Wastewater-based epidemiology may be useful for informing public health response to viral diseases like COVID-19 caused by SARS-CoV-2. We quantified SARS-CoV-2 RNA in wastewater influent and primary settled solids in two wastewater treatment plants to inform the preanalytical and analytical approaches and to assess whether influent or solids harbored more viral targets. The primary settled solids samples resulted in higher SARS-CoV-2 detection frequencies than the corresponding influent samples. Likewise, SARS-CoV-2 RNA was more readily detected in solids using one-step digital droplet (dd)RT-PCR than with two-step RT-QPCR and two-step ddRT-PCR, likely owing to reduced inhibition with the one-step ddRT-PCR assay. We subsequently analyzed a longitudinal time series of 89 settled solids samples from a single plant for SARS-CoV-2 RNA as well as coronavirus recovery (bovine coronavirus) and fecal strength (pepper mild mottle virus) controls. SARS-CoV-2 RNA targets N1 and N2 concentrations correlated positively and significantly with COVID-19 clinically confirmed case counts in the sewershed. Together, the results demonstrate that measuring SARS-CoV-2 RNA concentrations in settled solids may be a more sensitive approach than measuring SARS-CoV-2 in influent.

Analysis of environmental DNA (eDNA) to identify macroorganisms and biodiversity has the potential to significantly augment spatial and temporal biological monitoring in aquatic ecosystems. Current monitoring methods relying on the physical identification of organisms can be time consuming, expensive, and invasive. Measuring eDNA shed from organisms provides detailed information on the presence and abundance of communities of organisms. However, little is known about eDNA shedding and decay in aquatic environments. In the present study, we designed novel Taqman qPCR assays for three ecologically and economically important marine fish-Engraulis mordax (Northern Anchovy), Sardinops sagax (Pacific Sardine), and Scomber japonicas (Pacific Chub Mackerel). We subsequently measured fish eDNA shedding and decay rates in seawater mesocosms. eDNA shedding rates ranged from 165 to 3368 pg of DNA per hour per gram of biomass. First-order decay rate constants ranged from 0.055 to 0.101 per hour. We also examined the size fractionation of eDNA and concluded eDNA is both intra- and extracellular. Finally, we derived a simple mass-balance model to estimate fish abundance from eDNA concentration. The mesocosm-derived shedding and decay rates inform the interpretation of eDNA concentrations measured in environmental samples and future use of eDNA as a monitoring tool.

Health-relevant microorganisms present in natural surface waters and engineered treatment systems that are exposed to sunlight can be inactivated by a complex set of interacting mechanisms. The net impact of sunlight depends on the solar spectral irradiance, the susceptibility of the specific microorganism to each mechanism, and the water quality; inactivation rates can vary by orders of magnitude depending on the organism and environmental conditions. Natural organic matter (NOM) has a large influence, as it can attenuate radiation and thus decrease inactivation by endogenous mechanisms. Simultaneously NOM sensitizes the formation of reactive intermediates that can damage microorganisms via exogenous mechanisms. To accurately predict inactivation and design engineered systems that enhance solar inactivation, it is necessary to model these processes, although some details are not yet sufficiently well understood. In this critical review, we summarize the photo-physics, -chemistry, and -biology that underpin sunlight-mediated inactivation, as well as the targets of damage and cellular responses to sunlight exposure. Viruses that are not susceptible to exogenous inactivation are only inactivated if UVB wavelengths (280-320 nm) are present, such as in very clear, open waters or in containers that are transparent to UVB. Bacteria are susceptible to slightly longer wavelengths. Some viruses and bacteria (especially Gram-positive) are susceptible to exogenous inactivation, which can be initiated by visible as well as UV wavelengths. We review approaches to model sunlightmediated inactivation and illustrate how the environmental conditions can dramatically shift the inactivation rate of organisms. The implications of this mechanistic understanding of solar inactivation are discussed for a range of applications, including recreational water quality, natural treatment systems, solar disinfection of drinking water (SODIS), and enhanced inactivation via the use of sensitizers and photocatalysts. Finally, priorities for future research are identified that will further our understanding of the key role that sunlight disinfection plays in natural systems and the potential to enhance this process in engineered systems. Environmental signicanceThe manuscript provides a comprehensive synthesis of the current understanding of the mechanisms by which sunlight causes damage to microorganisms, ultimately leading to inactivation. This topic is important for understanding the fate and transport of microbiological contaminants in all sunlit surface waters, including fresh and marine ecosystems, as well as engineered treatment systems.

Freshwater mussels are vital components of stream ecosystems, yet remain threatened. Thus, timely and accurate species counts are critical for proper conservation and management. Mussels live in stream sediments and can be challenging to survey given constraints related to water depth, flow, and time of year. The use of environmental DNA (eDNA) to monitor mussel distributions and diversity is a promising tool. Before it can be used as a monitoring tool, however, we need to know how much eDNA mussels shed into their environment and how long the eDNA persists. Here, we present a novel application of eDNA to estimate both the presence/absence and abundance of a freshwater mussel species, Lampsilis siliquoidea. The eDNA shedding and decay rates reported within are the first for freshwater mussels. We determined that eDNA shedding was statistically similar across mussel densities, but that first-order decay constants varied between experimental treatments. Finally, we effectively modeled downstream transport of eDNA and present a model that can be used as a complementary tool to estimate mussel density. Our results suggest that eDNA has the potential to be a complementary tool to survey mussels and enhance current efforts to monitor and protect freshwater mussel biodiversity.