F-specific RNA Bacteriophages Research Articles

Disinfection Efficiency of Peracetic Acid (PAA): Inactivation of Coliphages and Bacterial Indicators in a Municipal Wastewater Plant

The aim of the study was to assess the efficiency of low doses of peracetic acid against viral and bacterial indicators in wastewater and to evaluate if the treatment allows regulatory requirements to be satisfied. A total of 31 samplings were carried out, each involving the collection of secondary effluent and of effluent disinfected with 1.2 or 1.5 mg 1−1 of peracetic acid (contact time 20 minutes). In each sample were measured: somatic coliphages, F-specific RNA bacteriophages, Escherichia coli, total and faecal coliforms, enterococci. Peracetic acid disinfection showed significant differences between the reductions of the microorganisms tested: E. coli showed the highest reduction (1.78 and 2.43 Log respectively with 1.2 and 1.5 mg 1−1 of peracetic acid) and phages the lowest (ranging between 0.52 and 0.60 Log). Only a concentration of 1.5 mg 1−1 of peracetic acid would enable the effluent to be discharged into surface waters in compliance with Italian regulations. The variability of microbial resistance against the peracetic acid disinfection treatment, underlines the importance of assessing disinfection efficiency by using more than one indicator microorganism. The detection of E. coli could be usefully accompanied by tests for more resistant microorganisms such as enterococci or coliphages. In conclusion, peracetic acid can be used for the disinfection of effluents even at low doses, with the advantage of reducing costs and preventing the formation of significant amounts of genotoxic by-products.

Field investigations on the survival of Escherichia coli and presence of other enteric micro-organisms in biosolids-amended agricultural soil

To measure the survival of enteric micro-organisms in agricultural soil amended with conventional and enhanced treated biosolids in relation to environmental and edaphic conditions. Escherichia coli, Salmonella and F-specific RNA bacteriophage were enumerated in sludge and amended soil. Salmonella was not detected and only small numbers of bacteriophages were found in conventional, dewatered mesophilic anaerobically digested biosolids (DMAD). Neither organism was detected in soil. Escherichia coli numbers in soil increased with DMAD application compared with the unamended control, or soil receiving enhanced treated, thermally dried digested (TDD) and composted (CPT) biosolids. Empirical statistical models were developed summarizing the relationship between soil temperature, moisture content and time and E. coli populations. Background numbers of E. coli declined with increasing soil temperature and decreasing soil moisture responding to seasonal patterns in environmental conditions. Time following application was the only significant explanatory variable of E. coli numbers and decay in DMAD-amended soil. E. coli are an indigenous component of the microbial community in field soil and populations increased in cool, moist soil during autumn-winter and declined in warm, dryer soil during spring-summer. Enhanced treated biosolids were not a source of E. coli, but reduced the size of the indigenous population possibly by stimulating the activity of predatory and competing soil flora because of the organic substrate input from sludge. Conventionally treated biosolids increased E. coli numbers in soil. However, introduced bacteria declined rapidly and survival was limited to 3 months, irrespective of the timing of sludge application or environment. The results provide assurance that residual numbers of pathogens applied to soil in treated biosolids decay to background values well within cropping and harvesting restrictions imposed when sewage sludge is spread on farmland.

Evaluation of Two Library-Independent Microbial Source Tracking Methods To Identify Sources of Fecal Contamination in French Estuaries

In order to identify the origin of the fecal contamination observed in French estuaries, two library-independent microbial source tracking (MST) methods were selected: (i) Bacteroidales host-specific 16S rRNA gene markers and (ii) F-specific RNA bacteriophage genotyping. The specificity of the Bacteroidales markers was evaluated on human and animal (bovine, pig, sheep, and bird) feces. Two human-specific markers (HF183 and HF134), one ruminant-specific marker (CF193'), and one pig-specific marker (PF163) showed a high level of specificity (>90%). However, the data suggest that the proposed ruminant-specific CF128 marker would be better described as an animal marker, as it was observed in all bovine and sheep feces and 96% of pig feces. F RNA bacteriophages were detected in only 21% of individual fecal samples tested, in 60% of pig slurries, but in all sewage samples. Most detected F RNA bacteriophages were from genotypes II and III in sewage samples and from genotypes I and IV in bovine, pig, and bird feces and from pig slurries. Both MST methods were applied to 28 water samples collected from three watersheds at different times. Classification of water samples as subject to human, animal, or mixed fecal contamination was more frequent when using Bacteroidales markers (82.1% of water samples) than by bacteriophage genotyping (50%). The ability to classify a water sample increased with increasing Escherichia coli or enterococcus concentration. For the samples that could be classified by bacteriophage genotyping, 78% agreed with the classification obtained from Bacteroidales markers.

Somatic coliphages and bacterial indicators of bathing water quality in the beaches of Gipuzkoa, Spain

Monitoring the quality of the bathing waters of Gipuzkoa (the Basque Country, Spain) makes it possible to assess the suitability of its 15 beaches for bathing throughout each season. In 1998, the parameters E. coli, somatic coliphages (SOMCPH) and F-specific RNA bacteriophages (FRNAPH) were incorporated into the bathing water quality monitoring system. This enabled the study of the link between bacterial and viral indicators as well as the analysis of the ratios between both types of indicators in waters with different levels of pollution. Although bacterial indicators (total coliforms (TC) and faecal coliforms (FC)) and enterococci showed a strong correlation between them, the correlations between the viral indicators and between the viral and bacterial indicators were weaker, though significant in all cases. The ratio between SOMCPH and E. coli indicates that at low levels of bacterial pollution (E coli <100 MPN/100 ml) SOMCPH outnumber E coli. In contrast, at higher levels of pollution (E coli >100 MPN/100 ml), SOMCPH numbers are lower than those of E-coli. The data reveal the presence of viral indicators in waters classified as suitable for bathing by the European Directive and alert us to their suitability.

Inactivation of Poliovirus 1 and F-Specific RNA Phages and Degradation of Their Genomes by UV Irradiation at 254 Nanometers

Several models (animal caliciviruses, poliovirus 1 [PV1], and F-specific RNA bacteriophages) are usually used to predict inactivation of nonculturable viruses. For the same UV fluence, viral inactivation observed in the literature varies from 0 to 5 logs according to the models and the methods (infectivity versus molecular biology). The lack of knowledge concerning the mechanisms of inactivation due to UV prevents us from selecting the best model. In this context, determining if viral genome degradation may explain the loss of infectivity under UV radiation becomes essential. Thus, four virus models (PV1 and three F-specific RNA phages: MS2, GA, and Qbeta) were exposed to UV radiation from 0 to 150 mJ.cm-2. PV1 is the least-resistant virus, while MS2 and GA phages are the most resistant, with phage Qbeta having an intermediate sensitivity; respectively, 6-log, 2.3-log, 2.5-log, and 4-log decreases for 50 mJ.cm-2. In parallel, analysis of RNA degradation demonstrated that this phenomenon depends on the fragment size for PV1 as well as for MS2. Long fragments (above 2,000 bases) for PV1 and MS2 fell rapidly to the background level (>1.3-log decrease) for 20 mJ.cm-2 and 60 mJ.cm-2, respectively. Nevertheless, the size of the viral RNA is not the only factor affecting UV-induced RNA degradation, since viral RNA was more rapidly degraded in PV1 than in the MS2 phage with a similar size. Finally, extrapolation of inactivation and UV-induced RNA degradation kinetics highlights that genome degradation could fully explain UV-induced viral inactivation.

Development of real-time RT-PCR methods for specific detection of F-specific RNA bacteriophage genogroups: Application to urban raw wastewater

F-specific RNA bacteriophages have been classified into four genogroups (GI, GII, GIII and GIV). It was suggested that two of these genogroups are more frequent in human excreta (GII and GIII) and the two other (GI and GIV) are specific for animal excreta. Real-time RT-PCR methods using TaqMan MGB probe were developed to detect the four genogroups. Primers and probes of each specific RT-PCR were designed to target all sequenced bacteriophages belonging to one genogroup, without cross-reactivity with other genogroups. These four methods showed detection limits ranging between 0.01 and 10 PFU/mL and PCR efficiencies ranging between 87 and 95%. The newly methods were tested in urban raw wastewater. Genogroups I and II were detected in all samples ( n = 7); GIII in six samples and GIV was never detected. GI was predominant in one sample, in which the quantity of Cryptosporidium and Giardia was, respectively, three and eight times higher than the mean values. Because GI is mainly observed in animals, it was hypothesized that this increase was due to an animal input. The use of F-specific RNA phage genotyping to estimate the origin of faecal pollution requires appropriate validation. In this context, real-time RT-PCR will undoubtedly be useful.

Integrated Analysis of Established and Novel Microbial and Chemical Methods for Microbial Source Tracking

Several microbes and chemicals have been considered as potential tracers to identify fecal sources in the environment. However, to date, no one approach has been shown to accurately identify the origins of fecal pollution in aquatic environments. In this multilaboratory study, different microbial and chemical indicators were analyzed in order to distinguish human fecal sources from nonhuman fecal sources using wastewaters and slurries from diverse geographical areas within Europe. Twenty-six parameters, which were later combined to form derived variables for statistical analyses, were obtained by performing methods that were achievable in all the participant laboratories: enumeration of fecal coliform bacteria, enterococci, clostridia, somatic coliphages, F-specific RNA phages, bacteriophages infecting Bacteroides fragilis RYC2056 and Bacteroides thetaiotaomicron GA17, and total and sorbitol-fermenting bifidobacteria; genotyping of F-specific RNA phages; biochemical phenotyping of fecal coliform bacteria and enterococci using miniaturized tests; specific detection of Bifidobacterium adolescentis and Bifidobacterium dentium; and measurement of four fecal sterols. A number of potentially useful source indicators were detected (bacteriophages infecting B. thetaiotaomicron, certain genotypes of F-specific bacteriophages, sorbitol-fermenting bifidobacteria, 24-ethylcoprostanol, and epycoprostanol), although no one source identifier alone provided 100% correct classification of the fecal source. Subsequently, 38 variables (both single and derived) were defined from the measured microbial and chemical parameters in order to find the best subset of variables to develop predictive models using the lowest possible number of measured parameters. To this end, several statistical or machine learning methods were evaluated and provided two successful predictive models based on just two variables, giving 100% correct classification: the ratio of the densities of somatic coliphages and phages infecting Bacteroides thetaiotaomicron to the density of somatic coliphages and the ratio of the densities of fecal coliform bacteria and phages infecting Bacteroides thetaiotaomicron to the density of fecal coliform bacteria. Other models with high rates of correct classification were developed, but in these cases, higher numbers of variables were required.

Effect of high hydrostatic pressure on four genotypes of F-specific RNA bacteriophages

The pressure responses of four genotypes of F-specific RNA bacteriophages, f2, GA, Qbeta and SP, were evaluated with respect to pressure magnitude, treatment temperature and suspending medium. The pressure responses were studied with respect to pressure magnitude (350 to 600 MPa), treatment temperature (-10 to 50 degrees C) and suspending media. Phages f2 and GA had much higher pressure resistances than Qbeta and SP. Pressure resistances of Qbeta and SP were enhanced with increase in salt concentrations in the range of 350 to 600 MPa from -10 to 50 degrees C in PBS. Qbeta and SP had greater pressure resistances when suspended in phosphate-buffered saline (PBS) with added glucose (5%, w/w), UHT whole milk and Dulbecco's Modified Eagle's Medium plus 10% fetal bovine sera than they did in PBS. Two surfactants, sucrose laurate and monolaurin, and one chelating agent, ethylenediamine tetraacetic acid (EDTA), increased the pressure resistance of Qbeta and SP, but had modest effect on either f2 or GA. Four representative F-specific RNA bacteriophages, f2 (serotype I), GA (serotype II), Qbeta (serotype III) and SP (serotype IV) showed different resistances to hydrostatic pressure in the range of 350-600 MPa. This study screened for practical surrogates of HAV for validation of commercial high hydrostatic pressure processing.

Occurrence of bacterial indicators and bacteriophages infecting enteric bacteria in groundwater in different geographical areas

The aim of this research was to determine the suitability of coliphages (bacteriophages) for assessing the microbial quality of groundwater. The number of several bacterial indicators (faecal coliforms, Escherichia coli, enterococci and spores of sulfite-reducing clostridia) and bacteriophages (somatic coliphages, F-specific RNA bacteriophages and bacteriophages infecting Bacteroides fragilis) were determined in groundwater of aquifers in various geographical areas. Results show that the relative abundance, determined as percentages of positive detections, of the bacterial indicators and bacteriophages varies depending on the aquifer. A single bacterial indicator may not be enough to assess microbiological quality in certain aquifers. One bacterial indicator and a bacteriophage parameter provide more information than two bacterial indicators. Coliphages (CPH) provide different information from that provided by bacterial indicators on the microbial quality of groundwater in different geographical areas. Easy, fast and inexpensive methods for the detection of CPH are feasible in both industrialized and developing countries.

Inactivation of viruses, bacteria, spores and protozoa by ultraviolet irradiation in drinking water practice: a review

The inactivation rate constants (k-value) of pathogenic viruses, bacteria and protozoa and indicator or model microorganisms for monochromatic UV253.7 radiation have been calculated from literature studies describing experiments of seeded microorganisms in collimated beam apparatus with low-pressure lamps. Results show that data of most studies fitted well in the first-order Chick-Watson disinfection kinetics. Viruses require the highest UV fluence and the double-stranded DNA adenovirus is by far the most persistent species with an UV fluence requirement of 125 mJ/cm2 for 3 log inactivation. Translation of these k-values to full-scale UV systems requires accurate fluence assessment in these continuous flow systems with fluence distribution, variations in water quality (UV transmittance) and operational aspects like lamp age and fouling. Determination of the reduction equivalent fluence or REF with seeded model organisms is an accepted methodology to validate UV systems. Frequently used model organisms F-specific RNA phage MS2 and spores of Bacillus subtilis have low k-values. For validation of systems with low fluence requirement to inactivate UV sensitive organisms, alternative model organisms are suggested. Further research is needed to verify the potential of REF verification of full-scale systems by monitoring inactivation of indigenous spores (anaerobic or aerobic) with large volume sampling.

Enteroviruses and Bacteriophages in Bathing Waters

A new procedure for detecting and counting enteroviruses based on the VIRADEN method applied to 10 liters of seawater was examined. It improved the efficiency of detection by taking into account both the number of positive isolations and numbers found with traditional methods. It was then used to quantify viruses in bathing waters. A number of bacterial indicators and bacteriophages were also tested. Cultivable enteroviruses were detected in 55% of the samples, most of which complied with bacteriological criteria. In contrast, viral genomes were only detected in 20% of the samples by reverse transcription-PCR. Somatic coliphages outnumbered all other indicators. F-specific RNA phages were detected in only 15% of the samples, whereas phages infecting Bacteroides thetaiotaomicron were detected in 70% of samples. A numerical relationship between the numbers of enteroviruses and the numbers of enterococci and somatic coliphages was observed. In situ inactivation experiments showed that viruses persisted significantly longer than the bacterial indicators. Only somatic coliphages and bacteriophages infecting Bacteroides persisted longer than the viruses. These results explain the numbers of enteroviruses and indicators in bathing waters attending the numbers usually found in sewage in the area. Somatic coliphages show a very good potential to predict the risk of viruses being present in bathing waters.

Enumeration of bacteriophages in water by different laboratories of the European Union in two interlaboratory comparison studies

As part of a European project on bacteriophages in bathing waters two interlaboratory comparison studies were carried out (May 1997 and March 1998). During these studies phage reference materials as well as naturally polluted standard samples were analysed in 16 European laboratories. Three groups of bacteriophages were tested using standardised methods: somatic coliphages, F-specific RNA-phages and phages of Bacteroides fragilis. Many of the participating laboratories applied one or more of the phage methods for the first time, after a one-week training session in a central laboratory. Nevertheless, the values of repeatability ( r = 1.35–1.38 calculated on log 10-scale) and reproducibility ( R = 1.52–2.04 calculated on log 10-scale) when analysing phage reference materials were close to the theoretical optimum for a Poisson distribution. When analysing the naturally polluted samples more variation in results within and between laboratories was found ( r = 1.63–2.34; R = 3.10–5.72), in comparison with the results obtained with the pure phage reference materials.

Tracking the origin of faecal pollution in surface water: an ongoing project within the European Union research programme

The objectives of this study are to generate knowledge about methods to track the sources of faecal pollution in surface waters, with the aim of having one or a few easy procedures applicable to different geographic areas in Europe. For this, a first field study using already proposed methods (genotypes of F-specific RNA bacteriophages, bacteriophages infecting Bacteroides fragilis, phenotypes of faecal coliforms and enterococci, and sterols) has been done in five areas representing a wide array of conditions in Europe. The present faecal indicators (faecal coliforms, enterococci, sulfite reducing clostridia and somatic coliphages) have also been included in this first field study. At the same time some emerging methods have been settled or adapted to water samples and assayed in a limited number of samples. The results of this first field study indicate that no single parameter alone is able to discriminate the sources, human or non-human, of faecal pollution, but that a 'basket' of 4 or 5 parameters, which includes one of the present faecal indicators, will do so. In addition, numerical analysis of the data shows that this 'basket' will allow the successful building of predictive models. Both the statistical analyses and the studied predictive models indicate that genotype II of F-specific RNA bacteriophages, the coprostanol and the ratio coprostanol: coprostanol+epicoprostanol are, out of the studied parameters, those with a greater discriminating power. Either because unsuccessful adaptation of the methods to water samples or because the preliminary assays in water samples indicated low discriminating capability, only three (sorbitol-fermenting bifidobacteria, some species of bifidobacteria detected by PCR with specific primers and phages infecting Bacteroides tethaiotaomicron) of the newly assayed methods have been considered for a second field study, which is currently underway. Expectations are that these new tools will minimize the number of parameters in the 'basket', or at least minimize the difficulty in assaying them.

Comparison of bacteriophage and enteric virus removal in pilot scale activated sludge plants

The aim of this experimental study was to determine comparatively the removal of two types of bacteriophages, a somatic coliphage and an F-specific RNA phage and of three types of enteric viruses, hepatitis A virus (HAV), poliovirus and rotavirus during sewage treatment by activated sludge using laboratory pilot plants. The cultivable simian rotavirus SA11, the HAV HM 175/18f cytopathic strain and poliovirus were quantified by cell culture. The bacteriophages were quantified by plaque formation on the host bacterium in agar medium. In each experiment, two pilots simulating full-scale activated sludge plants were inoculated with viruses at known concentrations, and mixed liquor and effluent samples were analysed regularly. In the mixed liquor, liquid and solid fractions were analysed separately. The viral behaviour in both the liquid and solid phases was similar between pilots of each experiment. Viral concentrations decreased rapidly following viral injection in the pilots. Ten minutes after the injections, viral concentrations in the liquid phase had decreased from 1.0 +/- 0.4 log to 2.2 +/- 0.3 log. Poliovirus and HAV were predominantly adsorbed on the solid matters of the mixed liquor while rotavirus was not detectable in the solid phase. In our model, the estimated mean log viral reductions after 3-day experiment were 9.2 +/- 0.4 for rotavirus, 6.6 +/- 2.4 for poliovirus, 5.9 +/- 3.5 for HAV, 3.2 +/- 1.2 for MS2 and 2.3 +/- 0.5 for PhiX174. This study demonstrates that the pilots are useful models to assess the removal of infectious enteric viruses and bacteriophages by activated sludge treatment. Our results show the efficacy of the activated sludge treatment on the five viruses and suggest that coliphages could be an acceptable indicator of viral removal in this treatment system.

Reduction of bacterial indicators and bacteriophages infecting faecal bacteria in primary and secondary wastewater treatments.

To compare the suitability of various bacterial and viral indicators to assess the removal of faecal micro-organisms by primary and secondary wastewater treatment processes. The numbers of several bacterial indicators [faecal coliforms (FC), enterococci (ENT) and sulphite-reducing clostridia (SRC)] and bacteriophages (somatic coliphages, F-specific RNA phages and bacteriophages infecting Bacteroides fragilis strain RYC2056) were determined in incoming raw sewage and effluents from various primary and secondary wastewater treatment processes in several geographical areas. Reductions in the numbers of indicators were calculated as log10 reductions. Processes based on removal and mild disinfection, showed no significant differences in the elimination of any of the indicators tested or between geographical areas. In contrast, treatment processes that include strong microbial inactivation, such as lime-aided flocculation and lagooning, showed significant differences between the log10 reductions of the various micro-organisms studied, FC showing the highest reduction and spores of SRC and phages infecting B. fragilis the lowest. The microbial elimination performance of treatment processes based principally on removal and mild disinfection can be evaluated with a single indicator. In contrast, processes with additional disinfecting capabilities require more than one indicator for accurate evaluation of the treatment; bacteriophages are good candidates for use as second indicators. Bacteriophages provide additional information for the evaluation of microbial elimination in some treatment plants. The easy, fast and cheap methods available for phage determination are feasible both in industrialized and developing countries.

Removal of F-specific RNA bacteriophages in artificial recharge of groundwater - a field study

Artificial recharge of groundwater offers a semi-natural means to produce raw water for drinking-water plants. Surface water works are increasingly being replaced by artificial groundwater works in Finland. Two municipalities, one serving 30,000 and the other 170,000 inhabitants, have considered filtering river water through eskers for the production of potable water. In this study the removal of bacteriophages during infiltration of river water was estimated, for the evaluation of treatment adequacy in a field study. A 5-m-deep column of sand was constructed and used to mimic the percolating phase in infiltration. An artificial esker was constructed on the riverbank by isolating a 2-m-wide, 2-m-deep and 18-m-long bed of coarse sand with plastic. The sand bed represented the saturated zone. River water was pumped at a rate of 40 L/h to the sand column. The river water was spiked with F+ specific RNA phage MS2 by adding phage suspension during one week at an average concentration of 4.3 x 10(9) PFU/mL. Samples for phage assays were taken during one month, from four sampling sites, on the basis of detention time as estimated by a tracer experiment with sodium chloride. The median count of MS2 for percolated water was 2.4 x 10(5) PFU/mL, representing a 96.7% reduction. During the passage of 6 m in the saturated zone, a further reduction of 98.5% occurred. During the passage from 6 m to 12 m the additional reduction was 99.97%. The overall reduction was between 6 and 7 log10 units. The removal of MS2 phages was rather efficient, although the esker material was coarse, mainly sandy, gravel.

Detection of enteric viruses in shellfish from the Norwegian coast.

Common blue mussels (Mytilus edulis), horse mussels (Modiolus modiolus), and flat oysters (Ostrea edulis) obtained from various harvesting and commercial production sites along the Norwegian coast were screened for the presence of norovirus by a real-time reverse transcription (RT)-nested PCR assay and for possible indicators of fecal contamination, i.e., for F-specific RNA bacteriophages (F-RNA phages) by plaque assay and for human adenoviruses and human circoviruses by nested PCR assay. The aims were to obtain relevant information for assessing the risk of transmission of enteric viruses by shellfish and to investigate the potential of various indicator viruses in routine screening. Noroviruses were detected in 6.8% of the samples, and the indicators were detected in 23.8% (F-RNA phages), 18.6% (adenoviruses), and 8.0% (circoviruses) of the samples. A seasonal variation was observed, with the exception of circoviruses, with more positive samples in the winter. A positive correlation was found between F-RNA phages and noroviruses. However, F-RNA phages were present in only 43% of the norovirus-positive samples. The results show that mussels from the Norwegian coast can constitute a risk of infection with enteric viruses and that routine testing of samples may be justified. Advantages and disadvantages of various options for screening are discussed.

Standardised evaluation of the performance of a simple membrane filtration-elution method to concentrate bacteriophages from drinking water

The bacteriophage elution procedure described further after adsorption to acetate–nitrate cellulose membrane filters allows better recovery of phages concentrated from 1 l of water than elution proceduresm used previously. The improvement is due to the combined effect of the eluent (3% (w/v) beef extract, 3% (v/v) Tween 80, 0.5 M NaCl, pH 9.0) and the application of ultrasound instead of agitation or swirling. Average recovery of somatic coliphages, 82±7%, was the greatest, and that of phages infecting Bacteroides fragilis, 56±8%, the lowest, with intermediate values for F-specific and F-specific RNA bacteriophages. Thus, the method allowed recovery of over 56% for all the phages suggested as surrogate indicators. The method was then validated according to an International Standardisation Organisation validation standard procedure and implemented in routine laboratories, which obtained reproducible results.

Water and mass budgets of a vertical-flow constructed wetland used for wastewater treatment

To estimate the nutrient and organic matter (Biological Oxygen Demand (BOD 5) and Chemical Oxygen Demand (COD)) removal capacity of a constructed vertical-flow wetland in The Netherlands, a water and nutrient budget study was conducted. Also bacterial water quality enhancement was measured. The system had a Phragmites australis vegetation and comprised four parallel compartments of 0.25 ha each, which were loaded sequentially with sewage from recreational facilities. Annual loading rates were moderate and were estimated to be 16 700 kg COD ha −1; 6700 kg BOD 5 ha −1; 2400 kg N ha −1; and 335 kg P ha −1. The removal efficiencies for COD (81%) and BOD 5 (96%) were high. Almost all Escherichia coli and F-specific RNA bacteriophages (>99%) were removed from the wastewater during transport. The removal efficiencies for nitrogen (30%) and phosphorus (24%) were much lower. Nutrient removal was the result of plant uptake and harvesting (15% of total N input, 10% of total P input), denitrification (8% of total N input), sedimentation and accumulation of organic matter in the soil (7% of N total input, 14% of total P input). Removal efficiencies for N and P could be increased by harvesting the Phragmites vegetation in October, rather than the current harvesting practice in December. This vertical-flow wetland appeared to be P-saturated after 15 years of operation. The use of sandy sediments with better P-adsorbing properties is advocated as a critical issue for the design of these systems. Further, the groundwater eutrophication resulting from the poor functioning of the drainage systems has shown that a good hydraulic separation of vertical-flow systems from the subsurface is an important prerequisite for their proper functioning.

Survival of bacterial indicator species and bacteriophages after thermal treatment of sludge and sewage.

The inactivation of naturally occurring bacterial indicators and bacteriophages by thermal treatment of a dewatered sludge and raw sewage was studied. The sludge was heated at 80 degrees C, and the sewage was heated at 60 degrees C. In both cases phages were significantly more resistant to thermal inactivation than bacterial indicators, with the exception of spores of sulfite-reducing clostridia. Somatic coliphages and phages infecting Bacteroides fragilis were significantly more resistant than F-specific RNA phages. Similar trends were observed in sludge and sewage. The effects of thermal treatment on various phages belonging to the three groups mentioned above and on various enteroviruses added to sewage were also studied. The results revealed that the variability in the resistance of phages agreed with the data obtained with the naturally occurring populations and that the phages that were studied were more resistant to heat treatment than the enteroviruses that were studied. The phages survived significantly better than Salmonella choleraesuis, and the extents of inactivation indicated that naturally occurring bacteriophages can be used to monitor the inactivation of Escherichia coli and Salmonella.