## A LTERNATE TEST PROCEDURE (ATP) VALIDATION STUDY REPORT

For


## Free and Total Chlorine Using Disposable “Planar” Reagent-Filled Cuvettes

Revised 8/ 15/1 3

HACH COMPANY 5600 LINDBERGH DRIVE

LOVELAND, CO 805378

Page 1 of 341.0 Background

The objective of this study is to evaluate Hach’s disposable Planar Reagent- Filled Cuvette (PRFC) me thod for the measurement of free, total, and combined free and total chlorine (Hach Method s 10260, 10270 and 10280) against the USEPA reference method SM -4500 Cl G for approval and use in USEPA regulatory drinking water programs. The method s are applicable to measure chlorine (Free and/ or total) between 0.2 and 2.0 mg/L Cl 2 . The methods use disposable planar reagent

- filled cuvettes (PRFCs) that are based on the standard (approved) DPD ( N, N

- diethyl -p-p henylenediamine) reaction (See Figure 1). The absorbance of the Wurster Dye is measured at 510 nm and the intensity of the absorbance is directly proportional to the concentration of chlorine. The DPD

- filled planar cuvettes, when interfaced with the pumping and optical system of the instrument, act as a sampling, mixing and measurement vessel. The PRFCs are in the form factor similar to a popsicle stick (~85 mm x11 mm x 1 mm, see Figure 2) and contain all of the required reagents to perform a DPD based chlorine test (Free and Total).

Figure 1: Mechanism for the reaction of DPD with Chlorine

Figure 2 : Schematic of the disposable reagent

- filled planar cuvette for Free and Total Chlorine (Note that the amount of DPD placed on the chip for the 30μL sample is in exact proportion to the amount of DPD placed in the Hach Powder Pillows for a 10 mL sample)

Page 2 of 34For this ATP study a prototype benchtop instrument was used to make the chlorine

measurements (Figure 3). It is envisioned that the commercial version of this prototype will contain up to 4 slots to make up to 4 simultaneous measur ements using PRFCs. This will allow drinking water facilities the capability to measure other colorimetric paramete rs in the field beyond chlorine in approximately the same amount of time it take s them t o perform a single chlorine measurement today. F igure 4 shows a schematic for the future embodiment of the instrument that would have 4 individual ports for

measurement

. The future system will use the same cuvettes, wavelength, optical path, and mixing protocol as prototype instrument used in this study.

Figure 3: Prototype breadboard setup used to make the free and total chlorine measurements for the ATP s tudy

Page 3 of 34Figure 4 : Future Industrial design of a h and -held 4 port instrument

Chlorine measurements were performed by drawing sample into the cuvette, mixing the sample with the pre

- filled reagent(s), followed by an optical measurement at 510 nm after the reaction is complete across the width of the cuvette (11 mm optical path). Sample measurement, mixing, timing, and measurement are all controlled by a pumping system within the instrument. The only step that needs to be performed to make a mea surement is to connect the PRFC(s) into the instrument and place it in the water and draw in the sample (The instrument uses a conductivity probe to determine when the chips have been dipped in the sample). The rest of the measurement is controlled by the system.

Page 4 of 34The detailed approach to make a free and total chlorine meas urement using PRFCs is as follows:

1) Draw 30 μL of sample into the cuvette. 2) Mix the sample with the DPD and buffer 3) Wait 30 seconds for the reaction to be complete 4) Draw the sample into the optical path and take and optical measurement at 510 nm for free chlorine 5) Move the sample up to mix with the potassium iodide 6) Wait 180 seconds for complete reaction of monochloramine (Also dichloramine and trichloramine , if present)

. 7) Draw the sample back into the optical path to make a measurement for total chlorine 8) Move the sample up to mix with the ascorbic acid to bleach out the Wurster’s Dye 9) Wait 1 minute 10) Draw the sample back into the optical path and make a measurement to zero out any imperfection s from the cuvette and blank on the sample. Total Testing ti me for Free and Total Chlorine~4.5 minutes Note: In step 7 the blanking is performed after the measurement to improve analytical performance. If the sample was zeroed prior to reaction with the D PD it was noticed that the chlorine demand of the polystyrene cuvette impacted analytical performance.

The method was compared to the standard Hach DPD methods (0.2-2.0 mg/L Cl 2 ) for free and total chlorine in a 3 lab ATP study (Hach method numbers 8021 (Free) and 8167 (Total)). The method was found to perform well in both high and low ionic strength matrices between pH 6 and 8.8. The method also generated MDL s for free chlorine of 0.023 mg/L and total chlorine of 0.027 mg/L.

1.1 Method Justification

The most widely used EPA approved method for the measurement of chlorine in the field (SM 4500 Cl- G) involves the use of powder pillows and colorimeter. Powder pillowbased methods are very dependent on technique and can be prone to inter-o perator error. Errors in wavelength selection, sample measurement, reagent addition, mixing, and/or timing can cause variability in the chlorine measurement. Scratched cuvettes can also cause problems in measurements. The proposed method using disposabl e PRFC s automates the entire process from sampling to measurement eliminating all of the pot ential areas for operator error because each planar cuvette is barcoded to identify the method, all of the reagents for the test are contained in the cuvette, and the mixing and timing of the sample reaction are controlled by the instrument.

1.2 Method Equivalency

The QC acceptance criteria for the method were determined based on analytical perf ormance relative to SM 4500 Cl G (DPD powder pillow method) for both free Cl 2

Page 5 of 34and total Cl 2 . Method performance was based on side- by -side studies against DPD powder pillows using 3 analytical performance metrics: 1) MDLs in low and high ionic strength matrices 2) Precision and accuracy of the method at the ~2.0 mg/L Cl 2 in low and high ionic strength matrices 3) Spike recoveries relative to SM4500- Cl G (DPD powder pillow) in ground water and surface water matrices at various pH values (3 different ground water matrices and 3 different surface water matrices from 6 differen t locations tested in 3 different laboratories) 4) Monochloramine interference on a free chlorine measurement relative to the approved method The recoveries that are calculated for this method were calculated against the results generated by Hach Free and Total chlorine powder pillows (SM4500- Cl G)

. This was due to the difficulty of preparing accurate and stable chlorine standards. Chlorine standards are inherently unstable and degrade over time causing issues with repetitive precision based tests such as MDL studies. For our studies t he same sample was used for both the reagent- filled cuvette tests and the powder pillow tests. 10 mL s ample s were prepared and m easured with the disposable cuvettes first (30 μL) immediately follo w ed by the powder pillow tests (~10 mL) . Both samples were reacted with DPD (either in the chip or with the powder pillow) within 30 seconds to ensure the same concentration was measured using both methods. This eliminated the variable of chlorine degradation between meas urements and allowed for better correlation between the methods.

Below is a summary of the pooled data from the three different labs that were normalized to a single concentration for each specific test . Note: Due to the instability of chlorine standard each location was not able to prepare exactly the same concentration samples for each test.

Test % recovery for Free Cl chip % recovery for Total Cl chip MDL (mg/L) or
%RSD Free Cl2

PRFC MDL (mg/L) or %
RSD Free Cl2
Powder Pillow MDL (mg/L) or
%RSD Total Cl2

PRFC MDL (mg/L) or %
RSD Total Cl2
Powder Pillow 0.2 mg/L Low Ionic Strength (MDL) 97% 85% 0.019 0.019 0.025 0.050 0.2 mg/L High Ionic Strength (MDL) 89% 93% 0.025 0.020 0.033 0.040 2 mg/L Low Ionic Strength 103% 93% 2.30% 2.50% 2.88% 2.25% 2 mg/L-High Ionic Strength 102% 92% 5.22% 3.38% 4.15% 4.02%

GW Matrix Sample 48% 291% below detection
limit below detection
limit below detection
limit below detection
limit GW Matrix Spike (Spike to ~2 mg/L) 104% 95% 1.27% 1.25% 0.88% 0.62%

SW Matrix Sample 75% 94% below detection
limit below detection
limit below detection
limit below detection
limit SW Matrix Spike (Spike to ~2 mg/L) 103% 97% 2.54% 2.25% 1.41% 0.86% GW Matrix pH 6 (Spike to ~2 mg/L) 105% 98% 2.00% 1.34% 1.46% 1.00% GW Matrix pH 7.4 (Spike to ~2 mg/L) 104% 97% 1.16% 1.86% 0.69% 0.56% GW Matrix pH 8.2 (Spike to ~2 mg/L) 103% 96% 1.40% 2.01% 1.56% 0.49% GW Matrix pH 8.8 (Spike to ~2 mg/L) 103% 95% 2.43% 1.24% 1.43% 0.69% SW Matrix pH 6 (Spike to ~2 mg/L) 105% 97% 1.32% 1.80% 2.00% 1.07% SW Matrix pH7.4 (Spike to ~2 mg/L) 104% 96% 2.69% 2.12% 1.07% 0.64% SW Matrix pH 8.2 (Spike to ~2 mg/L) 103% 95% 3.86% 1.77% 3.30% 1.19% SW Matrix pH 8.8 (Spike to ~2 mg/L) 104% 95% 1.72% 1.40% 4.67% 3.26% 

## Pooled Data (All sites normalized)

Page 6 of 34The pooled results for the planar re agent- filled cuvette (PRFC) tests compared favorably in many individual tests for both free and total chlorine. Based on this data it wa s determined that the method is “equally effective” in testing free and total chlorine when compared to the reference method. We also believe that the method will be significantly improved upon once automated production equipment is used to manufacture th e PRFCs and the commercial version of the instrument is available.

1.3 Analytes

Free Chlorine [CAS# 7782-50-5] Total Chlorine (Can be composed of one or more of the following) Monochloramine [CAS# 14611-51-9] Dichloramine [CAS# 3400-09-7] Tri -Chloroamin e [CAS# 10025 -85-1]

2.0 Study Implementation

The Hach Company was responsible for setting up the ATP study plan with collaboration from Gly nda Smith and Steve Wendelken of the EPA. The test plans were executed at 3 facilities using 2 different matrices at each facility (Ground water and Surface water). The facilities were: 1) Hach Company Laboratories (Loveland, CO surface water and Greely

, CO ground water. 5/3/2011-5/16/2011

a. Hach Company laboratories were selected because to the simplified logistics and to reduce cost 2) Test America labs (2 labs , Cedar Falls and Phoenix) were selected through their corporate manager who found labs that had available time to pe rform the analyses. Each lab was paid $60/hour for their work. Each study took approximately 40 hours at each laboratory to complete

a. Cedar Falls 5/23/2011-5/27/2011 and 7/10/2011-7/13/2011 (Samples: Cedar Falls, IA ground w ater and Chicago, IL surface wa ter) b. Phoenix Test America Laboratory 5/16/201-5/20/2011 (Fond du Lac, WI ground water and Phoenix, AZ surface water) The labs were supplied with 2 Hach prototy pe PRFC measurement systems, disposable PRFCs to run the tests, reagents to prepare standards, a Hach Pocket Colorimeter, Hach free and total Chlorine powder pillows, and Hach cuvettes for the colorimeters.

2.1 Study Schedule

Hach Loveland Lab Studies: 5/3/2011-5/16/2011 Cedar Falls, IA Test America Laboratory 5/23/2011-5/27/2011 and 7/10/2011-7/13/2011 Phoenix, AZ Test America Laboratory 5/16/201-5/20/2011

2.2 Sample Collection

• Samples were collected following standard USEPA sampling procedures

• Samples measured in Loveland, CO

Page 7 of 34o Surface Water: Loveland Tap water samples were collected directl y from the taps in the Hach Loveland facility

o Ground Water: Greeley, CO ground water was collected from Hach employee’s well directly outside of his house and directly transported to the Hach Laboratories

• Samples measured in Cedar Falls IA

o Surface Water: Chicago, IL surface water samples were collected from at 190 South LaSalle Street, 25th Floor Chicago, IL 60603 and shipped to the Hach Laboratories and then subsequently shipped to the Cedar Falls, IA Test America Laboratory

o Ground Water: Cedar Falls, IA ground water samples were collected directly from the tap at the Cedar Falls, IA Test America Laboratory

• Samples measured in Phoenix, AZ

o Surface Water: Phoenix, AZ surface water samples were collected directly from the tap at the Phoenix, AZ Test America Laboratory

o Ground Water: Fond du Lac, WI ground water samples were collected at 45 S. Main St., Fond du Lac, WI 54935 and directly shipped to the Phoenix Test America Laboratories All samples that were shipped to the labs did not have any chlorine residual by the time they reached the test laboratories due to the stability of free chlorine.

2.3 Types of Analyses Performed The matrix of tests that were performed at each site is below.

Page 8 of 34All free chlorine tests were performed using a free chlorine standa rd prepared from stock 6%-8% sodium hypochlorite. All total chlorine tests were performed using a monochloramine standard that was prepared by mixing the appropriate amount of ammonium chloride standard with a free chlorine standard prepared from sodium h ypochlorite. The monochloramine interference test was performed by measuring the free chlorine result of a 2.0 mg/L monochloramine standard.

2.4 Study Plan Deviations T here were some issues that were encountered during the testing with the prototype system (instrument and cuvettes) . There were several tests that were performed that the software deemed “bad” tests and did not record those results. There were also some communication issues between the computers and the “prototype b readboards” that happened sporadically. These issues seemed to cause issues for the technicians running the tests with keeping count of how many tests they performed. In al l, there were 18 total tests (9 pairs ) that were missed out of 912 tests (456 pairs). We believe that in most cases the combination of the relatively high frequency of system errors with the complicated test plans caused Hach DPD
Free Chlorine
Powder
Pillow Hach DPD
Total Chlorine
Powder
Pillow Hach Free
Chlorine (Disposable PRFC) Hach Total
Chlorine (Disposable PRFC)
Standard Verification 0 0 0 0 0.2 mg/L Low Ionic Strength 7 7 7 7 0.2 mg/L High Ionic Strength 7 7 7 7

2 mg/L Low Ionic Strength 7 7 7 7 2 mg/L-High Ionic Strength 7 7 7 7 GW Matrix Sample 4 4 4 4 GW Matrix Spike (Spike to ~2 mg/L) 4 4 4 4 SW Matrix Sample 4 4 4 4 SW Matrix Spike (Spike to ~2 mg/L) 4 4 4 4

Monochlormaine Interference at 2 mg/L NH2Cl 4 4 4 4 GW Matrix pH 6 (Spike to ~2 mg/L) 4 4 4 4 GW Matrix pH 7.4 (Spike to ~2 mg/L) 4 4 4 4 GW Matrix pH 8.2 (Spike to ~2 mg/L) 4 4 4 4 GW Matrix pH 8.8 (Spike to ~2 mg/L) 4 4 4 4 SW Matrix pH 6 (Spike to ~2 mg/L) 4 4 4 4

SW Matrix pH7.4 (Spike to ~2 mg/L) 4 4 4 4

SW Matrix pH 8.2 (Spike to ~2 mg/L) 4 4 4 4 SW Matrix pH 8.8 (Spike to ~2 mg/L) 4 4 4 4
Total # of Tests 64 64 64 64

Page 9 of 34the technicians to miscount the tests that they ran at different facilities. That being said we believe that the 89 2 data points that were generated in the test plan should suffice as a very comprehensive data set. The individual deviations are outlined below: 1) Loveland , CO : There were 4 missing reading from the Loveland, CO total chlorine set of tests. There were t wo 2.0 mg/L high ionic strength spikes, one 2.0 mg/L low ionic strength spike, and one 2.0 mg/L surface water spike. The reason these tests were not performed was there were communication issues with the “prototype breadboard instrument” that made the technician running the samples assume the test had been performed. It was an oversight by the technician running the samples. 2) Phoenix , AZ : One missing reading was found in the Phoenix data (2.0 mg/L high ionic strength spike). The reason this data point is missing is unknown. It is assumed the operator who performed the analysis miscounted how many replicates that he ran and missed by one. Since it was a single data point out of the 151 pairs of tests (302 total tests) performed in Phoenix we don’t believ e it should have much impact on the interpretation of the data. 3) Cedar Falls IA: All of the total chlorine data in Cedar Falls had to be rerun on a second date because the preparation of th e monochloramine standard was performed wrong. A large excess of a mmonia was added to the free chlorine standard, which made the total chlorine standard mostly di- and tri- chloramines rather than monochloramine. Although this did not impact the results it did not represent what would traditionally be measured in drinking water systems that use monochloramine as a disinfectant. Therefore, we decided to re

- run the tests at a later date to ensure a more homogeneous set of data between the three sites.

There were 4 missing total chlorine data points in the second set of te sts performed in Cedar Falls. They were all 2.0 mg/L spikes of ground water that were pH adjusted (2 pH 7.4, 1 pH 6.0, and 1 pH 8.2). The reasons that these samples were missing were oversights by the technician performing the tests. During the second s ets of test there were several communication and analyses issues the led to invalid results that were not reported by the prototype system. Because of these different hardware issues it is believed that the technician performing the analyses lost count of the different tests he was performing.

There also was one missing free chlorine test (2.0 mg/L spike into low ionic strength water). We believe this was an oversight by the technician performing the tests.

There was a major outlier in the low ionic strength 2 mg/.L total c hlorine spiked samples. The disposable PRFC test reported a value of 0.05 mg/L for a ~2.0 mg/L total chlorine spike. The best rationale for this outlier was that the chip did not have DPD in the sample channel of the PRFC and therefore no color was generated to measure.

3.0 Method Procedure and Data

Page 10 of 34Method is attached

.
3.1 Validation Study Demonstration Data All data is summarized below from each individual site for each specific analyte (Free or total chlorine). Tables 7 and 8 show compiled sets of data with all data normalized to the same concentration (Each site did not necessarily spike to exactly 2 mg/L so the data was normalized to 2.0 mg/L for all of the spike concentrations)

Table 1: Loveland Free Chlorine Data 

# LOVELAND FREE Cl2 # of reps Hach Disposable Chips Free
Chlorine

Ave Conc Hach DPD

Free
Chlorine
Powder
Pillow Ave

Conc Free
Chlorine
Hach PPA Chip % Recovery Free
Chlorine
Hach PPA Chip % RSD (MDL For 0.2 mg/L standards) Free Chlorine Hach
Powder Pillow % RSD

(MDL For 0.2 mg/L standards)

0.2 mg/L Low Ionic Strength 7 0.21 0.22 96% 0.014 0.012 0.2 mg/L High Ionic Strength 7 0.26 0.27 96% 0.025 0.017 2 mg/L Low Ionic Strength 7 1.96 1.89 104% 0.5% 3.2% 2 mg/L-High Ionic Strength 7 1.80 1.79 100% 7.2% 2.8% GW Matrix Sample 4 0.01 0.02 33% 12.2% 0.0% GW Matrix Spike (Spike to ~2 mg/L) 4 1.91 1.81 105% 1.5% 0.0% SW Matrix Sample 4 0.43 0.42 103% 5.4% 3.6% SW Matrix Spike (Spike to ~2 mg/L) 4 2.03 1.94 105% 1.7% 3.4% GW Matrix pH 6 (Spike to ~2 mg/L) 4 2.08 1.93 108% 3.5% 0.5% GW Matrix pH 7.4 (Spike to ~2 mg/L) 4 2.05 1.95 105% 1.2% 2.1% GW Matrix pH 8.2 (Spike to ~2 mg/L) 4 2.04 1.98 103% 1.7% 3.7% GW Matrix pH 8.8 (Spike to ~2 mg/L) 4 2.01 1.90 106% 4.1% 1.6% SW Matrix pH 6 (Spike to ~2 mg/L) 4 2.11 2.00 106% 0.8% 1.4% SW Matrix pH7.4 (Spike to ~2 mg/L) 4 2.06 2.00 103% 4.2% 3.0%

SW Matrix pH 8.2 (Spike to ~2 mg/L) 4 2.10 2.06 102% 1.9% 3.2% SW Matrix pH 8.8 (Spike to ~2 mg/L) 4 2.04 1.96 104% 2.4% 1.7%

Page 11 of 34Table 2: Phoenix Free Chlorine Data

Table 3: Cedar Falls Free Chlorine Data 

# PHOENIX

FREE Cl2 # of reps Hach Disposable Chips Free
Chlorine

Ave Conc Hach DPD

Free
Chlorine
Powder
Pillow Ave

Conc Free
Chlorine
Hach PPA Chip % Recovery Free
Chlorine
Hach PPA Chip % RSD (MDL For 0.2 mg/L standards) Free Chlorine Hach
Powder Pillow % RSD

(MDL For 0.2 mg/L standards)

0.2 mg/L Low Ionic Strength 7 0.23 0.21 111% 0.022 0.025 0.2 mg/L High Ionic Strength 7 0.18 0.20 90% 0.042 0.038 2 mg/L Low Ionic Strength 7 1.97 1.87 106% 3.9% 3.0% 2 mg/L-High Ionic Strength 6 1.94 1.84 106% 1.2% 1.8% GW Matrix Sample 4 0.01 0.05 16% 15.8% 54.2% GW Matrix Spike (Spike to ~2 mg/L) 4 2.00 1.90 106% 1.0% 2.0% SW Matrix Sample 4 2.02 1.89 107% 1.8% 1.5% SW Matrix Spike (Spike to ~2 mg/L) 4 1.95 1.86 105% 0.3% 0.8% GW Matrix pH 6 (Spike to ~2 mg/L) 4 0.39 0.37 106% 1.5% 2.6% GW Matrix pH 7.4 (Spike to ~2 mg/L) 4 2.03 1.91 107% 1.0% 0.6% GW Matrix pH 8.2 (Spike to ~2 mg/L) 4 1.99 1.89 105% 1.9% 0.5% GW Matrix pH 8.8 (Spike to ~2 mg/L) 4 1.99 1.91 104% 1.6% 1.6% SW Matrix pH 6 (Spike to ~2 mg/L) 4 2.05 1.98 103% 2.0% 2.6% SW Matrix pH7.4 (Spike to ~2 mg/L) 4 1.97 1.90 104% 2.7% 2.2% SW Matrix pH 8.2 (Spike to ~2 mg/L) 4 1.96 1.89 104% 7.0% 0.5% SW Matrix pH 8.8 (Spike to ~2 mg/L) 4 1.95 1.84 106% 1.5% 1.5%



# CEDAR
FALLS FREE Cl2 # of reps Hach Disposable Chips Free
Chlorine

Ave Conc Hach DPD

Free
Chlorine
Powder
Pillow Ave

Conc Free
Chlorine
Hach PPA Chip % Recovery Free
Chlorine
Hach PPA Chip % RSD (MDL For 0.2 mg/L standards) Free Chlorine Hach
Powder Pillow % RSD

(MDL For 0.2 mg/L standards)

0.2 mg/L Low Ionic Strength 7 0.18 0.21 85% 0.033 0.040 0.2 mg/L High Ionic Strength 7 0.17 0.21 81% 0.023 0.024 2 mg/L Low Ionic Strength 6 1.90 1.88 101% 1.1% 0.9% 2 mg/L-High Ionic Strength 7 1.83 1.81 101% 5.8% 5.0% GW Matrix Sample 4 0.63 0.68 94% 3.9% 0.9% GW Matrix Spike (Spike to ~2 mg/L) 4 1.95 1.91 102% 1.6% 1.2% SW Matrix Sample 4 0.00 0.03 15% 35.3% 40.0% SW Matrix Spike (Spike to ~2 mg/L) 4 1.81 1.82 99% 4.6% 2.5% GW Matrix pH 6 (Spike to ~2 mg/L) 4 1.84 1.83 101% 1.0% 0.7% GW Matrix pH 7.4 (Spike to ~2 mg/L) 4 1.86 1.84 101% 1.6% 2.8% GW Matrix pH 8.2 (Spike to ~2 mg/L) 4 1.94 1.91 101% 0.8% 0.9% GW Matrix pH 8.8 (Spike to ~2 mg/L) 4 1.90 1.89 100% 1.4% 0.7% SW Matrix pH 6 (Spike to ~2 mg/L) 4 1.93 1.85 104% 1.3% 1.8% SW Matrix pH7.4 (Spike to ~2 mg/L) 4 1.92 1.83 105% 1.1% 1.6% SW Matrix pH 8.2 (Spike to ~2 mg/L) 4 1.88 1.84 102% 1.3% 1.1% SW Matrix pH 8.8 (Spike to ~2 mg/L) 4 1.89 1.85 102% 1.7% 1.5%

Page 12 of 34Table 4: Loveland Total Chlorine Data

Table 5: Phoenix Total Chlorine Data 

# LOVELAND TOTAL Cl2 # of reps Hach Disposable Chips Total

Chlorine

Ave Conc Hach DPD
Total
Chlorine
Powder
Pillow Ave

Conc Total
Chlorine
Hach PPA

Chip % Recovery Total
Chlorine
Hach PPA Chip % RSD (MDL For 0.2 mg/L standards) Total Chlorine
Hach Powder
Pillow % RSD (MDL For 0.2 mg/L standards)

0.2 mg/L Low Ionic Strength 7 0.16 0.20 82% 0.010 0.017 0.2 mg/L High Ionic Strength 7 0.16 0.19 85% 0.015 0.022 2 mg/L Low Ionic Strength 6 1.97 2.02 98% 3.5% 1.0% 2 mg/L-High Ionic Strength 5 1.66 1.78 93% 1.8% 1.1% GW Matrix Sample 4 0.03 0.06 52% 285.5% 110.9% GW Matrix Spike (Spike to ~2 mg/L) 4 1.66 1.81 92% 2.1% 0.8% SW Matrix Sample 4 0.29 0.33 86% 9.4% 1.2% SW Matrix Spike (Spike to ~2 mg/L) 3 1.83 1.85 99% 0.9% 0.3% GW Matrix pH 6 (Spike to ~2 mg/L) 4 1.69 1.74 97% 1.8% 1.4% GW Matrix pH 7.4 (Spike to ~2 mg/L) 4 1.78 1.84 97% 5.7% 0.7% GW Matrix pH 8.2 (Spike to ~2 mg/L) 4 1.76 1.83 96% 1.4% 0.5% GW Matrix pH 8.8 (Spike to ~2 mg/L) 4 1.70 1.81 94% 1.5% 1.0% SW Matrix pH 6 (Spike to ~2 mg/L) 4 1.77 1.80 99% 1.5% 0.6% SW Matrix pH7.4 (Spike to ~2 mg/L) 4 1.74 1.76 99% 1.0% 0.7%

SW Matrix pH 8.2 (Spike to ~2 mg/L) 4 1.69 1.73 98% 4.0% 1.2% SW Matrix pH 8.8 (Spike to ~2 mg/L) 4 1.64 1.70 96% 1.6% 0.9%



# PHOENIX TOTAL Cl2 # of reps Hach Disposable Chips Total

Chlorine

Ave Conc Hach DPD
Total
Chlorine
Powder
Pillow Ave

Conc Total
Chlorine
Hach PPA

Chip % Recovery Total
Chlorine
Hach PPA Chip % RSD (MDL For 0.2 mg/L standards) Total Chlorine
Hach Powder
Pillow % RSD (MDL For 0.2 mg/L standards)

0.2 mg/L Low Ionic Strength 7 0.20 0.21 96% 0.027 0.018 0.2 mg/L High Ionic Strength 7 0.19 0.20 95% 0.023 0.012 2 mg/L Low Ionic Strength 7 1.92 1.96 98% 0.9% 0.7% 2 mg/L-High Ionic Strength 7 1.79 1.92 93% 0.9% 0.8% GW Matrix Sample 4 0.04 0.01 708% 6.3% 11.5% GW Matrix Spike (Spike to ~2 mg/L) 4 1.85 1.90 97% 0.5% 0.6% SW Matrix Sample 4 0.50 0.48 104% 0.6% 0.4% SW Matrix Spike (Spike to ~2 mg/L) 4 1.76 1.86 94% 1.6% 1.4% GW Matrix pH 6 (Spike to ~2 mg/L) 4 1.88 1.89 99% 5.1% 2.6% GW Matrix pH 7.4 (Spike to ~2 mg/L) 4 1.83 1.89 97% 0.8% 0.5% GW Matrix pH 8.2 (Spike to ~2 mg/L) 4 1.81 1.90 95% 1.4% 0.7% GW Matrix pH 8.8 (Spike to ~2 mg/L) 4 1.80 1.90 95% 0.5% 0.7% SW Matrix pH 6 (Spike to ~2 mg/L) 4 1.81 1.93 94% 2.2% 1.8% SW Matrix pH7.4 (Spike to ~2 mg/L) 4 1.75 1.89 93% 0.5% 0.8% SW Matrix pH 8.2 (Spike to ~2 mg/L) 4 1.70 1.87 91% 3.0% 0.4% SW Matrix pH 8.8 (Spike to ~2 mg/L) 4 1.69 1.84 92% 0.5% 1.6%

Page 13 of 34Table 6: Cedar Falls Total Chlorine Data 

# CEDAR

FALLS TOTAL Cl2 # of reps Hach Disposable Chips Total

Chlorine

Ave Conc Hach DPD
Total
Chlorine
Powder
Pillow Ave

Conc Total
Chlorine
Hach PPA

Chip % Recovery Total
Chlorine
Hach PPA Chip % RSD (MDL For 0.2 mg/L standards) Total Chlorine
Hach Powder
Pillow % RSD (MDL For 0.2 mg/L standards)

0.2 mg/L Low Ionic Strength 7 0.14 0.18 78% 0.035 0.099 0.2 mg/L High Ionic Strength 7 0.17 0.17 98% 0.047 0.069 2 mg/L Low Ionic Strength 7 1.62 1.93 84% 36.7% 2.8% 2 mg/L-High Ionic Strength 7 1.80 1.98 91% 3.5% 0.4% GW Matrix Sample 4 0.10 0.09 115% 1.8% 1.4% GW Matrix Spike (Spike to ~2 mg/L) 4 1.93 2.00 97% 1.4% 0.7% SW Matrix Sample 4 0.12 0.13 94% 81.5% 68.3% SW Matrix Spike (Spike to ~2 mg/L) 4 1.90 1.96 97% 1.8% 0.7% GW Matrix pH 6 (Spike to ~2 mg/L) 3 1.84 1.91 96% 1.1% 0.3% GW Matrix pH 7.4 (Spike to ~2 mg/L) 2 1.87 1.93 97% 0.4% 0.8% GW Matrix pH 8.2 (Spike to ~2 mg/L) 3 1.86 1.93 96% 2.1% 0.3% GW Matrix pH 8.8 (Spike to ~2 mg/L) 4 1.82 1.90 96% 2.0% 0.5% SW Matrix pH 6 (Spike to ~2 mg/L) 4 1.94 1.98 98% 2.3% 0.5% SW Matrix pH7.4 (Spike to ~2 mg/L) 4 1.93 1.97 98% 1.6% 0.5% SW Matrix pH 8.2 (Spike to ~2 mg/L) 4 1.91 1.98 97% 1.7% 1.9% SW Matrix pH 8.8 (Spike to ~2 mg/L) 4 1.95 2.03 96% 9.0% 6.5%

Page 14 of 34Table 7: All sites Normalized Free Chlorine Data

Table 8: All sites Normalized Total Chlorine Data Test Number of Samples % recovery for Free Cl chip MDL (mg/L) or
%RSD Free Cl2 Chip MDL (mg/L) or %
RSD Free Cl2 PP 0.2 mg/L Low Ionic Strength (MDL) 21 97% 0.019 0.019 0.2 mg/L High Ionic Strength (MDL) 21 89% 0.025 0.020 2 mg/L Low Ionic Strength 20 103% 2.30% 2.50% 2 mg/L-High Ionic Strength 20 102% 5.22% 3.38%

GW Matrix Sample 12 48% below detection
limit below detection
limit GW Matrix Spike (Spike to ~2 mg/L) 12 104% 1.27% 1.25%

SW Matrix Sample 12 75% below detection
limit below detection
limit SW Matrix Spike (Spike to ~2 mg/L) 12 103% 2.54% 2.25% GW Matrix pH 6 (Spike to ~2 mg/L) 12 105% 2.00% 1.34% GW Matrix pH 7.4 (Spike to ~2 mg/L) 12 104% 1.16% 1.86% GW Matrix pH 8.2 (Spike to ~2 mg/L) 12 103% 1.40% 2.01% GW Matrix pH 8.8 (Spike to ~2 mg/L) 12 103% 2.43% 1.24% SW Matrix pH 6 (Spike to ~2 mg/L) 12 105% 1.32% 1.80% SW Matrix pH7.4 (Spike to ~2 mg/L) 12 104% 2.69% 2.12% SW Matrix pH 8.2 (Spike to ~2 mg/L) 12 103% 3.86% 1.77% SW Matrix pH 8.8 (Spike to ~2 mg/L) 12 104% 1.72% 1.40% 

# Pooled Data (All sites normalized)

Test Number of Samples % recovery for Total Cl chip MDL (mg/L) or
%RSD Total Cl2 Chip MDL (mg/L) or %
RSD Total Cl2 PP 0.2 mg/L Low Ionic Strength (MDL) 21 85% 0.025 0.050 0.2 mg/L High Ionic Strength (MDL) 21 93% 0.033 0.040 2 mg/L Low Ionic Strength 20 98% 23.13% 2.24% 2 mg/L-High Ionic Strength 19 92% 4.15% 4.02%

GW Matrix Sample 12 290% below detection
limit below detection
limit GW Matrix Spike (Spike to ~2 mg/L) 12 95% 0.88% 0.62%

SW Matrix Sample 12 94% below detection
limit below detection
limit SW Matrix Spike (Spike to ~2 mg/L) 11 97% 1.41% 0.86% GW Matrix pH 6 (Spike to ~2 mg/L) 11 98% 1.46% 1.00% GW Matrix pH 7.4 (Spike to ~2 mg/L) 10 97% 0.69% 0.56% GW Matrix pH 8.2 (Spike to ~2 mg/L) 11 98% 1.56% 0.49% GW Matrix pH 8.8 (Spike to ~2 mg/L) 12 95% 1.43% 0.69% SW Matrix pH 6 (Spike to ~2 mg/L) 12 96% 2.00% 1.07% SW Matrix pH7.4 (Spike to ~2 mg/L) 12 97% 1.07% 0.64% SW Matrix pH 8.2 (Spike to ~2 mg/L) 12 95% 3.30% 1.19% SW Matrix pH 8.8 (Spike to ~2 mg/L) 12 95% 4.67% 3.26% 

# Pooled Data (All sites normalized)

Page 15 of 34Table 9: Monochloramine Interference

3.1.1 Calibration

In future embodiments of the system each disposable PRFC will be barcoded so that the instrument can identify the parameter, range, and calibration associated with the reagent lots . Th erefore, for this ATP study, slightly different calibration curves were used for each lot of PRFCs. The specific calibration equations for each site are listed below. Each calibration was linear from 0 -2.0 mg/L Cl 2
. Loveland Free Chlorine: Concentration=2.971*abs Loveland Total Chlorine: Concentration=3.299*abs+0.01 Phoenix Free Chlorine: Concentration=2.928*abs Phoenix Total Chlorine: Concentration=3.281*abs+0.022 Cedar Falls Free Chlorine : Concentration=2.928*abs Cedar Falls Total Chlorine : Concentration=3.281*abs+0.022

3.1.2 Initial Demonstration of Capability MDL’s were determined using 7 standards at about 0.2 mg/L Cl 2 . Precision was defined by the %RSD at about 2.0 mg/L Cl 2 . Accuracy was defined as the % recovery relative to the DPD powder pillow method. All of the data is summarized in Tables 1-8.

3.1.3 QC Controls

Due to the instability of chlorine standards QC controls were n ot used in this study.

3.1.4 Precision and Accuracy

Precision was defined by the %RSD at about 2.0 mg/L Cl 2 . Accuracy was defined as the % recovery relative to the DPD powder pillow method. All of the data is summarized in Tables 1 -8.

3.2 Holding Time/Storage Stability

A holding time study was not required for this ATP study.

4.0 Data Analysis and Discussion

All testing that was performed on the Hach Di sposable PRFCs were performed on prototype bench top equipment that incorporated the same opti cs and pumping system that will be incorporated into the commercial hand-held system. It is the goal of Hach to 

## MONOCHLORAMINE
INTERFERENCE PRFC Powder pillow
Average Monochloramine Interference (12 measeurements) 6.3% 4.0%

Page 16 of 34commercialize a hand -held system that has the capability to perform 4 simultaneous analyses using the same measurement paradigm as the breadboard instrument. Hach understands that to receive EPA approval the 4-slot han dheld system will have to undergo similar testing that was performed in this study to ensure similar analytical performance

Standard and Spike Preparation Unfortunately b ecause of the instab ilities of the chlorine standards and spikes we could not evaluate the recoveries of the disposable cuvette and powder pillow methods relative to a known spike concentration. Therefore, all re covery calculati ons were based on the recovery of the PRFCs relative the results obtained by the Hach powder pillow method (SM4500- Cl G)

. To minimize drift of the prepared spiked samples the samples were run on t he PRFC system and then immediately run using the Hach powder pillow method. Results are discussed below.

Free Chlorine MDL’s in Low and High Ionic Strength Water The MDL for the disposable cuvettes compared well against the powder pillow

tests in both low and high ionic strength waters. For the pooled data both methodologies (PRFCs and Powder pillows) obtained the same MDL (0.19 mg/L Cl 2 ) in low ionic strength water . In high ionic strength water the PRFC method obtained a pooled MDL of 0.025 mg/L and the powder pillow method obtained an MDL of 0.020 mg/L. Based on this data we consider the PRFC method equally sensitive for detecting free chlorine.

Spike Recoveries/Accuracy in Low and High Ionic Strength Water There was very good a greement betwee n the PRFC method and the Hach powder pillows for low and ionic strength water that were spiked to approximately 2 mg/L free Cl 2 . The pooled average recovery for the 2 mg/L Cl 2 spiked samples were 103% in low ionic strength and 102% in high ionic strength. Based on this data we consider the PRFC method equally accurate at measuring free chlorine in low and high ionic strength water.

Matrices The pooled avera ge spike recovery for all matrices spiked to ~ 2 mg/L free chlorine samples was 103.9% (N=120 sampl es including SW and GW with all pH adjustments ; spike recovery was calculated relative to the average powder pillow results). The %RSD for the same pooled samples was 2.1% for the PRFC tests and 1.7% for the powder pillow test (N=120 samples). Based on t his data we consider the PRFC method equally effective at recovering and detecting free chlorine in different matrices at different pH values.

Monochloramine Interference on a Free Chlorine Test The free chlorine interference from a 2.0 mg/L monochloramine standard was 4.0% for the free chlorine powder pillow and 6.3% for the PRFC’s. This difference is likely due to the powder pillow measurement being made

Page 17 of 34immediately after dissolution of the pow der and the chip measurement being made at ~30 seconds. If the powder pillow measurement was made at ~20 seconds it would be expected to have a slightly lower monochloramine interference.

Total Chlorine MDL’s in Low and High Ionic Strength Water Th e MD L for the PRFC method compared well against the powder pillow tests. For the pooled data the PRFC method obtained an MDL of 0.025 mg/L Cl 2 and the Hach powder pillow method obtained an MDL of 0.05 mg/L Cl 2 . In high ionic strength water the PRFC method obtained a pooled MDL of 0.033 mg/L and the powder pillow method obtained an MDL of 0.040 mg/L. Based on this data we consider the PRFC method equally sensitive for detecting total chlorine.

Spike Recoveries /Accuracy in Low and High Ionic Strength Water Th ere was ve ry good agreement between the PRFC method and Hach powder pillows for low and ionic strength water that were spiked to approximately 2 mg/L total Cl 2 . The pooled average recovery for the 2 mg/L Cl 2 spiked samples were 92% in low ionic strength and 93% in high ionic strength. Based on this data we consider the PRFC method equally accurate at measuring total chlorine in low and high ionic strength water.

Matrices The pooled average spike recovery for all matrix spiked 2 mg/L total chlorine sample s was 96.5% (N=114 samples included SW and GW with all pH adjustment; spike recovery was calculated relative to the average powder pillow results). The %RSD for the same pooled samples was 2.1% for the PRFC tests and 1.3% for the powder pillow test (N=114 samples). Based on this data we consider the PRFC method equally effective at recovering and detecting total chlorine in different matrices at different pH values.

5.0 Conclusions The Hach disposable cuvette method compared very well against the approved powder pillow method. We expect the analytical performance to improve significantly once t he reagents are deposited on to the disposable cuvettes with an automated manufacturing line and th e instrume nt is commercial ready and out of the prototype phase. Once the manufacturing line is commissioned and the hand-held instrume nt is in the final form factor Hach plan s on generating another set of data at a single laboratory to verify the hand- held system performance in addition to providing the EPA with a system to evaluate internally.

Page 18 of 34

### APPENDIX A. VALIDATION STUDY PLAN

The spreadsheet below describes the validation study plan that was executed at each facility.



# General testing procedure for Hach

c hips (P rovided to the testing labs )

Overview: This is intended to be a guide for operation of the PPA instrument for outside technical personnel. The detailed test plan is covered in a separate document. This Hach DPD
Free Chlorine
Powder
Pillow Hach DPD
Total Chlorine
Powder
Pillow Hach Disposable Chips Free
Chlorine Hach Disposable Chips Total

Chlorine 0.2 mg/L Low Ionic Strength 7 7 7 7 0.2 mg/L High Ionic Strength 7 7 7 7

2 mg/L Low Ionic Strength 7 7 7 7 2 mg/L-High Ionic Strength 7 7 7 7 GW Matrix Sample 4 4 4 4 GW Matrix Spike (Spike to ~2 mg/L) 4 4 4 4 SW Matrix Sample 4 4 4 4 SW Matrix Spike (Spike to ~2 mg/L) 4 4 4 4

Monochlormaine Interference at 2 mg/L NH2Cl 4 4 4 4 GW Matrix pH 6 (Spike to ~2 mg/L) 4 4 4 4 GW Matrix pH 7.4 (Spike to ~2 mg/L) 4 4 4 4 GW Matrix pH 8.2 (Spike to ~2 mg/L) 4 4 4 4 GW Matrix pH 8.8 (Spike to ~2 mg/L) 4 4 4 4 SW Matrix pH 6 (Spike to ~2 mg/L) 4 4 4 4

SW Matrix pH7.4 (Spike to ~2 mg/L) 4 4 4 4

SW Matrix pH 8.2 (Spike to ~2 mg/L) 4 4 4 4 SW Matrix pH 8.8 (Spike to ~2 mg/L) 4 4 4 4
Total # of Tests 64 64 64 64

Page 19 of 34contains information that may be considered “helpful hints” on standard handling and instrument operation. 

### Background: Drinking water is purified in the US by a series of steps that involve mechanical (settling) means. Water is chemically decontaminated by one of two methods: direct chlorination or reaction with monochloramine. The tests for the two primary residuals (Cl 2 or NH 2 Cl) are termed “free” and “total”, and the sum of the two methods is termed

“ free and total ”. Hach “powder pillow” test methods (Hach method 8021) are the standard for most drinking water systems in the US, so the new PPA chip method (Hach method 10260) results will be compared to the powder pillow method results. As a rule during the testing procedure, samples should be tested by both methods whenever possible and as tempora lly close together as possible. 

### Handling of standards: Aqueous chlorine standards are inherently unstable. In aqueous solutions chlorine gas is in equilibrium with hypochlorous acid and its conjugate base hypochlorite. When the solution is left open to the atmosphere, chlorine gas will leave the solution, causing a downward drift in the concentration. Thus, chlorine solutions should be made as close to measurement time as possible and kept tightly covered. Samples should be read on the instrument (aka “bre adboard”), then immediately read with a powder pillow. Tap water has residual chlorine/monochloramine content when it comes out of the faucet. Like all chlorine solutions, the chlorine content of tap water goes down with time. Time in pipes will also caus e the chlorine content to drop; therefore when a draw of tap water is needed, the tap water should be run full open for at least five minutes before a sample is taken to insure consistency between runs. Additionally, large volumes of water (>1000mL) should be used. The bulk of samples this size (and the increased volume/surface area ratio) helps keep the chlorine concentration relatively constant over the life of the experiment. Gloves should be used at all times when hands will be in contact with the solutions or with the chips, as skin oils have a chlorine demand. 

### Spiking calculations: Spiking solutions requires calculations based on the initial concentration of the standard solution. Each batch of standard solution will be different and the exact

concen tration of the standard is listed on the vial label. This is the concentration that should be used in calculations. The most basic equation for calculating the volume needed of a concentrated standard to reach a particular end concentration of a diluted

st andard is M 1 V 1 = M 2 V 2 where M 1 = concentration of the concentrated standard (in mg/L), M 2 = concentration of the diluted solution (the solution being spiked)(in mg/L), V 1 = volume of the concentrated standard (in mL), and V 2 = the target concentration of the spiked solution (in mL). The equation is rearranged to isolate the volume of the concentrated standard (that which will be measured and added to the solution being spiked): V 1 = M 2 V 2 / M 1 So to spike a 10 mL DI water sample with a 0.2 mg/L spike using a 65 mg/L standard, the equation will read:

Page 20 of 34V 1 = (0.2 mg/L)(10 mL) / (65 mg/L) = 0.031 mL = spiking volume It is important to remember that drinking water samples will have some residual chlorine. The residual chlorine must be taken into account whe n calculating the additional chlorine needed. For example, Loveland, CO tap water has 0.4 mg/L Cl 2 residual. Thus to spike to 1.75 mg/L requires only an additional 1.35 mg/L Cl 2 from the spike. Before testing on chips, run a powder pillow test using the c alculated spike to be sure that the spike will give the desired concentration (within 0.05 mg/L). It has been observed that occasionally the calculated volume does not give the target spike; this may be due to a release of chlorine immediately upon opening the ampule. If the initial test does not give the desired concentration, adjust the spiking volume. After the chlorine standard is opened it will immediately start to lose chlorine. Therefore, on

- the -fly adjustments to the volume are required; as a rule of thumb, if the chlorine content is 10% low on a run, the spike should be increased for the next run. 

### Spiking and Data Acquisition Procedure: General procedure: Obtain ~1000-1500mL of the unspiked water to be tested (e.g. distilled (low ionic strength) water, high ionic strength water, surface water, ground water, pH adjusted water) and place it in a large beaker between the two breadboards. Have a pipetteman (and fresh tip for each water type), co lorimeter vial and cap, small plastic sample cup, and Excel spreadsheet ready. 1) Withdraw 10.0 mL unspiked water from the beaker with the pipetteman and place it in the colorimeter vial. 2) Using good laboratory practices open the chlorine standard ampule (or in the case of total chlorine the monochloramine standard). After each withdrawal cap the ampule with parafilm, taking care to avoid getting cut by the sharp edges of the ampule. 3) With a micropipetteman withdraw the calculated amount of chlorine and add
it to the unspiked water. Cap the vial and shake vigorously for 2-3 seconds.

a. For the first run, it is advisable to get a reading of chlorine content using a powder pillow to be sure that the chlorine content from the spike is correct.
b. If the powder pillow reading show that the chlorine content of the spikes solution is correct, wash the vial 3x with DI water and restart the procedure, moving on to the next step. 4) Check to make sure that the instrument is reading “present sample”. 5) Pour the contents of the vial into the sample cup. 6) Lift the cup up to the chip until the chip is touching the bottom of the cup.

a. The instrument will read “remove sample” 7) Remove the sample. 8) Repeat the presentation of the sample on the second instrument. 9) Pour the sample back into the vi al. 10) Set a zero for the pocket colorimeter by placing the vial in the colorimeter, returning the cover, and pressing the “zero” button (on the left).

Page 21 of 3411) Open the appropriate powder pillow (free or total chlorine) (see below for procedure for using the powder pillow) and add it to the vial. Cap and mix the
vial. 12) After the prescribed wait period (30 seconds for Free chlorine, 3 min for Total chlorine) take a measurement using the pocket colorimeter. Be sure that the white diamond on the vial is facing you. 13) Rinse the vial 3x with DI water. Use the same vial for all measurements.


### Using the instrument (breadboard) : The PPA instrument is intended to be straightforward to use. There are only a few steps: opening the chip bag, preparing the instrument, placing the chip in the slot, presenting the sample to the chip, removing the sample from the chip, and reading the output. Here are a few pointers for the operation of the instrument: 1) Opening the chip bag. Wear gloves whenever handling or preparing to handle chips. To avoid cutting either the chip or the bag of desiccant, be sure to push the solids inside the bag to one side. Using a pair of scissors, make a cut lengthwise along the opposite side. Cutting the chip will compromise its integrity; cutting the desiccant bag s imply makes a mess. 2) Preparing the instrument. Using the dropdown menu, select the parameter to be tested (Free Chlorine, Total Chlorine, or Free and Total). Several fields need to be entered before or during the measurement.

a. Before the measurement
i. Operator
ii. Chip lot-this is found on the outside of the bag, and should start
with either V2A or C2A
iii. Chip number-this is found on the back of the chip and should have a letter (A -J) and a number (1- 22).
iv. Test type - this is entered in the comment field, and should be the name of the test as indicated on the test plan spreadsheet (e.g. Matrix spike, 0.5ppm).
b. During the measurement
i. Powder pillow reading.
ii. It is critical that the p owder pillow reading be entered in the EPA for PPA software for each chip before hitting “O K” to accept the reading for the chip. 3) Placing the chip in the slot. The instrument should read “Insert chip”. At the bottom of the instrument, find two parallel metal rods protruding from the box. The back of the chip (the side with the ridge and the chip number) will be placed against these rods. Push the chip up into the instrument until it fits snugly in the instrument-a soft click can be heard when it is properly seated. At the bottom of

Page 22 of 34the chip is an angle that should have its lowest end on the right side of the chip. Once the chip is inserted, click on the “OK” button. 4) Presenting the sample to the chip. Once the chip is installed the instrument will read “present sample”. At this time the sample (prepared just previously) should be placed in a small cup. The cup (with sample) is lifted to cover the entirety of the angled portion of the bottom of the chip. 5) Removing the sample from the chip. When the instrument has drawn the sample, it will read “remove sample” and the cup should be withdrawn immediately. The instrument will not begin reading until the sample has been removed. At this point the sample should be transferred to a vial. The appropriate powder pillow is added and a measurement taken on the pocket colorimeter for comparison.

After the breadboard reading is complete, a message saying “enter comment and remove chip” will appear. 6) Reading the output. The concentration will appear in large numbers. This should be recorded in the excel spreadsheet, along with the associated powder pillow
value for that test. 7) Removing the chip. Pull down on the chip and it will dislodge. Be sure to pull straight down. Once the chip is out, place it in the used chips bag to be returned to
Hach. 8) Wipe the metal rods dry with a clean laboratory paper towel, such as a K imwipe.



### Powder Pillows: Hach method 8021 consists of foil pouches containing all the reagents for a given test, a cuvette, and a colorimeter. The procedure for using the powder pillows is straightforward. First obtain 10.0 mL of sample water and place it in the cuvette. Check the powder pillow to make sure it is the appropriate test (e.g. total chlorine or free chlorine), then hold the pouch by the top (crinkled edge) and flick it to make sure the contents are at the bottom of the pillow. Cut the top off the pillow close to the crinkled edge and push the two sides together to form a pouring spout (this is called pooching the pouch). Pour the contents into the cuvette, attach the cover to the cuvette and swirl vigorously for approximately 10 seconds, or un til all the solid is dissolved. If a sonicator is available, a 4-5 second sonication will remove bubbles and give more reliable readings. For the FREE CHLORINE test, wipe the vial with a clean towel/Kimwipe and measure the color in the colorimeter between 30 and 60 seconds after the pillow contents have been added. For the TOTAL CHLORINE test, wipe the vial with a clean towel/Kimwipe, and take a measurement 3 minutes after the pillow contents have been added. Be sure that the reading has stabilized by taking three measurements. If the reading doesn’t change, write the reading in the notebook. If the reading changes, continue to read until three identical readings are obtained.

Page 23 of 34

# Detailed Testing S chedule (Provided to the Laboratories)

Monday PM: Begin “Free Chlorine” testing A. Low ionic strength standards, high and low spike:
1. Obtain ~500 mL distilled or deionized water (DI water).
2. Prepare two instruments. Instructions can be found in the document “Testing Procedure for Hach chips”.

a. The basic installation of the instrument will be performed by the Hach representative.
b. Sample to sample preparation of the instrument involves inserting a new chip and entering data in the required fields: operator, chip lot number, chip ID, and test type (e.g. Low ionic, 1.9p pm).

c. In the dropdown menu of Test Types in the upper left of the screen (Free, Total, or Free + Total), select “ Free ”.
d. One breadboard should be marked C2A and the other V2A. Chips with the formulation C2A will be run exclusively on the C2A breadboard, and V2A chips on the V2A breadboard.
i. Chip formulations are marked on the white label on the outside of the chip bag.
ii. Note that in the Excel spreadsheet there are separate entries for C2A and V2A chips.
3. Calculate the needed volume of high concentration (60-75 p pm) Cl 2 (in ampoules provided by Hach) using the equation:

M 1 V 1 = M 2 V 2 V 1 = M 2 V 2 / M 1 V 1 = ( 0.2 mg/L )(10 mL) / (65 mg/L) = 0.031 mL = spiking volume (typically the practical value of the needed volume is 38 -40 uL) V 1 = ( 1.9 mg/L )( 10 mL) / (65 mg/L) = 0.292 mL = spiking volume (typically the practical value of the needed volume is ~340- 350 uL)

4. Check the validity of the calculation by spiking the calculated volume into 10.0 mL DI water, then performing a powder pillow test. Adjust the spike volume according to the results of the powder pillow test.
5. Repeat step 4 until the desired spike concentration is attained (0.2 mg/L or 1.9 mg/L)(concentrations should be ±10% of target value; record exact concentrations measured).

Page 24 of 346. Pipette 10.0 mL of DI water into the cuvette. Spike with the volume of concentrated Cl2 determined in Step 4. Cap and shake vigorously for 3
seconds.
7. Pour the contents into a plastic sample cup.
8. Present the sample to the instruments (C2A and V2A) while the instruments are reading “present sample”. This is done by lifting the sample cup from underneath the chip until the instrument beeps and reads “remove sample”. Repeat the sample presentation on the second breadboard.
9. Pour the contents of the sample from the cup back into the cuvette.
10. Add the appropriate powder pillow (Free chlorine), cap and shake for 5
seconds.
11. If a sonication bath is available, immerse the cuvette into the bath deep enough to cover the sample but not the cover.
12. Wipe the cuvette clean wi th a laboratory towel (e.g. Kimwipe).
13. Read the concentration using the pocket colorimeter; the reading must be taken within 60 seconds (preferably 30 seconds) of addition of the powder to the sample. Record the value in both the Excel spreadsheet and the instrument (in the box marked “powder pillow value”)
14. Wash the cuvette 3x with DI water.
15. When the instrument reads “enter comment and remove chip” pull the chip out by pulling straight down on the end of the chip.
16. Wipe the metal posts dry with a paper towel (i.e. Kimwipe).
17. Repeat this process to acquire seven 0.2 mg/L data points and seven 1.9 mg/L data points using DI water as the matrix.
18. If the instrument reads “Bad Chip”, repeat the experiment with a new chip. Chips marked “bad chip” do NOT count toward th e data point total.

B. Ground water matrix (procedure will be repeated for surface water matrix)
1. Obtain a 1000-1500 mL sample of ground water.
2. Prepare the instrument as previously.
3. Measure 10.0 mL of ground water into a plastic sample cup.
4. Follow steps 8-18 above.
5. Acquire four data points by repeating the above procedure.
6. When complete cover the water sample with Parafilm for use Tuesday AM for matrix spiking.

C. Prepare high ioni c strength water for Tuesday.

1. To a 1 L volumetric flask add 1.40 g of NaHCO 3 , 1.48 g Na 2 SO 4 , and 1.54 g NaCl. Dilute to volume with deionized water. Prepare 4L of high ionic strength water

.

D. At the end of Monday there should be 18 entries in the Excel spreadsheet.

Page 25 of 34Tuesday AM: A. High ionic strength standards, high and low spike:
1. Obtain ~500 mL high ionic strength water.
2. Prepare two instruments:

a. Sample to sample preparation of the instrument involves inserting a new chip and entering the required fields: operator, chip lot, chip ID, and test type. 3. Calculate the needed volume of high concen tration (60 -75 ppm) Cl 2 (in ampoules provided by Hach using the equation:

M 1 V 1 = M 2 V 2 V 1 = M 2 V 2 / M 1 V 1 = ( 0.2 mg/L )(10 mL) / (65 mg/L) = 0.031 mL = spiking volume (typically the practical value of the needed volume is 38- 40 uL) V 1 = ( 1.9 mg/L )( 10 mL) / (65 mg/L) = 0.292 mL = spiking volume (typically the practical value of the needed volume is ~340-350 uL)

4. Check the validity of the calculation by spiking the calculated volume into 10.0 mL high ionic strength water, then performing a powder pillow test. Adjust the spike volume according to the results of the powder pillow test.
5. Repeat step 4 until the desired spike concentration is attained (0.2 mg/L or 1.9 mg/L) (concentrations should be ±10% of target value; record exact
concentrations measured ).
6. Pipette 10.0 mL of high ionic strength water into the cuvette. Spike with the
volume of concentrated Cl 2 determined in Step 4. Cap and shake vigorously
for 3 seconds.
7. Pour the contents into a plastic sample cup.
8. Present the sample to the instruments ( C2A and V2A) while the instruments are reading “present sample”. This is done by lifting the sample cup from underneath the chip until the instrument beeps and reads “remove sample”.
9. Pour the contents of the sample from the cup back into the cuvette.
10. Add the appropriate powder pillow (Free chlorine), cap and shake for 5
seconds.
11. If a sonication bath is available, immerse the cuvette into the bath deep enough to cover the sample but not the cover.
12. Wipe the cuvette clean with a laboratory towel (e.g. Kimwipe ).
13. Read the concentration using the pocket colorimeter; the reading must be taken within 60 seconds (preferably 30 seconds) of addition of the powder to the sample. Record the value in both the Excel spreadsheet and the instrument (in the box marked “powder pillow value”)
14. Wash the cuvette 3x with DI water.

Page 26 of 3415. When the instrument reads “enter comment and remove chip” pull the chip out by pulling straight down on the end of the chip.
16. Wipe the metal posts dry with a paper towel (i.e. Kimwipe).
17. Repeat this process to acquire seven 0.2 mg/L data points and seven 1.9 mg/L data points using high ionic strength water as the matrix.
18. If the instrument reads “Bad Chip”, repeat the experiment with a new chip. Chips marked “bad chip” do NOT count toward the data point total.

B. Ground water 1.9 mg/L spike
1. Repeat the above procedure substituting ground water for high ionic strength

water.

Tuesday P M: A. Ground water pH study
1. Obtain 1500 mL of ground water (the sample used earlier can be reused).
2. Measure the pH.
3. Determine if the p H is closer to 6.0 or 8.8.

a. If the pH is closer to 6.0, adjust the full 1500 mL to pH 6.0 using the 0.1 M HCl. Be sure to have active stirring during the pH adjustment.
b. If the pH is closer to 8.8, adjust the full 1500 mL to pH 8.8 using the 0.1 M NaOH. Be sure to have active stirring during the pH adjustment.
4. Once the pH has been adjusted withdraw a 10.0 mL sample and place it in the

cuvette. 5. Spike the sample with concentrated Cl 2 (as done previously) to 1.9 mg/L. Cover the cuvette and shake the sample.
6. Pou r the sample into the plastic sample cup.
7. Present the sample to each breadboard.
8. Replace the sample into the cuvette, add a Free chlorine powder pillow, and read the concentration using the pocket colorimeter.
9. Rinse the cuvette 3x with DI water.
10. Record the powder pillow data in the instrument and in the Excel spreadsheet (along with the breadboard data).
11. Collect 4 data points at the pH 6.0/8.8 point by repeating steps 5 through 11.
12. Adjust the pH to the next closest target pH on the Excel spreadsheet.

a. If the solution pH is at 6.0, add 0.1 M NaOH to raise the pH to 7.4.
b. If the solution pH is at 8.8, add 0.1 M HCl to lower the pH to 8.2.
13. Repeat steps 5 -11 to acquire four data points at the new pH.
14. Continue to adjust the pH until all four pH’s (6.0, 7.4, 8.2, and 8.8) have four data points read.

B. Surface water matrix

Page 27 of 341. Follow the same procedure as for ground water (Monday PM, part B).
2. Allow the tap to run for at least 5 minutes before starting to fill the sample
container.
3. Acquire four data points for the surface w ater matrix.

Wednesday AM: A. Surface water spike
1. Obtain 1500 mL of fresh surface tap water, being sure to run the tap for 5 minutes before collecting the sample.
2. Spike with concentrated Cl 2 (as done earlier) to a concentration of 1.9 mg/L.
Be sure to account for the Cl 2 concentration found in the tap water.

a. For example, Loveland, CO tap water has 0.4 mg/L Cl 2 . Thus, to reach 1.9 mg/L requires only a 1.5 mg/L spike.
3. Obtain breadboard and powder pillow readings as done earlier: spike the sample, transfer to the sample cup, present to breadboard C2A, present to breadboard V2A, run powder pillow test, record data on breadboard and spreadsheet.
B. Surface water pH study.
1. Follow the procedure for the ground water pH study, substituting surface (tap) water for the ground water. As with the spike above, the starting Cl 2 concentration will need accounting.
2. Four data points will need to be acquired for each of the 4 pH solutions (6.0, 7.4, 8.2, and 8.8).

At the completion of the surface water pH study, the entire “Free Chlorine” sheet in the Excel spreadsheet should be completed. Wednesday PM : Begin “Total Chlorine” testing A. Low ionic strength standards, high and low spike:
1. Prepare a high concentration monochloramine standard.

a. Gather a 1000 mL beaker, 1.0 mL bleach, 60 mg NH 4 Cl,and 0.141 mL
0.1M NaOH .
b. Fill the beaker to the 1000 mL mark with DI H 2 O.

c. Add the NH 4 Cl and stir 2 minutes after all solid has dissolved.
d. Add the NaOH.

e. Add 1.0 mL of bleach over 5 minutes, with vigorous stirring.
f. Allow the solution to stir for 30 minutes.

g. This solution will have a concentration of ~50 mg/L. The exact concentration will vary slightly depending on the exact concentration
of the bleach.
2. Obtain ~500 mL distilled or deionized water (DI water).
3. Prepare two instruments. Instructions can be found in the document “Testing Procedure for Hach chips”. As a review,

Page 28 of 34a. In the dropdown menu of Test Types in the upper left of the screen (Free, Total, or Free + Total), select “ Tota l ”.
b. Sample to sample preparation of the instrument involves inserting a new chip and entering data in the required fields: operator, chip lot number, chip ID, and test type (e.g. Low ionic, 1.9ppm).

c. One breadboard should be marked C2A and the other V2A. Ch ips with the formulation C2A will be run exclusively on the C2A breadboard, and V2A chips on the V2A breadboard.
ii. Chip formulations are marked on the white label on the outside of the chip bag.
iii. Note that in the Excel spreadsheet there are separate entries f or C2A and V2A chips.
4. Calculate the needed volume of high concentration monochloramine standard made in step 1. The concentration of the standard will be approximately 50 mg/L.

M 1 V 1 = M 2 V 2 V 1 = M 2 V 2 / M 1 V 1 = ( 0.2 mg/L )(10 mL) / (50 mg/L) = 0.040 mL = spiking volume V 1 = ( 1.9 mg/L )(10 mL) / (50 mg/L) = 0.380 mL = spiking volume

5. Check the validity of the calculation by spiking the calculated volume into 10.0 mL DI water, then performing a powder pillow test with a Total chlorine powder pillow (note that t he Total powder pillow requires 3 minutes wait before measuring). Adjust the spike volume according to the results of the powder pillow

test.
6. Repeat step 5 until the desired spike concentration is attained (0.2 mg/L or 1.9 mg/L) (concentrations should be ± 10% of target value; record exact concentrations measured).
7. Pipette 10.0 mL of DI water into the cuvette. Spike with the volume of concentration monochloramine determined in Step 4. Cap and shake vigorously
for 3 seconds.
8. Pour the contents into a plastic sample cup.
9. Present the sample to the instruments (C2A and V2A) while the instrument is reading “present sample”. This is done by lifting the sample cup from underneath the chip until the instrument beeps and reads “remove sample”. Repeat the sample presentation on the second breadboard.
10. Pour the contents of the sample from the cup back into the cuvette.
11. Add the appropriate powder pillow (Total chlorine), cap and shake for 5 seconds.
12. If a sonication bath is available, immerse the cuvette into the bath deep enough to cover the sample but not the cover.
13. Wipe the cuvette clean with a laboratory towel (e.g. Kimwipe).

Page 29 of 3414. Read the concentration using the pocket colorimeter; the reading must be taken 3 minutes after addition of the powder to the sample. Record the value in both the Excel spreadsheet and the instrument (in the box marked “powder pillow value”)
15. Wash the cuvette 3x with DI water.
16. When the instrument reads “enter comment and remove chip” pull the chip out by pulling straight down on the end of the chip.
17. Wipe the metal posts dry with a paper towel (i.e. Kimwipe).
18. Repeat this process to acquire seven 0.2 mg/L data points and seven 1.9 mg/L data points using DI water as the matrix.
19. If the instrument reads “Bad Chip”, repeat the experiment with a new chip. Chips marked “bad chip” do NOT count toward the data point total.

E. Ground water matrix (procedure will be repeated for surface water matrix)
1. Obtain a 1000-1500 mL sample of ground water.
2. Prepare the instruments as previously.
3. Measure 10.0 mL of ground wa ter into a plastic sample cup.
4. Follow steps 7-18 above.
5. Acquire four data points by repeating the above procedure.
6. When complete cover the water sample with Parafilm for use Thursday AM for matrix spiking.

F. At the end of Wednesday there should be 18 entrie s in the Excel “Total” spreadsheet, and the “Free” spreadsheet should be completed.

T hursday AM: High ionic strength water, low and high spikes: 1. Obtain ~500 mL high ionic strength water.
2. Prepare two instruments:
b. Sample to sample preparation of the instrument involves inserting a new chip and entering the required fields: operator, chip lot, chip ID, and test type. 3. Calculate the needed volume of high concentration monochloramine standard (prepared Wednesday afternoon, approx. 50 mg/mL) using the equation:

M 1 V 1 = M 2 V 2 V 1 = M 2 V 2 / M 1 V 1 = ( 0.2 mg/L )(10 mL) / (50 mg/L) = 0.040 mL = spiking volume V 1 = ( 1.9 mg/L )(10 mL) / (50 mg/L) = 0.380 mL = spiking volume

4. Check the validity of the calculation by spiking the calculated volume into 10.0 mL high ionic strength water, then performing a powder pillow test. Adjust the spike volume according to the results of the powder pillow test.

Page 30 of 345. Repeat step 4 until the desired spike concentration is attained (0.2 mg/L or 1.9 mg/L) (concentrations should be ±10% of targe t value; record exact concentrations measured).
6. Pipette 10.0 mL of high ionic strength water into the cuvette. Spike with the volume of monochloramine standard determined in Step 4. Cap and shake vigorously for 3 seconds.
7. Pour the contents into a plastic sample cup.
8. Present the sample to the instruments (both C2A and V2A) while the instrument is reading “present sample”. This is done by lifting the sample cup from underneath the chip until the instrument beeps and reads “remove sample”.
9. Pour the contents of the sample from the cup back into the cuvette.
10. Add the appropriate powder pillow (Total chlorine), cap and shake for 5
seconds.
11. If a sonication bath is available, immerse the cuvette into the bath deep enough to cover the sample but not the cover.
12. Wipe the cuvette clean with a laboratory towel (e.g. Kimwipe).
13. Read the concentration using the pocket colorimeter; the reading must be taken 3 minutes after addition of the powder to the sample. Record the value in both the Excel spreadsheet and the instrument (in the box marked “powder pillow value”)
14. Wash the cuvette 3x with DI water.
15. When the instrument reads “enter comment and remove chip” pull the chip out by pulling straight down on the end of the chip.
16. Wipe the metal posts dry with a paper towel (i.e. Kimwipe).
17. Repeat this process to acquire seven 0.2 mg/L data points and seven 1.9 mg/L data points using high ionic strength water as the matrix.
18. If the instrument reads “Bad Chip”, repeat the experiment with a new chip. Chips marked “bad chip” do NOT coun t toward the data point total.

C. Ground water 1.9 mg/L spike
1. Repeat the above procedure substituting ground water for high ionic strength

water. 2. Acquire four data points, using a fresh spiked sample for each reading.

T hursday P M: A. Ground water pH study
1. Obta in 1500mL of ground water (the sample used earlier can be reused).
2. Measure the pH.
3. Determine if the pH is closer to 6.0 or 8.8.

Page 31 of 34d. If the pH is closer to 6.0, adjust the full 1500 mL to pH 6.0 using the 0.1 M HCl. Be sure to have active stirring during the pH adjustment.

e. If the pH is closer to 8.8, adjust the full 1500 mL to pH 8.8 using the 0.1 M NaOH. Be sure to have active stirring during the pH adjustment.
4. Once the pH has been adjusted withdraw a 10.0 mL sample and place it in the

cuvette. 5. Spike the sample with monochloramine (as done previously) to 1.9 mg/L. Cover the cuvette and shake the sample.
6. Pour the sample into the plastic sample cup.
7. Obtain breadboard and powder pillow readings as done earlier: spike the sample, transfer to the sample cup, present to breadboard C2A, present to breadboard V2A, run powder pillow test, record data on breadboard and spreadsheet.
8. Replace the sample into the cuvette, add a Total chlorine powder pillow, and read the concentration using the pocket colorimeter.
9. Rinse the cuv ette 3x with DI water.
10. Record the powder pillow data in the instrument and in the Excel spreadsheet (along with the breadboard data).
11. Collect 4 data points at the pH 6.0/8.8 point by repeating steps 4 through 11.
12. Adjust the pH to the next closest target pH on the Excel spreadsheet.

a. If the solution pH is at 6.0, add 0.1 M NaOH to raise the pH to 7.4.
b. If the solution pH is at 8.8, add 0.1 M HCl to lower the pH to 8.2.
13. Repeat steps 4 -11 to acquire four data points at the new pH.
14. Continue to adjust the pH until all four pH’s (6.0, 7.4, 8.2, and 8.8) have four data points read.

Surface water matrix
1. Follow the same procedure as for ground water (Monday PM, part B), substituting the monochloramine standard for the concentrated chlorine.

2. Allow the tap to run for at least 5 minutes before starting to fill the sample
container.

3. Acquire four data points for the surface water matrix.

Page 32 of 34Friday AM: A. Surface water spike
1. Obtain 1500 mL of fresh surface tap water, being sure to run the tap for 5 minutes before collecting the sample.
2. Spike with monochloramine standard (as done earlier) to a concentration of 1.9 mg/L (concentrations should be ±10% of target value; record exact concentrations measured). Be sure to account for the Cl 2 concentration found in the tap water. For exa mple, Loveland, CO tap water has 0.4 mg/L Cl 2
. Thus, to reach 1.9 mg/L requires only a 1.5 mg/L spike.
3. Obtain breadboard and powder pillow readings as done earlier: spike the sample, transfer to the sample cup, present to breadboard C2A, present to breadboard V2A, run powder pillow test, record data on breadboard and spreadsheet.
4. Surface water pH study.
5. Follow the procedure for the ground water pH study, substituting surface (tap) water for the ground water. As with the spike above, the starting Cl 2 concent ration will need accounting.
6. Four data points will need to be acquired for each of the 4 pH solutions (6.0, 7.4, 8.2, and 8.8).

B. Monochloramine interference:
1. In the dropdown menu of Test Types in the upper left of the screen (Free, Total, or Free + Total), select “ Free + Total ”.
2. To a 10.0 mL sample of DI water, add enough monochloramine standard to reach a concentration of 1.9 mg/mL (concentrations should be ±10% of target value; record exact concentrations measured).
3. Present the sample and acquire four dat a points as done earlier.

At the completion of the surface water pH study, the entire “Free Chlorine” sheet in the Excel spreadsheet should be completed.

Page 33 of 34

### APPENDIX B. SUPPORTING DATA

B.1 Raw Data All raw data is attached in the 3 files (Loveland data.xlsx, Phoenix data.xlsx, and Cedar Falls data.xlsx). Within each file the raw data f ro m the instrum ent can be found.

B.2 Example Calculations Sample concentrations were determined by the PRFC system and by the pocket colorimeters. There was no need to calculate the concentrations of the samples.

B.3 Waveforms for a typical set of total chlorine data at 2.0 mg/L

Page 34 of 34