This research aimed to address the knowledge gaps in the literature regarding the removal of waterborne pathogens (viruses and protozoa) by modified multistage slow sand filtration. In the current study, two pilot-scale multistage slow sand filtration systems were operated continuously for over two years. The pilot systems treated agricultural- and urban-impacted raw river water of variable quality with turbidity peaks over 300 NTU and seasonal cold temperatures <2??C. The first system (Pilot 1) consisted of two independent trains that included pre-ozonation, shallow-bed upflow gravel roughing filtration, and shallow-bed slow sand filtration. Pilot 1 was a pilot-scale version of an innovative, commercially available full-scale system. The second system (Pilot 2) included a full-depth upflow gravel roughing filter, a full-depth slow sand filter, and a second shallow-depth slow sand filter in series. The SSFs of both pilots were operated at high hydraulic loading rates (typically 0.4 m/h) at the upper limit of the literature recommended range (0.05 to 0.4 m/h). Both pilot systems provided excellent turbidity removal despite the high filtration rates. Effluent turbidity of all multistage SSF pilot systems were within the regulated effluent limits in Ontario for full-scale SSFs (below 1 NTU at least 95% of the time and never exceeded 3 NTU), despite raw water turbidity peaks over 100 NTU. The roughing filters contributed to approximately 60-80% of the full-train turbidity removal, compared to and 20-40% for the slow sand filters. On average, the second slow sand filter in pilot 2 provided almost no additional turbidity removal. The slow sand filter run lengths were short because of frequent high raw water turbidity, with about 50-80% of the runs in the range of 1-3 weeks. To prevent excessive SSF clogging and maintenance, filtration rates should be decreased during periods of high turbidity. Seven Cryptosporidium and Giardia challenge tests were conducted on the slow sand filters of both pilot systems at varying filtration rates (0.4 or 0.8 m/h), temperatures (2 to 25??C), and biological maturities (4 to 20 months). Removal of oocysts and cysts were good regardless of sand depth, hydraulic loading rate, and water temperature in the ranges tested. Average removals in the SSFs ranged from 2.6 to >4.4 logs for Cryptosporidium oocysts and ranged from >3.8 to >4.5 logs for Giardia cysts. This was consistent with findings in the literature, where oocyst and cyst removals of >4 logs have been reported. Cryptosporidium oocyst removals improved with increased biological maturity of the slow sand filters. At a water temperature of 2??C, average removal of oocysts and cysts were 3.9 and >4.5 logs, respectively, in a biologically mature SSF. Doubling the filtration rate from 0.4 to 0.8 m/h led to a marginal decrease in oocyst removals. Sand depths in the range tested (37-100 cm) had no major impact on oocyst and cyst removals, likely because they are removed primarily in the upper section of slow sand filter beds by straining. In general, good oocyst and cyst removals can be achieved using shallower slow sand filter bed depths and higher filtration rates than recommended in the literature. There are very few studies in the literature that quantify virus removal by slow sand filtration, especially at high filtration rates and shallow bed depths. There are no studies that report virus removal by slow sand filtration below 10??C. As such, 16 MS2 bacteriophage challenge tests were conducted at varying water temperatures (<2 to >20??C) and filtration rates (0.1 vs. 0.4 m/h) between February and June 2006 on biologically mature slow sand filters with varying bed depths (40 vs. 90 cm). Biologically mature roughing filters were also seeded with MS2. Average MS2 removals ranged from 0.2 to 2.2 logs in the SSFs and 0.1 to 0.2 logs in the RFs under all conditions tested. Virus removal by slow sand filtration was strongly dependant on hydraulic loading rate, sand depth, and water temperature. Virus removal was greater at a sand depth of 90 cm vs. 40 cm, at an HLR of 0.1 m/h vs. 0.4 m/h, and at warm (20-24??C) vs. cold (<2-10??C) water temperatures when sufficient warm water acclimation time was provided. Increased sand depth likely increased MS2 removal because of greater detention time for predation and greater contact opportunities for attachment to sand grains and biofilms. A lower HLR would also increase MS2 removal by increasing detention time, in addition to decreasing shear and promoting attachment to filter media and biofilms. Greater MS2 removal at warmer water temperatures was attributed to improved biological activity in the filters. Schmutzdecke scraping was found to have only a minor and short-term effect on MS2 removals. Virus removal can be optimized by providing deep SSF beds and operating at low filtration rates. Virus removal may be impaired in cold water, which could affect the viability of using SSF/MSF at northern climates if communities do not use disinfection or oxidation. As a stand-alone process, slow sand filtration (with or without roughing filtration) may not provide complete virus removal and should be combined with other treatment processes such as disinfection and oxidation to protect human health.

Thesis advisor: Gregory R. Chiklis / Thesis advisor: Kathleen Dunn / In molecular diagnostics, the polymerase chain reaction (PCR) is used to amplify small amounts of nucleic acids found in patient samples, allowing for detection of diseases within hours of infection. This early detection allows medical professionals to diagnose and treat patients with greater success. It is crucial that internal controls, such as NATtrol™-treated microorganisms, are used in these PCR assays to avoid false-negative results and ensure accurate diagnosis of patients. NATtrol™ treatment renders microorganisms non-infectious while leaving them fully intact with their complete RNA or DNA genomes. Therefore, NATtrol™-treated microorganisms can be used in PCR as full-process internal controls that are spiked into patient samples and co-extracted and co-amplified within the sample. If the spiked NATtrol™ control returns expected results on the test, then the patient sample result can also be trusted. Here, we performed studies to validate the use of NATtrol™-treated MS2 virus as a universal full-process internal molecular control. In these studies, a quantitative, real-time, reverse-transcription PCR (qRT-PCR) assay was performed on the Roche LightCycler 480 instrument. Studies included working range validation, limit of detection, within-run precision, between-run precision, real-time stability, freeze-thaw (transport) stability, and open-vial (use-life) stability. All studies demonstrated the precision and stability of the MS2 NATtrol™ molecular control. / Thesis (BS) — Boston College, 2014. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: College Honors Program. / Discipline: Biology Honors Program. / Discipline: Biology.

This research aimed to address the knowledge gaps in the literature regarding the removal of waterborne pathogens (viruses and protozoa) by modified multistage slow sand filtration. In the current study, two pilot-scale multistage slow sand filtration systems were operated continuously for over two years. The pilot systems treated agricultural- and urban-impacted raw river water of variable quality with turbidity peaks over 300 NTU and seasonal cold temperatures <2°C. The first system (Pilot 1) consisted of two independent trains that included pre-ozonation, shallow-bed upflow gravel roughing filtration, and shallow-bed slow sand filtration. Pilot 1 was a pilot-scale version of an innovative, commercially available full-scale system. The second system (Pilot 2) included a full-depth upflow gravel roughing filter, a full-depth slow sand filter, and a second shallow-depth slow sand filter in series. The SSFs of both pilots were operated at high hydraulic loading rates (typically 0.4 m/h) at the upper limit of the literature recommended range (0.05 to 0.4 m/h). Both pilot systems provided excellent turbidity removal despite the high filtration rates. Effluent turbidity of all multistage SSF pilot systems were within the regulated effluent limits in Ontario for full-scale SSFs (below 1 NTU at least 95% of the time and never exceeded 3 NTU), despite raw water turbidity peaks over 100 NTU. The roughing filters contributed to approximately 60-80% of the full-train turbidity removal, compared to and 20-40% for the slow sand filters. On average, the second slow sand filter in pilot 2 provided almost no additional turbidity removal. The slow sand filter run lengths were short because of frequent high raw water turbidity, with about 50-80% of the runs in the range of 1-3 weeks. To prevent excessive SSF clogging and maintenance, filtration rates should be decreased during periods of high turbidity. Seven Cryptosporidium and Giardia challenge tests were conducted on the slow sand filters of both pilot systems at varying filtration rates (0.4 or 0.8 m/h), temperatures (2 to 25°C), and biological maturities (4 to 20 months). Removal of oocysts and cysts were good regardless of sand depth, hydraulic loading rate, and water temperature in the ranges tested. Average removals in the SSFs ranged from 2.6 to >4.4 logs for Cryptosporidium oocysts and ranged from >3.8 to >4.5 logs for Giardia cysts. This was consistent with findings in the literature, where oocyst and cyst removals of >4 logs have been reported. Cryptosporidium oocyst removals improved with increased biological maturity of the slow sand filters. At a water temperature of 2°C, average removal of oocysts and cysts were 3.9 and >4.5 logs, respectively, in a biologically mature SSF. Doubling the filtration rate from 0.4 to 0.8 m/h led to a marginal decrease in oocyst removals. Sand depths in the range tested (37-100 cm) had no major impact on oocyst and cyst removals, likely because they are removed primarily in the upper section of slow sand filter beds by straining. In general, good oocyst and cyst removals can be achieved using shallower slow sand filter bed depths and higher filtration rates than recommended in the literature. There are very few studies in the literature that quantify virus removal by slow sand filtration, especially at high filtration rates and shallow bed depths. There are no studies that report virus removal by slow sand filtration below 10°C. As such, 16 MS2 bacteriophage challenge tests were conducted at varying water temperatures (<2 to >20°C) and filtration rates (0.1 vs. 0.4 m/h) between February and June 2006 on biologically mature slow sand filters with varying bed depths (40 vs. 90 cm). Biologically mature roughing filters were also seeded with MS2. Average MS2 removals ranged from 0.2 to 2.2 logs in the SSFs and 0.1 to 0.2 logs in the RFs under all conditions tested. Virus removal by slow sand filtration was strongly dependant on hydraulic loading rate, sand depth, and water temperature. Virus removal was greater at a sand depth of 90 cm vs. 40 cm, at an HLR of 0.1 m/h vs. 0.4 m/h, and at warm (20-24°C) vs. cold (<2-10°C) water temperatures when sufficient warm water acclimation time was provided. Increased sand depth likely increased MS2 removal because of greater detention time for predation and greater contact opportunities for attachment to sand grains and biofilms. A lower HLR would also increase MS2 removal by increasing detention time, in addition to decreasing shear and promoting attachment to filter media and biofilms. Greater MS2 removal at warmer water temperatures was attributed to improved biological activity in the filters. Schmutzdecke scraping was found to have only a minor and short-term effect on MS2 removals. Virus removal can be optimized by providing deep SSF beds and operating at low filtration rates. Virus removal may be impaired in cold water, which could affect the viability of using SSF/MSF at northern climates if communities do not use disinfection or oxidation. As a stand-alone process, slow sand filtration (with or without roughing filtration) may not provide complete virus removal and should be combined with other treatment processes such as disinfection and oxidation to protect human health.

Même si les traitements thermiques ou la désinfection par les oxydants ont démontré leur efficacité virucide, les mécanismes liés à la perte du caractère infectieux ne sont pas connus. Ceci pose un réel problème d’interprétation de la présence de génome viral en matière de risque infectieux dans les aliments. Ce travail de thèse a pour objectif d’étudier l’évolution des propriétés de surface (charge et hydrophobie) de virus modèles, bactériophage MS2 et norovirus murin, au cours de l’inactivation par la chaleur, l’hypochlorite de sodium et l’ozone. Pour nos deux virus, nous démontrons l’existence d’une température critique au-delà de laquelle la particule virale se déstructure en libérant son génome. Un simple traitement à la RNase permettrait alors de ne détecter que des virus infectieux par biologie moléculaire. Le traitement thermique implique aussi une augmentation de l’hydrophobie soulignant des modifications conformationnelles de la capside. L’hypochlorite de sodium ne modifie que peu les propriétés de surface mais des phénomènes d’oxydation ont lieu au niveau de la capside puisque la charge du bactériophage MS2 est légèrement modifiée. Ces modifications diminuent la résistance thermique du virus. Nous démontrons un effet synergique de l’hypochlorite de sodium et la chaleur sur le bactériophage MS2 (inactivation, RNase et hydrophobie). Quant à l’ozone gazeux, nous soulignons son intérêt pour le traitement virucide des aliments fragiles. Ainsi, ce travail précise les mécanismes d’inactivation des virus et ouvre de nouvelles perspectives tant pour discriminer les virus infectieux et non-infectieux que pour proposer l’exploration de nouveaux traitements technologiques / Although heat treatments or disinfections by oxidants have proven their virucidal efficiencies, mechanisms related to the loss of infectivity are not known. This statement could lead to a misinterpretation of the presence of viral genome on infection risk for humans in food matrices. This thesis aimed to study the evolution of surface properties (charge and hydrophobicity) for model viruses, bacteriophage MS2 and murine norovirus, during the heat, sodium hypochlorite and ozone inactivations. For both viruses, the existence of a critical temperature beyond which the viral particle was disrupted and released its genome was demonstrated. Simple treatment with RNase would then only detect infectious virus by molecular biology. The heat treatment also involved a transient increase in the hydrophobicity which highlighted conformational changes of the viral capsid. Sodium hypochlorite slightly modified the surface properties but oxidation phenomena occurred onto capsid since the bacteriophage MS2 charge has changed a little. These changes decreased the thermal resistance of the virus. Synergistic effects of both sodium hypochlorite and heat were observed on the inactivation of MS2 phages, the sensitivity of their genome to RNases and the increase in hydrophobicity of remaining infectious particles. Regarding gaseous ozone, we underlined its interest in the case of virucidal treatment of fragile food matrices. Therefore, this work specified the virus inactivation mechanisms and opened up new perspectives to discriminate infectious from non-infectious viruses but also to propose the exploration of new technological processes