Grantee Research Project Results

2022 Progress Report: Human Enteric Viruses and Viral Surrogates as Measures of Water Reuse Potential from Centralized and Decentralized Wastewater Treatment

EPA Grant Number: R840259
Title: Human Enteric Viruses and Viral Surrogates as Measures of Water Reuse Potential from Centralized and Decentralized Wastewater Treatment
Investigators: Heufelder, George , Baumgaertel, Brian , Olmsted, Emily Michele , Regan, Kathleen , Pancorbo, Oscar C
Institution: Barnstable County Department of Health and Environment and Massachusetts Department of Environmental Protection
EPA Project Officer: Ludwig-Monty, Sarah
Project Period: August 1, 2021 through May 10, 2025
Project Period Covered by this Report: August 1, 2021 through July 31,2022
Project Amount: $1,239,655
RFA: Viral Pathogen and Surrogate Approaches for Assessing Treatment Performance in Water Reuse (2021) RFA Text | Recipients Lists
Research Category: Water Treatment

Objective:

This study endeavors to identify decentralized and centralized wastewater treatment processes capable of removing human enteric viruses that allow safe reuse of the treated wastewater and further to evaluate the use of five surrogates as possible indicators for presence of human enteric viruses following treatment processes. The use of tested surrogate measures is being validated by parallel measurements of selected human enteric viruses using state-of-the-art, validated analytical methods.

Progress Summary:

This project is successfully leveraging the assets and talents of two regional agencies: Massachusetts Alternative Septic System Test Center (MASSTC) for the full-scale test locations of candidate technologies, collection of samples and processing of culturable bacteriophage indicators, and Wall Experiment Station of Massachusetts Department of Environmental Protection (MassDEP-WES) for development of state-of-the-art assay techniques for human enteric virus, advanced instrumentation, and laboratory expertise to assay samples for human enteric virus genomic material.

To date, over 55 sampling events have been conducted. Over 900 assays for male-specific and somatic coliphage, and indicator bacteria have been completed with associated, field measurements and chemistry measurements at MASSTC

Over 691 samples for PCR analyses have been successfully sampled, concentrated, kept at -80oC and transported to MassDEP-WES.
MassDEP-WES is evaluating a new digital PCR platform recently launched by Qiagen that is much faster and more economical than the proposed Bio-Rad’s ddPCR. The Qiagen dPCR instrument, purchased using MassDEP-WES capital funds, is being used to validate the methods for the EPA STAR Grant targets and will be used to compare performance to the proposed ddPCR methods.
Sources of Bovine Corona (RNA) Virus (Merck Bovilis vaccine) and Bovine alpha-Herpes (DNA) Virus (Zoetis BOVI-SHIELD IBR vaccine) and their respective RT-ddPCR and ddPCR primer-probes have been secured for use as processing (i.e., extraction) and analysis controls.
High levels of the surrogates human-associated Bacteroides HF-183 target gene sequence, CrAssphage, and pepper mild mottle virus fecal indicators have been detected in nucleic acid extracts of the MASSTC sewage samples used for molecular method development and will be used for Limit of Detection (LOD) and Limit of Quantitation (LOQ) determinations by ddPCR and RT-ddPCR; wastewater (sewage) dilution factors will be used for these determinations since no appropriate commercial products of purified target nucleic acid are available for these wastewater indicators/surrogates.

Future Activities:

MASSTC will identify and test two additional decentralized system technologies.

MassDEP-WES will complete all ddPCR and RT-ddPCR assay validations for each of the six target microbes and the two viral vaccine (RNA and DNA) positive controls in 3-4 weeks providing that the human norovirus GI RNA standard arrives in November 2022.

MassDEP-WES will begin analysis of -80°C frozen MASSTC samples in January 2023 after the updated QAPP and Method SOPs for sample extraction and ddPCR / RT-ddPCR analyses have been completed and approved. The LOD and LOQ determinations will have been completed and the Bovine Herpes Virus ddPCR Assay reagents (i.e., primer-probe set) and vaccine will have been determined to lack interferences with other project targets (i.e., RNA or DNA) and primer-probe sets.

Journal Articles:

No journal articles submitted with this report: View all 1 publications for this project

Supplemental Keywords:

Innovative/Alternative Technologies, onsite septic systems, bioreactors, EPA Region 1, soil treatment, media filters, human enteric viral pathogens, viral surrogates

Progress and Final Reports:

Original Abstract

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.