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The risk of infection constituted by enteroviruses detected in drinking water supplies analysed in this study were assessed. Coxsackie B viruses (CBV) were used as a model in these assessments. A high proportion of Coxsackie B virus infections are asymptomatic. However, clinical manifestations may range from mild, undifferentiated febrile illness or upper respiratory tract infection to a severe, systemic and sometimes fatal disease of sensitive populations. Dose-response studies suggested that an exponential model best describes infectivity of CBV. The analysis of 172 samples of treated drinking water supplies described in this study revealed the presence of CBVs in 11% (water treatment unit A) and 16% (water treatment unit B) of the samples. This incidence of CBV was used as a basis for risk assessment. The results indicated that the drinking water supplies concerned constitute a risk of CBV infection of 3.91 × 10-3 (unit A) and 7.4 × 10-3 (unit B) per year. The estimated risk of infection are about an order of magnitude higher than the yearly acceptable risk of one infection per 10,000 consumers proposed for drinking water supplies.

To meet newly proposed bromate standards, ozone doses should be decreased, thus also decreasing the risk of renal cell cancer, but the risk of infection with Cryptosporidium parvum will increase at the same time. The present study was designed to evaluate and balance these two risks, using a probabilistic risk assessment, which involves calculating the disease burden, expressed in the number of disability adjusted life-years (DALY) as developed by Havelaar et al. In the case of Neuilly-sur-Marne ozone contactors, four ozone doses were studied at 5°C and at 22°C. Results showed a sharp decrease of the disease burden with the application of ozone, and then a slight increase as the ozone dose was increased. Minimal DALYs were obtained with ozone doses of 1.5 mg/L at 22°C and 2.5 mg/L at 5°C. Nevertheless, these two ozone doses do not comply with the 10 μg/L bromate standard, as an average of 12 at 5°C and 11 at 22°C are produced.

As part of the EU project “Bacteriophages in Bathing Waters” (January 1996 - June 1999) research was carried out to optimise the method for detection and enumeration of somatic coliphages in water as described in ISO/CD 10705-2 of August 1995. It was concluded that this draft ISO standard needed to be amended in certain aspects. For determining the viable count of the host culture WG5 Escherichia coli, a membrane filtration technique should be used instead of spread plate technique as the latter gives lower and less reproducible results. A freshly prepared inoculum culture of host strain WG5 should be used instead of a frozen inoculum culture as freezing of the inoculum culture is found to negatively influence the phage counts. The double agar layer method (DAL) is preferred to the single agar layer method (SAL) for performing the phage analysis as the DAL method gives higher phage counts than the SAL method.

As part of a prospective cohort study among triathletes to determine a relationship between the microbiological quality of fresh bathing water and the risk of acquiring an intestinal infection, the exposure of the triathletes to microbiological contaminants was assessed. Waters were collected at seven triathlons (swimming course 1–1.5km) held in the summer of 1993 and 1994 to have a range of water qualities. All were influenced by sewage effluents, most also by agricultural run-off. Samples were collected several weeks before the event to establish a sampling programme (1993) and during the actual exposure of the triathletes (1993 and 1994) and examined for thermotolerant coliforms alone (samples preceding the event) and for E. coli, faecal enterococci, Staphylococcus aureus, F-specific RNAphages, enteroviruses (1993 and 1994) and for thermophilic Campylobacter, Salmonella, Aeromonas, Plesiomonas shigelloides and Pseudomonas aeruginosa (1993). The samples taken in the weeks before the exposure showed significant differences in thermotolerant coliform concentration between locations, depths and times. Also during swimmer exposure, significant differences occurred in microorganism levels at the different sampling points over the swimming course. As the triathletes swam as a group, they were exposed to approximately the same water at the same time. The geometric mean concentration was used to characterise each site. In the epidemiological study, the risk of an intestinal infection correlated with the concentration of thermotolerant coliforms and E. coli but not with the other parameters. The geometric mean concentration of thermotolerant coliforms at the triathlons ranged from 11–330/100mL and 54–1,200/100mL E. coli. Ranking of the seven sites by faecal pollution level, based on the geometric mean concentration of a faecal indicator, resulted in a different ranking for each indicator. At the fresh water sites studied, only the ratio between the geometric mean density of E. coli and thermotolerant coliforms was constant. The ratio between the other parameters related to faecal pollution (faecal enterococci, F-specific RNA phages, enteroviruses) varied considerably. Water quality standards relating to faecal pollution can only be based on parameters that show a significant correlation with risk of intestinal illness.

Shellfish are frequently contaminated by Campylobacter spp, presumably originating from faeces from gulls feeding in the growing or relaying waters. The possible health effects of eating contaminated shellfish were estimated by quantitative risk assessment. A paucity of data was encountered necessitating many assumptions to complete the risk estimate. The level of Campylobacter spp in shellfish meat was calculated on the basis of a five-tube, single dilution MPN and was strongly season-dependent. The contamination level of mussels (

The development of microbiological reference materials (RMs) has succesfully led to the certification of six materials (CRMs) and to the production of a wider range of non-certified reference materials. Both materials (RM and CRM) are prepared in the same way. They consist of gelatin capsules filled with milk powder artificially contaminated with a selected test strain. The difference between an RM and a CRM is the degree of control of the materials. Because of this difference the RMs and CRMs differ largely in price and application. An RM is intended to be used in daily quality control (QC) of microbiological (routine) analysis. For this purpose the use of a control chart can be very useful. A CRM is intended to be used incidentally, e.g. for validation of a (new) method, or for a complete check of the performance of the laboratory and technicians. The certified value of a microbiological CRM is method dependent. A user of a CRM will compare their mean results with the given certified value and confidence limits. With a so-called power calculation technique the optimal number of capsules and replicates for a CRM were calculated.

This pilot study was carried out to determine the relationship between microbiological water quality parameters and the occurrence of health complaints among triathletes. Data were collected at an Olympic distance triathlon (n=314) and a run-bike-run (n=81; controls for exposure to fresh water). At the time of the triathlon, the concentrations of Escherichia coli , thermotolerant coliforms, faecal streptococci, entero- and reoviruses, F-specific RNA phages, Salmonella, Campylobacter, Aeromonas, Plesiomonas shigelloides, Pseudomonas aeruginosa and Staphylococcus aureus were examined over the swimming course. Information on the occurrence of health complaints during the competition and in the week thereafter was collected through a written questionnaire. The results show that triathletes and run-bike-runners are comparable with respect to factors other than water exposure (age, sex, training history, physical stress, lower intestinal health complaints during the competition) that may influence the occurrence of health complaints in the week after the competition. Triathletes and run-bike-runners reported gastro-intestinal (7.7% vs 2.5%), respiratory (5.5% vs 3.7%), skin/mucosal (2.6% vs 1.2%), general (3.5% vs 1.2%) and total symptoms (14.8% vs 7.4%) in the week after the event. The health risks for triathletes for all symptom groups are not significantly higher than for run-bike-runners. The geometric mean concentration of faecal indicator bacteria is relatively low: E. coli 170/100 ml; faecal streptococci 13/100 ml, enteroviruses were present at concentrations of 0.1/l. The group of triathletes was homogeneusly and relatively intensely exposed to water; they all swam in the same body of water at the same time and 75% reported to have swallowed freshwater. It was concluded that this study design is suitable to study the relationship between health complaints and microbiological water quality. In the summers of 1993 and 1994, a study will be carried out concerning several run-bike-runs and triathlons in freshwaters of different quality.

Risk-based evaluations of the hygienic quality of drinking water require accurate data on removal and inactivation of pathogens by different steps of the treatment chain. The continuing trend to reduce chemical disinfection leads to an increased interest in the effect of other processes, based on physical removal or biological inactivation. This study reports data on the removal and inactivation of entero- and reoviruses by three such processes. For comparison, data on a variety of model organisms are also reported. All studies were carried out in the winter period because the concentration of viruses is then at its maximum, and the reducing capacities of the processes are at their minima. Storage in three reservoirs in series (average detention time 7 months) reduced the concentration of enteroviruses by a factor of 400-1,000, river bank filtration was highly effective, reducing enteroviruses by a factor of at least 10,000. The effect of coagulation/flocculation/sedimentation/filtration processes was highly variable, and was better when rapid sand filtration was included. The removal of F-specific RNA bacteriophages most closely followed that of viruses in these three processes.

UV inactivation experiments of microorganisms have been performed and published by various workers for decades. Resulting data even of the same species of microorganisms may show important differences in UV-susceptibility. The reasons for these varying results could be found either in different biological conditions like culturing methods for preparing the test organisms or in technical problems regarding UV-irradiation equipment and dose measurement. Therefore three groups working on UV inactivation performed a collaborative study to find out which influences could be responsible for varying results in laboratory UV experiments. Each working group had developed a laboratory UV irradiation apparatus, which differed in technical construction and method for UV dose measurement. For our study we used as a test organism spores of Bacillus subtilis ATCC 6633 which were cultured in large quantity, freeze-dried and stored for all following experiments. Thereby we established controlled biological conditions. The first series of experiments in 1992 showed that differences in inactivation curves did occur, related especially to dose distribution in irradiation vessels, in irradiation geometry and in partial shadowing of UV light. Subsequently the irradiation procedure and methods for dose measurement were improved resulting in consistent, reproducible and comparable results. The equation of the regression curve was: log (N/N0) = −0.013 D + 0.18. A 2 log reduction would require a dose of 169 ± 11 J/m2, a 3 log reduction 241 ± 9 J/m2, respectively (level of significance: a = 0.05). In recent years bioassay methods have been suggested in order to evaluate UV-disinfection plants. Therefore it will be of increasing public health interest to ensure the quality of laboratory UV irradiation devices used for calibration of test-organisms for these bioassays.

The general concept of measurement errors is applied to quantitative bacteriological counts on membrane filters or agar plates. The systematic errors of these methods are related to the growth characteristics of the medium (recovery of target organisms and inhibition of non-target organisms) and to its differential characteristics (sensitivity and specificity). Factors that influence the precision of microbiological counts are the variation between replicates, within samples, between operators and between laboratories. It is also affected by the linearity of the method, the verification rate and, where applicable, the number of colonies subcultured for verification. Repeatability (r) and reproducibility (R) values can be calculated on the logarithmic scale.

F-specific RNA (FRNA) bacteriophages can be assayed in wastewater by simple and rapid methods. Their inactivation by UV radiation follows first-order kinetics and relatively simple formulas can describe the effect of UV absorbance by wastewater and the wavelength-dependent killing efficiency of polychromatic lamps. Because the organisms can be grown in high concentrations, biological calibration of full-scale reactors requires only relatively small volumes of phage culture. Naturally occurring FRNA-phages were more resistant to UV than somatic coliphages, Escherichiacoli and faecal streptococci. The inactivation rate constant was almost equal to that of reoviruses, which underlines the suitability of FRNA-phages as a process indicator for UV inactivation of viruses. A pure culture of MS2 was inactivated at a rate which was almost twice that of naturally occurring FRNA-phages, indicating the necessity of designing reactors for practical applications on field-data rather than laboratory experiments.

Reference materials for water microbiology were prepared by spray-drying milk, artificially contaminated with a known test strain. The resulting highly contaminated milk powder was mixed with sterile milk powder to a contamination level of 2000-3000 cfu/g. Gelatin capsules were filled with the mixture (0.2 g/capsule) to produce the reference materials. Test strains used were: WR1 Escherichia coli, WR3 Enterobacter cloacae, WR63 Enterococcus faecium and WR51 Staphylococcus spec. Optimalization of the mixing procedure and aging of the highly contaminated milk powder resulted in relatively homogeneous and stable reference materials. Short-time challenge tests at “high” temperatures (30 and 37 °C) may be predictive for long-term stability at “low” temperatures (4-6 °C). Reference materials with strain WR3 were stable for one week at 30 °C and for at least 6 months at 4-6 °C. Possible applications of the reference materials are: quality control of routine measurements, comparison of the efficiency of different culturing methods and as a standardized sample in collaborative studies.

The development of microbiological reference materials (RMs) has succesfully led to the certification of six materials (CRMs) and to the production of a wider range of non-certified reference materials. Both materials (RM and CRM) are prepared in the same way. They consist of gelatin capsules filled with milk powder artificially contaminated with a selected test strain. The difference between an RM and a CRM is the degree of control of the materials. Because of this difference the RMs and CRMs differ largely in price and application. An RM is intended to be used in daily quality control (QC) of microbiological (routine) analysis. For this purpose the use of a control chart can be very useful. A CRM is intended to be used incidentally, e.g. for validation of a (new) method, or for a complete check of the performance of the laboratory and technicians. The certified value of a microbiological CRM is method dependent. A user of a CRM will compare their mean results with the given certified value and confidence limits. With a so-called power calculation technique the optimal number of capsules and replicates for a CRM were calculated.

As part of a prospective cohort study among triathletes to determine a relationship between the microbiological quality of fresh bathing water and the risk of acquiring an intestinal infection, the exposure of the triathletes to microbiological contaminants was assessed. Waters were collected at seven triathlons (swimming course 1–1.5km) held in the summer of 1993 and 1994 to have a range of water qualities. All were influenced by sewage effluents, most also by agricultural run-off. Samples were collected several weeks before the event to establish a sampling programme (1993) and during the actual exposure of the triathletes (1993 and 1994) and examined for thermotolerant coliforms alone (samples preceding the event) and for E. coli , faecal enterococci, Staphylococcus aureus , F-specific RNAphages, enteroviruses (1993 and 1994) and for thermophilic Campylobacter, Salmonella, Aeromonas, Plesiomonas shigelloides and Pseudomonas aeruginosa (1993). The samples taken in the weeks before the exposure showed significant differences in thermotolerant coliform concentration between locations, depths and times. Also during swimmer exposure, significant differences occurred in microorganism levels at the different sampling points over the swimming course. As the triathletes swam as a group, they were exposed to approximately the same water at the same time. The geometric mean concentration was used to characterise each site. In the epidemiological study, the risk of an intestinal infection correlated with the concentration of thermotolerant coliforms and E. coli but not with the other parameters. The geometric mean concentration of thermotolerant coliforms at the triathlons ranged from 11–330/100mL and 54–1,200/100mL E. coli . Ranking of the seven sites by faecal pollution level, based on the geometric mean concentration of a faecal indicator, resulted in a different ranking for each indicator. At the fresh water sites studied, only the ratio between the geometric mean density of E. coli and thermotolerant coliforms was constant. The ratio between the other parameters related to faecal pollution (faecal enterococci, F-specific RNA phages, enteroviruses) varied considerably. Water quality standards relating to faecal pollution can only be based on parameters that show a significant correlation with risk of intestinal illness.

Risk-based evaluations of the hygienic quality of drinking water require accurate data on removal and inactivation of pathogens by different steps of the treatment chain. The continuing trend to reduce chemical disinfection leads to an increased interest in the effect of other processes, based on physical removal or biological inactivation. This study reports data on the removal and inactivation of entero- and reoviruses by three such processes. For comparison, data on a variety of model organisms are also reported. All studies were carried out in the winter period because the concentration of viruses is then at its maximum, and the reducing capacities of the processes are at their minima. Storage in three reservoirs in series (average detention time 7 months) reduced the concentration of enteroviruses by a factor of 400-1,000, river bank filtration was highly effective, reducing enteroviruses by a factor of at least 10,000. The effect of coagulation/flocculation/sedimentation/filtration processes was highly variable, and was better when rapid sand filtration was included. The removal of F-specific RNA bacteriophages most closely followed that of viruses in these three processes.

Model organisms can be used to detect the possible presence of pathogens (index-function) or to assess the performance of a treatment process (indicator-function). To evaluate the index-function of FRNA-phages their ecology was studied. These phages were shown to be absent from feces of humans, dogs, cows, horses and to occur in relatively low numbers only in feces of pigs and calves. High counts were obtained from feces of broiler chickens (103-107 pfu/g). In various types of wastewater, counts were usually between 103 and 104 pfu/ml. These high counts could not be explained by direct fecal input. Multiplication of FRNA-phages in the environment seemed unlikely because of the necessity of bacterial host-strains to bear F-pili. These are only produced at temperatures above 30°C. It was shown however, that multiplication of FRNA-phages can also occur if a host-cell pregrown at 37°C (e.g. in the warm-blooded intestine) is transferred to environmental temperatures. Hence, the presence of FRNA-phages in environmental samples is indirectly associated with fecal pollution

F specific RNA (FRNA) bacteriophages are attractive models for the study of the fate of animal viruses in water treatment processes and pure cultures (MS2, f2) have frequently been used in model experiments. The use of indigenous FRNA phages has been hampered by the lack of selective enumeration methods. Usually, bacteriophages in sewage are enumerated by counting the number of plaques produced in a lawn of an Escherichia coli host strain. Both somatic and F specific phages will contribute to the count. We report the development of a simple and highly selective method, based on a specially constructed host strain of Salmonella typhimurium (WG 49, phage type 3 Nalr (F'42 lac :: Tn5)). Counts on this host strain normally originate exclusively from F specific phages, because there are only few somatic Salmonella phages in most sewage samples. FRNA phage counts from natural sewage samples using this host strain are more reliable and higher than those obtained on E. coli hosts. F specific bacteriophages were detected in all sewage samples investigated in numbers ranging from 102 − 5 × 104 pfu/ml. Data are presented on the number and types of F specific phages in mixed domestic/industrial sewage; in poultry processing wastewater and in hospital wastewater. The potential of F specific phages as water quality indicators is discussed.

Removal of four groups of indicator bacteria and two groups of bacteriophages in a detention pond receiving Combined Sewer Overflows (CSO) were measured. Also the survival of the bacteria in the sediments was studied. The overall removal rate of the bacteria tended to be somewhat higher than the removal rate for phages. Most values were in the range of 0.7 - 1.0 per day (e-basis). Sedimentation was of limited importance except for a short period following the overflow event. Dilution contributed in a reduction of 0.3 - 0.4 per day and die-off rates were about 0.3 - 0.6 per day, for phages maybe as low as 0.2 per day. Survival of the bacteria in the sediment was much better than in the water, net loss rates were about 0.1 per day, resulting in a more or less continuous contamination of the sediment with pathogenic bacteria as represented by indicator organisms.