A Two-Tier Effect-Based Water Bioactivity Surveillance System.

Title: A Two-Tier Effect-Based Water Bioactivity Surveillance System. ABSTRACT. Chemical polluƟon of water threatens public health worldwide, linked to ~1.4 million deaths/year. Man-made chemicals have surged from ~1,000 in 1900 to over 350,000 today, with annual producƟon exceeding 1,000 million metric tons. Many remain untested, complicaƟng water safety oversight. ConvenƟonal water tesƟng focuses on a few known contaminants, lacks biological context, and fails to capture the synergisƟc effects of complex mixtures. To address these gaps, we propose a two-Ɵer approach—WASANET—built on Attagene’s FACTORIAL platiorm. The FACTORIAL assay quanƟtaƟvely measures mulƟple transcripƟon factor (TF) acƟviƟes, generaƟng an Aggregate Response Index (ARI) that reflects overall bioacƟvity of water samples. Validated under the U.S. EPA ToxCast program and recognized by a White House Tibbetts Award, this platiorm provides a uniform scale for comparing diverse water sources. Tier I emphasizes rapid, cost-effecƟve triage screening to idenƟfy “hot spots” via a high-throughput FACTORIAL ARI assay, enhanced with key xenobioƟc-metabolizing enzymes for broader detecƟon. Tier II combines targeted chemical analyses with the FACTORIAL assay to pinpoint the specific contaminants driving observed bioacƟvity, enabling more effecƟve remediaƟon strategies. SPECIFIC AIM 1: Build the infrastructure for Tier I water safety surveillance. 1A. Develop a single-step, high-throughput FACTORIAL ARI assay. 1B. Enhance metabolic capabiliƟes using criƟcal metabolic enzymes. 1C. Establish standardized workflows for sample handling and data management. SPECIFIC AIM 2: Develop Tier II methodology to idenƟfy the most impactiul pollutants. 2A. Integrate the FACTORIAL assay with chemical analyses to locate criƟcal contaminants. 2B. Validate Tier II using samples with known pollutant profiles. SPECIFIC AIM 3: Deploy a prototype WASANET system in collaboraƟon with water authoriƟes and researchers in CA, NC, and IA, evaluaƟng both environmental and residenƟal sources. By delivering quanƟtaƟve ARI scores, WASANET permits clear risk comparisons across sites and helps prioriƟze hotspots for in-depth invesƟgaƟon. IdenƟfying key toxicants further supports cost-effecƟve remediaƟon and targeted regulatory measures. This high-content strategy bridges criƟcal gaps in convenƟonal water tesƟng, providing acƟonable data on both known and hidden hazards. The proposed work will demonstrate WASANET’s feasibility, setting a foundaƟon for widespread adopƟon and improved public health protecƟon. Overall, WASANET offers a transformaƟve leap in water safety monitoring worldwide.