327.0-1

METHOD 327.0 DETERMINATION OF CHLORINE DIOXIDE AND CHLORITE ION 

IN DRINKING WATER USING LISSAMINE GREEN B AND 

HORSERADISH PEROXIDASE WITH DETECTION BY 

VISIBLE SPECTROPHOTOMETRY

EPA 815-R-05-008

Revision 1.1

May 2005

Teri A. Dattilio and Barry V. Pepich, Shaw Environmental, Inc.

David J. Munch and Patricia S. Fair, US EPA, Office of Ground Water and

Drinking Water

Zsolt Körtvélyesi and Gilbert Gordon, Miami University

TECHNICAL SUPPORT CENTER

OFFICE OF GROUND WATER AND DRINKING WATER

U. S. ENVIRONMENTAL PROTECTION AGENCY

CINCINNATI, OHIO 45268
327.0-2

METHOD 327.0

DETERMINATION OF CHLORINE DIOXIDE AND CHLORITE ION IN DRINKING WATER

USING LISSAMINE GREEN B AND HORSERADISH PEROXIDASE WITH DETECTION 

BY VISIBLE SPECTROPHOTOMETRY

1. SCOPE AND APPLICATION

1.1 This is a spectrophotometric method for the analysis of chlorine dioxide and chlorite* in

finished drinking waters. It is primarily intended to be used at drinking water utilities in

conjunction with daily monitoring requirements.

1.2 The experimentally determined Detection Limits (DLs) for chlorine dioxide and chlorite

are provided in Section 17, Table 1. The DL is defined as the statistically calculated

minimum concentration that can be measured with 99% confidence that the reported

value is greater than zero. Detection Limits are technique and instrument dependent. (1)

The DL differs from, and is lower than, the Minimum Reporting Level (MRL) 

(Sect. 3.10). Method performance was demonstrated over a combined concentration

range of 0.2 mg/L to 2.2 mg/L. Precision and accuracy data are presented in Section 17,

Tables 1-3.

1.3 Chlorine dioxide is a volatile, reactive gas. Care was taken during method development

to minimize steps that could result in the loss of chlorine dioxide during analysis. 

Analyst technique can affect method precision and accuracy.

2. SUMMARY OF METHOD

2.1 A drinking water sample is collected headspace-free in a 16-mL amber glass vial. A

second sample is collected and sparged with an inert gas to remove all traces of chlorine

dioxide and then transferred to a second 16-mL amber vial. A third vial is filled with

reagent water. A 1.0-mL aliquot of water is removed from each vial, and a 1.0-mL

aliquot of a concentrated citric acid buffer containing glycine is added. The samples are

capped and gently mixed, and then a second 1.0-mL aliquot is removed and a 1.0-mL

aliquot of a Lissamine Green B (LGB)/Horseradish Peroxidase (HRP) reagent is added. 

The HRP catalyzes the conversion of chlorite to chlorine dioxide. Chlorine dioxide (2,3)

rapidly oxidizes the LGB, reducing its absorption in the red region of the visible spectrum

in proportion to the chlorine dioxide concentration. A visible spectrophotometer is

(4,5)

used to measure the absorbance of the reagent water blank and sample absorbance at 

633 nm, which is the absorbance maximum for LGB in the citric acid/glycine buffer. The

absorbance difference between the reagent water blank and the samples is used to

calculate the concentrations of chlorine dioxide, using an external standard calibration

____________________________

*Throughout this document chlorite ion (ClO2) is referred to as ‘chlorite’ for simplicity, even though the –

use of ‘chlorite ion’ is more accurate. 
327.0-3

curve, determined using chlorite standards. The unsparged sample is used to determine

the total chlorite and chlorine dioxide concentration and the sparged sample is used to

determine the chlorite concentration. The chlorine dioxide concentration is the difference

between these two values.

3. DEFINITIONS

3.1 ANALYSIS BATCH – A set of samples that is processed and analyzed together and

includes no more than 10 Field Samples. An analysis batch must also include at least one

Method Blank and all required QC samples. The Method Blanks and QC samples do not

contribute to the maximum Field Sample total of 10.

3.2 CALIBRATION STANDARD (CAL) – A solution prepared from the primary dilution

standard solution and/or stock standard solution. The CAL solutions are used to calibrate

the instrument response with respect to analyte concentration.

3.3 CONTINUING CALIBRATION CHECK (CCC) – A calibration standard containing the

method analyte(s), which is analyzed periodically to verify the accuracy of the existing

calibration for those analytes.

3.4 DETECTION LIMIT (DL) – The minimum concentration of an analyte that can be

identified, measured, and reported with 99% confidence that the analyte concentration is

greater than zero. This is a statistical determination of precision (Sect. 9.2.3), and

accurate quantitation is not expected at this level.(1)

3.5 FIELD DUPLICATES (FD1 and FD2) – Two separate samples collected at the same time

and place under identical circumstances and treated exactly the same throughout field and

laboratory procedures. Analyses of FD1 and FD2 give a measure of the precision

associated with sample collection and storage, as well as laboratory procedures.

3.6 LABORATORY FORTIFIED SAMPLE MATRIX (LFSM) – An aliquot of an environmental sample to which known quantities of the method analytes are added in the

laboratory. The LFSM is analyzed exactly like a sample, and its purpose is to determine

whether the sample matrix contributes bias to the analytical results. The background

concentrations of the analytes in the sample matrix must be determined in a separate

aliquot and the measured values in the LFSM corrected for background concentrations.

3.7 LABORATORY FORTIFIED SAMPLE MATRIX DUPLICATE (LFSMD) – A second

aliquot of the field sample used to prepare the LFSMD, fortified, processed and analyzed

identically. The LFSMD is used instead of the Field Duplicate to access method

precision when the occurrence of target analytes is low.

3.8 MATERIAL SAFETY DATA SHEET (MSDS) – Written information provided by

vendors concerning a chemical’s toxicity, health hazards, physical properties, fire, and

reactivity data, including storage, spill, and handling precautions.
327.0-4

3.9 METHOD BLANK (MB) – An aliquot of reagent water that is free from all method

analytes and is processed and analyzed with the samples in the analysis batch. The

absorbance value of the MB is used to calculate the absorbance change caused by the

reaction of chlorine dioxide with LGB.

3.10 MINIMUM REPORTING LEVEL (MRL) – The minimum concentration that can be

reported as a quantitated value for a target analyte in a sample following analysis. This

concentration can be no lower than the concentration of the lowest continuing calibration

standard for that analyte and can be used only if acceptable quality control criteria for this

standard are met.

3.11 PRIMARY DILUTION STANDARD (PDS) SOLUTION – A solution containing the

analytes prepared in the laboratory from stock standard solutions and diluted as needed to

prepare calibration solutions and other needed analyte solutions.

3.12 REAGENT WATER – A high-purity water, typically either distilled or deionized, that is

free from organic and inorganic contaminants and all method analytes. 

3.13 STOCK STANDARD SOLUTION (SSS) – A concentrated solution containing one or

more method analytes prepared in the laboratory using assayed reference materials or

purchased from a reputable commercial source.

4. INTERFERENCES

4.1 Free available chlorine reacts with LGB to discolor the solution. Glycine reacts with the

free available chlorine to form chloroaminoacetic acid, thereby eliminating the potential

for this type of interference.

4.2 Laboratory glassware can potentially exhibit a demand for chlorine dioxide. While this

(5)

was not observed during method development, it could bias the analytical results and lead

to poor accuracy and/or precision. This potential problem can be avoided if sample

collection vials are cleaned and reused as described in Section 8.1.2.

4.3 Chlorite concentrations are calculated after the samples are sparged to remove chlorine

dioxide. Samples that are not efficiently sparged to remove the chlorine dioxide will

yield chlorite values that are higher than the true value.

5. SAFETY

5.1 The toxicity or carcinogenicity of each reagent used in this method has not been precisely

identified; each chemical compound should be treated as a potential health hazard, and 

exposure to these chemicals should be minimized. The laboratory is responsible for

maintaining a current awareness file of OSHA regulations regarding the safe handling of

the chemicals specified in this method. A reference file of MSDSs should also be made
327.0-5

available to all personnel involved in the chemical analysis. Additional references to

laboratory safety are available.

(6-8)

5.2 This method does not require the preparation of chlorine dioxide standards, which could

have inhalation hazards. It also does not require the preparation of chlorite standards

from a recrystallized solid standard, which can have an explosion hazard. Because of the

significant potential dangers associated with these materials, they are not recommended

for use in conjunction with this method. The chlorite standard cited in Section 7.3.1 does

not pose an explosion hazard.

6. EQUIPMENT AND SUPPLIES

6.1 VISIBLE SPECTROPHOTOMETER – A single- or double-beam spectrophotometer that

uses a monochromator to make accurate absorbance measurements at discrete

wavelengths (Agilent Model 8453 or equivalent) equipped with a standard, rectangular 

1-cm path length cell is recommended. The spectrophotometer must have a linear

response and acceptable photometric accuracy up to an absorbance value (A) of 1.6.

6.1.1 Nondispersive spectrophotometers (e.g., filter-based, light emitting diode-based,

etc.) with alternate cuvette shapes and pathlengths are allowed as long as they

meet the method QC requirements. These devices must have filters with

transmission maximum near 633 nm and must have a linear response and

acceptable photometric accuracy up to an absorbance value of 1.6.

6.1.2 Longer pathlength cells may be used for applications that require lower DLs;

however, the concentration of the Buffered LGB Stock Solution (Sect. 7.2.4)

must be adjusted accordingly. Shorter pathlengths are not recommended.

6.2 CUVETTES – A matched set of 1-cm glass cuvettes with polytetrafluoroethylene (PTFE)

stoppers (Fisher Cat. #: 22200-350 or equivalent). Alternate path lengths and/or cell

shapes are allowed as long as the method QC criteria are met.

6.3 ANALYTICAL BALANCE – Used to accurately weigh analytical standard materials

(±0.1 mg sensitivity).

6.4 TOP LOADING BALANCE – Used to accurately weigh reagents (±10 mg sensitivity).

6.5 WEIGHING BOATS – Plastic, disposable - used to weigh reagents.

6.6 WEIGHING FUNNEL – Used to weigh and transfer LGB and HRP (Fisher Cat. #:

19958-3 or equivalent).

6.7 PIPETTES – Use 0.5-mL and 1.0-mL Class A, volumetric pipettes (Fisher Cat. #: 13-

651-11 or equivalent) and/or variable volume single channel pipettes with disposable

plastic tips.
327.0-6

6.7.1 Use a variable volume, single channel pipette (Brinkman Eppendorf Research

Pro Pipette, Fisher Cat. #:21-378-84 or equivalent) with a volume of 500 to

5000 L to deliver volumes of 1.0 mL or larger.

6.7.2 Use a variable volume, single channel pipette (Brinkman Eppendorf Research

Pro Pipette, Fisher Cat. #:21-378-83 or equivalent) with a volume of 50 to 

1000 L to deliver volumes less than 1.0 mL.

6.8 VIALS – 16-mL amber glass vials with PTFE-lined screw caps (Fisher Cat. #: 03-391-8E

or equivalent) used to collect samples.

6.9 ERLENMEYER FLASKS – 500 mL and 250 mL glass, used to prepare reagents.

6.10 BOTTLES – Amber glass bottles with PTFE-lined screw caps or opaque, high-density

polyethylene (HDPE) bottles of various sizes to store reagents and standards.

6.11 BEAKERS – 100 mL (Fisher Cat.#: 02-540H or equivalent), used to collect and sparge

samples for chlorite analyses.

6.12 VOLUMETRIC FLASKS – Class A, of varying sizes. 

6.13 SPARGING APPARATUS – A cylinder of nitrogen or helium equipped with a regulator

that is attached to an appropriate length of Tygon tubing and a disposable, Pasteur pipette.

Glass gas-dispersion tubes with fritted cylinders or disks may also be used as long as they

are rinsed between uses. 

7. REAGENTS AND STANDARDS

7.1 INTENDED USE OF REAGENTS AND STANDARD SOLUTIONS – Sections 7.2 and

7.3 describe reagents, solvents and standards that are compatible with a 1-cm rectangular

cuvette. In this configuration, and with these reagents, this method has a linear range of

0.2 - 2.2 mg/L total chlorite and chlorine dioxide. If longer pathlength cells are used to

obtain concentration data at lower levels, the Buffered LGB Stock Solution (Sect. 7.2.4)

must be adjusted accordingly.

7.2 REAGENTS AND SOLVENTS – Reagent grade or better chemicals should be used. 

Unless otherwise indicated, all reagents should conform to the specifications of the

Committee on Analytical Reagents of the American Chemical Society , where such (9)

specifications are available. Other grades may be used, provided it is first determined that

the reagent is of sufficiently high purity to permit its use without lessening the quality of

the determination.

7.2.1 REAGENT WATER – Purified water (typically either deionized or distilled)

which does not contain any chlorine dioxide or chlorite or compounds that

absorb light at 633 nm. The absorbance of the reagent water at 633 nm for a 
327.0-7

1-cm pathlength (against a cuvette containing air) should be 5 milli-absorbance

units (mA) or less.

7.2.2 CONCENTRATED CITRIC ACID/GLYCINE BUFFER – Add 9.0 g of

675 3 2

trisodium citrate dihydrate (C O H Na •2H O) (Sigma Cat. #: C3434 or

equivalent) 676 2 2, 5.0 g disodium hydrogencitrate sesquihydrate (C O H Na •1.5H O)

(Sigma Cat. #: 35908-4 or equivalent) and 1.0 g glycine (Sigma Cat. #: G7162

or equivalent) to a 250-mL Erlenmeyer flask. Add 125 mL of reagent water and

mix thoroughly. Add one drop of chloroform (or about 50 μL) (Aldrich HPLC

Grade, Cat. #: 36,692-7 or equivalent) and store this solution at # 6 °C. 

7.2.2.1 Care must be taken to ensure that all crystalline material is

completely dissolved either by using a stir bar or by swirling the

contents of the flask manually for several minutes.

7.2.2.2 The pH of the concentrated buffer stock solution should be

confirmed with a pH electrode to be in the pH range of 6.0 - 6.2 upon

dilution by adding a 1.0-mL aliquot of the buffer to 15 mL of reagent

water. The pH of the concentrated stock solution may be adjusted, if

nece 676 2 2ssary, by adding small amounts of the C O H Na •1.5H O to

675 3 2

lower the pH value or C O H Na •2H O to increase the pH value.

7.2.2.3 Chloroform acts as an antimicrobial agent. If the buffer solution

turns cloudy, which is indicative of microbial growth, it must be

discarded.

7.2.3 LISSAMINE GREEN B (LGB) CONCENTRATED STOCK SOLUTION IN

REAGENT WATER – Accurately weigh 240 mg of LGB (Aldrich Cat. #: 

19958-3 or equivalent) in a glass weighing funnel (Sect. 6.6). Transfer the LGB

to a 250-mL wide neck volumetric flask with reagent water and dilute to the

mark using reagent water. Add a small PTFE stirbar and allow the solution to

stir overnight. Transfer the solution to a 500-mL amber glass bottle which has a

PTFE-lined cap and store this solution at # 6 °C. If stored properly, this

solution should be stable for 2 months. 

7.2.3.1 The minimum acceptable purity for the LGB solid is 70%. Higher

purity is recommended. Although Aldrich specifies that their LGB

has a minimum purity of 60%, the lot of LGB used in this work was

73% pure according to the manufacturer. This information can be

obtained from Aldrich by contacting a technical service

representative. 

7.2.3.2 Confirm that the LGB solid is at least 70% pure as follows. Add 

6 mL of the Concentrated Citric Acid/Glycine Buffer (Sect. 7.2.2) to

a 100-mL volumetric flask. Fill the flask about half full with reagent

water and then add a 1.0-mL aliquot of the LGB Concentrated Stock
327.0-8

Solution. Fill the flask to the mark with reagent water and mix

thoroughly. Allow the flask to equilibrate at room temperature for

15 - 20 minutes, and then mix again. Transfer an aliquot of this

solution to a cuvette with a 1-cm pathlength. The absorbance of this

solution must be $ 0.99. 

7.2.4 BUFFERED LGB STOCK SOLUTION – Prepare a dilution of the LGB

Concentrated Stock Solution in the Concentrated Citric Acid/Glycine Buffer. 

The concentration of this solution should be confirmed prior to mixing it

with the Horseradish Peroxidase (HRP) Stock Solution to prepare the

Combined LGB/HRP Reagent (Sect. 7.2.6). This step will eliminate waste

of the HRP enzyme, which is the most expensive reagent in the method. 

The procedure below is used to determine that the Buffered LGB Stock Solution

has been prepared at the appropriate concentration so that when it is used in the

procedure it will yield a Method Blank absorbance in the required absorbance

range of 1.5 ± 0.1.

7.2.4.1 For LGB with a purity of 73%, transfer 40 mL of the LGB

Concentrated Stock Solution (Sect. 7.2.3) to a wide neck 100-mL

volumetric flask. Add 6 mL of the Concentrated Citric Acid/Glycine

Buffer (Sect. 7.2.2), dilute to the mark with reagent water, and mix

thoroughly. For LGB that has lower purity, the volume of the

Concentrated Stock Solution can be estimated using the equation

where LGB V is the volume of the LGB Concentrated Stock Solution

LGB

required, and P is the purity of the LGB (obtained from the

manufacturer) expressed as a whole number.

7.2.4.2 Confirm that the Buffered LGB Stock Solution concentration is in

the correct range as follows. Fill a 16-mL vial with reagent water,

remove 1.0 mL of the water and add 1.0 mL of the of the

Concentrated Citric Acid/Glycine Buffer (Sect. 7.2.2). Cap the vial

and shake. Remove 0.5 mL of the buffered reagent water and add

0.5 mL of the Buffered LGB Stock Solution. Recap the vial and mix

the contents thoroughly by shaking. Transfer an aliquot of this

solution to a 1-cm cuvette and determine the absorbance at 633 nm. 

The absorbance of this solution at 633 nm must be in the range of

1.5 ± 0.1. If not, the Buffered LGB Stock Solution should be

reprepared from the LGB Concentrated Stock Solution. The

appropriate volume of the LGB Concentrated Stock Solution can be

calculated using the equation
327.0-9

where Vreq is the volume of the LGB Concentrated Stock Solution

added that is required to prepare the new solution, V is the volume of

the LGB Concentrated Stock Solution originally added (40 mL in

Sect. 7.2.4.1), and Ameas is the measured absorbance.

7.2.4.3 If the analyst is using the method to determine concentrations

< 0.2 mg/L, a longer pathlength cuvette is required. The Buffered

LGB Stock Solution concentration must be adjusted accordingly so

that the absorbance in the longer pathlength cuvette, when checked

as described in Section 7.2.4.2, is in the absorbance range of 

1.5 ± 0.1. To calculate the amount of LGB Concentrated Stock

Solution required for the solution, simply divide the recommended

volume in Section 7.2.4.1 by the pathlength.

7.2.4.4 This stock solution is used to prepare the Combined LGB/HRP

Reagent and is not preserved with chloroform. This solution should

not be kept or stored for more than one day. 

7.2.5 BUFFERED HORSERADISH PEROXIDASE (HRP) STOCK SOLUTION –

Weigh out 60 - 70 mg of Type II HRP (Sigma Cat. #: P8250 or equivalent)

using a glass weighing funnel (Sect. 6.6) and an analytical balance. Transfer the

HRP to a wide-neck, 50-mL volumetric flask by washing it from the funnel with

reagent water. The HRP is a lyophalized powder that can be lost during transfer

if care is not taken to minimize air currents. Carefully rinse the walls of the

funnel with reagent water to ensure complete transfer. Add 6 mL of the

Concentrated Citric Acid/Glycine Buffer (Sect. 7.2.2) and then dilute the flask

contents to the mark with reagent water and mix thoroughly. 

7.2.5.1 The HRP must have a minimum activity of 150 units per mg of solid

using the purpurogallin method. The Type II HRP sold by Sigma (10)

meets this criterion. According to Sigma, their HRP is stable for up

to 1 year if stored as a solid at # -20 °C. Other sources of HRP (11)

may also be suitable, but they have not been evaluated.

7.2.5.2 This stock solution is used to prepare the Combined LGB/HRP

Reagent and is not preserved with chloroform, and therefore should

not be kept or stored for more than one day. 

7.2.6 COMBINED LGB/HRP REAGENT – Confirm the concentration of the

Buffered LGB Stock Solution according to Section 7.2.4.2 prior to performing

this step. This will prevent wasting the Buffered HRP Stock Solution, which is
327.0-10

the most expensive reagent in this method. Combine 50 mL of the Buffered

LGB Stock Solution (Sect. 7.2.4) with the entire volume of Buffered HRP Stock

Solution (Sect. 7.2.5) in an amber glass bottle with a PTFE-lined cap. Do not

rinse the 50-mL volumetric flask containing the Buffered HRP Stock Solution to

transfer the remaining droplets as quantitative transfer of this reagent is not

necessary. The volume of the combined reagent is sufficient for approximately

100 tests (e.g., blanks, samples, calibrations, etc.). Add one drop of chloroform

(or about 50 μL). This solution may be used for 14 days if stored at # 6 °C.

7.2.7 HELIUM OR NITROGEN ($99.995%) – UHP Grade or purer, used to sparge

samples to remove chlorine dioxide.

7.3 STANDARD SOLUTIONS – Because chlorite is converted to chlorine dioxide using the

HRP reagent, this method uses chlorite standards to calibrate for both chlorine dioxide

and chlorite (Sect. 2.1). Chlorite standard solutions may be prepared from certified,

commercially available solutions or from solid compounds. Preparation of chlorite

standards from a recrystallized solid of high purity (95%+) is not recommended as this

form of chlorite can pose an explosion hazard. Solution concentrations listed in this

section were used to develop this method and are included as an example. Even though

stability times for standard solutions are suggested in the following sections,

laboratories should use standard QC practices to determine when Standard

Solutions described in this section should be replaced.

7.3.1 CHLORITE STOCK STANDARD SOLUTION (1000 mg/L) – Certified

chlorite standard solutions are commercially available (Absolute Standards, Cat.

# 54109 or equivalent). Commercial standards should be stored at # 6 °C in an

amber glass bottle or high density polyethylene (HDPE) container to prolong

standard life. If stored properly, this solution should be stable for 6 months.

7.3.2 CHLORITE PRIMARY DILUTION STANDARD (PDS) (10 mg/L) – This

solution is used to prepare Calibration Standards and Continuing Calibration

Check Standards. Partially fill a 500-mL volumetric flask with reagent water. 

Add 5.0 mL of the Chlorite Stock Standard Solution (Sect. 7.3.1) using a pipette

and fill the flask to the mark using reagent water. Invert and shake the flask

several times to ensure that the solution is completely mixed and then transfer

the flask contents to an amber bottle with a PTFE-lined cap or an HDPE

container. This solution should be stored at # 6 °C. If stored properly, this

solution should be stable for 3 months.

7.3.3 CALIBRATION STANDARDS – At least three Calibration Standards are

required to establish an initial calibration curve. Five Calibration Standards are

recommended. The concentration of the Calibration Standards must span the

intended reporting range. An example of the dilutions that were used to prepare

the standards used in the development of this method is included in the table

below. All Calibration Standards are to be prepared from the Chlorite Primary

Dilution Standard (Sect. 7.3.2) using Class A volumetric or variable-volume
327.0-11

pipettes and volumetric flasks. Transfer an appropriate aliquot of the Chlorite

PDS (10 mg/L) (Sect. 7.3.2) to the appropriate flask and dilute to the mark with

reagent water. Mix the contents of the flask thoroughly and transfer to an amber

glass bottle or opaque plastic container. These solutions should be stored at 

# 6 °C. If stored properly, these solutions should be stable for 3 months.

Chlorite PDS 

Volume

(mL)

Final 

Volume

(mL)

Calibration

Std. Conc.

(mg/L)

10 500 0.20

25 500 0.50

50 500 1.00

75 500 1.50

100 500 2.00

7.3.4 CONTINUING CALIBRATION CHECK (CCC) STANDARDS – Calibration

Standards prepared above may also be used as CCC Standards.

7.3.5 LFSM FORTIFICATION SOLUTION (100 mg/L) – This solution is used to

fortify Field Samples for the Laboratory Fortified Sample Matrix requirement

(Sect. 9.5). Partially fill a 100-mL volumetric flask with reagent water. Add 

10.0 mL of the Chlorite Stock Standard Solution (Sect. 7.3.1) using a pipette,

and then fill the flask to the mark using reagent water. Invert and shake the

flask several times to ensure the solution is completely mixed and then transfer

the contents of the flask to an amber bottle with a PTFE-lined cap or an HDPE

container. This solution should be stored at #6 °C. If stored properly, this

solution should be stable for 3 months.

8. SAMPLE COLLECTION, PRESERVATION, AND STORAGE

8.1 SAMPLE VIAL PREPARATION

8.1.1 VIAL SELECTION – The procedure and reagent concentrations were developed

using the vials listed in Section 6.8. The manufacturer lists the volume of these

vials to be 16 mL. The procedure used to select appropriate vials is described

below. If alternate vials are used that have an average volume that differs from

16 mL by more than 10%, reagent concentrations and volumes must be adjusted

accordingly.
327.0-12

8.1.1.1 Select an appropriate number of vials based on anticipated sample

load requirements. Vials that do not have appropriate volumes will

be rejected during this process, so begin the selection process with

10-15% more vials than needed. Vials may be reused after cleaning. 

Method development was conducted with approximately 30 vials.

8.1.1.2 Label and cap each 16-mL vial and determine the tare weight with

the PTFE-lined cap. Fill the vial with water and recap. Dry the

external walls of the vial and confirm that there is no headspace by

inverting the vial and looking for an air bubble. Weigh the full vial

and record the water weight. Convert the water weight to volume of

the vial by assuming 1.0 g of water is equal to 1.0 mL.

8.1.1.3 Determine the average volume for the vials tested, and select the

vials that are closest in volume. Discard any vial that has a volume

that is greater than 1% above or below the average value. During

development of this method, the average volume was 16.4 mL and

vials were accepted that were in the volume range of 16.4 ± 0.1 (or

within 0.63%). Approximately 10% of the vials were rejected.

8.1.2 VIAL CLEANING – Vials may be reused. Prior to reuse they should be

thoroughly cleaned with soap and water and rinsed several times with reagent

water. Vials (without caps) can be dried in an oven at 140 °C. Reusing vials

may help avoid any potential for bias associated with chlorine dioxide

demand of the glassware (Sect. 4.2).

8.2 SAMPLE COLLECTION

8.2.1 Prior to sample collection, remove the aerator (if present) to minimize loss of

chlorine dioxide during sample collection. Open the tap and allow the system to

flush until the water temperature has stabilized (usually 3-5 minutes). Reduce

the tap flow just before collecting the samples.

8.2.2 Two aliquots must be collected at each sampling point if both chlorine dioxide

and chlorite are to be determined. Both aliquots require sample processing to

halt the continued formation and/or reproportionation of the method analytes. 

Two pipette tips (or pipettes) will be required for this procedure. Tips can be

used repeatedly to perform the same functions with each Field Sample. This is

not expected to cause significant contamination due to carryover. However,

care should be taken to ensure that fluid is not retained in (or on) the pipette tip

prior to performing the next transfer. These pipette tips (or pipettes) are given

numbers for clarity. The pipette tips are summarized in the table below with

their function. These tips may be saved for use in Section 11.
327.0-13

Pipette Tip # Used to

1 Remove sample and remove buffered sample

2 Transfer citric acid/glycine buffer

8.2.3 FIELD SAMPLE COLLECTION TO DETERMINE CHLORINE DIOXIDE

AND CHLORITE – Fill the 16-mL vial completely so that no headspace

remains after capping the vial. This is most easily done by slightly overfilling

the vial. Invert the capped vial to ensure that the vial does not contain an air

bubble (or headspace). Recollect the sample if a bubble is noted. Remove a

1.0-mL aliquot of the Field Sample from the vial using pipette tip 1 and add a

1.0-mL aliquot of the Concentrated Citric Acid/Glycine Buffer (Sect. 7.2.2)

using pipette tip 2. Free available chlorine is removed in this step. Gently mix

the vial by inverting it three times. Clearly label the sample. This aliquot will be

used to determine the total chlorine dioxide and chlorite concentration.

Note: When removing the 1.0-mL aliquot from the vial, carefully place the

pipette tip into the vial in such a way that the sample does not overflow out of

the vial. As sample is withdrawn into the pipette, the tip should be gently

lowered so that it is always below the surface of the sample.

8.2.4 FIELD SAMPLE COLLECTION TO DETERMINE CHLORITE – Collect a

second aliquot (approximately 40 mL) in an open container with a large surface

area such as a 100-mL beaker. Containers like bottles are not efficiently sparged

even over long times and should not be used. This aliquot will be used to

determine the chlorite concentration and must be sparged immediately after

collection. Field Samples that are not completely sparged will yield chlorite

concentrations that are high.

8.2.4.1 Sparge the sample using a constant flow of nitrogen or helium gas

until the chlorine dioxide is removed. This can be accomplished

using a lecture bottle or gas cylinder with a regulator equipped with a

suitable length of Tygon tubing attached to a Pasteur pipette or a gasdispersion tube with a fritted cylinder or disk (Sect. 6.13). A new

pipette should be used for each sample. Glass gas-dispersion tubes

should be rinsed thoroughly between samples.

8.2.4.1.1 Sparging a sample contained in a beaker with nitrogen at

a flow rate of 250 mL/minute for 15 minutes using a

Pasteur pipette removed over 97% of the chlorine

dioxide.

8.2.4.1.2 Laboratories should confirm that their procedure used to

sparge chlorine dioxide from Field Samples is sufficient

by analyzing a sample known to contain high levels of
327.0-14

chlorine dioxide ( > 2 mg/L) and evaluating its removal

at various sparge times.

8.2.4.2 Transfer an aliquot of the sparged Field Sample to a 16-mL vial. 

Invert the capped vial to ensure that it is completely filled. If an air

bubble is noted, uncap the vial and add additional sparged sample as

necessary. Remove a 1.0-mL aliquot of the sparged Field Sample

from the vial using pipette tip 1 and add a 1.0-mL aliquot of the

Concentrated Citric Acid/Glycine Buffer (Sect. 7.2.2) using pipette

tip 2. This step removes any free available chlorine from the sample. 

Gently mix the vial by inverting it three times. 

8.2.5 QC SAMPLES – Field Duplicates require collection of an extra 16-mL vial as

per Section 8.2.3 and an additional sparged sample as per Section 8.2.4. A

portion of the Field Sample sparged in Section 8.2.4 should be used for the

Laboratory Fortified Sample Matrix.

8.3 SAMPLE STORAGE – Field Samples and Field Duplicates should be analyzed as soon

as possible. Samples may be held for up to 4 hours if collected properly and stored in a

cooler at #10 °C.

9. QUALITY CONTROL

9.1 Each laboratory using this method is required to operate a Quality Control (QC) program. 

The requirements of this program consist of an Initial Demonstration of Capability (IDC),

and subsequent analysis in each analysis batch of a Continuing Calibration Check

Standard (CCC). A Laboratory Fortified Sample Matrix (LFSM) and a Field Duplicate

Sample are recommended weekly. This section details the specific requirements for each

QC parameter. The QC criteria discussed in the following sections are summarized in

Section 17, Tables 5 and 6. These criteria are considered the minimum acceptable QC

criteria, and laboratories are encouraged to institute additional QC practices to meet their

specific needs.

9.2 INITIAL DEMONSTRATION OF CAPABILITY (IDC) – Requirements for the Initial

Demonstration of Capability are described in the following sections. The full IDC must

be conducted prior to the analysis of any Field Samples each time a new analyst uses the

method. Sections 9.2.1 and 9.2.2 must be repeated each time a new LGB Concentrated

Stock Solution (Sect. 7.2.3) is prepared. An acceptable Initial Calibration (Sect. 10.2)

must be conducted prior to performing the IDC.

9.2.1 INITIAL DEMONSTRATION OF ACCURACY – Process and analyze five

highest-level Calibration Standards and five reagent water Method Blanks

through the procedure described in Section 11.2. Calculate the chlorite

concentration for each Calibration Standard as described in Section 12. The
327.0-15

average concentration for the five replicates must be ± 30% of the fortified

value.

9.2.2 INITIAL DEMONSTRATION OF PRECISION – Using the same set of

replicate data generated for Section 9.2.1, calculate the standard deviation and

relative standard deviation (RSD) of the replicate values. The RSD is calculated

using the equation

where 

S is the standard deviation for the replicate values,

and is the average value for the replicates. 

The RSD of the results of the replicate analyses must be less than 20%.

9.2.3 DETECTION LIMIT (DL) DETERMINATION – Prepare and analyze at least

seven replicate CCCs at a chlorite concentration estimated to be near the DL

using the procedure described in Section 11.2 for total chlorite and chlorine

dioxide. For a 1-cm pathlength, the CCCs must be fortified with chlorite in the

concentration range of 0.20 to 0.35 mg/L or at a concentration no more than 3-

to 5-times the calculated DL. Calculate the DL using the equation

DL (n-1,1-"=0.99) = S×t

where 

(n - 1, 1 - " = 0.99) t is the Student’s t value for the 99% confidence level

with n-1 degrees of freedom, 

n is the number of replicates, 

and S is the standard deviation of replicate analyses.

 Selected t values can be found in the table below.

Number of Replicates 7 8 9 10 11

Student’s t value 3.143 2.998 2.896 2.821 2.764

9.2.3.1 The DL for this method is a function of pathlength and analyte

concentration. Longer pathlengths will result in improved sensitivity

and should decrease the DLs for chlorine dioxide and chlorite. DLs

for a 1-cm pathlength calculated by fortifying reagent water with

chlorite in the concentration range of 0.20 to 0.35 mg/L should be 

# 0.25 mg/L.
327.0-16

9.2.3.2 Detection Limit is a statistical determination of precision only. If (1)

the DL replicates are fortified at a low enough concentration, they

may result in a calculated DL that is higher than the fortified

concentration. Therefore no precision and accuracy are specified for

the DL.

9.3 CONTINUING CALIBRATION CHECK (CCC) STANDARDS – A mid-level CCC is

prepared as described in Section 7.3.4 and analyzed with each analysis batch or each day

samples are analyzed. For a 1-cm pathlength, this CCC should have a chlorite

concentration near 1.0 mg/L. The calculated chlorite concentration for the CCC

(Sect.12.5) must be ± 30% of the expected value. If this criterion is not met, the Field

Sample(s) should be re-collected and reanalyzed after establishing acceptable CCC

recovery (Sect. 10.3.2).

9.4 FIELD DUPLICATE (FD) – A Field Duplicate is recommended weekly for both chlorine

dioxide and chlorite. Calculate the relative percent difference (RPD) between the Field

Sample (FD1) and the Field Duplicate (FD2) as shown below. The RPD for samples with

concentrations greater than the lowest calibration standard should not exceed 30%. The

RPD at concentrations at or near the lowest calibration standard (at or below 0.30 mg/L

for a 1-cm pathlength) should not exceed 50%.

If the RPD for the Field Sample and the Field Duplicate falls outside the designated

range, and the laboratory performance for that analyte meets the QC performance

criterion in the CCC, the recovery is judged to be matrix biased. The result for that

analyte in the Field Sample is labeled “suspect/matrix” to inform the data user that the

results are suspect due to matrix effects.

9.5 LABORATORY FORTIFIED SAMPLE MATRIX (LFSM) – A Laboratory Fortified

Sample Matrix is recommended weekly.

9.5.1 Collect a Field Sample for the LFSM as described in Section 8.2.4 and process

it as described in Section 11.4. Fortify the Field Sample using the LFSM

Fortification Solution (Sect. 7.3.5) with a chlorite concentration that is greater

than the expected concentration based on historical levels, being careful not to

exceed the upper limit of the initial calibration curve. This should be done in a

manner that does not increase the sample volume by more than 1%.

9.5.2 Calculate the recovery (R) for each analyte using the equation
327.0-17

where A is the measured concentration in the fortified sample, B is the measured

concentration in the unfortified sample, and C is the fortification concentration.

9.5.3 Recoveries from samples fortified at or above their native concentration should

range between 70 and 130% unless the samples were fortified at low levels (e.g.,

total concentrations at or below 0.30 mg/L) where 50 to 150% recoveries are

acceptable. If the accuracy of the LFSM falls outside the designated range, and

the laboratory performance for chlorite is shown to be in control (e.g., the QC

criterion for the CCC was met), the recovery is judged to be matrix biased. The

result for that analyte in the unfortified sample is labeled “suspect/matrix” to

inform the data user that the results are suspect due to matrix effects.

10. CALIBRATION

10.1 An acceptable initial calibration must be established before Field or QC Samples are

analyzed. After initial calibration is successful, a Continuing Calibration Check (CCC) is

required during each analysis batch or each day samples are analyzed.

10.2 INITIAL CALIBRATION – An initial calibration curve must be generated prior to

conducting the IDC and prior to analyzing any Field Samples. An initial calibration is

also required after preparing new Combined LGB/HRP Reagent as described in Section

7.2.6 (at least every 2 weeks). An initial calibration curve is established by plotting the

absorbance differences ()A) between the Method Blank and a number of Calibration

Standards versus chlorite concentrations in the Calibration Standards. Studies conducted

during method development indicated that calibration curves generated for chlorite and

chlorine dioxide using the Combined LGB/HRP Reagent (Sect. 7.2.6) were similar

enough to allow the calibration for both analytes using chlorite. This procedure is

described below.

10.2.1 Prepare at least three Chlorite Calibration Standards that span the intended

reporting range as described in Section 7.3.3. Five Calibration Standards are

recommended.

10.2.1.1 The high-level calibration standard should not exceed a

concentration of 2.2 mg/L. At higher concentrations, the response

becomes nonlinear.

10.2.2 Fill a 16-mL sample vial with each Calibration Standard and process each vial

along with at least one Method Blank (MB) according to the procedure outlined

in Section 11.2. Two MBs are recommended.

10.2.3 Calculate )A at 633 nm between the MB and each Calibration Standard. If two

MBs are used, use the average MB absorbance value for this calculation.
327.0-18

10.2.4 Plot )A for each calibration standard as a function of chlorite concentration (or

chlorine dioxide concentration, since chlorite is converted to chlorine dioxide

prior to reacting with LGB). This is a procedural calibration technique, where

the calibration standards are processed identically to the Field and QC Samples. 

This eliminates the need to adjust the Calibration Standard concentrations to

account for dilutions made during the procedure.

10.2.5 Fit the data using a linear regression and determine the slope and y intercept

using the equation

)A = 2 Slope[ClO ] + Int. –

where )A is the absorbance difference between the blank and the sample,

2

[ClO ] is the chlorite concentration in mg/L, and Slope and Int. are the slope –

and y intercept for the linear fit, respectively. 

10.2.6 The r value for the linear fit should be 0.98 or better. Use the equation for the 2

line to calculate chlorine dioxide and/or chlorite concentration.

10.2.7 Check the Initial Calibration by calculating the concentrations of the Calibration

Standards that were used to create the calibration curve. Use the linear

regression fit formula for the calculation. Each calculated concentration, except

the calculated concentration for the lowest Calibration Standard, should

calculate to be 70-130% of its expected value. The lowest Calibration Standard

should calculate to be 50-150% of its expected value. Laboratories that have

difficulty achieving these criteria will have trouble meeting the QC requirements

summarized in Section 9.

10.3 CONTINUING CALIBRATION CHECK (CCC) – A Continuing Calibration Check is

required with each Analysis Batch or each day samples are analyzed. This QC sample is

processed with the Method Blank(s) and Field Sample(s) in the Analysis Batch to ensure

that the data collected in the Analysis Batch are valid.

10.3.1 Process a CCC in an identical manner to the Field Samples as described in

Section 11.2. The CCC concentration should be near the mid-point of the initial

calibration range. Calibration Standards used to prepare the initial calibration

curve may be used for this purpose. For a 1-cm pathlength, the CCC should

have a chlorite concentration at or near 1.0 mg/L.

10.3.2 Calculate the CCC concentration using the initial calibration curve generated

above (Sect. 10.2) as described in Section 12. The CCC response must be in the

range of ± 30% of the expected value. If this criterion is not met, then all data

for the Field Samples in the Analysis Batch are invalid. Remedial action should

be taken which could require establishing a new initial calibration and may

require the preparation of new reagents (the HRP and LGB solutions have the
327.0-19

shortest shelf lives). Field Samples should be re-collected and reanalyzed after

re-establishing acceptable QC performance.

11. PROCEDURE

11.1 As noted in Section 2.1, this procedure involves a two-step process for the determination

of chlorine dioxide and chlorite in drinking waters. The first step, described in Section

11.2, uses the Combined LGB/HRP Reagent to convert chlorite to chlorine dioxide to

measure the total concentration of both analytes. This procedure is used to process Field

Samples, Field Duplicates, and CCCs. In the second step, described in Section 11.3,

samples are sparged to remove the chlorine dioxide, and are then analyzed to determine

the chlorite concentration. Chlorite is not removed during sparging. This procedure is

used to process Field Samples and Field Duplicates. (The chlorine dioxide concentration

is calculated by subtracting the chlorite concentration determined in the second step from

the total concentration determined in the first step.) Section 11.4 describes the processing

of LFSMs, which require a slight modification to the procedure described in Section 11.3.

11.1.1 This is a headspace-free procedure. Care taken to minimize headspace will help

the analyst to achieve the QC performance criteria summarized in Section 9.

11.2 TOTAL CHLORINE DIOXIDE AND CHLORITE PROCEDURE USED TO PROCESS

FIELD SAMPLES, FIELD DUPLICATES AND CCCs

11.2.1 The unsparged samples collected in the 16-mL vials are prepared and analyzed

using the procedure described in this section. If samples have been stored (Sect.

8.3), they should be allowed to equilibrate to room temperature.

11.2.2 Fill a 16-mL vial with reagent water and label it as the Method Blank. One MB

is required in each Analysis Batch (# 10 Field Samples). Two MBs are

recommended.

11.2.3 Fill a 16-mL vial with a CCC solution prepared according to the procedure

described in Section 7.3.4.

11.2.4 This procedure requires three separate pipette tips (or pipettes). Each tip will be

used repeatedly to perform the same function with each Field and QC sample.

This is not expected to cause significant contamination due to carryover. 

However, care should be taken to ensure that fluid is not retained in (or on) the

pipette tip prior to performing the next transfer. These pipette tips (or pipettes)

are given numbers for clarity. The pipette tips are summarized in the table

below with their function.
327.0-20

Pipette Tip # Used to

1 Remove sample and remove buffered sample

2 Transfer citric acid/glycine buffer

3 Transfer HRP/LGB mixed reagent

11.2.5 Remove a 1.0-mL aliquot from the CCC and MBs using pipette tip 1. Add a

1.0-mL aliquot of the Concentrated Citric Acid/Glycine Buffer (Sect. 7.2.2) to

the CCC and MBs using pipette tip 2.

Note: Field Samples and Field Duplicates are buffered with the Concentrated

Citric Acid/Glycine Buffer at the time of collection to remove the free available

chlorine.

11.2.6 Cap the CCC and MB vials and gently mix their contents by inverting them

three times. This step buffers the samples to a uniform pH.

11.2.7 Process all Field Samples, Field Duplicates, CCCs and MBs at the same time

through the remainder of the procedure described in this section. Be careful not

to agitate the samples any more than necessary. Uncap the vial and remove an

additional 1.0-mL aliquot using pipette tip 1. Immediately add 1.0-mL of the

Combined LGB/HRP Reagent (Sect. 7.2.6) using pipette tip 3. Cap and mix the

contents of the vial thoroughly.

11.2.8 Check the vial for headspace by inverting it and looking for an air bubble. If the

vial contains air bubbles, the sample must be re-collected (or reprepared in the

case of a CCC or MB).

11.2.9 Allow the samples to react at room temperature for at least 20 minutes, but

preferably not longer than 40 minutes. Loss of color during this period is

normal and is a result of the reaction between LGB and chlorine dioxide.

11.2.10 Thoroughly mix the contents of each vial and then transfer an aliquot to a 1-cm

cuvette. The cuvette should first be rinsed with reagent water or an aliquot of the

sample. Wipe the outside of the cuvette to remove water droplets and/or finger

prints. Use the same cuvette, to measure the visible absorbance at 633 nm for

the MB(s), Field Sample(s), Field Duplicate (if processed), and CCC

minimizing the time between measurements.

11.3 CHLORITE PROCEDURE USED TO PROCESS FIELD SAMPLES AND FIELD

DUPLICATES

11.3.1 This procedure may be used as a stand-alone procedure for the determination of

chlorite or in conjunction with the procedure in Section 11.2 for the
327.0-21

determination of chlorine dioxide as well. The sparged samples are analyzed

using the procedure described in this section.

11.3.2 Collect, sparge, and preserve each FS or FD to remove chlorine dioxide and free

available chlorine as described in Section 8.2.4. 

11.3.3 If samples have been stored (Sect. 8.3), they should be allowed to equilibrate to

room temperature.

11.3.4 Process the Field Samples and Field Duplicates through all steps described in

Sections 11.2.7 through 11.2.10.

11.4 PROCESSING LABORATORY FORTIFIED SAMPLE MATRICES (LFSMs)

11.4.1 This procedure is used for the processing of Laboratory Fortified Sample

Matrices.

11.4.2 Collect, sparge, and preserve an FS as described in Section 8.2.4. The sparged

sample should be poured into two 16-mL vials. One vial is analyzed for chlorite

using the procedure described in Section 11.3 and the other vial is fortified and

analyzed according to the procedure described below.

11.4.3 Fortify the Field Sample with the LFSM Fortification Solution (Sect. 7.3.5) that

is greater than the expected chlorite concentration based on historical

information. This will require a fourth pipette tip that must not be used for

other purposes. Small amounts of the LFSM Fortification Solution can

contaminate other Field or QC samples from carryover because this

solution is highly concentrated.

11.4.3.1 Be careful not to exceed the upper concentration of the initial

calibration curve when calculating the fortification level. 

Fortification levels should be in the range of 1 - 2 times the native

level so that the chlorite concentration will be within the linear range

of the initial calibration curve.

11.4.3.2 As an example, to fortify a Field Sample in a 16.4-mL vial with 

1.0 mg/L of chlorite, one would need to add 0.0164 mg (1.0 mg/L x

0.0164 L). This would require a 164- L aliquot of the LFSM

Fortification Solution ( 0.0164 mg/0.100 mg/mL = 0.164 mL or

164 L).

11.4.3.3 If small volumes of the LFSM Fortification Solution are used (1% of

the vial volume or less), this aliquot can be delivered without first

removing a same-size aliquot to make room for reagents. If this

technique is used the analyst must be careful to insert the pipette tip

beneath the surface of the vial during delivery of the aliquot and to
327.0-22

check the pipette tip after delivery of the aliquot to ensure that the

entire volume has been delivered. Cap the vial once the fortification

is complete and mix thoroughly.

11.4.4 Process the LFSM according to the steps described in Sections 11.2.7 through

11.2.10. 

12. DATA ANALYSIS AND CALCULATION

12.1 Chlorine dioxide and chlorite concentrations are calculated using an initial calibration

curve that is generated using chlorite standards. Slopes for both analytes are similar and,

therefore, no conversion factor is required in order to calculate the chlorine dioxide

concentrations. 

12.2 Generate an initial calibration curve according to Section 10.2 by processing at least

three, but preferably five, initial calibration standards through the entire procedure

outlined in Section 10.2.

12.3 Establish an Initial Calibration curve as described in Section 10.2. Plot )A against the

undiluted concentration of the calibration standards.

12.3.1 Samples processed according to the method generated initial calibration curves

with slopes in the range of 0.33 - 0.38. Different vial volumes and/or reagent

volumes will cause the slopes to differ from this value.

12 2 2 .4 ClO AND ClO CALCULATION: Determine the absorbance difference for each Field –

and QC Sample processed according to Section 11.2, and calculate the total chlorine

dioxide and chlorite concentration using the slope and intercept determined in Section

10.2. If more than one MB is prepared and analyzed in the Analysis Batch, use the

average MB absorbance when determining the absorbance difference for each sample.

12.5 ClO2 CALCULATION: Determine the absorbance difference for each Field and QC –

Sample processed according to Sections 11.3 and 11.4, and calculate the chlorite

concentration using the slope and intercept determined in Section 10.2. If more than one

MB is prepared and analyzed in the Analysis Batch, use the average MB absorbance

when determining the absorbance difference for each sample.

12.6 ClO2 CALCULATION: Calculate the chlorine dioxide concentration for each Field and

QC sample by subtracting the chlorite concentration (Sect. 12.5) from the total chlorite

plus chlorine dioxide concentration (Sect. 12.4).

12.7 Quantitate only those values for which the combined chlorine dioxide and chlorite

concentration falls within the concentration range defined by the lowest and highest

initial calibration standards.
327.0-23

12.7.1 Pathlength and reagent concentrations were carefully optimized during method

development. Analyte concentrations above 2.2 mg/L are in a concentration

range that is not linear if the reagent concentrations specified in this method for

a 1-cm pathlength are used. For this reason, analysts must not extrapolate

beyond the calibration range nor should the initial calibration range be extended

beyond this concentration.

12.7.2 Field Samples that exceed this concentration (2.2 mg/L) should be re-collected

and carefully diluted by a factor of 2 being careful to minimize unnecessary

sample agitation that will result in the loss of chlorine dioxide.

12.7.3 Analytical results obtained from dilutions should be considered estimated

values.

12.8 Analyte concentrations are reported in mg/L usually to two significant figures.

13. METHOD PERFORMANCE

13.1 PRECISION, ACCURACY, AND DETECTION LIMITS – Detection Limits for chlorite

and chlorine dioxide are presented in Section 17, Table 1 together with method precision

and accuracy in reagent water. Single laboratory precision and accuracy are presented in

Table 2 for a chlorinated surface water and in Table 3 for a chlorinated ground water.

13.2 SAMPLE STORAGE STABILITY STUDIES – A storage stability study was conducted

by fortifying 0.8 mg/L of chlorine dioxide and 1.0 mg/L of chlorite into a chlorinated

drinking water sample and analyzing at 0, 2, and 4 hours after storage at 10 °C. The data

in Table 4 illustrate that chlorite concentrations are stable over a 4-hour time period when

glycine is present to eliminate the free available chlorine residual.

14. POLLUTION PREVENTION

14.1 For information about pollution prevention that may be applicable to laboratory

operations, consult “Less is Better: Laboratory Chemical Management for Waste

Reduction” available from the American Chemical Society’s Department of Government

Relations and Science Policy, 1155 16th Street NW, Washington, D.C., 20036.

15. WASTE MANAGEMENT

15.1 The analytical procedures described in this method generate relatively small amounts of

waste since only small amounts of reagents and solvents are used. The matrices of

concern are finished drinking water. However, the Agency requires that laboratory waste

management practices be conducted consistent with all applicable rules and regulations,

and that laboratories protect the air, water, and land by minimizing and controlling all
327.0-24

releases from fume hoods and bench operations. Also, compliance is required with any

sewage discharge permits and regulations, particularly the hazardous waste identification

rules and land disposal restrictions. For further information on waste management,

consult “The Waste Management Manual for Laboratory Personnel” also available from

the American Chemical Society at the address in Section 14.1.

16. REFERENCES

1. Glaser, J.A.; Foerst, D.L.; McKee, G.D.; Quave, S.A.; Budde, W.L. "Trace Analyses for

Wastewaters," Environ. Sci. Technol. 1981, 15, 1426-1435.

2. Hollenberg, P. F.; Rand-Meir, T.; Hager, L. P. “The Reaction of Chlorite with

Horseradish Peroxidase and Chloroperoxidase,” J. Biol. Chem.1974, 249, 5816-5825.

3. Hewson, W. D.; Hager, L. P. “Mechanism of the Chlorination Reaction Catalyzed by

Horseradish Peroxidase with Chlorite,” J. Biol. Chem.1979, 254, 3175-3181.

4. Chiswell,B.; O’Hallaran,K.R. “Use of Lissamine Green B as a Spectrophotometric

Reagent for the Determination of Low Residuals of Chlorine Dioxide” The Analyst, 1991,

116, 657-661.

5. Hoffman, R.; Andrews, R.C.;Ye, Q. “Comparison of Spectrophotometric Methods for

Measuring Chlorine Dioxide in Drinking Water,” Environmental Technology, 1998, 19,

761-773. 

6. “OSHA Safety and Health Standards, General Industry,” (29CFR1910). Occupational

Safety and Health Administration, OSHA 2206, (Revised, Jan. 1976).

7. “Carcinogens-Working with Carcinogens,” Publication No. 77-206, Department of

Health, Education, and Welfare, Public Health Service, Center for Disease Control,

National Institute of Occupational Safety and Health, Atlanta, Georgia, August 1977.

8. “Safety In Academic Chemistry Laboratories,” 3 Edition, American Chemical Society rd

Publication, Committee on Chemical Safety, Washington, D.C., 1979.

9. “Reagent Chemicals,” Sixth Edition, American Chemical Society Specifications, Official

from Jan. 1, 1982, American Chemical Society, S, Washington, D. C., 1981.

10. Peroxidase description, Sigma catalog 2002-2003, page 1635 and references contained

therein.

11. Personal communication with Technical Representative at Sigma (1-800-521-8956).
327.0-25

17. TABLES, DIAGRAMS, FLOWCHARTS, AND VALIDATION DATA

TABLE 1: PRECISION AND ACCURACY AND DETECTION LIMITS FOR CHLORINE

DIOXIDE AND CHLORITE IN REAGENT WATER

Fortification

Concentration Chlorite Chlorine Dioxide

ClO2

–

(mg/L)

ClO2

(mg/L)

Recovery

(%)

RSD

(%)

DLa

(mg/L)

Recovery

(%)

RSD

(%)

DLa

(mg/L)

 0.25

not

fortified 112 12 0.11

not

fortified

not

fortified

not

fortified

 0.25 0.95 118 8.5 0.078 116 3.6

not

calculated

not

fortified 0.26

not

fortified

not

fortified

not

calculated 102 5.1 0.042

1.0 0.26 103 2.9

not

calculated 124 16 0.16

1.0 0.94 98.5 3.2

not

calculated 111 4.8

not

calculated

Based on seven replicates analyzed over one day. Detection Limits were calculated as follows: a

DL (n-1,1-"=0.99) = S×t

where

(n-1,1-" = 0.99) t = Student’s t value for the 99% confidence level with n-1

degrees of freedom

n = number of replicates, and

S = standard deviation of replicate analyses.
327.0-26

TABLE 2: PRECISION AND ACCURACY FOR CHLORITE AND CHLORINE DIOXIDE 

IN A CHLORINATED SURFACE WATERa

Fortification Level Chlorite Chlorine Dioxide

ClO2

–

(mg/L)

ClO2

(mg/L)

Recovery

(%)

RSD

(%)

Recovery

(%)

RSD

(%)

1.0 not fortified 107 4.2 not fortified not fortified

not fortified 0.82 not fortified not fortified 100 1.9

1.0 0.82 109 3.7 91.3 8.9

2.0 not fortified 105 1.4 not fortified not fortified

not fortified 1.94 not fortified not fortified 96.9 1.0

The drinking water used in these studies had a free available chlorine concentration of 0.9 mg/L. 

a
327.0-27

The drinking water used in these studies had a free available chlorine concentration of a

0.8 mg/L 3 and a hardness of 325 mg/L (CaCO ).

TABLE 3: PRECISION AND ACCURACY FOR CHLORITE AND CHLORINE DIOXIDE

IN A CHLORINATED GROUND WATERa

Fortification Level Chlorite Chlorine Dioxide

ClO2

–

(mg/L)

ClO2

(mg/L)

Recovery

(%)

RSD

(%)

Recovery

(%)

RSD

(%)

1.0 not fortified 110 4.4 not fortified not fortified

not fortified 0.90 not fortified not fortified 92.0 3.3

1.0 0.90 107 1.6 93.8 3.0

2.0 not fortified 100 2.7 not fortified not fortified

not fortified 2.0 not fortified not fortified 110 1.7
327.0-28

TABLE 4: STORAGE STABILITY FOR CHLORITE AND CHLORINE DIOXIDE IN A

CHLORINATED SURFACE WATER SAMPLEa

Holding

Time

(Hours)

Chlorite Fortified 

at 1.0 mg/L

Chlorine Dioxide and

Chlorite Fortified at a Total

Concentration of 1.8 mg/Lb

Recovery

(%)

RSD

(%)

Recovery

(%)

RSD

(%)

0 83.3 2.5 97.3 1.4

2 100 6.6 98.1 1.4

4 99.3 2.4 98.1 2.8

The drinking water used in these studies had a free available chlorine concentration of 0.9 mg/L. 

a 

Chlorine dioxide and chlorite were fortified into the chlorinated drinking water sample using b 

concentrated standards just prior to preserving the samples so that the concentrations in the

surface water were 0.80 and 1.0 mg/L, respectively. 
327.0-29

TABLE 5: INITIAL DEMONSTRATION OF CAPABILITY (IDC) REQUIREMENTS

Method

Reference

Requirement Specification and

Frequency

Acceptance Criteria

Section

9.2.1

Initial Demonstration

of Accuracy

Prior to the analysis of any

Field Samples by a new

analyst and each time a new

LGB Concentrated Stock

Solution is prepared, process

five highest-level calibration

standards. Calculate the

average concentration for

five standards.

Mean concentration must be

within ± 30% of the fortified 

value.

Section

9.2.2

Initial Demonstration

of Precision

Using the same data from

Section 9.2.1, calculate the

RSD.

RSD must be < 20%.

Section

9.2.3

Detection Limit 

Determination

Prior to the analysis of any

Field Samples by a new

analyst, prepare and analyze

a minimum of seven

replicate CCCs fortified at a

chlorite concentration of

0.20 - 0.35 mg/L if using the

1-cm pathlength procedure. 

Calculate the DL using the

equation in Section 9.2.3.

DL should be # 0.25 mg/L

when using the 1-cm

pathlength procedure. 
327.0-30

TABLE 6: QUALITY CONTROL REQUIREMENTS (SUMMARY)

Method

Reference

Requirement Specification and Frequency Acceptance Criteria

Section 8.3 Sample

Storage

Analyze samples as soon as

possible. Samples may be stored at

# 10 °C for up to 4 hours 

Sample results are valid only if

samples are analyzed within

holding time.

Section

10.2

Initial

Calibration

Calibrate with at least three

calibration standards prior to the

IDC and each time a new

Combined LGB/HRP Reagent is

prepared. 

Five calibration standards are

recommended.

Highest level for the 1-cm

pathlength procedure must not

exceed 2.2 mg/L.

When each calibration standard

is calculated as an unknown

using the calibration curve, the

result should be 70-130% of the

expected value for all except

the lowest standard, which

should be 50-150% of the

expected value. 

Sections

9.3 and

10.3

Continuing

Calibration

Check (CCC)

Verify initial calibration by

analyzing a mid-level CCC each

day samples are analyzed. 

 The CCC should be at or near 1.0

mg/L for the 1-cm pathlength

procedure.

The calculated chlorite

concentration must be ± 30% of

the expected value.

Section 9.4 Field

Duplicate (FD)

An FD is recommended weekly.

Calculate RPD according to

Section 9.4.

RPD should not exceed 30%

for concentrations above the

low-level calibration standard.

RPD should not exceed 50%

for concentrations at or near the

low-level calibration standard

(at or below 0.3 mg/L for the 

1-cm pathlength procedure).

Section 9.5 Laboratory

Fortified

Sample Matrix

(LFSM)

An LFSM is recommended weekly.

Calculate Recovery according to

Section 9.5.

Recovery should be 70 - 130%

except for samples fortified at

low levels (total concentrations

at or below 0.3 mg/L for the 

1-cm pathlength procedure),

where the Recovery should be

50 - 150%.