Lume Sensor ATP Validation Study Design
& Statistical Analysis Plan

1. Background & Regulatory Context

1.1 The Alternate Test Procedure Program

The EPA Alternate Test Procedure (ATP) program, codified at 40 CFR 136.4 (nationwide) and 40 CFR 136.5 (limited-use, facility-specific), provides a pathway for developers to submit new analytical methods for regulatory approval as alternatives to methods listed in 40 CFR Part 136. These are the required methods for Clean Water Act compliance reporting. This document describes the study design and statistical analysis plan for a limited-use ATP application under 40 CFR 136.5, submitted by the City of Boulder Utilities to EPA Region 8.

1.2 Colorado Regulation 93 / 303(d)

Boulder Creek is listed as impaired under Colorado Regulation 93 (the state implementation of the federal 303(d) program for impaired waters) for E. coli exceedances. The applicable water quality standard is a geometric mean of 126 CFU/100 mL over a 30-day period, with a single-sample maximum of 235 CFU/100 mL. The City of Boulder Utilities currently monitors compliance at 6 designated monitoring locations on Boulder Creek using IDEXX Colilert as the reference method. This study will determine whether the Virridy Lume tryptophan-like fluorescence (TLF) sensor produces results statistically equivalent to Colilert at these compliance monitoring sites.

1.3 The Case for Continuous Monitoring

Current Reg 93 compliance monitoring on Boulder Creek relies on discrete grab samples processed by a laboratory with an 18–24 hour delay before results are available. This creates a structural monitoring gap: contamination events driven by storm runoff, upstream discharges, or wildlife activity may occur and resolve entirely between sampling events, leaving the public exposed without warning. The Lume sensor provides continuous, in-situ E. coli estimates at 15-minute intervals, enabling real-time detection of exceedance events and substantially increasing the density of compliance-relevant data.

1.4 Relationship to Existing Data

Virridy has previously collected 856 paired Lume–Colilert observations across multiple sites and waterbodies (global dataset, 2021–2025) (R² = 0.67, Cohen’s kappa = 0.82–0.84, MAPE = 7.1% in log-transformed space). This existing dataset is used to support the pre-submission consultation with EPA Region 8 and demonstrates technical feasibility, but does not constitute the formal regulatory validation dataset. The prospective study described in this document will generate the regulatory-grade paired dataset required for the 136.5 application, under a pre-specified, agreed study protocol.

2. Study Objectives & Regulatory Claim

2.1 Primary Objective

Demonstrate that the Virridy Lume tryptophan-like fluorescence sensor, using a multivariate linear regression model with TLF intensity, turbidity, and temperature as inputs, produces E. coli concentration estimates that are statistically equivalent to IDEXX Colilert results at Boulder Creek’s 6 Regulation 93 TMDL monitoring sites across all seasons, flow conditions, and concentration ranges relevant to Reg 93 compliance.

2.2 Secondary Objectives

• Demonstrate correct binary classification of samples above and below the Reg 93 geometric mean threshold (126 CFU/100 mL) with pre-specified sensitivity and specificity targets
• Characterize method precision (sensor-to-sensor reproducibility) across the 6 monitoring sites
• Determine the method detection limit (MDL) in both TLF units and translated CFU/100 mL
• Characterize known interferences (dissolved organic carbon, humic fluorescence, turbidity, temperature) and their effect on the prediction equation
• Generate a complete method document suitable for inclusion in a 40 CFR 136.5 application

2.3 Regulatory Claim Hierarchy

The primary regulatory claim is direct replacement of Colilert grab sampling for Reg 93 compliance reporting. Fallback claims are listed in order of preference if the primary claim cannot be fully supported by the dataset:

3. Method Description

3.1 Principle of the Method

The Lume sensor measures tryptophan-like fluorescence (TLF) in situ at 275 nm excitation and 340 nm emission. Tryptophan is an amino acid present in the cell walls of bacteria, including E. coli, and its fluorescence signal is a proxy for fecal contamination. The sensor simultaneously measures turbidity (NTU) and water temperature (°C) as co-variates. A multivariate linear regression model, fit to paired Lume–Colilert calibration data and validated on a held-out test set, converts the three sensor outputs to an E. coli concentration estimate in CFU/100 mL.

3.2 Prediction Equation

The regulatory prediction equation takes the following form, fit in log₁₀ space to account for the log-normal distribution of environmental E. coli concentrations:

Where TLF is in ppb tryptophan equivalents, turbidity is in NTU, and temperature is in °C. The model coefficients (β₀–β₃) will be estimated by ordinary least squares regression on the training partition of the study dataset (see Section 8). The fitted coefficients will be locked before evaluation on the holdout test set and will not be modified thereafter. The final coefficient values will be reported in the ATP application and become fixed parameters of the approved method.

3.3 Sensor Specifications

Parameter	Specification
TLF excitation wavelength	275 nm
TLF emission wavelength	340 nm
TLF units	ppb tryptophan equivalents
Turbidity measurement	Nephelometric (NTU)
Temperature measurement	°C (thermistor)
Measurement interval	15 minutes (configurable)
Deployment type	Submersible in-situ sensor
Data transmission	Cellular (primary); on-board SD card (backup)
Power	Solar or line power with battery backup
Operating temperature range	0–40 °C

3.4 Method Scope and Applicability

This method is applicable to freshwater surface water for the measurement of E. coli in the range of approximately 1–10,000 CFU/100 mL. The method is designed for continuous in-situ monitoring and is not applicable to discrete grab sample laboratory analysis. The method as validated is specific to Boulder Creek, Colorado under the 40 CFR 136.5 limited-use approval; nationwide applicability under 40 CFR 136.4 will require additional multi-site validation.

4. Study Design

4.1 Study Sites

The study will be conducted at the 6 City of Boulder Utilities TMDL compliance monitoring locations on Boulder Creek. These are the same sites at which Boulder currently collects Colilert grab samples for Regulation 93 reporting to CDPHE. Using existing compliance monitoring sites ensures that the validation dataset directly reflects the conditions and concentration ranges relevant to the regulatory claim.

Site	Description	Expected Range	Regulatory Significance
BC-Can	Upstream reference / background	Low (<10 CFU/100 mL)	Establishes baseline TLF and natural organic matter background; critical for MDL characterization
BC-Eben	Canyon mouth / Eben Fine Park	Low–moderate	Mixed urban/natural signal; entry point for canyon runoff
BC-13	13th Street compliance point	Variable — low to high	Central compliance point; demonstrates performance at and above the 126 CFU/100 mL Reg 93 threshold
BC-CU	CU campus compliance point	Moderate	Mixed urban stormwater signal; seasonal variability important
BC-30	30th Street compliance point	Moderate–low	Natural attenuation zone; demonstrates performance during dilution and recovery
BC-55	55th Street / watershed outlet	Variable	Integrates full watershed; irrigation diversions may affect flow dynamics seasonally

Note: Site descriptions and names are provisional. Exact site IDs, GPS coordinates, and Reg 93 permit associations to be confirmed with City of Boulder Utilities prior to study start.

4.2 Study Duration

The study will run for 12 months from the formal study start date (defined as the date all 6 sensors are deployed and the reference method sampling program is operating at the required frequency). Twelve months is the minimum duration required to capture all four seasons and, critically, two complete spring runoff events on Boulder Creek. Spring runoff (typically April–June) produces the highest E. coli concentrations of the year and is the period most likely to generate exceedances of the Reg 93 threshold. It must be represented in both the training and holdout partitions of the dataset.

4.3 Sensor Deployment

Task	Timing	Details
Deploy Lume sensors at all 6 sites	Pre-study	Each sensor co-located within 2 meters of the designated Colilert grab sample collection point at the same depth and cross-section position. GPS coordinates, installation photographs, and sensor serial numbers documented at deployment.
Firmware and model version lock	Study Day 0	Firmware version and prediction equation coefficients (once fit per Section 8) are locked for the study duration. Any firmware updates require a protocol amendment and notification to EPA Region 8.
Data pipeline validation	Week 1	Confirm all 6 sensors are transmitting reliably. Verify measurement interval, data completeness, and sensor-to-server latency. Automated alerts configured for signal dropout, out-of-range readings, and battery/power status.

4.4 Operators and Responsibilities

Party	Role
Virridy	Sensor deployment, maintenance, calibration verification, data management, and statistical analysis. Contracted to City of Boulder for the duration of the study.
City of Boulder Utilities	Applicant on the 136.5 ATP application. Oversight of the study. Coordinates Colilert grab sample collection at the 6 monitoring sites per existing Reg 93 SOPs.
Reference Laboratory	State-certified laboratory for E. coli analysis. Processes all Colilert samples for the study. [See Open Question B-1: confirm certified lab and willingness to increase sampling frequency]
CDPHE	State regulatory oversight. Reviews study design and provides letter of support for the 136.5 application.

5. Reference Method Protocol

5.1 Reference Method

The reference method for this study is IDEXX Colilert for E. coli quantification in freshwater, analyzed using the Quantitray/2000 MPN method per the manufacturer’s protocol and consistent with 40 CFR Part 136 Table IA. The specific Colilert formulation (standard 24-hour vs. Colilert 18-hour) will be confirmed with City of Boulder Utilities and will match the version currently used for Reg 93 compliance reporting. Both are EPA-approved; consistency with Boulder’s existing compliance program is required so that the ATP validation dataset is directly comparable to the regulatory baseline.

5.2 Sample Collection Protocol

Parameter	Requirement
Collection frequency	Minimum 3 times per week per site during the study period. Event-based enhanced sampling (see 5.3) increases this frequency during high-flow events.
Collection timing	Exact time of grab sample recorded to the minute by the collector. Matched against nearest Lume 15-minute reading (pairing window: ≤15 minutes; see Section 6).
Collection location	Within 2 meters of the Lume sensor, same depth and cross-section position.
Collection containers	Sterile, single-use 100 mL bottles per Part 136 / Colilert protocol.
Preservation and holding time	Per approved Colilert protocol: chilled at 4 °C, analyzed within 6 hours of collection.
Chain of custody	Formal chain of custody documentation for every sample, from collection through laboratory reporting. COC retained for minimum 10 years.
Field conditions log	At each sampling event: weather (clear/rain/snow/runoff), visual water clarity, estimated stream flow condition (baseflow/elevated/flood), any visible upstream disturbance, air temperature, and collector initials.
Laboratory duplicate	Every 10th paired sample: collect a field duplicate grab (two bottles from the same sampling event). Submitted blind to the laboratory. Provides reference method precision estimate (relative percent difference).

5.3 Enhanced Sampling During High-Flow Events

Spring runoff and storm events are the primary drivers of Reg 93 exceedances on Boulder Creek. The Lume’s continuous monitoring will automatically detect elevated TLF signals. When any Lume sensor reads above a pre-defined alert threshold (to be determined during initial deployment, approximately 2× baseline TLF), the Colilert sampling frequency at that site will be increased to daily for the duration of the event. This generates additional paired observations at high concentrations, which are the most important for demonstrating performance at and above the 126 CFU/100 mL threshold.

6. Paired Data Collection Protocol

6.1 Definition of a Valid Paired Observation

A paired observation is defined as a Colilert result and a Lume sensor reading that meet all of the following criteria:

1. The Colilert grab sample was collected within ±15 minutes of a Lume 15-minute measurement interval (i.e., the Lume reading is the single reading closest in time to sample collection, within one measurement interval)
2. The Lume sensor was operating normally at the time of the reading (no QC flags applied; see Section 7)
3. The Colilert sample was analyzed by the certified reference laboratory and has a valid COC
4. The grab sample was collected within 2 meters of the sensor at the same depth and cross-section
5. No documented maintenance activity (sensor cleaning, calibration, removal) was performed within 30 minutes of the reading

6.2 Minimum Sample Size Requirements

Metric	Minimum Target	Rationale
Total valid paired observations	≥600	6 sites × ≥100 per site. Substantially exceeds Colilert’s original multi-lab validation sample count. Provides sufficient power for equivalence testing and holdout evaluation.
Paired observations per site	≥80	Minimum for site-level performance statistics. Lower-bound sites may have fewer observations due to site-specific access constraints.
Observations above 126 CFU/100 mL	≥30 total (≥5 per site)	The Reg 93 threshold must be represented in the dataset. Classification performance at the threshold is meaningless without samples above it. Enhanced event sampling (Section 5.3) is designed to meet this requirement.
Observations above 235 CFU/100 mL	≥10 total	The Reg 93 single-sample maximum. High-end performance must be characterized.
Seasonal coverage	All 4 seasons at all 6 sites	Spring runoff, summer baseflow, fall recession, and winter low-flow represent distinct hydrologic and biological conditions. All must be represented.
Continuous data completeness	≥90% uptime per sensor per month	Demonstrates operational reliability of the method as deployed.

6.3 Data Recording and Management

All paired observations will be stored in a centralized, version-controlled database. Each record will include: site ID, sample date and time (UTC), Lume TLF reading (ppb), turbidity (NTU), temperature (°C), raw Lume E. coli estimate (CFU/100 mL), Colilert MPN result (CFU/100 mL), data quality flags, field conditions log entries, laboratory COC reference, and collector/operator ID. Raw data will be archived in original format with all transformations and QA flag applications fully documented and reproducible. Minimum retention: 10 years.

7. QA/QC Requirements

7.1 Sensor Calibration and Verification

QC Check	Frequency	Details
Field blank	Monthly (per site)	Submerge sensor in laboratory-grade DI water before and after each maintenance visit. TLF reading must be ≤[MDL to be determined]. Any reading above MDL triggers investigation and possible recalibration before data collection resumes.
Tryptophan standard verification	Monthly (per site)	Verify sensor response against a prepared tryptophan standard at a known concentration (e.g., 100 ppb). Acceptable response: within ±15% of the expected value. Out-of-range triggers recalibration.
Optical window cleaning	Bi-weekly	Clean optical window with approved cleaning solution. Verify post-cleaning blank reading before returning sensor to service.

7.2 Data Quality Flags

Flag	Trigger	Action
Fouling suspected	TLF drift >20% over 24 hours without corresponding Colilert change; post-cleaning blank >MDL	Flag all readings during fouling period. Exclude from paired dataset. Document in maintenance log.
Out-of-range turbidity	Turbidity >[upper operating limit NTU — to be determined from initial deployment]	Flag readings. High turbidity may saturate optical signal. Include flagged readings in sensitivity analysis but exclude from primary dataset.
Maintenance window	Any period during which sensor was removed, cleaned, or calibrated	Exclude all readings within ±30 minutes of maintenance activity from paired dataset.
Power interruption	Gap in data >30 minutes	Flag readings immediately before and after gap. Investigate cause. Exclude interpolated values from paired dataset.
Temperature out of range	Water temperature <0 °C or >35 °C	Flag readings. Outside characterized operating range. Include in sensitivity analysis; exclude from primary dataset.

7.3 Reference Method QC

QC Check	Frequency	Details
Laboratory field duplicate	Every 10th sample	Blind field duplicate submitted to certified laboratory. Acceptable lab RPD: ≤[to be confirmed with reference laboratory; typically ≤30% for environmental E. coli samples].
Laboratory positive control	Per laboratory QA plan	Certified laboratory runs positive and negative controls per their accredited QA plan. Control results reported with each analytical batch.
Colilert holding time compliance	Every sample	Sample analyzed within 6 hours of collection. Samples exceeding holding time excluded from paired dataset.

7.4 Data Completeness Requirements

For a calendar month’s data to count toward the study dataset, the following minimum completeness thresholds must be met: (a) ≥90% of expected Lume 15-minute readings must be present and unflagged for each site; (b) ≥80% of planned Colilert grab samples must have valid paired Lume readings within the ±15 minute pairing window. Months not meeting these thresholds will be documented and excluded from the primary dataset; if exclusion brings total paired observations below the Section 6.2 minimums, the study will be extended accordingly.

8. Model Development & Validation

8.1 Model Form

The prediction equation (Section 3.2) will be fit by ordinary least squares (OLS) regression to log₁₀-transformed E. coli concentrations. Below-detection-limit Colilert results will be treated as one-half the detection limit for regression purposes (standard left-censored data convention). The model form is additive with three predictors — TLF, turbidity, and temperature — and no site-specific or temporal covariates. No interaction terms will be included unless pre-specified below and justified by exploratory analysis on the training partition only.

8.2 Training / Holdout Split — Pre-Specified Protocol

Partition	Data Period	Purpose
Training set	Months 1–9 of the study (all 6 sites)	Fit the OLS regression coefficients (β0–β3). Model is built entirely on this partition. The coefficients are fixed after training and not updated.
Holdout test set	Months 10–12 of the study (all 6 sites)	Independent evaluation of the fixed model. All primary performance statistics (see Section 9) are calculated on this partition only. The holdout period is chosen to include spring runoff (if the study starts in January, this is the second spring; otherwise adjusted accordingly).

A temporal split is used rather than a random split because it mimics real-world deployment: the model is trained on historical data and applied forward in time. This is more conservative than a random split and eliminates any possibility of the training data “seeing” future conditions.

8.3 Model Acceptance Criteria (Training Phase)

Before the model is locked for holdout evaluation, it must meet the following minimum acceptance criteria on the training set:

• R² ≥ 0.50 (log₁₀-transformed concentrations)
• All three predictors (TLF, turbidity, temperature) must have non-zero coefficients with the expected sign (TLF positive; turbidity positive or negative depending on correlation with organic matter; temperature positive)
• No predictor with a statistically significant coefficient of opposite sign to that expected (would indicate collinearity or overfitting)
• Residuals approximately normally distributed (Shapiro-Wilk test, p > 0.05) and homoscedastic across the fitted range

If these criteria are not met on the training set, the model form will be reviewed (e.g., log₁₀ transformation of predictors, addition of one pre-specified interaction term: TLF × turbidity) with notification to EPA Region 8. The holdout set will not be examined until the model form is finalized.

8.4 Model Coefficient Reporting

The final fitted coefficients (β₀, β₁, β₂, β₃), standard errors, 95% confidence intervals, and p-values will be reported in the ATP application. The complete prediction equation with numerical coefficients will be included in the method documentation (Section 3.2) such that any laboratory can implement the method using a standard spreadsheet without proprietary software.

9. Statistical Analysis Plan

9.1 Primary Endpoint — Continuous Equivalence

Evaluated on the holdout test set only (Months 10–12). All analyses on log₁₀-transformed values unless otherwise specified.

Test	Method	Pre-Specified Threshold
Pearson correlation	r between log10(Lume) and log10(Colilert) on holdout set	r ≥ 0.80 [proposed; to be confirmed with EPA Region 8]
Coefficient of determination	R² on holdout set	R² ≥ 0.65 [proposed]
Mean absolute percentage error	MAPE in log-transformed space on holdout set	MAPE ≤ 15% [proposed]
Systematic bias	Geometric mean ratio: median(Lume/Colilert) on holdout set. Test for bias using paired t-test on log differences.	Geometric mean ratio between 0.67 and 1.50 (i.e., within a factor of 1.5×); paired t-test p > 0.05 for no significant bias [proposed]
Limits of agreement	Bland-Altman analysis: mean difference ± 1.96 SD of differences (log10 scale)	95% of differences within ±1.0 log10 unit (factor of 10×); reported for transparency [proposed]
Formal equivalence test	Two one-sided t-tests (TOST) on log10 differences. Equivalence margin: ±0.5 log10 units (≈ factor of 3×; comparable to Colilert’s own inter-lab variability of 20–30%)	Both one-sided tests significant at p < 0.05 within ±0.5 log10 equivalence margin [proposed]

9.2 Secondary Endpoint — Classification at Reg 93 Threshold

Binary classification performance at the Regulation 93 geometric mean threshold of 126 CFU/100 mL, evaluated on the holdout test set.

Metric	Definition	Pre-Specified Threshold
Sensitivity (true positive rate)	P(Lume ≥126 | Colilert ≥126)	≥0.85 [proposed — protective; missed exceedances are the higher public health risk]
Specificity (true negative rate)	P(Lume <126 | Colilert <126)	≥0.80 [proposed]
Cohen’s kappa	Agreement beyond chance on binary classification	≥0.70 [proposed — “substantial agreement”]
Overall accuracy	(TP + TN) / N	≥0.85 [proposed]
Area under ROC curve	AUC from continuous Lume predictions vs. binary Colilert threshold outcome	≥0.80 [proposed]
False negative rate	P(Lume <126 | Colilert ≥126)	≤0.15 [proposed — complement of sensitivity; protects against under-reporting exceedances]

Classification performance will also be evaluated at the Reg 93 single-sample maximum (235 CFU/100 mL) and reported as supplementary analyses. Confusion matrices for each site and season will be reported in the full comparability report.

9.3 Precision Analysis

Analysis	Data Source	Metric
Temporal within-sensor precision	Readings during stable baseflow periods (identified post-hoc by <5% CV over 24 hours)	Coefficient of variation (CV) of consecutive readings; characterizes instrument noise floor vs. real environmental signal.
Reference method precision	Field duplicate Colilert samples (every 10th sample)	RPD between field duplicates. Documents Colilert’s own measurement uncertainty at Boulder Creek, providing context for the equivalence margin.

9.4 Stratified Performance Analyses

All primary and secondary endpoint analyses will be repeated for the following stratifications, reported as supplementary analyses in the comparability report:

• By site (BC-Can, BC-Eben, BC-13, BC-CU, BC-30, BC-55): identifies site-specific performance differences or outliers
• By season (spring/summer/fall/winter): documents seasonal performance; spring runoff reported separately
• By flow condition (baseflow / rising limb / peak / recession): defined using USGS stream gauge data from the nearest Boulder Creek gauge
• By concentration range (<10 / 10–126 / 126–235 / >235 CFU/100 mL): performance at each regulatory decision threshold
• By turbidity range (<10 / 10–50 / >50 NTU): documents turbidity interference effects

9.5 Statistical Software and Reproducibility

All statistical analyses will be performed in R (version ≥4.3) or Python (scikit-learn, scipy, statsmodels). Analysis code will be version-controlled and provided to EPA Region 8 upon request. All results will be reproducible from the archived raw data using the provided code. No analyses will be performed in Excel alone; Excel may be used for data entry and visualization only.

10. Method Detection Limit

10.1 TLF Detection Limit

The instrument detection limit for TLF will be determined per 40 CFR Part 136 Appendix B: minimum 7 replicate measurements of a tryptophan standard near the expected detection limit, run in both laboratory (controlled tryptophan solution in DI water) and field (low-concentration upstream reference site BC-Can) conditions. MDL = t-value (α = 0.01, n−1 degrees of freedom) × standard deviation of replicates.

10.2 E. coli MDL (Translated)

The TLF MDL will be translated to an E. coli MDL in CFU/100 mL by applying the fitted prediction equation. The method reporting limit (MRL) will be set at 3× the MDL, consistent with standard analytical chemistry practice. Colilert results below the Lume MRL will be reported as <MRL in the paired dataset and treated as left-censored in regression analyses.

10.3 Relationship to Regulatory Thresholds

The MDL and MRL will be compared against the Reg 93 thresholds (126 CFU/100 mL geometric mean; 235 CFU/100 mL single-sample maximum) and the existing literature MDL of approximately 10 CFU/100 mL for TLF-based methods. The method must demonstrate that the MRL is substantially below the regulatory threshold to support the compliance monitoring claim.

11. Interferences & Limitations

11.1 Known Interferences

Interference	Mechanism	Mitigation in This Method
Dissolved organic carbon (DOC) / humic-like fluorescence	Humic and fulvic acids can fluoresce at wavelengths overlapping the TLF signal, causing false positive E. coli estimates	Partially mitigated by turbidity correction in the prediction equation. The magnitude of humic interference at Boulder Creek is characterized during the study by comparing TLF at low-E. coli / high-DOC conditions (e.g., late fall leaf litter input) against Colilert results.
High turbidity	Very high turbidity (>[threshold NTU]) can attenuate fluorescence signal (inner filter effect) or scatter light into detector	Turbidity is included as a model predictor. Readings above the identified interference threshold are flagged (Section 7.2). Performance at high turbidity conditions is characterized separately.
Temperature	TLF signal intensity is temperature-dependent; fluorescence efficiency decreases with increasing temperature	Temperature included as a model predictor. Study spans all four seasons, ensuring temperature effects are represented in the calibration dataset.
Algal fluorescence	Phytoplankton can produce fluorescence signals in the TLF spectral region, particularly during algal blooms	Algal events documented in field conditions log. Performance during identified algal bloom periods analyzed separately. Boulder Creek turbid spring runoff typically limits algal growth.
Biofouling	Biological growth on the optical window attenuates signal over time	Bi-weekly cleaning protocol (Section 7.1); fouling detection via daily drift monitoring (Section 7.2); maintenance rotation minimizes gaps.
Chlorinated effluent	WWTP effluent may carry chlorine residual that partially inactivates E. coli but does not immediately destroy TLF signal, causing over-prediction	Site BC-13 may receive residual-chlorinated effluent intermittently. Performance at BC-13 analyzed separately; any systematic bias at this site documented and disclosed.

11.2 Method Limitations

• The prediction equation is calibrated to Boulder Creek water chemistry. Performance at sites with substantially different DOC, humic content, or conductivity is not validated by this study and should not be assumed without site-specific evaluation.
• The method measures total TLF, not E. coli specifically. The prediction equation uses turbidity and temperature to correct for non-E. coli TLF sources, but cannot distinguish E. coli fluorescence from all potential interfering substances.
• The method does not distinguish viable from non-viable E. coli. Post-chlorination over-prediction (documented at BC-13) reflects this limitation.
• Sensor performance during ice formation or under ice cover has not been characterized. Data collected when water temperature is <1 °C should be considered provisional.

12. Deliverables & Schedule

12.1 Pre-Study Prerequisites

• All 6 Lume sensors deployed and transmitting at Boulder Creek TMDL monitoring sites
• Certified reference laboratory confirmed and under contract for increased Colilert sampling frequency
• Colilert version (standard / Colilert) confirmed and consistent with Boulder’s existing Reg 93 program
• This study design document submitted to and acknowledged by EPA Region 8 (study design pre-registration)
• Statistical analysis plan (Section 9) accepted by EPA Region 8 in writing, including pre-specified thresholds
• Chain-of-custody and field conditions logging protocols in place and tested

12.2 Study Timeline

12.3 Deliverables

Deliverable	Recipient	Timing
Study design document (this document)	EPA Region 8, CDPHE, City of Boulder	Pre-study
Quarterly interim data reports	EPA Region 8, CDPHE	Months 3, 6, 9
Model fitting report (locked coefficients)	EPA Region 8	Month 9
Full statistical analysis report (holdout)	EPA Region 8, CDPHE, City of Boulder	Month 13
Boulder Creek comparability report	EPA Region 8, CDPHE	Month 14
Complete method documentation (EPA format)	EPA Region 8	Month 15
40 CFR 136.5 application package	EPA Region 8	Month 16

13. Regulatory Engagement

Ongoing coordination with CDPHE and EPA Region 8 throughout the project. Front-loaded in Q1–Q2 to align study design with regulatory expectations before committing to the full field deployment. The regulatory context is Colorado Regulation 93—CDPHE’s 303(d) program for impaired waters—under which Boulder Creek carries E. coli impairment. The initial ATP application will be scoped to support Reg 93 compliance monitoring at Boulder’s six monitoring points.

13.1 CDPHE Initial Engagement

Task	Timing	Details
Introductory briefing to CDPHE Water Quality Control Division	Month 1	Present the Lume technology, existing Boulder Creek validation data, and ATP intent. Identify CDPHE staff lead for ongoing coordination.
Review Colorado Regulation 93 / 303(d) requirements for Boulder Creek	Month 1–2	Confirm the Reg 93 E. coli impairment listing and TMDL threshold (126 CFU/100 mL geometric mean) for Boulder Creek. Identify Boulder’s existing monitoring obligations at the 6 monitoring points and determine how continuous Lume data maps to Reg 93 compliance reporting requirements.
CDPHE feedback on study design	Month 2–3	Share proposed data collection protocol (see Section 17). Get CDPHE input on sample counts, seasonal coverage, and any Colorado-specific requirements.

13.2 EPA Region 8 Engagement

Task	Timing	Details
Pre-submission meeting request to EPA Region 8	Month 1	Contact EPA Region 8 Water Technical Unit in Denver. Request formal pre-submission consultation per ATP program guidance.
Prepare briefing package	Month 1–2	Package includes: technology summary, published validation data, proposed method scope (E. coli in freshwater and enterococci in marine water via TLF-based linear regression for recreational water monitoring), proposed study design, draft QA/QC framework, and specific questions for EPA.
Pre-submission meeting	Month 3–4	Formal meeting with EPA Region 8. Present briefing package, receive guidance on study design, statistical requirements, and any Region 8-specific considerations.
Parallel consultation with EPA OST (headquarters)	Month 3–4	Contact Lemuel Walker at EPA Office of Science and Technology (walker.lemuel@epa.gov, 202-566-1077). OST handles nationwide ATP review; early engagement ensures Region 8 and OST expectations are aligned.

13.3 Key Questions for Regulators

14. Field Study Workplan

The core data collection effort. Deploy Lume sensors at all 6 of the City of Boulder Utilities’ recreational water monitoring locations on Boulder Creek. Collect continuous TLF data paired with Boulder’s existing weekly Colilert E. coli grab samples.

14.1 Site Preparation & Deployment

Task	Timing	Details
Formalize MOU with City of Boulder Utilities	Month 1	Define roles, data sharing, access to compliance sites, sampling coordination, and liability. Boulder continues their existing Colilert program unchanged; Virridy adds sensor deployment.
Site survey of all 6 compliance locations	Month 1–2	Visit each site. Assess: mounting infrastructure, power availability, cellular/data connectivity, flow characteristics, security, and seasonal access constraints (ice, high water).
Sensor procurement & pre-deployment calibration	Month 1–2	Prepare 7 Lume sensors (6 primary + 1 spare for maintenance). Factory calibration with tryptophan standards. Document sensor serial numbers, firmware versions, and initial calibration data.
Install sensors at all 6 sites	Month 2–3	Staggered installation over 2–4 weeks. At each site: mount sensor, verify data transmission, confirm measurement interval (15-min default), photograph installation, record GPS coordinates. Begin continuous logging.
Validate data pipeline	Month 3	Confirm all 6 sensors are transmitting reliably to the Lume data platform. Set up automated data quality alerts (signal dropout, out-of-range readings, battery/power status).

14.2 Ongoing Data Collection

Task	Frequency	Details
Continuous Lume TLF measurement	Every 15 min	Automated. Each reading: TLF intensity (ppb), turbidity (NTU), temperature (°C), ML model E. coli estimate (CFU/100 mL), categorical classification, model confidence score.
Paired Colilert grab sample (Boulder’s program)	Weekly	Boulder staff collect grab samples at each recreational water monitoring site per their existing reporting schedule. Log exact sample collection time to match against nearest Lume reading.
Enhanced sampling during events	As needed	Increase Colilert grab frequency during high-flow events, storm runoff, and any known upstream contamination events. Target: 2–3× weekly during events to increase paired observations at high concentrations.
Field QC: blank verification	Monthly	Submerge sensor in DI water before and after each maintenance visit. Verify TLF reading is below MDL. Document any drift.
Sensor maintenance	Bi-weekly	Clean optical window, check mounting integrity, download local backup data, verify calibration against tryptophan standard. Log all maintenance activities with timestamps.
Data review & QA flagging	Weekly	Review incoming data for each site. Flag and document: fouling events, power interruptions, sensor malfunctions, outliers. Apply data quality flags per QA/QC protocol.

14.3 Data Collection Targets

Metric	Target	Rationale
Study duration	12–18 months minimum	Must span all four seasons to capture temperature, flow, and biological variability. Two full spring runoff cycles preferred.
Paired observations per site	≥52 (weekly × 12 months)	52 is the baseline from weekly Colilert. Enhanced event sampling should push this to 70–100+ per site.
Total paired observations (all sites)	≥400–600	6 sites × 70–100 each. Substantially exceeds typical EPA multi-lab requirements.
Concentration range	<1 to >1,000 CFU/100 mL	Must cover the full range relevant to recreational water quality criteria.
Continuous data completeness	≥90% uptime per sensor	Account for maintenance windows, fouling, and equipment failures. One spare sensor provides coverage during repairs.
Duplicate sensor precision data	≥12 one-week deployments	One per month across different sites = reproducibility data across conditions.

15. Limited-Use ATP Application

Facility-specific ATP approval under 40 CFR 136.5, submitted by the City of Boulder to EPA Region 8 for Colorado Regulation 93 / 303(d) compliance monitoring on Boulder Creek. This is the near-term regulatory milestone: a site-specific approval that allows Boulder to use Lume data in fulfillment of its Reg 93 impaired-waterbody monitoring obligations, with the TMDL-driven threshold of 126 CFU/100 mL geometric mean as the key compliance metric.

15.1 Application Preparation

Task	Timing	Details
Compile Boulder Creek paired dataset	Month 13–14	After 12 months of data collection. Merge Lume continuous data with Boulder’s Colilert results. Apply QA/QC flags. Generate site-specific performance statistics.
Prepare site-specific comparability report	Month 14–15	Statistical analysis from Workstream 4 (see ATP Overview) tailored to Boulder’s specific sites and permit conditions. Include seasonal breakdown, flow-condition breakdown, and performance at Boulder’s permit limit thresholds.
Draft limited-use application	Month 15–16	Application per 40 CFR 136.5 requirements. Includes: method description, comparability data, QA/QC protocol, proposed reporting framework.
City of Boulder internal review & approval	Month 16–17	Boulder Utilities reviews and approves the application. Boulder is the applicant under 136.5—Virridy provides technical support.
CDPHE pre-submission review	Month 16–17	Share draft with CDPHE for informal feedback before formal submission to EPA Region 8. CDPHE support strengthens the application.

15.2 Submission & Review

Task	Timing	Details
Submit to EPA Region 8	Month 17	City of Boulder submits the limited-use ATP application to the EPA Region 8 Administrator.
Respond to EPA information requests	Month 17–20	Anticipate questions and requests for additional data. Have supplementary analyses pre-computed.
EPA Region 8 approval	Month 18–21	If approved, EPA issues a limited-use ATP approval letter. Boulder can begin using Lume data for recreational water quality monitoring on Boulder Creek.

15.3 Post-Approval Operations

Task	Details
Compliance reporting protocol	Define how Boulder reports Lume data for recreational water quality monitoring. Agree on: averaging period, reporting frequency, data completeness thresholds, and backup procedure (revert to Colilert if sensor data completeness falls below threshold).
Parallel monitoring period	Continue Colilert sampling in parallel with Lume for an initial period (6–12 months) after limited-use approval. Builds confidence and provides ongoing comparability data.
Performance monitoring & reporting	Quarterly performance reports to EPA Region 8 and CDPHE during the parallel monitoring period. Document ongoing accuracy, precision, and any issues.

16. Next Steps: Colorado State-Wide Expansion Track A — Phase 2

After the Boulder Creek limited-use ATP is approved, expand the Lume’s recognized use to additional Colorado waterbodies listed under Reg 93 / 303(d). The goal is to build a body of multi-site, multi-condition evidence that supports both CDPHE statewide recognition and the nationwide freshwater ATP submission.

16.1 Additional Colorado Limited-Use ATPs

Task	Timing	Details
Identify target Colorado watersheds	Month 18–20	Select 2–4 additional Colorado 303(d)-listed waterbodies with active E. coli impairments and willing utility/agency partners. Geographic and hydrologic diversity strengthens the broader ATP submission (e.g., mountain streams, plains rivers, reservoirs).
Submit additional 40 CFR 136.5 applications	Month 21–24	Each new site submits its own facility-specific ATP application to EPA Region 8, using the Boulder Creek method documentation and comparability report as the technical foundation.

16.2 CDPHE State Method Recognition

Task	Timing	Details
Briefing to CDPHE on multi-site performance	Month 22–24	Present aggregated Colorado performance data across all approved sites. Propose CDPHE formally recognize the Lume method for E. coli monitoring on Colorado Reg 93 / 303(d) waterbodies.
CDPHE state guidance or policy adoption	Month 24+	Work with CDPHE to issue guidance that streamlines future Reg 93 monitoring approvals for the Lume across Colorado. Provides a state-level endorsement that supports the nationwide EPA submission.

17. Data Collection Protocol

Supplementary protocol details for paired data collection at each Boulder Creek site, covering elements not fully addressed in Sections 5–7 above. Designed to generate EPA-quality comparability data.

17.1 Colilert SOP Details

Parameter	Requirement
Colilert formulation	IDEXX Colilert (24-hour incubation at 35°C), matching Boulder’s existing Reg 93 compliance program. Must use consistent formulation throughout study. See Open Question B-2.
Quantification method	Quanti-Tray/2000 MPN per IDEXX protocol and 40 CFR Part 136.
Incubation control	Incubator temperature verified daily during all active incubation periods. Temperature logs retained as part of chain of custody.
Laboratory QC controls	Positive control (E. coli ATCC 25922 or equivalent) and negative control (sterile Colilert reagent in DI water) run with each analytical batch. Results reported with each batch.
MPN calculation	Per IDEXX Quanti-Tray/2000 MPN table. Below-detection results reported as <1 MPN/100 mL. Saturated Quanti-Tray (all wells positive) reported as >2419.6 MPN/100 mL.

17.2 Certified Laboratory Requirements

Requirement	Details
CLCP certification scope	Laboratory must hold current CLCP certification specifically for E. coli in surface water by the Colilert / Quanti-Tray method. Verify certification is active at study start and before each annual renewal period.
PT sample performance	Laboratory must demonstrate acceptable performance on proficiency testing (PT) samples for E. coli within 12 months prior to study start. PT results requested as part of laboratory selection.
Sample turnaround	Laboratory must commit to returning results within 24 hours of receipt to allow timely data management. Results provided as a digital report with sample IDs, collection times, and MPN values.

17.3 10-Year Data Archival

Category	Retention Requirement
Raw sensor data	All raw TLF, turbidity, temperature readings archived in original format. No deletion or overwrite. Minimum 10-year retention from study end date.
Laboratory Colilert reports	All original laboratory reports retained. Digital copies stored in version-controlled database. Physical copies retained by the City of Boulder per their existing record-keeping requirements.
Chain of custody records	All COC forms (paper originals and digital scans) retained for minimum 10 years. COC records are EPA records and may not be destroyed without agency notification.
QA/QC records	Calibration logs, maintenance logs, field blank results, equipment service records. 10-year minimum retention.
Analysis code and models	All statistical analysis scripts, model coefficient files, and data processing code version-controlled and archived. Must be sufficient to fully reproduce all results in the ATP application from raw data.

17.4 Chain of Custody Specifics

COC Element	Requirement
Sample identification	Each grab sample assigned a unique sample ID at the time of collection: [SiteID]-[YYYYMMDD]-[HHMM]-[CollectorInitials]. Label affixed to sample bottle before leaving the collection site.
Collector signature	COC form signed by collector at collection. Field conditions (weather, flow, turbidity estimate, any anomalies) recorded at time of collection.
Transfer documentation	COC accompanies sample from field to laboratory. Each transfer (e.g., field to cooler, cooler to lab intake) documented with date, time, and signature.
Laboratory receipt	Laboratory intake staff sign and timestamp COC upon receipt. Record condition of samples (temperature, ice presence, container integrity). Reject and document samples received outside holding time or temperature.
Holding time compliance	Sample must be analyzed (Colilert added) within 6 hours of collection. Holding time calculated from collection time recorded on COC, not laboratory receipt time.
Digital COC backup	Photograph or scan of completed COC uploaded to study database within 24 hours of laboratory receipt.