Ultraviolet Seawater Treatment Unit'

Transcription

1 APPLIED MICROBIOLOGY, Jan. 1969, p. 1-6 Copyright 1969 American Society for Microbiology Vol. 17, No. I Printed in U.S.A. Inactivation of Poliovirus Type 1 by the Kelly-Purdy Ultraviolet Seawater Treatment Unit' WILLIAM F. HILL, JR., FREDERICK E. HAMBLET, AND WILLIAM H. BENTON Gulf Coast Marine Health Sciences Laboratory, Environmental Control Administration, U.S. Public Health Service, Dauphin Island, Alabama Received for publication 15 October 1968 Three experiments were conducted to determine the effect of ultraviolet (UV) radiation on poliovirus-contaminated seawater. In two of the experiments, the effectiveness of the Kelly-Purdy UV Seawater Treatment Unit to inactivate poliovirus type 1 (T1) suspended in continuously flowing seawater was determined. In experiment 1, the observed survival ratio of poliovirus T, was 2.3 X 10-4 (99.98% reduction) in 15.7 sec. No virus was detected (<0.2 plaque-forming unit/ml) in 20.6 seconds. The calculated half-life value was 1.29 sec. In experiment 2, the observed survival ratio of poliovirus T, was 5.9 X 10-4 (99.94% reduction) in 11.7 sec. No virus was detected in 15.7 sec. The calculated half-life value was 1.37 sec. In experiment 3, a laboratory-controlled UV experiment designed to closely simulate the geometry of the continuously flowing seawater system, the observed survival ratios of poliovirus T1 were 9.7 X 10- (99.03 % reduction) and 3.6 X 10-4 (99.96% reduction) in 15 and 30 sec, respectively; the calculated half-life value was 2.38 sec. A statistically significant difference was found between the inactivation rates of poliovirus T, in the two test systems. This rate difference was attributed primarily to UV dosage and stirring effects. The data indicated that UV radiation effectively inactivated poliovirus T1 in flowing seawater. These results validate the efficacy of the Kelly-Purdy UV Seawater Treatment Unit for use in commercial depuration systems. The need for disinfecting continuously flowing seawater is related principally to the purification system for shellfish (deputation) in which natural seawater is used. The ultraviolet (UV) radiation treatment unit developed for this purpose and shown to effectively destroy coliform organisms is commonly referred to as the Kelly-Purdy UV Seawater Treatment Unit (15). Previous studies in our laboratory indicated that a model UV radiation unit could effectively and predictably inactivate poliovirus suspended in flowing turbid seawater (12). The model UV unit, however, was not adaptable to commercial size operations primarily because of the limited quantity of seawater that could be subjected to treatment; the maximal flow rate was 25 liters/min. The Kelly- Purdy UV unit, however, was considered to be of a design and capacity sufficient for application to commercial deputation systems (15). The seawater flow rate of this UV unit was approximately 140 to 150 liters/min. Because the ability of the IScientific Contribution no. 51, U.S. Public Health Service, Gulf Coast Marine Health Sciences Laboratory, Environmental Control Administration, Dauphin Island, Ala Kelly-Purdy UV unit to inactivate viruses had not been tested, it was decided to ascertain the effectiveness of this UV unit in inactivating poliovirus under maximal operational conditions. The findings would then be definitively applicable to the commercial situation. These experiments were conducted in a prototype commercial shellfish depuration system located in our wet laboratory. Poliovirus (vaccine strain) was selected as the model virus to minimize the infection hazards involved in wet laboratory operations. A laboratory UV experiment (lab-control UV experiment) was also conducted to establish base line information on the survival of poliovirus in seawater under more rigid experimental conditions. The information obtained from these experiments should not only answer the academic question of the suitability of UV radiation as a tool for inactivating viruses in seawater but also should validate the use of the Kelly-Purdy UV Seawater Treatment Unit in commercial depuration systems. In this paper, we report the results of these experiments.

2 2 HILL, HAMBLET, AND BENTON APPL. MICROBIOL. MATERIALS AND METHODS Virus. The LSc2ab strain of poliovirus type 1 (T,) was used throughout the study. The virus was propagated in HEp-2 cells. The virus stock was stored at -70 C until used. Cell culture. HEp-2 cells were used for plaque assays. The cells were grown in 32-oz (about 900 ml) screw-cap prescription bottles as a monolayer in a growth medium consisting of Eagle's basal medium (BME) and 10% calf serum with penicillin (100 units/ ml), streptomycin (100 jig/ml), neomycin (2001Ag/ml), and amphotericin B (1.0 ig/ml). Cell overlay medium was a maintenance medium consisting of BME and 5% chicken serum with % neutral red, 1.1% purified agar (Difco), and the above-named antibiotics. Plaque assay. The plaque assay procedure of Dulbecco and Vogt (6), as modified by Hsiung and Melnick (14), was used throughout the study. Plaques were permanently marked and counted daily as described by Berg et al. (2). Cell-monolayer bottles exhibiting the greatest number of plaques short of overcrowding were recorded and used for computations. For purposes of assay, serial 10-fold dilutions of virus were made in nutrient broth (10) and 1 ml of virus at each dilution level was inoculated onto three cell-monolayer bottles [3-oz (about 85 ml) prescription size]. Five replicate subsamples were assayed for surviving virus at each sampling point or timed interval. Counts were expressed as plaque-forming units (PFU) per ml. Statistical treatment. Mean virus plaque counts of untreated and UV-treated seawater from all experiments were transformed to loge survival ratios for each UV exposure time.these data were then subjected to regression analysis (19). From the analyses, the slope functions b and the standard deviation of the slope functions Sb were determined. The difference between the slopes of any two lines (inactivation curves) was tested by pooling the estimate of the variance available from each line. The slopes were then compared for statistical significance at the 5% fiducial probability level. Certain data were tested by the analysis of variance test. In these cases, all virus plaque counts were transformed to square roots before analysis. This transformation of data stabilizes the variance, which then approximates statistical normality (1, 5). Seawater supply. The seawater used in the Kelly- Purdy UV experiments was pumped into the laboratory from Dauphin Island Bay, Ala. The fiber glass intake lines extended 21 meters into the Bay and reached a maximal depth of 0.9 m below sea level. The flow of seawater was regulated by a polyvinylchloride ball valve. All seawater contact surfaces in the system were nonmetallic. The fixed seawater parameters coincident to the Kelly-Purdy UV experiments are shown in Table 1. Salinity was determined hydrometrically (20). Turbidity was determined spectrophotometrically in a Spectronic-20 colorimeter (9). In the lab-control UV experiment, the seawater was filter-sterilized and had a salinity of 21.8 parts per thousand; the ph was 8.0. TABLE 1. Seawater parameters of Kelly-Purdy UV experiments Expt ph Salinitya Turbidity Flow rate %00 O ppm liters/min a Parts per thousand Kelly-Purdy UV Seawater Treatment Unit. The UV treatment unit (Fig. 1) was described in detail elsewhere (15). Briefly, the two-section unit was fabricated of wood and coated with epoxy resin. The top bulbunit section contained thirteen 30-w UV bulbs (91 cm long) positioned equidistant and at right angles to the direction of flow. A reflective aluminum sheet covered the inside top of the bulb-unit section. The bottom section contained six wooden baffles (2.6 cm high) inserted at right angles to the direction of the flow to provide a rolling or stirring effect. The distance from the bulbs to the surface of the water was approximately 8 cm. The depth of the seawater was 0.9 cm at the top of the baffles and 3.5 cm at the baffle interspace. The UV bulbs were checked for intensity with a Westinghouse SM-600 UV meter before the experiments and after a 10-min warm-up period. Bulbs with an output intensity of less than 60% rated efficiency were replaced. The average UV intensity of the 13 bulbs was 68.8 and 77.2,uw/cm2 in experiments 1 and 2, respectively. UV radiation exposure chamber. The UV exposure chamber used in the lab-control UV experiment was designed to closely simulate the geometry of the Kelly-Purdy UV Seawater Treatment Unit. Viruscontaminated seawater was placed in petri dishes to a depth of 0.6 cm. The distance of the UV source (a single 30-w bulb, 91 cm long) from the surface of the seawater was 14 cm. The seawater was not stirred during exposure. The intensity of the UV bulb was 83 JAw/cm2. Kelly-Purdy UV experiments. The UV lights in the unit were turned on 10 min before the virus was added to the seawater. The seawater flow rate was adjusted to a maximum of 144 liters/min. Stock virus suspension was then added continuously to the flowing seawater at the intake line 12 meters from the influent end of the UV unit to effect thorough mixing. The mean input multiplicity of virus was approximately 900 and 1,400 PFU/ml of seawater in experiments 1 and 2, respectively. To equilibrate the virus-contaminated seawater throughout the UV unit before collecting samples, 4 min were allowed to elapse. Samples of untreated seawater at the influent end and samples of UV-treated seawater at points in juxtaposition to the center of the five baffles and at the effluent end of the UV unit were collected simultaneously (Fig. 1). Immediately after collection, all samples were diluted in nutrient broth and then frozen at -70 C before assay. Samples were so handled as to preclude photoreactivation. Lab-control UV experiment. Stock virus suspension

3 VOL. 17, 1969 INACTIVATION OF POLIOVIRUS IN SEAWATER 3 FIG. 1. Kelly-Purdy UV Seawater Treatment Unit. Samples were collected at the influent (INF), numbered baffles (I to 5), and the effluent (EFF). Arrows indicate direction offlow. was thawed immediately before use. A virus-pool was prepared in seawater by adding a mean input multiplicity of virus approximating 8,000 PFU/ml (final volume). Measured quantities of the virus-pool were then added to individual plastic petri dishes. The virus, in the open petri dishes, was exposed to irradiation for intervals of 5, 10, 15, 20, 25, or 30 sec; sampling time error was negligible. All samples, including the pool, were diluted in nutrient broth and then frozen at -70 C before assay. Samples were so handled as to preclude photoreactivation. RESULTS AND DISCUSSION Data that consist of integers, such as number of plaques in a cell monolayer or bacterial colonies in a plate count, frequently follow a Poissonian frequency distribution. The logarithm of the ratio of two Poisson variates approximates a normal frequency distribution. Thus, a geometric plot of survival data observed with viruses that produce plaques should manifest linearity. For purposes of this report, the logarithm of the mean virus plaque survival ratios was plotted against the UV exposure times and, thus, was treated as a linear or first-order kinetic reaction. This is a convenient way to graphically present survival data, recognizing, of course, that even data that closely fit straight lines are in reality pseudo first-order at best (11). The theoretical survival curve representing the lab-control UV experiment (unstirred seawater) was made by using the mean inactivation rate (k) calculated from the equation for a first-order reaction, Nt/No = e-kt, where N. is the virus plaque count at time zero, Nt is the virus plaque count at time t (time of UV exposure), and k is the inactivation rate (slope function). In the lab-control UV experiment, time was measured with a stop watch. The survival curve representing the Kelly-Purdy UV experiments (flowing seawater) was made by using the calculated slope function (b') as determined from a line of the form Y = b' X, where the intercept was constrained to pass through the origin. The best fit line was calculated by the equation: b' = XXY/1X2. The choice of b' rather than k as the slope function was based entirely on statistical inference. In the Kelly-Purdy UV experiments, UV exposure time was calculated by the following equalities: equation 1, Q = A V, where Q is the flow rate in cubic centimeters per second, A is the area (depth and width of the water column in square centimeters), and V is the velocity in centimeters per second; equation 2, D = VT, where D is the distance in centimeters, V is the velocity in centimeters per second (value from equation 1), and T is the time in seconds. The observed survival ratios in all the experiments are shown as plotted points, with each point representing the mean virus plaque count of five replicate counts (Fig. 2). Within the limits of experimental error, the observed data fit the theoretical first-order survival curves and indicate exponential inactivation of poliovirus in sea-

4 4 HILL, HAMBLET, AND BENTON APPL. MICROBIOL. 1ff I T - 1V 3- I= 'i 5 IE FIG. 2. Theoretical survival curves for poliovirus T, in unstirred (lab-control experiment) and flowing seawater (Kelly-Purdy experiments) exposed to UV radiation. water by UV radiation. Departure from linearity between the theoretical survival curves and the observed survival ratios was not considered significant. The results of the Kelly-Purdy UV experiments are shown in Table 2. In experiment 1, the observed survival ratios were 6.8 X 10-4 (99.93% reduction) and 2.3 X 104 (99.98% reduction) in 11.7 and 15.7 sec, respectively. No virus was recovered (<0.2 PFU/ml) in 20.6 sec. These calculated, average UV exposure times of 11.7, Replicate assays TABLE 2. Poliovirus multiplicities of flowing UVtreated seawater in the Kelly-Purdy experiments Calculated Expt avg Virus counta Survival ratio Reduction exposure time sec PFUlml I I X 10-' X X X <0. 2b 24.7 < , X X X < < <0.2 a Mean virus plaque count of five replicate subsamples. b No virus detected. 15.7, and 20.6 sec represented the sampling points at baffles 3, 4, and 5, respectively (Fig. 1). In experiment 2, the observed survival ratio was 5.9 X 10-4 (99.94% reduction) in 11.7 sec. No virus was recovered in 15.7 sec. These calculated UV exposure times of 11.7 and 15.7 sec represented the sampling points at baffles 3 and 4, respectively (Fig. 1). It should be noted that a virus multiplicity of only 1 PFU/ml was recovered in the 15.7-sec sample (baffle 4) from the five replicate subsamples in experiment 1, whereas no virus was recovered in the 15.7-sec sample from the five replicate subsamples in experiment 2. Regression analysis of the data from both experiments indicated no significant difference in the slope functions of the inactivation curves TABLE 3. Poliovirus multiplicities offlowing, untreated seawater Sampling pointsa Influent Effluent 1 9b I Meanc a See Fig. 1. b Virus plaque count: PFU/ml X 102. c Grand mean virus plaque count, 1,362.9.

5 VOL INACTIVATION OF POLIOVIRUS IN SEAWATER (P > 0.05). When the facts that the seawater was fairly turbid and that the flow rate was 144 liters/ min through the unit are considered, these results indicated not only a high level of reproducibility but also a high degree of effectiveness of the UV unit in inactivating poliovirus in flowing seawater. The results observed when the virus-contaminated seawater was sampled at each point in the Kelly-Purdy UV unit without UV radiation are shown in Table 3. The replicate virus plaque counts and the mean virus plaque counts at each sampling point were of the same order of magnitude. A two-factor design analysis of variance test (5) of the counts indicated that the virus plaque counts were not significantly different; i.e., replicate counts (within group), P > 0.05, and mean counts (between groups), P > Therefore, all of the observed differences in virus plaque counts could have occurred by chance alone greater than 5% of the time. The results of the lab-control UV experiment are shown in Table 4. The observed survival ratios were 9.7 X 10-s (99.03% reduction) and 3.6 X 10-4 (99.96% reduction) in 15 and 30 sec, respectively. When the regression analysis of these data was compared with the data of the Kelly-Purdy UV experiments, a significant difference in slope functions (P < 0.05) of the two survival curves TABLE 4. Poliovirus multiplicities of UV-treated seawater in laboratory control experiment Expsure Virus counta Survival ratio Reduction sec PFU/mt % 0 8, , X 107' X X X X 10-' X a Mean virus plaque count of five replicate subsamples. was indicated. This finding was expected and can be attributed to stirring effects and UV dosage. Of course, because of the flowing seawater and the multiple UV bulbs, a finite or precise determination of UV dosage as related to UV exposure time in the Kelly-Purdy UV experiments could not be directly made. Consequently, we would expect a statistical "error term" or bias to enter into the analytical calculations in relation to the UV dosage function. Remarkable agreement was nevertheless observed between the virus survival ratios of the two Kelly-Purdy UV experiments. Because the UV dosage as related to UV exposure time was measured directly and thus more rigidly in the lab-control UV experiment, greater precision was observed, as indicated by the standard deviation of the mean inactivation rate (standard error). The reaction constants of the three experiments are shown in Table 5. A comparison of the reaction constants indicated that considerable precision existed among all the experiments. Although perfect homoscedasticity of the slope functions (b' and/or k) was not achieved among the three experiments, the diminutive standard deviations observed would indicate a high degree of analytical precision and experimental control. This observation obviously substantiates the inherent value of replicate subsampling for reliably estimating the multiplicity of virus in the samples. The half-life values of poliovirus between the two Kelly-Purdy UV experiments were 1.29 and 1.37 sec for experiments 1 and 2, respectively; the time difference was only 0.08 sec. The half-life value of poliovirus in the lab-control UV experiment was 2.38 sec. Any influence of the seawater per se on the half-life values of poliovirus was ruled out in view of the time factor (16, 17). The results we observed, whereby inactivation of poliovirus T, infectivity followed first-order kinetics, were consistent with previous findings in our laboratory (13; W. F. Hill, Jr., et al., Bacteriol. Proc., p. 174, 1967) and with the findings of other investigators (3, 4, 7; J. Baron et al., Federation TABLE 5. Calculated slope functions (b'),a mean inactivation rates (k), and half-life ofpoliovirus T, exposed to UV radiation Standard Standard Expt Slope b' deviation -k sec' deviation k9 Half-life ofb' of 5 Kelly-Purdy Lab-control a b' = 2XY/2X2 of line Y = b'x where the intercept is constrained to pass through the or igin. b Half-life = /k. sec

6 6 HILL, HAMBLET, AND BENTON APPL. MICROBIOL. Proc., p. 557, 1959). It is recognized that departure from first-order kinetics has also been observed in virus survival curves (8, 11, 18; J. Baron et al., Federation Proc., p. 557, 1959). We, of course, did not expect to parallel the observations of other experimenters exactly, since obvious differences in experimental design would necessarily influence the results; i.e., virus suspending fluid, UV incident energy available, physical state of the virus (single virions versus aggregated virions), and UV absorption characteristics of the suspending fluid, to name a few. Nevertheless, we consider the results of the Kelly-Purdy UV experiments to have practical application in that effective virological disinfection of the seawater was achieved. We contend that a 103 survival ratio (.99.9% reduction) of the virus population represents effective inactivation. Consequently, our data provide direct supportive evidence that UV radiation is capable of producing desirable levels of treatment for seawater and that the Kelly-Purdy UV Seawater Treatment Unit efficaciously treats seawater to be used in commercial depuration systems. LITERATURE CITED 1. Bartlett, M. S The use of transformations. Biometrics 3: Berg, G., E. K. Harris, S. L Chang, and K. A. Busch Quantitation ofviruses by the plaque technique. J. Bacteriol. 85: Bishop, J. M., N. Quintrell, and G. Koch Poliovirus double-stranded RNA: inactivation by ultraviolet light. J. Mol. Biol. 24: Casto, B. C Effects of ultraviolet irradiation on the transforming and plaque-forming capacities of simian adenovirus SA7. J. Virol. 2: Dixon, W. J., and F. J. Massey, Jr Introduction to statistical analysis, 2nd ed. McGraw-Hill Book Co., Inc. New York. 6. Dulbecco, R., and M. Vogt Plaque formation and isolation of pure lines with poliomyelitis viruses. J. Exptl. Med. 99: Fogh, J Ultraviolet light inactivation of poliomyelitis virus. Proc. Soc. Expd. Biol. Med Galasso, G. J., and D. G. Sharp Effect of particle aggregation on the survival of irradiated vaccinia virus. J. Bacteriol. 90: Hach Chemical Co Turbidity test. Hach procedures for water and sewage analysis using B and L Spectronic-20 colorimeter, p Hach Chemical Co., Ames, Iowa. 10. Hamblet, F. E., W. F. Hill, Jr., and E. W. Akin Effect of plaque assay diluent upon enumeration of poliovirus type 1. Appl. Microbiol. 15: Hiatt, C. W Kinetics of the inactivation of viruses. Bacteriol. Rev. 28: Hill, W. F., Jr., F. E. Hamblet, and E. W. Akin Survival of poliovirus in flowing turbid seawater treated with ultraviolet light. Appl. Microbiol. 15: Hill, W. F., Jr., F. E. Hamblet, and W. H. Benton Effect of ultraviolet light on survival of selected enteroviruses in seawater. In R. J. Hammerstrom and W. F. Hill, Jr. (ed.), Proc. Gulf and South Atlantic States Shellfish Sanitation Res. Conf., Environmental Health Series, Public Health Service PubL No. 999-UIH Hsiung, G. D., and J. L. Melnick Plaque formation with poliomyelitis, coxsackie and orphan (Echo) viruses in bottle cultures of monkey epithelial cells. Virology 1: Kelly, C. B Disinfection of sea water by ultraviolet radiation. Am. J. Public Health 51:167G Magnusson, S., C. E. Hedstrom, and E. Lycke The virus inactivating capacity of sea water. Acta Pathol. Microbiol. Scand. 66: Matossian, A. M., and G. A. Garabedian Virucidal action of sea water. Am. J. Epidemiol. 85: Taylor, A. R., W. W. Kay, L. W. McLean, Jr., F. Oppenheimer, and F. D. Stimpert Effect of ultraviolet light on poliomyelitis virus. J. Immunol. 78: Youden, W. J Statistical methods for chemists, p John Wiley and Sons, Inc., London. 20. Zerbe, W. B., and C. B. Taylor Sea water temperature and density reduction tables. Special Publ. No U.S Department of Commerce, Washington, D.C.