The Lume: Continuous Microbial Water Quality Monitoring
The Lume is a submersible, in-situ water quality sensor developed by Virridy that provides continuous, real-time estimates of E. coli concentrations directly in rivers, streams, and other water bodies. Unlike conventional culture-based laboratory methods that require 24 hours to return results, the Lume reports a water quality reading every 60 seconds with no reagents, no sample collection, and no laboratory processing. It deploys permanently in the water column and transmits data continuously.
The Lume measures three parameters simultaneously: tryptophan-like fluorescence (TLF), turbidity, and water temperature. These inputs feed a multivariate linear regression model calibrated to the target site, producing a continuous E. coli concentration estimate (CFU/100 mL) and a categorical risk classification keyed to applicable regulatory thresholds.
Measurement Principle: Tryptophan-Like Fluorescence
The Lume detects tryptophan-like fluorescence (TLF) — a UV light signal produced by compounds associated with fecal contamination. When microbial pollution enters a waterway, tryptophan and related indole-class metabolites absorb light at approximately 275 nm and re-emit it at approximately 340 nm. The Lume measures this excitation–emission signal optically, in real time, from within the water column.
Coefficients are determined from paired Lume and culture-based reference observations at the target site. Once set, they are fixed and fully transparent — the equation is reproducible on a standard spreadsheet with no proprietary model dependencies.
Current Performance
The Lume has been evaluated against the IDEXX Colilert reference method — the same EPA-approved Part 136 method used by the City of Boulder for weekly recreational water quality reporting — across more than 850 paired observations spanning freshwater rivers, groundwater, drinking water, and coastal water environments.
The Boulder Creek dataset is of particular relevance to this ATP application: it is the same watershed, the same reference method, and the same regulatory context as the planned validation study. For full performance statistics including precision, bias, confusion matrices, and per-class analysis, see Existing Performance Data in the project plan below and the Lume–Colilert Comparability Report.
What is an Alternate Test Procedure?
The EPA's Alternate Test Procedure (ATP) program, governed by 40 CFR 136.4 and 40 CFR 136.5, allows developers to submit new or modified analytical methods for approval as alternatives to the methods listed in 40 CFR Part 136.[1] These are the required methods for Clean Water Act (CWA) compliance—specifically NPDES permit applications and discharge monitoring reports.
Currently, microbial water quality monitoring relies on culture-based grab sampling methods such as Colilert and membrane filtration. These methods require 24 hours for results, trained laboratory personnel, and provide only a single snapshot in time. For recreational water—swim beaches, rivers, and lakes—this means contamination events are detected too late to protect swimmers. An ATP approval for the Lume would establish tryptophan-like fluorescence (TLF) coupled with multivariate linear regression as a recognized method for continuous, real-time microbial water quality assessment, starting with recreational water monitoring where the public health impact is most immediate.
The Water Quality Monitoring Gap
An estimated 2 billion visits are made to U.S. beaches each year.[2] And across the country, hundreds of rivers and streams carry active E. coli impairment listings under 303(d) — yet monitoring relies on methods that are decades old, infrequent, and inherently delayed. The result is a monitoring gap that leaves swimmers exposed and makes it nearly impossible for regulators to track impairment in real time.
Beaches Monitored Nationally
Only ~3,600 coastal and Great Lakes beaches are systematically monitored under the EPA BEACH Act.[3] Tens of thousands of inland swimming sites at lakes, rivers, and reservoirs lack any federal monitoring program.[4]
Advisories & Closures Per Year
EPA’s BEACON database records thousands of advisory and closure actions annually at monitored beaches alone—each representing a potential public health exposure.[5]
Delay in Every Result
Culture-based methods require overnight incubation. By the time results are available, water conditions have often changed—advisories may be posted when water is safe, or not posted when it is unsafe.[6][7]
Estimated Annual Illnesses
Epidemiological modeling estimates approximately 90 million recreational waterborne illness cases per year in the U.S.[8]
What This Means in Practice
A family arrives at a river beach on a Saturday morning. A storm two days prior caused a contamination event. The most recent grab sample was taken Monday—five days ago—and showed safe levels. The next sample won’t be collected until the following Monday. Results from that sample won’t be available until Wednesday. The contamination event has come and gone entirely undetected.
Continuous monitoring with the Lume would have flagged elevated microbial indicators within minutes of the event, enabling same-day advisories and protecting the public in real time. EPA’s 2012 Recreational Water Quality Criteria acknowledged the limitations of culture-based methods and recommended moving toward faster monitoring approaches[6]—the ATP pathway provides the regulatory mechanism to make that transition.
Approval Pathways
The EPA provides two regulatory mechanisms for ATP approval: limited-use (40 CFR 136.5, facility-specific) and nationwide (40 CFR 136.4, via Methods Update Rule). Our strategy runs as two parallel tracks, both progressing from limited-use to nationwide.
Boulder Creek → Colorado State-Wide → Nationwide Freshwater
Phase 1: Boulder Creek facility-specific ATP under 40 CFR 136.5 — scoped to Colorado Regulation 93 / 303(d) compliance monitoring (E. coli, 126 CFU/100 mL geometric mean threshold), applied for by the City of Boulder Utilities in coordination with CDPHE and EPA Region 8.
- Phase 1: Boulder Creek limited-use ATP (Reg 93 / 303(d))
- Phase 2: Additional Colorado 303(d) sites + CDPHE state recognition
- Phase 3: Nationwide freshwater ATP (40 CFR 136.4, EPA OST)
Ocean Beaches → Coastal Limited-Use → Nationwide Coastal
A separate, independent track in partnership with the American Shore & Beach Preservation Association (ASBPA) at ocean beach sites in other states. Target analyte is enterococci, reference method is Enterolert. This track runs on its own timeline and is not a prerequisite for Track A approvals.
- Coastal beach sites — geographic diversity across ASBPA members
- Enterococci / Enterolert — marine indicator per EPA 2012 RWQC
- Feeds separate or combined nationwide coastal ATP submission
Validation Requirements
EPA published finalized protocols in 2018 for evaluating new and modified analytical methods.[9] Because the Lume introduces a fundamentally new determinative technique (fluorescence + multivariate linear regression rather than culture-based enumeration), it falls under the Protocol for Review and Validation of New Methods. The microbiological protocol (finalized 2010) also applies given the microbial analyte.[10]
Key Validation Elements
Multi-Laboratory Testing
Validation across multiple independent laboratories to demonstrate reproducibility. Each lab must independently deploy and calibrate the Lume sensor and compare results against approved reference methods.
Representative Matrices
Testing across diverse recreational water types: freshwater rivers and lakes (E. coli / Colilert), coastal and ocean beaches (enterococci / Enterolert), as well as stormwater-impacted and CSO-impacted receiving waters.
Comparative Data
Side-by-side comparison against approved Part 136 methods: Colilert for E. coli in freshwater, Enterolert for enterococci in marine water, and membrane filtration (EPA Methods 1600/1603), demonstrating statistical equivalence or superiority in detection performance.
Statistical Analysis
Formal comparability assessment following EPA's Appendix H procedures, including precision, accuracy, method detection limits, and matrix-specific performance metrics.
Method Documentation
Complete method write-up in standardized EPA format: procedural steps, sample handling, quality assurance/quality control (QA/QC) requirements, and performance criteria.
QC Protocol
Defined quality control procedures including calibration verification, positive and negative controls, field blanks, duplicate analyses, and acceptance criteria for each deployment.
Our Approach
The Lume already has a strong foundation of peer-reviewed validation data, with our most extensive dataset collected on Boulder Creek. Track A (Colorado freshwater) partners with the City of Boulder Utilities and CDPHE, starting with a Boulder Creek limited-use ATP for Regulation 93 / 303(d) compliance monitoring. Track B (coastal) partners with the American Shore & Beach Preservation Association (ASBPA) independently, targeting ocean beaches in other states.
Each track uses the appropriate EPA-recommended indicator and reference method for its water type:[6]
- Track A — Freshwater (Boulder Creek, Colorado rivers): E. coli paired against IDEXX Colilert
- Track B — Marine / coastal (ocean beaches via ASBPA): Enterococci paired against IDEXX Enterolert
Peer-reviewed publications establishing TLF-based linear regression as a viable method for microbial detection.[11][12] Over 94% categorical accuracy demonstrated on Boulder Creek and other sites. Validation across drinking water, freshwater rivers, and coastal environments. Two issued US patents covering the core technology. Existing Boulder Creek data includes R² = 0.67 and 7% MAPE in log-transformed space against Colilert.[11]
Working with CDPHE and EPA Region 8 in Denver to scope the ATP application around Colorado Regulation 93 / 303(d) compliance monitoring. Define analytes, reference methods, statistical thresholds, and study design requirements for the Boulder Creek limited-use approval. The Reg 93 / 303(d) framework — CDPHE’s impaired waters program — provides a concrete, well-defined regulatory context for the initial submission.
Install Lume sensors at City of Boulder’s 6 monitoring locations on Boulder Creek. Collect continuous TLF data paired with Boulder’s existing weekly Colilert E. coli grab samples. Boulder Creek is a CDPHE 303(d)-listed impaired waterbody with an E. coli geometric mean threshold of 126 CFU/100 mL under Reg 93. Data collection spans 12–18 months to cover seasonal variation, spring runoff, and storm events.
The City of Boulder submits a limited-use ATP application to EPA Region 8 under 40 CFR 136.5, scoped to Regulation 93 / 303(d) compliance monitoring on Boulder Creek. If approved, Boulder can use Lume data in fulfillment of its Reg 93 impaired-waterbody monitoring obligations — the first regulatory use of continuous TLF-based microbial monitoring under the Clean Water Act.
After Boulder Creek approval, expand to additional Colorado 303(d)-listed waterbodies through further limited-use ATPs at partner utilities. Pursue CDPHE state-level recognition of the Lume method for all Colorado Reg 93 / 303(d) compliance monitoring. Multiple Colorado sites provide the multi-laboratory evidence base needed for the nationwide freshwater ATP submission.
A separate, parallel track in partnership with the American Shore & Beach Preservation Association (ASBPA) at ocean beach sites in other states. Uses enterococci / Enterolert — not tied to the Colorado Reg 93 work. This track proceeds on its own timeline and leads to a coastal limited-use ATP, eventually feeding a combined nationwide submission with Track A.
Submit complete application package to EPA’s Office of Science and Technology under 40 CFR 136.4. Track A provides freshwater E. coli / Colilert data from Colorado sites; Track B provides marine enterococci / Enterolert data from ASBPA coastal sites. Track A (freshwater) may reach the nationwide threshold before Track B — a freshwater-first submission is viable, with a coastal amendment to follow. Nationwide approval establishes the Lume as a recognized 40 CFR Part 136 method across all CWA compliance monitoring categories.
Existing Evidence Base
The Lume’s ATP application builds on a substantial body of validation data, with our most extensive dataset from Boulder Creek—the same watershed where the regulatory validation study will take place.
Categorical Accuracy
Site-specific calibrated classification of microbial contamination risk across freshwater environments.[11]
Drinking Water Accuracy
Binary classification at 1 and 10 CFU/100 mL regulatory thresholds with Cohen’s kappa of 0.82–0.84.[13]
Boulder Creek MAPE
Mean absolute percentage error in log-transformed concentration space against Colilert on Boulder Creek—the same watershed as our ATP validation study.[11]
Seine River Accuracy
Binary E. coli classification on held-out test data from three sensors deployed along the Seine in Paris.[14]
Peer-Reviewed Papers
Published validation studies in Water Research, ES&T Water, Science of the Total Environment, and EarthArXiv.
Water Matrices
Validated across groundwater, freshwater rivers, drinking water, coastal/estuarine, and wastewater environments.
Regulatory Context
The EPA ATP program exists because analytical science advances faster than the rulemaking process can incorporate new methods. The Lume represents a fundamental shift from discrete, lab-processed grab sampling to continuous, in-situ monitoring—a transition that regulatory frameworks are increasingly prepared to support.
Key Regulatory References
40 CFR Part 136
Approved analytical methods for CWA compliance monitoring.
40 CFR 136.4
Procedures for nationwide ATP applications and approval.
40 CFR 136.5
Procedures for limited-use (facility-specific) ATP approvals.
40 CFR 136.6
Method modification flexibility that labs can exercise without ATP.
Colorado Regulation 93 / 303(d)
Colorado Regulation 93 is CDPHE’s implementation of the federal 303(d) program for impaired waters. Boulder Creek carries an active E. coli impairment listing under Reg 93, with a TMDL-driven geometric mean threshold of 126 CFU/100 mL. The City of Boulder Utilities monitors Boulder Creek weekly against this threshold using IDEXX Colilert. A limited-use ATP approval would allow the Lume’s continuous TLF data to supplement or replace the weekly grab sample as the compliance measurement — providing real-time visibility into impairment events rather than a weekly snapshot.
Colorado SB24-037 — Legislative Context
Colorado Senate Bill 24-037, Study Green Infrastructure for Water Quality Management, was signed into law by Governor Polis in May 2024. The law directs CU Boulder’s Mortenson Center and Colorado State University, in collaboration with CDPHE, to evaluate green infrastructure as an alternative approach to water quality management—identify pilot projects with municipal and utility partners, operate pilots for up to five years, and produce recommendations for legislative and administrative action. Virridy’s collaboration with the City of Boulder Utilities and CDPHE emerged directly from this legislative initiative. The Boulder Creek Lume deployment is one such pilot project. The ATP application process is a parallel track: if Lume data achieves regulatory approval under 40 CFR 136.5, it becomes admissible for Regulation 93 / 303(d) compliance reporting—transforming a research pilot into a formal compliance tool. SB24-037 project overview (CU Boulder Mortenson Center) →
Why Now
Several convergent trends make this the right moment to pursue ATP approval for continuous TLF-based monitoring:
- 303(d) / Reg 93 demand: Colorado has hundreds of 303(d)-listed waterbodies with E. coli impairments. A Boulder Creek limited-use approval creates a replicable template for Reg 93 compliance monitoring statewide.
- Regulatory momentum: EPA and state agencies are increasingly recognizing the limitations of infrequent grab sampling for protecting public health and are exploring continuous monitoring approaches.[6] CDC surveillance data document hundreds of recreational water-associated outbreaks and thousands of illnesses that current monitoring infrastructure fails to prevent.[15]
- Technology maturity: The Lume has moved beyond proof-of-concept to multi-site, multi-matrix validation with published, peer-reviewed performance data.
- Cost pressure: Utilities face rising costs for lab-based compliance monitoring. Continuous sensors dramatically reduce per-sample costs while increasing data density by orders of magnitude.
- Recreational water urgency: Beach closures and swim advisories are routinely issued 24–48 hours after contamination events have already passed, while swimmers may be exposed during the very events that trigger those advisories.[6][7] EPA’s 2012 Recreational Water Quality Criteria explicitly acknowledged the need for faster monitoring methods.[6]
- Industry demand: Major water quality instrumentation companies have identified recreational water monitoring as the highest-need application for real-time microbial sensors, contingent on EPA ATP approval as the key regulatory gate.
References
- U.S. EPA. Guidelines Establishing Test Procedures for the Analysis of Pollutants. 40 CFR Part 136. ecfr.gov/title-40/part-136
- Houston, J.R. (2008). The economic value of beaches — a 2008 update. Shore & Beach, 76(3), 22–26. Cited by NOAA Office for Coastal Management.
- U.S. EPA. National Beach Guidance and Required Performance Criteria for Grants. EPA-823-B-14-001. Beaches Environmental Assessment and Coastal Health (BEACH) Act of 2000, 33 U.S.C. §1346. epa.gov/beach-tech/beach-act
- U.S. Government Accountability Office (2007). Clean Water Act: Improved EPA Oversight Will Better Ensure That States’ Monitoring of Beach Water Quality Is Effective. GAO-07-591. gao.gov/products/gao-07-591
- U.S. EPA. BEACON — Beach Advisory and Closing Online Notification system. Annual notification season data. epa.gov/beach-tech/beacon
- U.S. EPA (2012). Recreational Water Quality Criteria. EPA 820-F-12-058. Office of Water. epa.gov/rwqc2012
- Boehm, A.B., et al. (2002). Decadal and shorter period variability of surf zone water quality at Huntington Beach, California. Environmental Science & Technology, 36(18), 3885–3892. doi:10.1021/es020524u
- DeFlorio-Barker, S., et al. (2018). Estimate of incidence and cost of recreational waterborne illness on a global scale. Environmental Health Perspectives, 126(10), 107101. doi:10.1289/EHP3888
- U.S. EPA (2018). Protocol for Review and Validation of New Methods for Regulated Organic and Inorganic Analytes in Wastewater Under EPA’s Alternate Test Procedure Program. EPA 821-B-18-001.
- U.S. EPA (2010). Protocol for the Evaluation of Alternate Microbiological Methods. Office of Science and Technology.
- Thomas, E.A., et al. (2024). Low-cost, continuous microbial water quality assessment using tryptophan-like fluorescence and machine learning. ES&T Water. doi:10.1021/acsestwater.4c00567
- Nowicki, S., et al. (2019). Tryptophan-like fluorescence as a measure of microbial contamination risk in groundwater. Science of the Total Environment, 646, 782–791. doi:10.1016/j.scitotenv.2018.07.274
- Thomas, E.A., et al. (2023). Continuous E. coli monitoring in drinking water using tryptophan-like fluorescence. Water Research.
- Thomas, E.A., et al. (2025). Real-time microbial water quality assessment of the Seine River using tryptophan-like fluorescence. EarthArXiv.
- Hlavsa, M.C., et al. (2024). Surveillance for waterborne disease outbreaks associated with drinking water and other nondrinking water exposures — United States, 2015–2020. MMWR Surveillance Summaries. cdc.gov/mmwr
Project Summary
This plan details Virridy’s path to EPA Alternate Test Procedure (ATP) approval for the Lume sensor, establishing tryptophan-like fluorescence (TLF) coupled with multivariate linear regression as a recognized analytical method under 40 CFR Part 136 for continuous microbial water quality monitoring. The plan runs as two independent tracks:
- Track A — Colorado Freshwater: Start with a Boulder Creek facility-specific (limited-use) ATP under 40 CFR 136.5, scoped to Reg 93 / 303(d) compliance monitoring (E. coli, geometric mean threshold 126 CFU/100 mL), in partnership with the City of Boulder Utilities and CDPHE. Scale to Colorado state-wide recognition, then to a nationwide freshwater ATP under 40 CFR 136.4.
- Track B — Coastal / ASBPA: A parallel, independent track in partnership with the American Shore & Beach Preservation Association (ASBPA) for coastal/marine applications in other states — enterococci as the target analyte, Enterolert as the reference method. This track proceeds on its own timeline and feeds a separate (or combined) nationwide coastal ATP submission.
| Track A — Colorado Freshwater | |
| Partner | City of Boulder Utilities · CDPHE |
| Initial Focus | Colorado Regulation 93 / 303(d) compliance monitoring — Boulder Creek, 6 monitoring sites (E. coli geometric mean threshold: 126 CFU/100 mL) |
| Reference Method | IDEXX Colilert (E. coli, freshwater) |
| State Regulator | Colorado Department of Public Health and Environment (CDPHE) |
| Federal Regulator | EPA Region 8, Denver |
| Track A Pathway | Phase 1: Boulder Creek limited-use (40 CFR 136.5) → Phase 2: Colorado state-wide → Phase 3: Nationwide freshwater (40 CFR 136.4) |
| Track B — Coastal (Parallel / Independent) | |
| Partner | American Shore & Beach Preservation Association (ASBPA) — coastal/marine sites, other states |
| Target Analyte | Enterococci (marine/coastal water) |
| Reference Method | IDEXX Enterolert |
| Track B Pathway | Coastal limited-use ATP → Nationwide coastal ATP (coordinates with Track A for combined 40 CFR 136.4 submission) |
| Existing Data | |
| Performance | 856 paired observations vs. Colilert. R² = 0.67, kappa 0.82–0.84. See Comparability Report |
Existing Performance Data
The Lume has 856 paired observations against the IDEXX Colilert reference method—the same EPA-approved Part 136 method used by the City of Boulder for weekly recreational water quality reporting. All datasets (field, laboratory, and drinking water) use Colilert as the consistent reference, providing a uniform basis for comparability. The existing data demonstrates strong agreement: R² = 0.67 and 7% MAPE on continuous regression, 92–95% balanced accuracy on categorical classification (kappa 0.82–0.84), and a conservative error direction that favors public health protection.
| Evaluation | Key Metric | Value | n |
|---|---|---|---|
| Continuous regression (Boulder Creek) | R² / MAPE | 0.67 / 7.12% | 38 |
| 3-class categorical (<10, 10–100, >100 MPN) | Bal. Acc / Kappa | 95% / 0.84 | 334 |
| Binary (threshold = 1 CFU/100 mL) | Accuracy / Kappa | 91% / 0.82 | 361 |
| Binary (threshold = 10 CFU/100 mL) | Accuracy / Kappa | 92% / 0.84 | 361 |
| Chlorine residual detection (binary) | Accuracy / Kappa | 85% / 0.70 | 66 |
For complete analysis including raw paired data, per-class performance, confusion matrices, chlorination effects, and method precision, see the Lume–Colilert Comparability Report.
Master Timeline
Two parallel tracks. Track A (Colorado freshwater): regulatory engagement → Boulder Creek field study → method documentation → statistical analysis → limited-use ATP (Boulder Creek) → Colorado state-wide expansion → nationwide freshwater ATP. Track B (Coastal / ASBPA): runs independently in other states, targeting enterococci at ocean beaches, and feeds the nationwide coastal ATP submission. Quarters shown are relative to project kickoff (Q1 = Months 1–3).
| Workstream | Q1 | Q2 | Q3 | Q4 | Q5 | Q6 | Q7 | Q8 | Q9 | Q10 |
|---|---|---|---|---|---|---|---|---|---|---|
| Track A — Colorado Freshwater (E. coli / Colilert) | ||||||||||
| 1. Regulatory Engagement | ||||||||||
| 2. Boulder Creek Field Study | ||||||||||
| 3. Method Documentation | ||||||||||
| 4. Statistical Analysis | ||||||||||
| 5. Limited-Use ATP — Boulder Creek | ||||||||||
| 5b. Colorado State-Wide Expansion | ||||||||||
| Track B — Coastal / ASBPA (Enterococci / Enterolert, parallel & independent) | ||||||||||
| 6. Coastal Beach Validation (ASBPA) | ||||||||||
| 7. Nationwide Recreational Water ATP | ||||||||||
| Key Milestones | ||||||||||
Active Planned Future Milestone
Workstream 3: Method Documentation
Write the complete analytical method in EPA standard format. Begins after EPA pre-submission meeting (Workstream 1) confirms requirements. Draft in parallel with data collection (Workstream 2).
3.1 Method Document Outline
| § | Section | Content |
|---|---|---|
| 1 | Scope and Application | Target analytes: E. coli in freshwater recreational water and enterococci in marine/coastal recreational water. Applicable concentration range (MDL to upper reporting limit). Applicable water matrices. Limitations and exclusions. |
| 2 | Summary of Method | TLF measurement at 275/340 nm excitation/emission. Multivariate linear regression: log₁₀(CFU/100 mL) = β₀ + β₁·TLF + β₂·Turbidity + β₃·Temperature. Output: continuous E. coli concentration estimate and categorical risk classification. |
| 3 | Definitions | TLF, tryptophan-like fluorescence, ppb tryptophan equivalents, categorical classification bins, calibration period, universal model, site-calibrated model, model version. |
| 4 | Interferences | Dissolved organic carbon (DOC), humic-like fluorescence (HLF), turbidity above threshold NTU, temperature effects, biofouling, optical window contamination. |
| 5 | Safety | No hazardous reagents. Standard field safety for in-water sensor deployment. Electrical safety for powered sensor housing. |
| 6 | Equipment and Supplies | Lume sensor specifications (optical, physical, electrical). Mounting hardware. Data transmission module. Tryptophan calibration standards. DI water for blanks. Maintenance tools. |
| 7 | Reagents and Standards | Tryptophan stock solution preparation. Calibration standard concentrations. Standard storage and shelf life. DI water specification for blanks. |
| 8 | Sample Collection | In-situ measurement: no sample collection, preservation, or transport required. Define sensor positioning relative to water surface, minimum submersion depth, flow velocity requirements. |
| 9 | Calibration | Factory calibration procedure. Field calibration verification (tryptophan standard check, blank verification). Site-specific ML model calibration requirements. Calibration frequency. Recalibration triggers. |
| 10 | Quality Control | Initial demonstration of capability (IDC). Ongoing QC: field blanks, calibration verification, duplicate sensor deployments, data completeness requirements. Corrective actions for QC failures. |
| 11 | Procedure | Step-by-step: site selection criteria, sensor deployment, initialization sequence, measurement interval configuration, data transmission verification, routine maintenance, data retrieval, decommissioning. |
| 12 | Data Analysis | ML model application: input features, model version, inference procedure. Output: E. coli concentration (CFU/100 mL), categorical classification, confidence interval. Reporting: time-stamped results, averaging period for compliance reporting. |
| 13 | Method Performance | MDL, precision (RSD from duplicate sensors), accuracy (bias vs. Colilert), comparability statistics. Matrix-specific performance tables. Reference to Boulder Creek validation data and published literature. |
| 14 | Waste Management | No waste generated during routine operation. Disposal of calibration standards per local regulations. Sensor end-of-life: electronic waste recycling. |
3.2 Documentation Tasks
| Task | Timing | Details |
|---|---|---|
| Obtain EPA method template / formatting requirements | Month 3–4 | From pre-submission meeting or EPA guidance documents. Ensure format matches what OST expects for review. |
| Draft Sections 1–8 (instrument and procedure) | Month 4–6 | These sections are largely defined by the existing sensor design. Can be drafted before validation data is complete. |
| Draft Sections 9–10 (calibration and QC) | Month 6–9 | Requires input from field QC data (Workstream 2). Define acceptance criteria based on observed sensor performance. |
| Draft Sections 11–14 (procedure, analysis, performance) | Month 9–15 | Section 13 (performance) requires complete validation data from Workstream 2 and statistical analysis from Workstream 4. |
| Internal review and revision | Month 15–16 | Technical review by Virridy team. External review by independent method documentation expert if budget allows. |
| CDPHE / EPA Region 8 review draft | Month 16–17 | Share draft with regulators for informal feedback before formal submission. |
Workstream 4: Statistical & Data Analysis
Formal statistical analyses required for the ATP submission, following EPA’s validation protocols and Appendix H comparability procedures.
4.1 Method Detection Limit (MDL)
| Task | Details |
|---|---|
| MDL determination per 40 CFR Part 136 Appendix B | Minimum 7 replicate measurements of a low-level standard (near expected detection limit). Calculate MDL = t-value × standard deviation. Perform in both laboratory (controlled tryptophan solution) and field (low-concentration site) conditions. |
| E. coli MDL (derived) | Translate TLF MDL to E. coli concentration MDL via the ML model. Report as CFU/100 mL. Compare against existing literature value of ~10 CFU/100 mL. |
4.2 Precision Analysis
| Task | Details |
|---|---|
| Sensor-to-sensor reproducibility | From duplicate sensor deployments (Workstream 2, monthly rotation): calculate relative percent difference (RPD), relative standard deviation (RSD), and coefficient of variation (CV) across all paired sensor observations. |
| Temporal precision | Assess variability of sensor readings during stable conditions (e.g., low-flow baseflow periods). Characterize instrument noise vs. real environmental signal. |
| Cross-site precision | Compare model performance metrics across all 6 Boulder Creek sites. Assess whether site-specific calibration is required or whether a universal Boulder Creek model is sufficient. |
4.4 Regression Model Documentation
| Task | Details |
|---|---|
| Equation documentation | Multivariate linear regression with fixed coefficients: log₁₀(CFU/100 mL) = β₀ + β₁·TLF + β₂·Turbidity + β₃·Temperature. Document: coefficient values, standard errors, confidence intervals, training dataset size, R², RMSE. |
| Coefficient transparency | Publish final coefficient values in the ATP method document. Equation is fully reproducible on a spreadsheet with no proprietary dependencies. |
| No versioning required | Coefficients are locked after the training phase and do not change. No retraining, versioning, or revalidation protocol required once the method is approved. |
| Universal model performance | Report performance with no site-specific calibration. The same equation applies across all 6 monitoring sites on Boulder Creek. |
Workstream 6: Coastal Beach Validation with ASBPA Track B — Parallel / Independent
A separate, independent track from the Colorado freshwater work, run in partnership with the American Shore & Beach Preservation Association (ASBPA) at ocean beach sites in other states. This track targets enterococci—the EPA-recommended indicator for marine and coastal recreational water—paired against Enterolert as the reference method.
6.1 ASBPA Partnership & Site Selection
| Task | Timing | Details |
|---|---|---|
| Formalize partnership with ASBPA | Month 15–16 | Define scope, site access, data sharing, and coordination. ASBPA provides access to monitored ocean beach sites and existing enterococci sampling programs. |
| Select 2–4 coastal beach sites | Month 16–17 | Target geographic diversity: Atlantic, Gulf, and/or Pacific coast. Sites should have existing Enterolert monitoring programs to maximize paired data collection efficiency. |
| Deploy Lume sensors at coastal sites | Month 17–18 | Install sensors at selected ocean beach monitoring locations. Adapt deployment protocols for saltwater/tidal conditions (biofouling, salinity interference characterization). |
6.2 Enterolert-Paired Data Collection
| Task | Timing | Details |
|---|---|---|
| Collect paired Lume TLF + Enterolert data | Month 18–24 | Continuous Lume readings paired with Enterolert (enterococci) grab samples from existing beach monitoring programs. Target: 6–12 months per site, covering seasonal variation and storm events. |
| Characterize marine-specific interferences | Month 18–24 | Assess salinity, algal fluorescence, and tidal cycle effects on TLF signal in marine water. Document any marine-specific calibration requirements. |
| Enterococci model development | Month 21–24 | Develop and validate ML model for enterococci estimation in marine water. May require marine-specific training data or transfer learning from the freshwater E. coli model. |
6.3 Coastal Comparability Analysis
| Task | Details |
|---|---|
| Enterococci regression & classification | Same statistical framework as freshwater E. coli analysis (Workstream 4), but with enterococci thresholds: 35 CFU/100 mL (EPA Beach Action Value), 70 CFU/100 mL (statistical threshold value). Compare against Enterolert and EPA Method 1600. |
| Cross-matrix performance summary | Unified report covering both freshwater (E. coli / Colilert) and marine (enterococci / Enterolert) validation. Demonstrates the Lume’s dual-indicator capability across the full range of recreational water types. |
Workstream 7: Nationwide ATP Submission
Full ATP application to EPA’s Office of Science and Technology for incorporation into 40 CFR Part 136 via the Methods Update Rule process. Both tracks feed this submission: Track A provides freshwater E. coli / Colilert data from Boulder Creek and additional Colorado sites; Track B provides marine enterococci / Enterolert data from ASBPA coastal sites. Note that Track A (freshwater) may reach the nationwide submission threshold before Track B; a freshwater-only nationwide application can be submitted first, with a subsequent coastal amendment.
7.1 Multi-Laboratory Expansion
Nationwide approval requires multi-laboratory validation. The Colorado state-wide expansion (Workstream 5b) provides multiple independent Colorado sites for freshwater data. ASBPA coastal sites provide independent marine validation.
| Task | Details |
|---|---|
| Recruit 2–3 additional validation sites | Recreational water sites in different states/EPA regions. Ideal: one inland lake/reservoir, one Great Lakes beach, one different climatic zone. Each site operates the Lume independently following the written method (Workstream 3). ASBPA coastal sites (Workstream 6) serve as additional independent validation. |
| Technology transfer & training | Train independent operators on sensor deployment, maintenance, calibration, and data management per the method documentation. Operators must be able to run the method without Virridy involvement. |
| Independent validation data collection | Each additional site collects 6–12 months of paired data (Lume + approved Part 136 method). Follows same protocol as Boulder Creek study. |
7.2 Application Package
| Component | Source |
|---|---|
| Complete method documentation | Workstream 3 |
| Boulder Creek freshwater validation report (6 sites, 12–18 months) — Track A Phase 1 | Workstreams 2 + 4 |
| Colorado state-wide multi-site validation reports — Track A Phase 2 | Workstream 5b |
| Coastal beach validation report (Enterolert-paired data) — Track B | Workstream 6 |
| Multi-laboratory validation reports | Workstreams 5b + 7.1 |
| Statistical comparability analysis (Appendix H) | Workstream 4 |
| MDL study report | Workstream 4.1 |
| ML model documentation & interpretability analysis (E. coli + enterococci) | Workstreams 4.4 + 6 |
| QA/QC protocol and field QC results | Workstreams 2 + 3 + 6 |
| Limited-use approval letter and post-approval performance data | Workstream 5 |
| Published peer-reviewed literature | Research publications |
7.3 Submission & Rulemaking
| Task | Details |
|---|---|
| Submit to EPA OST | Complete application package to the Office of Science and Technology, Office of Water. |
| EPA review | OST reviews the application against validation protocols. May request additional data, clarification, or supplementary analyses. Timeline: 6–18 months (variable). |
| Methods Update Rule | If accepted, EPA proposes including the Lume method in the next Methods Update Rule (MUR). Notice-and-comment rulemaking: Federal Register notice, public comment period (typically 60 days), EPA response to comments, final rule. |
| Post-approval support | Respond to public comments during rulemaking. Provide supplementary data if requested. Support EPA in developing any method-specific guidance documents. |
Risk Register
| Risk | Impact | Mitigation | |
|---|---|---|---|
| H | EPA classifies continuous monitoring output as incompatible with current permit compliance framework | Fundamental barrier. Limits value of ATP approval if continuous data can’t replace grab-sample-based compliance reporting. | Address in pre-submission meeting (Workstream 1). Propose specific compliance reporting frameworks. Engage CDPHE early. If needed, propose ATP for supplemental monitoring initially, with compliance use as future goal. |
| H | EPA requires multi-lab validation for limited-use approval (not just nationwide) | Delays limited-use timeline by 6–12 months. Additional cost and site recruitment needed. | Clarify in pre-submission meeting. If required, accelerate Workstream 6.1 (multi-lab recruitment) to run in parallel with Boulder data collection rather than sequentially. |
| H | Linear regression equation is not accepted as a determinative step in an analytical method | Fundamental challenge to the method framework. EPA has not previously evaluated a continuous in-situ sensor with a regression-based determinative step. | Emphasize that the linear regression equation is fully transparent, reproducible on a spreadsheet, and analogous to established correction equations in approved methods (e.g., turbidity corrections in UV absorbance methods). Raise in pre-submission meeting (E-5 open question). |
| M | Boulder Creek doesn’t span the full E. coli concentration range needed | Incomplete validation. EPA may require data at concentrations not naturally occurring at Boulder Creek sites. | Event-based enhanced sampling targets high concentrations (spring runoff, storms). 6 diverse sites increase range coverage. Existing laboratory Colilert data (n=334) already covers <10 to >100 MPN/100 mL. |
| M | Sensor fouling or reliability issues reduce data completeness below 90% | Weakens the dataset. EPA may question operational reliability of the method. | Bi-weekly maintenance schedule. One spare sensor for rapid swap. Automated fouling detection alerts. Robust mounting design for high-flow resilience. |
| M | Colilert reference method variability masks Lume performance | Wide Colilert confidence intervals make statistical comparability harder to demonstrate, even if the Lume is performing well. | Document Colilert method variability (literature and from duplicate grab samples). Frame Lume precision as comparable to or better than the reference method’s own precision (14% RPD for TLF duplicates vs. ≥26% for culture-based duplicates). |
| M | City of Boulder organizational changes affect partnership | Loss of champion, budget changes, or priority shifts could delay or derail the study. | Formalize MOU early. Ensure buy-in at management level, not just individual staff. Minimize Boulder’s operational burden (Virridy handles sensor deployment/maintenance; Boulder continues existing Colilert program unchanged). |
| L | EPA rulemaking timeline extends beyond 3–5 years for nationwide approval | Slow path to national market. Does not affect limited-use approvals. | This is expected. Limited-use approval (Workstream 5) provides regulatory value in the interim. ASBPA coastal validation (Workstream 6) builds evidence base in parallel. |
| L | Seasonal access constraints (ice, flooding) cause data gaps | Missing winter or spring runoff data weakens seasonal coverage. | Design sensor housing for winter deployment (heating element or insulated housing). For spring flooding: secure mounting above flood stage or plan for temporary sensor retrieval with rapid redeployment. |
Deliverables Checklist
Regulatory Engagement (Workstream 1)
- CDPHE introductory briefing deck
- EPA Region 8 pre-submission briefing package
- Pre-submission meeting summary and EPA guidance memo
- Confirmed study design parameters (sample counts, matrices, statistical thresholds)
Field Study (Workstream 2)
- Signed MOU with City of Boulder Utilities
- Site survey report (6 sites: locations, infrastructure, photos, GPS)
- Sensor deployment log (serial numbers, firmware, calibration certificates)
- Paired dataset: continuous Lume data + weekly Colilert results (12–18 months)
- Duplicate sensor precision dataset (12+ one-week deployments)
- Field QC records (blanks, calibration checks, maintenance logs)
- Data quality report with flagging summary
Method Documentation (Workstream 3)
- Complete EPA-format method document (Sections 1–14)
- Sensor hardware specification sheet
- ML model technical documentation
- QA/QC protocol document
- Standard operating procedure (SOP) for field deployment and maintenance
Statistical Analysis (Workstream 4)
- MDL study report (laboratory and field)
- Precision analysis report (sensor-to-sensor, temporal, cross-site)
- Accuracy and comparability report (Appendix H format)
- Matrix-specific performance tables
- ML model interpretability report (feature importance, SHAP analysis)
- Universal vs. site-calibrated model comparison report
Limited-Use Application (Workstream 5)
- Boulder Creek site-specific comparability report
- Limited-use ATP application (per 40 CFR 136.5)
- Compliance reporting framework (how Lume data appears in DMRs)
- Quarterly post-approval performance reports
Coastal Beach Validation (Workstream 6)
- Signed partnership agreement with ASBPA
- Coastal site survey report (2–4 ocean beach sites)
- Paired dataset: continuous Lume data + Enterolert enterococci results (6–12 months)
- Marine-specific interference characterization report
- Enterococci model validation report
- Cross-matrix (freshwater + marine) comparability summary
Nationwide Recreational Water ATP (Workstream 7)
- Multi-laboratory validation reports (2–3 additional sites)
- Dual-indicator (E. coli + enterococci) ATP application package to EPA OST
- Public comment response document (during rulemaking)
Partner With Us
We are building the regulatory case for continuous microbial water quality monitoring across two tracks. Track A starts with the City of Boulder Utilities on Boulder Creek (Colorado Reg 93 / 303(d) compliance monitoring) and expands to other Colorado utilities and impaired waterways. Track B targets coastal/marine monitoring with ASBPA at ocean beach sites in other states. We are seeking additional Colorado utilities with 303(d)-listed waterbodies, coastal beach operators, state regulators, and research partners interested in deploying the Lume alongside existing Colilert or Enterolert monitoring programs.
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