In vitro bioassays to monitor complex chemical mixtures at a carbon-based indirect potable reuse plant.

Potable water reuse technologies are used to treat wastewater to drinking water quality to help sustain a community's water resources. California has long led the adoption of potable water reuse technologies in the United States and more states are exploring these technologies as water resources decline. Reuse technologies also need to achieve adequate reductions in microbial and chemical contaminant risks to meet public health goals and secure public acceptance. In vitro bioassays are a useful tool for screening if reuse treatment processes adequately reduce toxicity associated with a range of chemical classes that are contaminants of concern. In this study, we used an aryl hydrocarbon receptor (AhR) and an estrogen receptor luciferase bioassay to detect the presence of dioxin-like and estrogenic compounds across a 3800 m3/d carbon-based indirect potable reuse plant that uses carbon-based treatment (SWIFT-RC). Our results demonstrate significant removal of dioxin-like compounds across the SWIFT-RC treatment train. Estrogenicity declined across the treatment train for some months but was extremely variable and low with many samples falling below the method quantification level; consequently, we were not able to reliably determine estrogenicity trends for SWIFT-RC. Comparing the bioanalytical equivalent concentrations detected in the SWIFT-RC water with established monitoring trigger levels from the state of California suggests that SWIFT-RC produced water that met the bioassay guidelines. The log total organic carbon concentration and AhR assay equivalent concentrations are weakly correlated when data across all SWIFT-RC processes are included. Overall, this research demonstrates the performance of in vitro bioassays at a demonstration-scale carbon-based IPR system and highlights both the potential utility and challenges associated with these methods for assessing system performance.

Validation of N95 Filtering Facepiece Respirator Decontamination Methods Available at a Large University Hospital.

BACKGROUND: Due to unprecedented shortages in N95 filtering facepiece respirators, healthcare systems have explored N95 reprocessing. No single, full-scale reprocessing publication has reported an evaluation including multiple viruses, bacteria, and fungi along with respirator filtration and fit. METHODS: We explored reprocessing methods using new 3M 1860 N95 respirators, including moist (50%-75% relative humidity [RH]) heat (80-82°C for 30 minutes), ethylene oxide (EtO), pulsed xenon UV-C (UV-PX), hydrogen peroxide gas plasma (HPGP), and hydrogen peroxide vapor (HPV). Respirator samples were analyzed using 4 viruses (MS2, phi6, influenza A virus [IAV], murine hepatitis virus [MHV)]), 3 bacteria (Escherichia coli, Staphylococcus aureus, Geobacillus stearothermophilus spores, and vegetative bacteria), and Aspergillus niger. Different application media were tested. Decontaminated respirators were evaluated for filtration integrity and fit. RESULTS: Heat with moderate RH most effectively inactivated virus, resulting in reductions of >6.6-log10 MS2, >6.7-log10 Phi6, >2.7-log10 MHV, and >3.9-log10 IAV and prokaryotes, except for G stearothermohphilus. Hydrogen peroxide vapor was moderately effective at inactivating tested viruses, resulting in 1.5- to >4-log10 observable inactivation. Staphylococcus aureus inactivation by HPV was limited. Filtration efficiency and proper fit were maintained after 5 cycles of heat with moderate RH and HPV. Although it was effective at decontamination, HPGP resulted in decreased filtration efficiency, and EtO treatment raised toxicity concerns. Observed virus inactivation varied depending upon the application media used. CONCLUSIONS: Both moist heat and HPV are scalable N95 reprocessing options because they achieve high levels of biological indicator inactivation while maintaining respirator fit and integrity.

Prevalence of Infection-Competent Serogroup 6 Legionella pneumophila within Premise Plumbing in Southeast Michigan.

Coinciding with major changes to its municipal water system, Flint, MI, endured Legionnaires' disease outbreaks in 2014 and 2015. By sampling premise plumbing in Flint in the fall of 2016, we found that 12% of homes harbored legionellae, a frequency similar to that in residences in neighboring areas. To evaluate the genetic diversity of Legionella pneumophila in Southeast Michigan, we determined the sequence type (ST) and serogroup (SG) of the 18 residential isolates from Flint and Detroit, MI, and the 33 clinical isolates submitted by hospitals in three area counties in 2013 to 2016. Common to one environmental and four clinical samples were strains of L. pneumophila SG1 and ST1, the most prevalent ST worldwide. Among the Flint premise plumbing isolates, 14 of 16 strains were of ST367 and ST461, two closely related SG6 strain types isolated previously from patients and corresponding environmental samples. Each of the representative SG1 clinical strains and SG6 environmental isolates from Southeast Michigan infected and survived within macrophage cultures at least as well as a virulent laboratory strain, as judged by microscopy and by enumerating CFU. Likewise, 72 h after infection, the yield of viable-cell counts increased >100-fold for each of the representative SG1 clinical isolates, Flint premise plumbing SG6 ST367 and -461 isolates, and two Detroit residential isolates. We verified by immunostaining that SG1-specific antibody does not cross-react with the SG6 L. pneumophila environmental strains. Because the widely used urinary antigen diagnostic test does not readily detect non-SG1 L. pneumophila, Legionnaires' disease caused by SG6 L. pneumophila is likely underreported worldwide.IMPORTANCEL. pneumophila is the leading cause of disease outbreaks associated with drinking water in the United States. Compared to what is known of the established risks of colonization within hospitals and hotels, relatively little is known about residential exposure to L. pneumophila One year after two outbreaks of Legionnaires' disease in Genesee County, MI, that coincided with damage to the Flint municipal water system, our multidisciplinary team launched an environmental surveillance and laboratory research campaign aimed at informing risk management strategies to provide safe public water supplies. The most prevalent L. pneumophila strains isolated from residential plumbing were closely related strains of SG6. In laboratory tests of virulence, the SG6 environmental isolates resembled SG1 clinical strains, yet they are not readily detected by the common diagnostic urinary antigen test, which is specific for SG1. Therefore, our study complements the existing epidemiological literature indicating that Legionnaires' disease due to non-SG1 strains is underreported around the globe.

Fate of the Urinary Tract Virus BK Human Polyomavirus in Source-Separated Urine.

Human polyomaviruses are emerging pathogens that infect a large percentage of the human population and are excreted in urine. Consequently, urine that is collected for fertilizer production often has high concentrations of polyomavirus genes. We studied the fate of infectious double-stranded DNA (dsDNA) BK human polyomavirus (BKPyV) in hydrolyzed source-separated urine with infectivity assays and quantitative PCR (qPCR). Although BKPyV genomes persisted in the hydrolyzed urine for long periods of time (T90 [time required for 90% reduction in infectivity or gene copies] of >3 weeks), the viruses were rapidly inactivated (T90 of 1.1 to 11 h) in most of the tested urine samples. Interestingly, the infectivity of dsDNA bacteriophage surrogate T3 (T90 of 24 to 46 days) was much more persistent than that of BKPyV, highlighting a major shortcoming of using bacteriophages as human virus surrogates. Pasteurization and filtration experiments suggest that BKPyV virus inactivation was due to microorganism activity in the source-separated urine, and SDS-PAGE Western blots showed that BKPyV protein capsid disassembly is concurrent with inactivation. Our results imply that stored urine does not pose a substantial risk of BKPyV transmission, that qPCR and infectivity of the dsDNA surrogate do not accurately depict BKPyV fate, and that microbial inactivation is driven by structural elements of the BKPyV capsid.IMPORTANCE We demonstrate that a common urinary tract virus has a high susceptibility to the conditions in hydrolyzed urine and consequently would not be a substantial exposure route to humans using urine-derived fertilizers. The results have significant implications for understanding virus fate. First, by demonstrating that the dsDNA (double-stranded DNA) genome of the polyomavirus lasts for weeks despite infectivity lasting for hours to days, our work highlights the shortcomings of using qPCR to estimate risks from unculturable viruses. Second, commonly used dsDNA surrogate viruses survived for weeks under the same conditions that BK polyomavirus survived for only hours, highlighting issues with using virus surrogates to predict how human viruses will behave in the environment. Finally, our mechanistic inactivation analysis provides strong evidence that microbial activity drives rapid virus inactivation, likely through capsid disassembly. Overall, our work underlines how subtle structural differences between viruses can greatly impact their environmental fate.

Biochemical and biological assays of endocrine disrupting compounds in various manure matrices.

Wastes generated by animal agriculture have complex matrices and present a significant challenge for achieving accurate measurements of estrogens. The objective of this study was to compare two different extraction methods and two different biochemical and biological estrogen assays on two dairy manure matrices. The two extraction methods studied in this research were a two-step base-toluene extraction and a one-step cyclohexane extraction. The two assays assessed were an enzyme-linked immunosorbent assay (ELISA) and a yeast estrogen screen (YES) bioassay. Estrogenic activity was measured directly as 17beta-estradiol (E2) through the ELISA method or as E2 equivalents (E2-eq) through the YES bioassay. Both extraction methods yielded reasonable recoveries from distilled water in the absence of matrix interferences. In manure samples, ELISA resulted in recoveries slightly higher than 100% in two types of dairy manures, but YES recoveries varied. The YES bioassay detected much higher estrogenic activities in dairy manure compared to the E2 concentrations measured by ELISA. The base-solvent extraction yielded higher E2 concentrations in dairy manure compared to the one-step cyclohexane extraction. These results suggest that manure matrices vary sufficiently that extraction methods must be optimized for specific assays utilized to quantify estrogens in manures.

Surface catalyzed Fenton treatment of bis(2-chloroethyl) ether and bis(2-chloroethoxy) methane.

This study examined the feasibility of using surface catalyzed Fenton treatment to remediate soil and groundwater contaminated by the chlorinated ethers, bis(2-chloroethyl) ether (BCEE) and bis(2-chloroethoxy) methane (BCEM). Parameters that affect the contaminant loss rate such as porewater pH, hydrogen peroxide concentration, and solid/water ratio were systematically evaluated. Batch reactors were set-up utilizing either contaminated or uncontaminated soil, obtained from an industrial site in Moss Point, MS, that was mixed with synthetic groundwater containing the contaminants of interest. The results show an increase in contaminant reduction with a decrease in pH, an increase in hydrogen peroxide concentration, or an increase in the solid/water ratio. For a similar set of conditions, contaminant reduction was greater for systems utilizing contaminated soil as compared to the systems containing uncontaminated soil. In addition, specific oxygen uptake rates (SOURs) were measured for biomass, collected from an activated sludge plant, exposed to different dilutions of untreated and surface catalyzed Fenton treated water to evaluate whether residual BCEE, BCEM, and their co-contaminants as well as their oxidation by-products were potentially inhibitory or can potentially serve as a substrate for the biomass. The measured SOURs show that the surface catalyzed Fenton treatment enhanced the biodegradability of the contaminated groundwater and served as a substrate for the biomass.

Impact of activated sludge-derived colloidal organic carbon on behavior of estrogenic agonist recombinant yeast bioassay.

The impact of size-fractionated colloidal organic carbon (COC) originating from a biological wastewater treatment facility on the sensitivity of the yeast estrogen screen (YES) bioassay containing the human estrogen receptor (hER) gene was evaluated. Dose-response curves of serially diluted 17beta-estradiol (E2), both in the presence and absence of COC, were generated by the YES bioassay. The concentration of E2 leading to a 50% YES response (effective concentration 50%, or EC50) was used to evaluate quantitatively the estrogenic activity of the different COC-E2 mixtures. The EC50 values for all COC size fractions, including COC-free samples (<1 kD), were statistically greater than the controls using Milli-Q water. Normalized EC50 values significantly increased as a function of COC concentration for the larger size fractions (>0.22 microm), but were not significantly affected by smaller COC material at environmental levels (1-5 mg/L), while both colloidal polysaccharide concentrations and colloidal fluorophores (measured at an excitation/emission wavelength pair of 350 nm/450 nm) appear to have an important role in the sensitivity of the YES bioassay. Estimates of the colloid-associated E2 fraction did not predict accurately increases in EC50 values. Matrix effects of the specific environment being tested with the YES bioassay need to be evaluated closely due to the sensitivity of the hER and reporter plasmid.

Activated sludge deflocculation in response to chlorine addition: the potassium connection.

Chlorination is often used to control filamentous bulking in activated sludge systems. Pure culture and mixed-liquor experiments showed that soluble potassium (K+) concentrations increased by 2.4 mg/L (80%) and 1.5 to 3.6 mg/L (11 to 30%) in the bulk liquid phase of pure and activated sludge cultures that were exposed to chlorine, relative to unchlorinated controls. Effluent turbidity and total suspended solids from settled mixed liquor increased significantly in both short-term batch and sequencing batch reactor experiments when chlorine mass load increased above 6 milligrams of chlorine per gram mixed liquor volatile suspended solids (mg Cl2/g MLVSS) in a single dose, which correlated with a localized chlorine concentration at the dose point of 10 mg/L as Cl2 or greater. The results support the hypothesis that the glutathione-gated potassium efflux (GGKE) bacterial stress response may contribute to increased effluent turbidity associated with high doses of mixed-liquor chlorination. It is suggested that potassium is a useful parameter to monitor at full-scale facilities when determining chlorine mass doses that should be used to control filaments and minimize increases in effluent turbidity.

Implicating the glutathione-gated potassium efflux system as a cause of electrophile-induced activated sludge deflocculation.

The glutathione-gated K(+) efflux (GGKE) system represents a protective microbial stress response that is activated by electrophilic or thiol-reactive stressors. It was hypothesized that efflux of cytoplasmic K(+) occurs in activated sludge communities in response to shock loads of industrially relevant electrophilic chemicals and results in significant deflocculation. Novosphingobium capsulatum, a bacterium consistent with others found in activated sludge treatment systems, responded to electrophilic thiol reactants with rapid efflux of up to 80% of its cytoplasmic K(+) pool. Furthermore, N. capsulatum and activated sludge cultures exhibited dynamic efflux-uptake-efflux responses very similar to those observed by others in Escherichia coli K-12 exposed to the electrophilic stressors N-ethylmaleimide and 1-chloro-2,4-dinitrobenzene and the reducing agent dithiothreitol. Fluorescent LIVE/DEAD stains were used to show that cell lysis was not the cause of electrophile-induced K(+) efflux. Nigericin was used to artificially stimulate K(+) efflux from N. capsulatum and activated sludge cultures as a comparison to electrophile-induced K(+) efflux and showed that cytoplasmic K(+) efflux by both means corresponded with activated sludge deflocculation. These results parallel those of previous studies with pure cultures in which GGKE was shown to cause cytoplasmic K(+) efflux and implicate the GGKE system as a probable causal mechanism for electrophile-induced, activated sludge deflocculation. Calculations support the notion that shock loads of electrophilic chemicals result in very high K(+) concentrations within the activated sludge floc structure, and these K(+) levels are comparable to that which caused deflocculation by external (nonphysiological) KCl addition.

Sorption of 17beta-estradiol and 17alpha-ethinylestradiol by colloidal organic carbon derived from biological wastewater treatment systems.

Sorption coefficients (K(COC)) between 17beta-estradiol (E2), 17alpha-ethinylestradiol (EE2) and size-fractionated colloidal organic carbon (COC) derived from two biological wastewater treatment facilities were quantified by fluorescence quenching. The two wastewater treatment systems included a full-scale activated sludge system (FSAS) and a membrane bioreactor (MBR). The K(COC) coefficients were highly variable and ranged between (<1 to 179) x 10(3) L/kgCOC for E2 and (<1 to 430) x 10(3) L/kgCOC for EE2 and were higher than expected from the analytes octanol-water partition coefficient. Correlations between the molar extinction coefficients measured at 280 nm (e280) and K(COC) coefficients were weak but stronger for E2 compared to EE2. Attempts at correlating sorption behavior with colloidal protein and polysaccharide concentrations were only marginally successful (r2 approximately 0.4). These low correlations suggest that aromatic content, protein, or polysaccharide concentration can not adequately explain E2 and EE2 sorption behavior to COC and that other fractions of the organic matter pool play an important role in binding. A substantial portion of the aqueous E2 and EE2 concentrations (up to 60%) may be associated with colloidal material, suggesting that COC may play a role in the fate and transport of E2 and EE2 during the activated sludge process.

Estrogen receptor agonist fate during wastewater and biosolids treatment processes: a mass balance analysis.

The estrogen receptor agonist fate of hexane extracts from various locations and phases (liquid and solid) within one pilot-scale and two full-scale wastewater treatment facilities were examined by use of the receptor-binding yeast estrogen screen (YES assay). Estrogenic activity was found in samples that contained a high concentration of biological solids and was particularly high in the suspended solid fraction from biosolids treatment facilities. Mass balances revealed that the estrogenic activity associated with the processed biosolids constituted between 5 and 10% of the influent estrogenic activity, while the treated liquid effluent prior to disinfection contained between 26 and 43%. Overall, this suggests that between 51 and 67% of the estrogenic activity contained in the influent wastewater was either biodegraded during the wastewater or biosolids treatment processes or was unavailable to the extraction/detection procedure. In both aerobic and anaerobic digestion, mass balances revealed an increase in estrogenic activity as treatment progressed and biosolids destruction occurred. The estrogenic activity associated with the solid phase decreased during mesophilic aerobic digestion. A correlation was observed between the estrogenicity of mixed liquor suspended solids and aerobic sludge age and suggests that wastewater treatment facilities can be designed and operated to enhance the sorption and removal of estrogenic compounds from the liquid phase.

Investigating a mechanistic cause for activated-sludge deflocculation in response to shock loads of toxic electrophilic chemicals.

It is hypothesized that a physiological bacterial stress response mechanism, called the glutathione-gated potassium efflux system, is a significant contributor to activated-sludge deflocculation caused by shock loads of toxic electrophilic chemicals. The results show significant potassium (K+) efflux from activated sludge flocs to the bulk liquid in response to sublethal (concentrations less than that required to reduce the specific oxygen uptake rate by 50%) shock loads of chloro-2,4-dinitrobenzene, N-ethylmaleimide, 2,4-dinitrotoluene, benzoquinone, and cadmium in a bench-scale sequencing batch reactor system. Electrophile-induced K+ efflux was correlated with significant deflocculation, as measured by an increase in effluent volatile suspended solids. The K+ efflux occurred immediately (within minutes) after toxin addition and preceded the observed increase in effluent turbidity. The transport of other cations, including sodium, calcium, magnesium, iron, and aluminum, either to or from the floc structure, was negligible as compared with K+ efflux, and cell lysis was determined to be minimal at the chemical shock loads applied. The current results are the first to suggest that activated-sludge upset (i.e., deflocculation) may be caused by a specific protective stress response in bacteria.