Critical Review on Bromate Formation during Ozonation and Control Options for Its Minimization.

Ozone is a commonly applied disinfectant and oxidant in drinking water and has more recently been implemented for enhanced municipal wastewater treatment for potable reuse and ecosystem protection. One drawback is the potential formation of bromate, a possible human carcinogen with a strict drinking water standard of 10 µg/L. The formation of bromate from bromide during ozonation is complex and involves reactions with both ozone and secondary oxidants formed from ozone decomposition, i.e., hydroxyl radical. The underlying mechanism has been elucidated over the past several decades, and the extent of many parallel reactions occurring with either ozone or hydroxyl radicals depends strongly on the concentration, type of dissolved organic matter (DOM), and carbonate. On the basis of mechanistic considerations, several approaches minimizing bromate formation during ozonation can be applied. Removal of bromate after ozonation is less feasible. We recommend that bromate control strategies be prioritized in the following order: (1) control bromide discharge at the source and ensure optimal ozone mass-transfer design to minimize bromate formation, (2) minimize bromate formation during ozonation by chemical control strategies, such as ammonium with or without chlorine addition or hydrogen peroxide addition, which interfere with specific bromate formation steps and/or mask bromide, (3) implement a pretreatment strategy to reduce bromide and/or DOM prior to ozonation, and (4) assess the suitability of ozonation altogether or utilize a downstream treatment process that may already be in place, such as reverse osmosis, for post-ozone bromate abatement. A one-size-fits-all approach to bromate control does not exist, and treatment objectives, such as disinfection and micropollutant abatement, must also be considered.

Quantitative Assessment of Microbial Pathogens and Indicators of Wastewater Treatment Performance for Safe and Sustainable Water Reuse in India.

Currently, there is no data on the molecular quantification of microbial indicators of recycled water quality in India. In this study, multiple microbial pathogens and indicators of water quality were evaluated at three wastewater treatment plants located in two Indian cities (New Delhi and Jaipur) to determine the treatment performance and suitability of recycled water for safe and sustainable reuse applications. Real-time polymerase chain reaction (PCR) was used for the rapid evaluation of six human pathogens and six microbial indicators of fecal contamination. Among the microbial indicators, pepper mild mottle virus (PMMoV), F+RNA-GII bacteriophage, Bacteroides thetaiotamicron, and four human pathogens (Norovirus genogroups I & II, Giardia, and Campylobacter coli) were detected in all of the influent samples analyzed. This work suggests that the raw influents contain lower levels of noroviruses and adenoviruses and higher levels of Giardia compared to those reported from other geographic regions. Overall, the efficacy of the removal of microbial targets was over 93% in the final effluent samples, which is consistent with reports from across the world. PMMoV and Giardia were identified as the best microbial targets, from the microbial indicators spanning across bacteria, bacteriophages, DNA/RNA viruses, and protozoan parasites, by which to evaluate treatment performance and recycled water quality in Indian settings, as they were consistently present at high concentrations in untreated wastewater both within and across the sites. Also, they showed a strong correlation with other microbial agents in both the raw influent and in the final effluent. These findings provide valuable insights into the use of culture-independent molecular indicators that can be used to assess the microbial quality of recycled water in Indian settings. IMPORTANCE Wastewater treatment plants (WWTPs) have rapidly increased in India during the last decade. Nonetheless, there are only a few labs in India that can perform culture-based screening for microbial quality. In the last 2 years of the pandemic, India has witnessed a sharp increase in molecular biology labs. Therefore, it is evident that culture-independent real-time PCR will be increasingly used for the assessment of microbial indicators/pathogens in wastewater, especially in resource-limited settings. There is no data available on the molecular quantitation of microbial indicators from India. There is an urgent need to understand and evaluate the performance of widely used microbial indicators via molecular quantitation in Indian WWTPs. Our findings lay the groundwork for the molecular quantitation of microbial indicators in WWTPs in India. We have screened for 12 microbial targets (indicators and human pathogens) and have identified pepper mild mottle virus (PMMoV) and Giardia as the best molecular microbiological indicators in Indian settings.

Design and operational considerations in response to Legionella occurrence in Las Vegas Valley groundwater.

Legionella occurrence monitoring is not required by United States Environmental Protection Agency (USEPA) drinking water regulations, and few occurrence studies exist for Legionella in source water or distribution systems. Legionella occurrence was monitored in Las Vegas Valley (Las Vegas, Nevada, USA) drinking water sources, including non-treated surface water, seasonal groundwater (61 wells, before and after chlorination), finished water (after treatment at water treatment facilities), and chlorinated distribution system water (at 9 reservoirs and 75 sample locations throughout the network). Legionella pneumophila was detected at least once at each of the wells sampled before chlorination, with an overall positivity rate of 38% (343/908). During well start-up (time<2 hours; turbidity>3 NTU), L. pneumophila concentrations averaged 2,792±353 MPN/100 mL, with a median of 105 MPN/100 mL, and range of <1 to 90,490 MPN/100 mL across 61 seasonally operated (typically April-October) groundwater wells. After initial flushing (turbidity<3 NTU), the average concentration decreased by more than two orders of magnitude to 24±3 MPN/100 mL but ranged from <1 to >2,273 MPN/100 mL. This trend indicates that stagnation (up to 391 days) contributed to greater initial concentrations, and flushing alone was incapable of complete L. pneumophila elimination. L. pneumophila concentration was significantly, positively correlated with total aqueous adenosine triphosphate (ATP) (p<0.00001, r=0.41-0.71), turbidity (p<0.00001, r=0.27-0.51), orthophosphate (p=0.35-0.076, r=0.51-0.59), and pump depth (p=0.032, r=0.40). During a full-scale assessment of chlorination (Ct=0.7 to 10.5 mg-min/L; T=26.6-28.1°C), substantial reduction of Legionella spp. (up to 2.5 logs) was observed; although, detectable concentrations were still measured. Extrapolating from a Chick-Watson model (log inactivation=0.28*(Ct); R2=0.87) constructed from the full-scale chlorination results, 3- and 4-log inactivation in Las Vegas Valley groundwater would require 10.8 and 14.3 mg-min/L, respectively; at least 3-log inactivation was required to bring Legionella spp. to below detection at the studied well. Chlorine exposure (Ct=0.1 to 10.9 mg-min/L) at most wells discharging directly to the distribution system was insufficient to fully inactivate Legionella spp. After discussing these findings with the state regulatory agency, direct-to-distribution wells (38 of 61 wells) remained out of operation; the distribution system, wells, and reservoirs were monitored for Legionella and chlorine residual, and additional treatment scenarios were identified for further evaluation. Legionella was either not detected or was well controlled in surface water, finished effluent from the drinking water treatment plant, chlorinated reservoirs, and the chlorinated distribution system. This study emphasizes the importance of utility-driven, non-regulatory research in order to protect public health and also identifies the need for greater occurrence monitoring and guidance for Legionella in groundwater supplies.

Ozone disinfection of waterborne pathogens and their surrogates: A critical review.

Viruses, Giardia cysts, and Cryptosporidium parvum oocysts are all major causes of waterborne diseases that can be uniquely challenging in terms of inactivation/removal during water and wastewater treatment and water reuse. Ozone is a strong disinfectant that has been both studied and utilized in water treatment for more than a century. Despite the wealth of data examining ozone disinfection, direct comparison of results from different studies is challenging due to the complexity of aqueous ozone chemistry and the variety of the applied approaches. In this systematic review, an analysis of the available ozone disinfection data for viruses, Giardia cysts, and C. parvum oocysts, along with their corresponding surrogates, was performed. It was based on studies implementing procedures which produce reliable and comparable datasets. Datasets were compiled and compared with the current USEPA Ct models for ozone. Additionally, the use of non-pathogenic surrogate organisms for prediction of pathogen inactivation during ozone disinfection was evaluated. Based on second-order inactivation rate constants, it was determined that the inactivation efficiency of ozone decreases in the following order: Viruses >> Giardia cysts > C. parvum oocysts. The USEPA Ct models were found to be accurate to conservative in predicting inactivation of C. parvum oocysts and viruses, respectively, however they overestimate inactivation of Giardia cysts at ozone Ct values greater than ∼1 mg min L-1. Common surrogates of these pathogens, such as MS2 bacteriophage and Bacillus subtilis spores, were found to exhibit different inactivation kinetics to mammalian viruses and C. parvum oocysts, respectively. The compilation of data highlights the need for further studies on disinfection kinetics and inactivation mechanisms by ozone to better fit inactivation models as well as for proper selection of surrogate organisms.

Norovirus detection in water samples at the level of single virus copies per microliter using a smartphone-based fluorescence microscope.

Norovirus is a widespread public health threat and has a very low infectious dose. This protocol presents the extremely sensitive mobile detection of norovirus from water samples using a custom-built smartphone-based fluorescence microscope and a paper microfluidic chip. Antibody-conjugated fluorescent particles are immunoagglutinated and spread over the paper microfluidic chip by capillary action for individual counting using a smartphone-based fluorescence microscope. Smartphone images are analyzed using intensity- and size-based thresholding for the elimination of background noise and autofluorescence as well as for the isolation of immunoagglutinated particles. The resulting pixel counts of particles are correlated with the norovirus concentration of the tested sample. This protocol provides detailed guidelines for the construction and optimization of the smartphone- and paper-based assay. In addition, a 3D-printed enclosure is presented to incorporate all components in a dark environment. On-chip concentration and the assay of higher concentrations are presented to further broaden the assay range. This method is the first to be presented as a highly sensitive mobile platform for norovirus detection using low-cost materials. With all materials and reagents prepared, a single standard assay takes under 20 min. Although the method described is used for detection of norovirus, the same protocol could be adapted for detection of other pathogens by using different antibodies.

Sediment re-suspension as a potential mechanism for viral and bacterial contaminants.

Pathogenic enteric viruses and bacteria tend to occur in higher concentrations and survive longer in aquatic sediments than suspended in the water column. Re-suspension of these organisms can result in a significant degradation of overlying water quality. Additionally, the re-suspension of microbial pathogens in artificial irrigation canals could endanger the consumption of fresh and ready-to-eat produce. Irrigation water has been implicated in numerous fresh produce outbreaks over the last 30 years. This study aimed to quantify the proportions of bacterial and viral re-suspension from sediment in a recirculating flume with varying velocities. MS2 coliphage and Escherichia coli were found to re-suspend at rates that were not significantly different, despite organism size differences. However, E. coli re-suspension rates from sand and clay were significantly different. This suggests that likely sediment-associated particles were recovered with the organisms attached. Similar re-suspension rates are hypothesized to be due to the dynamics of sediment transport, rather than that of the organisms themselves. This study also indicated that the re-suspension of sediment at very low velocities (e.g., less than 10 cm/s), could impact the microbiological quality of the overlaying water. Results from this study conclude that sediment could be a viable mechanism for irrigation water contamination.

Potential indicators of virus transport and removal during soil aquifer treatment of treated wastewater effluent.

Increased water demands have led to a notable interest in the use of treated wastewater for reuse. Typically, this results from the implementation of advanced treatment of final effluent from wastewater treatment plants prior to reuse for potable or non-potable purposes. Soil aquifer treatment (SAT) is a natural treatment process in which water from sources of varying quality is infiltrated into the soil to further improve its quality. The goal of this study was to determine the log10 reduction values (LRVs) of viruses naturally present in treated effluent and evaluate two potential indicators of virus removal and transport, pepper mild mottle virus (PMMoV) and crAssphage, during SAT of treated effluent. Groundwater was sampled at three wells with different attributes within the Sweetwater Recharge Facility (SWRF) in Tucson, AZ. These sites vary greatly in operational parameters such as effluent infiltration rates and wetting/drying cycles, which may influence virus removal efficiency. Detection of adenovirus, enterovirus, PMMoV, and crAssphage were determined by qPCR/RT-qPCR and log10 reduction values (LRVs) were determined. PMMoV and crAssphage were detected in groundwater associated with a set of recharge basins that exhibited shorter wetting/drying cycles and faster infiltration rates. LRVs for crAssphage and PMMoV at this site ranged from 3.9 to 5.8, respectively. Moreover, PMMoV was detected downflow of the SAT sites, indicating the potential degradation of microbial groundwater quality in the region surrounding managed aquifer recharge facilities. Overall, PMMoV and crAssphage showed potential as conservative process indicators of virus removal during SAT, particularly for attribution of LRV credits. Moreover, the detection of these viruses indicated the potential influence of wetting/drying cycles on virus removal by SAT, a parameter that has not yet been studied with respect to biological contaminants.

Smartphone-Based Paper Microfluidic Particulometry of Norovirus from Environmental Water Samples at the Single Copy Level.

Human enteric viruses can be highly infectious and thus capable of causing disease upon ingestion of low doses ranging from 100 to 102 virions. Norovirus is a good example with a minimum infectious dose as low as a few tens of virions, that is, below femtogram scale. Norovirus detection from commonly implicated environmental matrices (water and food) involves complicated concentration of viruses and/or amplification of the norovirus genome, thus rendering detection approaches not feasible for field applications. In this work, norovirus detection was performed on a microfluidic paper analytic device without using any sample concentration or nucleic acid amplification steps by directly imaging and counting on-paper aggregation of antibody-conjugated, fluorescent submicron particles. An in-house developed smartphone-based fluorescence microscope and an image-processing algorithm isolated the particles aggregated by antibody-antigen binding, leading to an extremely low limit of norovirus detection, as low as 1 genome copy/µL in deionized water and 10 genome copies/µL in reclaimed wastewater.

Novel Circular Rep-Encoding Single-Stranded DNA Viruses Detected in Treated Wastewater.

Here, we present the complete genome sequences of three circular replication-associated protein (Rep)-encoding single-stranded DNA (CRESS DNA) viruses detected in secondary treated and disinfected wastewater effluent. The discovered viruses, named wastewater CRESS DNA virus (WCDV)-1 to -3, represent novel viral species that seem to persist in wastewater effluent.

Variable non-linear removal of viruses during transport through a saturated soil column.

Reduction of viral surrogates (bacteriophage MS2 and murine norovirus-1 [MNV-1]) and viruses naturally present in wastewater (enteroviruses, adenoviruses, Aichi viruses, reovirus, pepper mild mottle virus) was studied in a long-term experiment simulating soil-aquifer treatment of a non-disinfected secondary treated wastewater effluent blend using a 4.4 m deep saturated soil column (95% sand, 4% silt, 1% clay) with a hydraulic residence time of 15.4 days under predominantly anoxic redox conditions. Water samples were collected over a four-week period from the column inflow and outflow as well as from seven intermediate sampling ports at different depths. Removal of MS2 was 3.5 log10 over 4.4 m and removal of MNV-1 was 3 log10 over 0.3 m. Notably, MNV-1 was removed to below detection limit within 0.3 m of soil passage. In secondary treated wastewater effluent, MNV-1 RNA and MS2 RNA degraded at a first-order rate of 0.59 day-1 and 0.12 day-1, respectively. In 15.4 days, the time to pass the soil column, the RNA-degradation of MS2 would amount to 0.8 log10, and in one day that of MNV-1 0.3 log10 implying that attachment of MNV-1 and MS2 to the sandy soil took place. Among the indigenous viruses, genome copies reductions were observed for Aichi virus (4.9 log10) and for pepper mild mottle virus (4.4 log10). This study demonstrated that under saturated flow and predominantly anoxic redox conditions MS2 removal was non-linear and could be described well by a power-law relation. Pepper mild mottle virus was removed less than all of the other viruses studied, which substantiates field studies at managed aquifer recharge sites, suggesting it may be a conservative model/tracer for enteric virus transport through soil.