

D*

D*

100

1000

10000

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log

(E.c

oli/1

00m

l)

E2

-D*-

D*--

D*D*

C--

--C

D*D* D*

D* D*

C

D*

100

1000

10000

100000

log

(E.c

oli/1

00m

l)

Appendix___________________________________________________________________________________________________________________________ 104

Appendix IV. Sample event diagrams A: Birs

Appendix___________________________________________________________________________________________________________________________ 105

12.03.

BB

C

B

1

10

100

1000

10000

B0 B1 B2 B EI

20.03

-

- - - -

1

10

100

1000

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100000

B0 B1 B2 B AI B EI

27.03.

----

1

10

100

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10000

B0 B1 B2 B3

29.03.

- - - - -

1

10

100

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10000

B0 B1 B2 B3 B T

03.04.

C

B - - -

1

10

100

1000

10000

B0 B1 B2 B3 B V

11.04.

---

C

C*

1

10

100

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100000

B0 B1 B2 B3 B V

16.04.

----

-

-

1

10

100

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B0 B1 B2 B3 B aw B V

23.04.

C*

C

C* -B - -

-

1

10

100

1000

10000

B0 B1 B2 B EI B3 B V B T B4

02.05.

B

C C

-

B - - -

1

10

100

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10000


Appendix___________________________________________________________________________________________________________________________ 106

05.05.

-------

-

1

10

100

1000

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100000


06.05.

--------

1

10

100

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07.05.

C C

C

C

-

- - -

1

10

100

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14.05.

-----

-

-

1

10

100

1000

10000


21.05.2007

CC C - C - -

-

1

10

100

1000

10000


30.05.

-

- - - - - -

1

10

100

1000

10000

100000


04.06.

D*

D*

C D* - - -

1

10

100

1000

10000

100000


18.06.

---D*CD*D*

1

10

100

1000

10000

100000


25.06.

---CC

D*

C

1

10

100

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100000


Appendix___________________________________________________________________________________________________________________________ 107

02.07.

C

D*

D*C - -

D*

1

10

100

1000

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100000


09.07.

CD*

D* C - - D*

1

10

100

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16.07.

B

D*

C C* - - D*

1

10

100

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log

(E.c

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30.07.

C--D*D*CD*

1

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Appendix___________________________________________________________________________________________________________________________ 108

B: Ergolz

20.03.

CCCC C

1

10

100

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10000

E0 E1 E2 E zw E3

27.03.

---

--

1

10

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E0 E1 E2 E3 E nF

29.03.

---

-

-

1

10

100

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100000

E0 E1 E2 E4 E nF

03.04.

---C

C*

110

1001000

10000100000

E0 E1 E2 E zw E3

11.04.

D*

C

-

- - -

1

10

100

1000

10000

100000

E0 E1 E2 E3 E4 E nF

16.04.

BB

CC

B

1

10

100

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100000

E0 E1 E2 E zw E3

23.04.

B

D*D*

- - - -

1

10

100

1000

10000

100000

E0 E1 E2 E zw E3 E zw 2 E4

02.05.

----

D*D*

C*

1

10

100

1000

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100000

E0 E1 E2 E3 E nF E bb E mb

05.05.

-

- -

-- - -

1

10

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100000


Appendix___________________________________________________________________________________________________________________________ 109

06.05.

------

-

1

10

100

1000

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100000


07.05.

----

D*D*

C

1

10

100

1000

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100000


14.05.

---

--

-

-

1

10

100

1000

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100000


21.05.

-C*

D* D*

C*-

1

10

100

1000

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100000

E0 E1 E2 E3 E4 E mb

30.05.

-----

-

-

1

10

100

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100000


04.06.

---C*

D*

D*

B

1

10

100

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100000


Appendix___________________________________________________________________________________________________________________________ 110

18.06.

---D*D*

D*

C

1

10

100

1000

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100000


25.06.

---B

D*D*

B

1

10

100

1000

10000

100000


02.07.

--D*D*D*D*D*

1

10

100

1000

10000

100000


09.07.

--D*D*

D*D*D*

1

10

100

1000

10000

100000


16.07.

--C*C*

D*D*

C*

1

10

100

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log

(E.c

oli/1

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30.07.

--D*D*D*D*

D*

1

10

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100000


Appendix_____________________________________________________________________ 111

Appendix V. Tables ‚influence of SCPs’ Table XYZ: Influence of SCP for Salmonella at the Ergolz

Analysis results Frequency Frequency of influence Both places negative 2 (12.5%) E0 positive, E1 negative 1 (6.25%)

3x no influence of SCP (18.75%)

Both places positive 7 (43.75%) E0 negative, E1 positive 6 (37.5%)

13x negative influence of SCP (81.25%)

Table XYZ: Influence of SCP for Salmonella at the Birs

Analysis results Frequency Frequency of influence Both places negative 6 (40%) B0 positive, B1 negative 3 (20%)

9x no influence of SCP (60%)

Both places positive 1 (6.7%) B0 negative, B1 positive 5 (33.3%)

6x negative influence of SCP (40%)

Table XYZ: Influence of SCP for EHEC at the Ergolz

Analysis results Frequency Frequency of influence Both places negative 1 (7.6%) B0 positive, B1 negative 0




Table XYZ: Influence of SCP for EHEC at the Ergolz

Analysis results Frequency Frequency of influence Both places negative 1 (7.1%) E0 positive, E1 negative 0




Table XYZ: Influence of SCP for Pseudomonas at the Ergolz

Analysis results Frequency Frequency of influence Both places negative 1 (7.7%) E0 positive, E1 negative 0




Table XYZ: Influence of SCP for Pseudomonas at the Birs

Analysis results Frequency Frequency of influence Both places negative 0 E0 positive, E1 negative 1 (7.7%)


Both places positive 12 (92.3%) E0 negative, E1 positive 0


Appendix_____________________________________________________________________ 112

Table XYZ: Influence of SCP for Rotavirus at Ergolz



Both places positive 3 (23.1%) E0 negative, E1 positive 0


Table XYZ: Influence of SCP for Rotavirus at the Birs

Analysis results Frequency Frequency of influence Both places negative 7 (50%) B0 positive, B1 negative 0

7x no influence of SCP (50%)


7x negative influence of SCP (50%)

Table XYZ: Influence of SCP for Norovirus at the Ergolz





Table XYZ: Influence of SCP for Norovirus at the Birs

Analysis results Frequency Frequency of influence Both places negative 8 (57.2%) B0 positive, B1 negative 1 (7.1%)




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