Wastewater residence times in sewage systems are typically less than 24 h. Although ϕ6 and MHV had T 90 values of 7—13 h in unpasteurized wastewater at 25 °C, the T 90 values increase to 28—36 h at 10 °C. Human enveloped viruses excreted in feces may therefore reach wastewater treatment plants in an infective state, especially in cool climates. Local outbreaks and global pandemics of enveloped viruses excreted in feces or urine are therefore relevant for wastewater utilities.

Inactivation kinetics of the enveloped viruses MHV, ϕ6, and Ebolavirus in pasteurized or gamma-irradiated wastewater have been reported previously. (11−13) In our experiments, the two enveloped viruses lost infectivity at a significantly slower rate in pasteurized wastewater compared to unpasteurized wastewater, except for the case of MHV at 25 °C ( Figure 1 SI Table S3 ). The most pronounced effect occurred with ϕ6, which had a first-order inactivation rate constant (±SD) of 0.317 (±0.022) hin unpasteurized wastewater and 0.044 (±0.004) hin pasteurized wastewater at 25 °C. A statistically significant difference in the inactivation kinetics of the nonenveloped viruses was not observed in pasteurized wastewater and unpasteurized wastewater; this may be due to the fact that our experiments were stopped before 90% of the nonenveloped viruses were inactivated. Discrepancies in inactivation kinetics in sterilized and nonsterilized wastewater have been reported previously for nonenveloped viruses, (46) and may be due to bacterial extracellular enzyme activity and protozoan or metazoan predation. (47,48) Overall, the results suggest that unpasteurized wastewater samples should be employed for survivability tests when feasible.

Figure 1. Virus survival in wastewater and pasteurized wastewater at 10 and 25 °C. Viruses were spiked into wastewater to final concentrations of 3 × 10 4 PFU mL –1 for MHV and 5—8 × 10 5 PFU mL –1 for MS2, T3 and ϕ6. Error bars represent the standard deviations of replicates from wastewater samples collected on different days ( n = 3). SI Table S3 summarizes corresponding rate constants and estimated T 90 values.

Inactivation of the two enveloped viruses (MHV and ϕ6) and nonenveloped virus MS2 in unpasteurized and pasteurized wastewater at 10 and 25 °C followed first-order kinetics ( Figure 1 SI Table S3 ), with inactivation proceeding faster for the enveloped viruses. In unpasteurized wastewater at 25 °C, the T(±SD) values for MHV and ϕ6 were 13 (±1) and 7 (±0.4) hours, respectively, and 121 (±36) hours for MS2 ( SI Table S3 ). The nonenveloped T3 virus survived much longer than the other virus surrogates with no significant decrease in infectivity observed within the 48 h experiments for both temperatures ( Figure 1 ). This is consistent with long survival times reported for tailed phages in adverse conditions. (45) The inactivation kinetics of the enveloped viruses were significantly (< 0.0001) slower in wastewater at 10 °C compared to 25 °C ( SI Figure S4 ), with T(±SD) values of 36 (±5) and 28 (±2) hours for MHV and ϕ6 at 10 °C, respectively ( SI Table S3 ). Like T3, MS2 inactivation was not statistically different at the two temperatures (= 0.1813) within the tested time scale ( SI Figure S4 ).

Comparison of Virus Partitioning in Wastewater

The measured concentrations of infective MHV and ϕ6 in the solids-removed wastewater samples immediately after spiking, mixing, and centrifuging, were consistently lower than the theoretical concentrations based on the amount of viruses spiked into the sample ( SI Figure S1 ). Approximately 47% of the spiked MHV and 77% of the spiked ϕ6 were recovered in the centrate of the solids-removed wastewater. This is compared to a nearly 100% recovery of the nonenveloped viruses MS2 and T3. Nearly all of the MHV was recovered when it was spiked into PBS and centrifuged in the same manner ( SI Figure S1 ). This suggests that a fraction of the enveloped viruses (53% MHV and 23% ϕ6) were rapidly inactivated in the solids-removed wastewater. A pronounced initial decrease in infective virus concentration was previously observed when Ebola virus was added to pasteurized wastewater. (12) In those experiments, the number of infective Ebola viruses decreased rapidly over the first 24 h (∼2-log loss) and then stabilized at a much slower inactivation rate over the subsequent 7 days. Similar biphasic inactivation kinetics have also been observed with nonenveloped viruses, which were attributed to subpopulations of viruses with varied susceptibilities to solution chemistry or temperature. (38) In our partitioning experiments, we chose to normalize measured concentrations in the wastewater and solids-removed wastewater samples over time to concentrations measured in solids-removed samples immediately after they were spiked with viruses, mixed, and centrifuged. We felt this approach was justified because the behaviors of the persistent subpopulations are of most interest for real wastewater systems.

k 1 ). MHV, ϕ6, and MS2 concentrations decreased significantly over a three-day period in the solids-removed wastewater samples ( Figure 2 ) and the resulting rate constants were assumed to equal virus inactivation rates in the liquid fraction of wastewater ( eq 1 ). (38) When the viruses were spiked in wastewater samples containing solids, the normalized MHV and ϕ6 concentrations in the wastewater liquid phase (in centrate after centrifugation) decreased rapidly in the first hour, and then eventually decreased at the same rate as virus inactivation in the solids-removed sample ( Figure 2 ). The MS2 concentration in the wastewater liquid phase decreased rapidly at first, and then slowed to a rate that was faster than MS2 inactivation in the solids-removed sample ( Figure 2 ). No significant decay of T3 was observed in the solids-removed wastewater samples or the liquid phase of wastewater samples.

Figure 2 Figure 2. Adsorption and inactivation kinetics and model simulations for enveloped viruses (MHV and ϕ6) and nonenveloped viruses (MS2 and T3) in 4 °C wastewater. Viruses were spiked into wastewater and solids-removed wastewater samples to final concentrations of 5 × 104 PFU mL–1 for MHV, and 6—8 × 105 PFU mL–1 for MS2, T3 and ϕ6. C l * and C l,ww * are nondimensional concentrations of infective viruses in the solids-removed sample centrates and wastewater sample centrates, respectively. Both values were normalized to the initial measured virus concentration in the solids-removed sample centrates. No significant decline in T3 infectivity was observed within 36 h. Error bars represent the range of data from duplicate experiments conducted in wastewater samples collected on different days (n = 2).

Based on these results, the MHV and ϕ6 sorption kinetics can be best described by a noninstantaneous quasi-equilibrium adsorption model in which the virus sorption to wastewater solids does not occur instantaneously and the inactivation rates in the wastewater solid and liquid phases are equal ( SI Table S4 ). A similar model was used to describe bacteriophage λ sorption kinetics with sand. (38) In comparison, MS2 behavior is best described by the noninstantaneous quasi-equilibrium adsorption and surface sink model. In this model, virus inactivation is faster in the solid phase than in the liquid phase ( SI Table S4 ); a similar model was proposed for the interaction of bacteriophage MS2 and PRD1 with sediments. (49) Bacteriophage T3 could not be modeled due to the nonsignificant decreases in infective viruses measured over the experiment time scale.

–1. These models predict that 26% of MHV, 22% of ϕ6, and 6% of MS2 adsorbed to wastewater solids at equilibrium ( Figure 3 SI Table S4 ). Although the T3 virus kinetics could not be modeled, < 5% of the spiked T3 had partitioned to the wastewater solids at the end of the 36 h experiment; this suggests that like MS2, T3 partitions overwhelmingly to the liquid fraction of wastewater ( Figure 2 ). The equilibrium percentages reported here are not representative for all wastewaters because wastewater solids concentrations vary widely. It should be noted that our wastewater solid concentrations were typical for medium-strength municipal wastewaters (37) SI Table S1 ) with an average TSS value of 235 mg L

Figure 3 Figure 3. Models for adsorption and inactivation kinetics of enveloped viruses (MHV and ϕ6) and nonenveloped viruses (MS2) in 4 °C wastewater. ξ 1 * represents the fraction of viruses inactivated in liquid fraction of wastewater; ξ 2 * represents the fraction of viruses reversibly adsorbed to wastewater solids; ξ 3 * represents the fraction of viruses inactivated on the solid surface.

The partitioning results for MS2 and T3 are consistent with an early observation that wastewater solids are poor at absorbing enteric viruses. (50) Wastewater solids tend to be negatively charged, as is MS2 (isoelectric point = 3.9). The isoelectric point for T3 has not been reported, but the similar T2 and T4 viruses have isoelectric points <6. (51) A study on the adsorption of four nonenveloped viruses to various solid surfaces demonstrated that long-ranged electrostatic interactions and hydrophobic effects between the virus capsid proteins and the sorbent surfaces dictated adsorption, with short-ranged van der Waals and steric interactions playing less important roles. (52) Similar work has not been conducted for enveloped viruses, and the impact that the surface phospholipids and various membrane proteins have on partitioning remains elusive.

Despite the poor sorption of nonenveloped enteric viruses to wastewater solids, some enteric viruses have been observed in primary settled solids in high concentrations. (36,53) In such cases, the viruses were likely released into wastewater within or strongly associated with fecal solids and never reached equilibrium between the liquid and solid fractions. When excreted in watery diarrhea or urine, the viruses would more likely reach equilibrium. Our results suggest that if allowed to reach equilibrium, enveloped viruses more strongly associate with wastewater solids than nonenveloped viruses. Consequently, enveloped viruses would be removed to a greater extent than nonenveloped viruses in primary wastewater treatment. More enveloped and nonenveloped viruses will need to be tested to confirm the results obtained with the two enveloped and two nonenveloped model viruses.