And Ionizing (Gamma) Radiation: a Comparative Approach

INACTIVATION OF BACTERIOPHAGES IN WATER BY MEANS OF NON-IONIZING(UV-253.7 nm) AND IONIZING (GAMMA) RADIATION: A COMPARATIVE APPROACH

REGINA SOMMER1*, WALTER PRIBIL1, SILVIA APPELT1, PETER GEHRINGER2, HELMUT ESCHWEILER2, HERMANN LETH2, ALEXANDER CABAJ3 and THOMAS HAIDER4 1 Hygiene Institute University of Vienna, Kinderspitalgasse 15, 1095, Vienna, Austria; 2 Austrian Research Center Seibersdorf, Austria; 3 Institute of Medical Physics and Biostatistics, University of Veterinary Medicine Vienna, Austria and 4 Institute of Environmental Hygiene, University of Vienna, Austria

(First received 14 March 2000; accepted in revised form 9 January 2001)

Abstract}The inactivation behaviour of the bacteriophages PHI X 174 (ssDNA virus), MS2 (ssRNA virus) and B40-8 (dsDNA) toward non-ionizing (UV-253.7 nm) as well as to ionizing radiation (gamma radiation) was studied in order to evaluate their potential as viral indicators for water disinfection by irradiation. Previous findings of the high UV-253.7 nm resistance of MS2 were confirmed whereas an unexpected high sensitivity to gamma radiation compared to the two other phages was found. On the other hand, PHI X 174 revealed an enhanced UV sensitivity but a high resistance to ionizing radiation. B40-8 had an intermediate position between the other two bacteriophages relative to both types of radiation. As expected, the data of E. coli reconfirmed the unreliability of fecal indicator bacteria for the purpose of predicting responses of viruses to water treatment. In UV disinfection the influence of water matrix may be adequately controlled by considering the UV (253.7 nm) absorption of the water whereas so far no such parameter has existed for the influence of the water quality on ionizing irradiation with respect to the scavenger concentration. # 2001 Elsevier Science Ltd. All rights reserved

Key words}UV radiation, ionizing radiation, inactivation, bacteriophages, disinfection, water

INTRODUCTION formation of photoproducts. The most prominent Irradiation treatment has been proved to be a photoproducts are the dimers of thymine, a powerful tool in inactivating human pathogenic pyrimidine base. Another very clearly defined UV- microorganisms in water, waste water and sludge, induced damage in the DNA is the bacterial spore in food and medical products (Lagunas-Solar, 1995; photoproduct. The changes in the nucleic acid Farooq et al., 1993). One of the main advantages of lead to the so-called reproductive cell death of the such treatment consists of the fact that since microorganisms (Harm, 1980). The individual UV irradiation is a physical process, no chemicals have resistance of a microorganism depends on the UV to be added. In order to understand these techniques fluence applied and its ability to protect from and to repair damages. The unit of the UV fluence we have to consider that microbial inactivation by 2 means of non-ionizing radiation (e.g. UV-253.7 nm) is J m . compared to ionizing radiation (e.g. gamma rays, The microbicidal mechanism of ionizing radiation electron beams), respectively, are each based on is not yet fully understood. The nucleic acids are entirely different mechanisms. likely to be the main target, but in viruses the coat UV plants, equipped with low pressure mercury also plays an important role (von Sonntag, 1987; lamps, emitting mainly UV radiation with a Mahnel et al., 1980). The microorganisms may be wavelength of 253.7 nm, are widely used in the affected either by direct (single and double strand disinfection of both drinking and waste water. UV- breaks of the nucleic acids) or by indirect reaction 253.7 nm is predominately absorbed by the nucleic (radicals originating from water, mainly OH free acids as the main target and causes damage by the radicals, H atoms and solvated electrons eaqu, generated by radiolysis either intra- or extra cellu- larly). The effect of the indirect attack by radicals *Author to whom all correspondence should be addressed. may be reduced by the presence of scavengers such as Tel.: +43-1-40490-79452; fax: +43-1-40490-9794; bicarbonate, but can also be enhanced by the e-mail: [email protected] presence of oxygen (von Sonntag, 1987). The direct

3109 3110 Regina Sommer et al. effect is not scavengeable. The unit for the absorbed of 4 log of microbial pathogens is demanded to dose is Gy (J kg1). ensure safe drinking water (US EPA, 1989). Bacterial viruses such as somatic coliphages, F-specific coliphages and phages infecting Bacter- oides fragilis have been proposed as indicators for MATERIALS AND METHODS human pathogenic, water-transmittable viruses to Bacteriophages}propagation and enumeration monitor the quality of water (drinking, recreational MS2 (F-specific bacteriophage), PHI X 174 (somatic and waste water) as well as to evaluate water coliphage) and B40-8 (phage infecting Bacteriodes fragilis) treatment and disinfection (Anon, 1991; Havelaar and the bacterial host strains (Salmonella typhimurium WG et al., 1991; Jofre et al., 1986). The search for viral 49, E. coli WG5 and Bacteroides fragilis HSP-40) were provided by the National Institute of Health and Environ- indicators became necessary, since numerous studies ment (Bilthoven, Netherlands), the Institute Pasteur de Lille showed that viruses are fairly resistant both to (France) and the University of Barcelona (Spain). natural factors in the environment (sunlight, tem- Bacteriophage MS2 was propagated in the host strain perature, antagonists) and to chemical and physical E. coli K-12 Hfr and enumerated by the double agar layer treatment. The bacterial indicators usually applied procedure according to ISO/CD 10705-1/1997, using the strain Salmonella typhimurium WG49. Bacteriophage PHI are not appropriate for predicting the viral quality of X 174 was grown in the bacterial host strain E. coli WG5 water (Tree et al., 1997; Dizer et al., 1993; Havelaar and analysed by the double agar layer technique, following et al., 1991; Anon, 1991; Sommer et al., 1989). the protocol of ISO 10705-2/1997. The propagation and Therefore the fecal indicator bacteria have to be detection of phage B40-8 by the double agar layer method was carried out in host strain B. fragilis HSP-40 under regarded as inadequate for predicting viral responses anaerobic culture conditions according to ISO/WD to treatment. 10705-4/1997. The compositions of the nutrient broths used However, the accurate knowledge of the charac- for the propagation of the bacteriophages are shown in teristics of possible indicators is a prerequisite for Table 1. After propagation of the phages in the bacterial their reliable application in water hygiene. Therefore hosts, suspended in the respective broth, bacteria were inactivated by adding chloroform (5 ml CHCl3 per 50 ml we chose representatives of the three phage groups broth) and removed by centrifugation. The supernatant proposed as viral indicators as mentioned above. representing the phage stock solution was stored at 4–68C. Moreover, these phages were subject of an extensive Immediately before use, phage stock solutions were passed international study about the feasibility of phages through a 0.2 mm membrane filter (Millex-GV, Millipore) to remove virus aggregates and were then diluted in tap water for the monitoring of the bathing water quality (drinking water, City of Vienna). The chemical main (European Community, 1999). characteristics of the tap water are indicated in Table 2. The aim of our study was to investigate the The final concentrations of the phage suspensions after 6 7 inactivation behaviour of three selected bacterio- dilution (1 ml phage stock to 1 l tap water) were 10 to 10 phages, namely PHI X 174 (somatic coliphage), MS2 plaque forming units (PFU) per ml. (F-specific coliphage) and B40-8 (phage infecting UV irradiation device and performance of UV-253.7 nm Bacteroides fragilis) regarding non-ionizing (UV- inactivation experiments 253.7 nm) as well as ionizing radiation (gamma A board mounted with 10 low-pressure mercury UV radiation) in order to evaluate their potential as viral lamps (EK 36; length 500 mm, wavelength 253.7 nm, ozone indicators for water disinfection by irradiation free, Katadyn) was horizontally suspended over the treatment. Since the dose of ionizing and the fluence irradiation vessel. An aperture (100 mm 150 mm) was of UV radiation cannot be directly compared due to fixed directly below the UV lamps, providing a bundle of quasi parallel radiation to the irradiation vessel. The test their entirely different inactivation mechanism, we suspensions (volume 25 ml) were irradiated in sterile petri chose a 4 log reduction of the phages as a measure for dishes (diameter 90 mm) and placed on a magnetic stirrer comparison. In drinking water treatment a reduction during permanent mixing. The depth of the layer was 4 mm.

Table 1. Composition of the nutrient broths used for phage propagation (according to ISO/CD 10705-1/1997; ISO/CD 10705-2/1997 and ISO/WD 10705-4/1997)a

Components of the nutrient broth for the propagation of phage

MS2 PHI X 174 B40-8

Yeast extract (1 g l1) Yeast extract (3 g l1) Yeast extract (2 g l1) Trypticase peptone (10 g l1) Peptone (10 g l1) Casein peptone (10 g l1) Meat extract (12 g l1) Meat peptone (10 g l1) Glucose (1 g l1) Glucose (1.8 g l1) Hemin (0.01 g l1) l-Cystein (0.5 g l1) NaCl (8 g l1) NaCl (3 g l1) NaCl (5 g l1) 1 1 CaCl2 (0.3 g l ) CaCl2 (0.05 g l ) 1 1 Na2CO3 (0.75 g l )Na2CO3 (2.65 g l ) 1 1 MgCl2 (0.60 g l ) MgSO47H2O (0.12 g l ) pH: 7.2 0.2 pH: 7.2 0.2 pH: 6.8 0.5 a During the preparation of the test suspensions of the phages, 1 ml of the respective phage stock solution is added to 1 l of tap water. Inactivation of bacteriophages by irradiation 3111

The UV fluence of 253.7 nm was measured on-line with a sample was removed from its position in the irradiation research radiometer (IL 1700 SED 240, International Light, chamber (after reaching the assigned radiation dose), USA), taking into consideration the absorption at 253.7 nm another bottle filled with the suspension was put into the of the test suspension and the reflection at the surface as empty space before irradiation was started again. Accord- previously described (Sommer et al., 1995). The fluence rate ingly, the same irradiating conditions for all samples were was about 1 W m2. The irradiation equipment was guaranteed. The gamma doses were obtained by varying the evaluated for reliability in an international laboratory trial exposure time. All experiments were carried out at room for UV irradiation devices (Sommer et al., 1995). The UV temperature. The observed increase of temperature in the fluence was varied by the exposure time. Experiments were irradiated samples (about 28C at the highest dose of 800 Gy) performed at room temperature. was negligible for the inactivation of the microorganisms.

Gamma irradiation device and performance of gamma Analysis of data inactivation experiments All experiments were repeated at least twice and The experiments were carried out in an AECL concentrations were measured in duplicate. Mean values ‘‘Gammacell 220’’ Cobalt-60 source having an average dose of each analysis were calculated as to the concentrations of 1. rate of about 0.96 Gy s during trial time. Dose rate the phages in both the non irradiated (No) and the calibration was established by a Fricke dosimeter. The half irradiated (N) samples (numbers of plaque forming units 1 value layer of this radiation in water is 110 mm. per ml, PFU ml ). Data were converted to lg10 values and The phage suspensions were irradiated in 50 ml glass the reductions lg(N/No) calculated. Values below the bottles with glass stoppers. Before starting the experiments, quantitative detection limit (DL: 20 PFU ml1 equivalent a fixed irradiation geometry for six bottles inside the to log 1.30) are indicated in the figures by the term 5DL. irradiation chamber was tested for homogeneity of the Reduction of bacteriophages versus UV fluence and the applied dose, using Bacillus subtilis spores as biodosimeter. dose of gamma irradiation were used to determine the This geometry was maintained during all gamma irradiation inactivation curve parameters. The functions of inactivation experiments, based on the following procedure: Whenever a were calculated by using a curve-fitting program (see Table 3 for equations).

Table 2. Main chemical characteristics of the tap water (drinking water, City of Vienna) RESULTS

Parameter Value UV inactivation The UV inactivation behaviour of the three pH 7.6 Conductivity (mScm2) 285 bacteriophages in tap water differed considerably 1 Oxidizability (KMnO4-demand, mg l )3.0(Fig. 1). A 4 log reduction was achieved for the most TOC (mg l1)0.8 1 susceptible phage PHI X 174 at a UV fluence of Ammonia (mg l ) 50.02 2 Nitrate (mg l1)4.6100 J m and for phage B40-8 at a fluence of 2 Nitrite (mg l1) 50.01 290 J m ; however, for the most resistant strain 1 Calcium (mg l ) 44.0 MS2 a UV fluence of 750 J m2 (obtained by Magnesium (mg l1)9.0 Chloride (mg l1)1.7extrapolation) was needed (Table 4). The UV Sulphate (mg l1) 11.0 inactivation kinetics of PHI X 174 and MS2 followed Hydrogen carbonate (mg l1) 159.0 Oxygen (mg l1)8a first order reaction, demonstrating a single-hit process. Surprisingly, this was not the case for B40-8,

2 1 2 1 Table 3. Curve parameters k, k1 (slope; m J ), d (shoulder width; decadic logarithm unit 1), k2 (slope of the tailed part of the curve; m J ), a (intercept of the tailing with the ordinate, decadic logarithm, unit 1) for the inactivation of the bacteriophages MS2, PHI X 174 and B40-8 by irradiation treatments in tap watera

Irradiation treatment Test strain k or k1 d k2 a

Gamma radiation PHI X 174 0.005386 Gy1 0.8234 0 0 B40-8 0.01147 Gy1 0 0.002828 Gy1 0.005323 MS2 0.03129 Gy1 0.5149 0 0 UV radiation PHI X 174 0.04013 m2 J1 00 0 B40-8 0.01679 m2 J1 0.8477 0 0 MS2 0.005391 m2 J1 00 0 a The functions of the inactivation curves were: N lg ¼ lgð10kH0 Þ first order kinetics ðwithout shoulder; without tailingÞ; No hi N d lg ¼ lg 1 ð1 10kH0 Þ10 curve with shoulder; without tailing; No N 10k1H0 þ a 10k2H0 lg ¼ lg curve without shoulder with tailing; No 1 þ a 2 where H0 means ﬂuence (J m ). 3112 Regina Sommer et al.

Fig. 1. UV inactivation (253.7 nm) of the bacteriophages PHI X 174, B40-8 and MS2 in tap water. Data of E. coli are given as a reference.

Table 4. Virus characteristics and irradiation doses (UV and ionizing radiation) required for a reduction of 4 log of the bacteriophages MS2, PHI X 174 and B40-8 in tap watera

Irradiation condition MS2 PHI X 174 B40-8

UV fluence (J m2) 750 (1.0) 100 (7.5) 290 (2.6) Gamma dose (Gy) 140 (6.4) 900 (1.0) 610 (1.5) Virus characteristics Nucleic acid ssRNA ssDNA dsDNA Weight (MDa) 1.8 2.0 34.5b Particle weight (MDa) 3.6 6.0 } Size (head diameter) 18 nm 15 nm 60 nm Polyhedral, naked Polyhedral, naked Polyhedral, naked No tail No tail Flexible tail Bacterial host F-specific E. coli WG5 Bacteroides F+Salmonella fragilis HSP-40 Typhimurium (Anaerobic cultivation) WG49 a The figures in parenthesis indicate the sensitivity of the phage under the two types of irradiation relative to the most resistant bacteriophage; (ss), single-stranded; (ds), double-stranded; and (MDa), Megadalton. b Corresponding to a DNA length of 51.7 kilobases (Puig and Girone´ s, 1999).

where the plot reduction versus UV fluence contained investigated. While for MS2 a gamma dose of about a shoulder. This may indicate, that more than one hit 140 Gy was sufficient for a 4 log reduction, PHI X or the damage of more than one target is necessary 174 needed a gamma dose of about 900 Gy for the for inactivation. The characteristics of the function of same effect. The dose requirement of B40-8 for a 4 log the inactivation curves are shown in Table 3. The reduction lay in between the two others (Table 4). In data of E. coli are inserted for comparison. As may the inactivation curves of MS2 and PHI X 174, a be seen from Fig. 1, E. coli was even more UV- shoulder was observed while the curve of B40-8 sensitive than bacteriophage PHI X 174 (4 log demonstrated a tailing at higher doses. Accordingly, reduction: 70 J m2). none of the inactivation curves of the three gamma- irradiated phages in tap water followed a first order Inactivation by gamma radiation kinetics (Table 3). Figure 2 presents the inactivation curves in water as a function of the absorbed dose for the three DISCUSSION phages, MS2, PHI X 174 and B40-8, as gamma irradiation is applied. The corresponding data for To our knowledge no comparative study on the E. coli are inserted for comparison. The results influence of ionizing and non-ionizing radiation on obtained were somewhat unexpected. There were bacteriophages has so far been published. Unexpect- great differences in the sensitivity of the three phages edly, the radiation treatments caused entirely Inactivation of bacteriophages by irradiation 3113

Fig. 2. Inactivation of the bacteriophages PHI X 174, B40-8 and MS2 in tap water by gamma irradiation as a function of the absorbed dose. Data of E. coli are given as a reference. (5DL) below detection limit.

different inactivation patterns for the individual bacterial, protozoan or eukaryotic cells, their UV bacteriophages tested. inactivation does not by any means follow first order kinetics. This is demonstrated in the dsDNA phage UV irradiation B40-8 with its inactivation curve consisting of a shoulder followed by an exponential decline. The Major differences in UV resistance were found shoulder at low fluences, representing a threshold among the three viruses. The extremely high UV fluence, is an indication that more than only one hit resistance of phage MS2, as compared to the other or the damage of more than only one target may be two phages, was in accordance with previous find- necessary for the inactivation (Harm, 1980). The ings. A reduction of 4 log was obtained in our study double-strandedness of the DNA in B40-8 seems to at a fluence of 750 J m2 as compared to about be the likely reason for the shoulder in the UV 800 J m2 (Havelaar et al., 1991) and 650 J m2 inactivation curve (Harm, 1980). (Meng and Gerba, 1996). Battigelli et al. (1993) reported more than 300 J m2 for a 1 log reduction. One may speculate that the type of nucleic acid in Gamma radiation MS2, the RNA, is the reason for the high UV The experiments were performed with an initial resistance. Contrary to DNA, RNA does not contain virus concentration of about 1.0 106 viruses per ml. thymine but uracil, which may be less photoreactive. Due to the known molecular weight of the virion of However, this cannot be the sole explanation since MS2 and PHI X 174 amounting to about 3.6 and other RNA viruses with a structure and size very 6.0 MD, it is possible to express the virus concentra- similar to MS2, e.g. poliovirus, are rather UV tion in terms of g viruses per litre of water (Table 4). sensitive. A 4 log reduction of poliovirus can be The corresponding concentrations are therefore achieved at fluences between 220 and 350 J m2 5.98 109 gl1 for MS2 and 9.97 109 gl1 for (Harris et al., 1987; Sommer et al., 1989; Battigelli PHI X 174. No data about the molecular weight of et al., 1993; Meng and Gerba, 1996). In contrast, the virion of B40-8 are yet available. It is obvious DNA viruses exist with high UV resistance as that for the irradiation of such highly diluted adenoviruses which are double-stranded and aqueous solutions a direct effect of radiation can 60–80 nm in dimension (Meng and Gerba, 1996). most likely be neglected. Even when taking into Therefore, a combined effect of size and type of the account that the viruses are not dissolved but just virion as well as of the nucleic acid is most likely suspended in water, this statement should, never- responsible for the individual resistance of viruses. theless be true. This hypothesis is supported by a The ssDNA virus PHI X 174 was highly UV study describing gamma inactivation of PHI X 174 sensitive; a fluence of 100 J m2 caused a 4 log suspended in 1% tryptone broth where this virus was reduction. Therefore, this bacteriophage cannot be irradiated frozen at 608C and dried at 30–408C taken as a reliable surrogate for pathogenic viruses in (Lemke and Sinskey, 1975). Under these conditions studies dealing with UV disinfection. Although the direct effect of radiation is certainly responsible viruses have a rather simple structure compared to for inactivation. In both cases the dose requirement 3114 Regina Sommer et al. for a 2 log reduction was about 17 kGy. Contrary to as explanation for the differing gamma sensitivity this, we observed a 2 log reduction of PHI X 174 because the broth used for cultivation of the suspended in tap water already with a gamma dose of phage B40-8 contains much more efficient OH free about 520 Gy (Fig. 2). This is about 30 less in terms radical scavengers than the broth for the cultivation of dose! Accordingly, the low concentration of the of phage PHI X 174 does (Table 1). Accordingly, a suspended viruses and the low dose requirement for lower dose requirement for ionizing radiation as inactivation may be taken as a clear indication that in compared to B40-8 inactivation may be expected. the present study we have to deal with an indirect Nevertheless, the dose requirement for the single- effect of radiation, implying that radicals formed stranded DNA phage PHI X 174 inactivation is during water radiolysis are mainly responsible for clearly higher than that found for the double- inactivation. Among these radicals OH free radicals stranded DNA-phage B40-8 inactivation (Fig. 2). and solvated electrons are the main products formed Mahnel et al. (1980) already mentioned that conflict- in almost equal amounts of 2.8 107 mol J1 while ing data on gamma-irradiation resistance exist just 0.57 107 mol J1 of H atoms are formed between viruses with single-stranded and those with (von Sonntag, 1987). double-stranded nucleic acids. Therefore the reason Postulating the indirect effect of radiation as the for the higher resistance of PHI X 174 compared to dominating mechanism, one has to carefully consider the two other phages found in the present study is not the presence of other solutes also able to react with at all clear. To make confusion even greater, the OH free radicals, the so-called scavengers. The Eisenstark et al. (1986) found that MS2 phage is tap water used contained bicarbonate in a concentra- more resistant to OH free radical attack than PHI X tion of 159 mg l1, corresponding to a scavenger 174 if radicals are generated by hydrogen peroxide capacity which is the product of the reaction rate and near-ultraviolet radiation. However, the scaven- constant for the reaction with OH free radicals and gers present in the system were not taken into the scavenger concentration of about 2.55 104 s1. consideration at all. Accordingly, a direct compar- Based on these data, the system would be classified as ison of their results with those presented in our study a low-scavenged one with regard to OH free radicals. is not possible. However, spiking the tap water with 1 ml of the Another interesting fact found in this study phage stock solution introduced additional scaven- demonstrated that the phage B40-8, contrary to the gers originating from the nutrient solution of the two other phages, showed a tailing in the inactivation phage cultivation. These phage-specific nutrient curve at higher gamma doses. This may be taken as solutions contain organic material in the order of an another indication that B40-8 inactivation takes 10–30 mg l1 (more details see Table 1); yet there is place in a highly scavenged system (von Sonntag, also some chloroform (as a component of the phage 2000). Furthermore, an oxygen effect can not be stock) added in the order of about 3 mg l1. Through excluded, but this has as yet to be investigated. these organic compounds an additional scavenging of the OH free radicals certainly takes place. It is, however, difficult to quantify the contribution of the Comparison of the microbicidal efficacy of UV different broths regarding OH free radical scavenging and gamma radiation because their organic compounds are chemically not For two reasons the microbicidal efficacy of non- sufficiently defined and their reaction rate constants ionizing and ionizing radiation cannot be compared are not known. directly and absolutely. Primarily this is due to their In a highly scavenged system, DNA and RNA entirely different inactivation mechanism: The in- have to be considered as the major targets of damage activation by UV-253.7 nm radiation has a direct (von Sonntag, 1987). Comparing the reaction effect on the nucleic acids whereas irradiation by rate constants of DNA with RNA as to their ionizing radiation under experimental conditions response to aqueous OH free radicals, DNA given constitutes an indirect effect of radiation. These (k[DNA+OH]=4 108 l mol1 s1) is 2.5 times less different mechanisms are also reflected in the defini- sensitive (k[RNA+OH]=1 109 l mol1 s1). This tion of the respective fluence or dose (J m2 for the would explain the lower dose requirement for the UV irradiation; J kg1(=Gy) for the irradiation with inactivation of the phage MS2 as compared to PHI X ionizing radiation). In addition, the influence of the 174. However, this does by no means explain the water matrix comes into place: In UV-253.7 nm significant difference in radioresistance observed disinfection the influence of the water matrix may between PHI X 174 and B40-8. This distinct be adequately controlled by considering the UV- difference is rather unexpected because in both 253.7 nm absorption of the water; so far no such phages the nucleic acid is the DNA; however, the parameter exists for the influence of the water quality DNA in PHI X 174 is single-stranded whereas the on the ionizing irradiation with respect to the DNA in B40-8 is double-stranded. Further distinc- scavenger concentration. The huge influence of the tions between these two phages are deviations in size matrix on the virus inactivation has been demon- and weight as well as evidence of a tail in phage strated by Sullivan et al. (1971): The radioresistance B40-8 (Table 4). The water matrix also cannot serve of poliovirus III suspended in protein-rich Eagles Inactivation of bacteriophages by irradiation 3115

MEM was approximately 5 times higher than sorption (253.7 nm)}may be adequately consid- suspended in distilled water. ered in calculating the fluence but no such However, the relative biological effectiveness} parameter exists for describing the influence of expressed as the dose requirement of the different the water quality on ionizing irradiation with irradiation treatments for a defined reduction of a respect to the scavenger concentration. Thus it particular virus; this may serve as a valuable tool for will be necessary to investigate the rate of comparison. We chose a 4 log reduction of the phages microbial inactivation for each of the suspending as a measure for comparison since in water disinfec- media (e.g. drinking water or waste water). tion a reduction of 4 log of microbial pathogens is demanded to ensure safe drinking water (US EPA, Acknowledgements}The authors gratefully acknowledge 1989). 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