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 Kortvelyesi and Gilbert Gordon, Miami University
                         TECHNICAL SUPPORT CENTER
               OFFICE OF GROUND WATER AND DRINKING WATER
                  U. S. ENVIRONMENTAL PROTECTION AGENCY
                            CINCINNATI, OHIO 45268

                                     327.0-1

-------
                                       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
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.(1) Detection Limits are technique and instrument dependent.
             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.(2>3) Chlorine dioxide
             rapidly oxidizes the LGB, reducing its absorption in the red region of the visible spectrum
             in proportion to the chlorine dioxide concentration/4'5' A visible spectrophotometer is
             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 (C1O2 ) is referred to as 'chlorite' for simplicity, even though the
use of 'chlorite ion' is more accurate.

                                           327.0-2

-------
             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 envi-
             ronmental 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.

             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.

                                           327.0-3

-------
       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.(5) While this
             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

                                            327.0-4

-------
             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 polytetrafiuoroethylene (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.

                                           327.0-5

-------
             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 |uL 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 |uL 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(9), where such
             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

                                           327.0-6

-------
        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
        trisodium citrate dihydrate (C6O7H5Nv2H2O) (Sigma Cat. #: C3434 or
        equivalent), 5.0 g disodium hydrogencitrate sesquihydrate (C6O7H6Na21.5H2O)
        (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 uL) (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
                  necessary, by adding small amounts of the C6O7H6Na21.5H2O to
                  lower the pH value or C6O7H5Na32H2O 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

                             327.0-7

-------
                  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 VLGB is the volume of the LGB Concentrated Stock Solution
                  required, and PLGB 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
                              327.0-8

-------
                       req     added
                                       15
                                    ^   meas
                  where Vreq is the volume of the LGB Concentrated Stock Solution
                  that is required to prepare the new solution, Vadded is the volume of
                  the LGB Concentrated Stock Solution originally added (40 mL in
                  Sect.  7.2.4.1), andAmeas 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.(10)  The Type II HRP sold by Sigma
                  meets this criterion. According to Sigma, their HRP is stable  for up
                  to 1 year if stored as a solid at <  -20 C.(11) Other sources of HRP
                  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
                              327.0-9

-------
               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 uL).  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 (HOPE) 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 HOPE
               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

                                    327.0-10

-------
                     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)
10
25
50
75
100
Final
Volume
(mL)
500
500
500
500
500
Calibration
Std. Cone.
(mg/L)
0.20
0.50
1.00
1.50
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 HOPE
                      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.
                                          327.0-11

-------
        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).

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.
                              327.0-12

-------
Pipette Tip #
1
2
Used to
Remove sample and remove buffered sample
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 gas-
                  dispersion 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

                              327.0-13

-------
                                           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.

        .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
                                           327.0-14

-------
        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
                         RSD= ^x 100%
                                X
                   S is the standard deviation for the replicate values,
                   and X 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 =
        where
                   *(n -1, i - <* = 0.99) ^ me 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
Student's t value
7
3.143
8
2.998
9
2.896
10
2.821
11
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.
                              327.0-15

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

                                  \FD1- FD2\
                                (FDL+

       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 1 1 .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
                                   (A- B)
                              R =  	x 100%
                                     C
                                    327.0-16

-------
                      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 (AA) 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 AA at 633 nm between the MB  and each Calibration Standard.  If two
                      MBs are used, use the average MB absorbance value for this calculation.
                                            327.0-17

-------
       10.2.4   Plot AA 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

                                         AA = Slope[Cl(V] +  Int.

               where AA is the absorbance difference between the blank and the sample,
               [C1O2 ] 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 r2 value for the linear fit should be 0.98 or better. Use the equation for the
               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
                                     327.0-18

-------
                      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.
                                           327.0-19

-------
Pipette Tip #
1
2
3
Used to
Remove sample and remove buffered sample
Transfer citric acid/glycine buffer
Transfer HRP/LGB mixed reagent
11.3
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.

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
                                    327.0-20

-------
               determination of chlorine dioxide as well. The sparged samples are analyzed
               using the procedure described in this section.

       1 1 .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.

       1 1.3.3   If samples have been stored (Sect. 8.3), they should be allowed to equilibrate to
               room temperature.

       1 1 .3.4   Process the Field Samples and Field Duplicates through all steps described in
               Sections 11.2.7 through 11.2.10.

1 1 .4   PROCESSING LABORATORY FORTIFIED SAMPLE MATRICES (LFSMs)

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

       1 1 .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.

               1 1 .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.

               1 1 .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-|uL aliquot of the LFSM
                         Fortification Solution (0.0164 mg/0.100 mg/mL = 0.164 mL or
                         164
               1 1.4.3.3   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


                                     327.0-21

-------
                                 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 AA 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.

       12.4   C1O2 AND C1O2 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   C1O2 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   C1O2 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.
                                            327.0-22

-------
              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


                                           327.0-23

-------
             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,"/. 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," 3rd Edition, American Chemical Society
             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).
                                           327.0-24

-------
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
C1O2
(mg/L)
0.25
0.25
not
fortified
1.0
1.0
C1O2
(mg/L)
not
fortified
0.95
0.26
0.26
0.94
Chlorite
Recovery
(%)
112
118
not
fortified
103
98.5
RSD
(%)
12
8.5
not
fortified
2.9
3.2
DLa
(mg/L)
0.11
0.078
not
calculated
not
calculated
not
calculated
Chlorine Dioxide
Recovery
(%)
not
fortified
116
102
124
111
RSD
(%)
not
fortified
3.6
5.1
16
4.8
DLa
(mg/L)
not
fortified
not
calculated
0.042
0.16
not
calculated
      "Based on seven replicates analyzed over one day. Detection Limits were calculated as follows:

                    DL =
                    where
                              t(n-u- = 0.99) = 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.
                                        327.0-25

-------
TABLE 2:   PRECISION AND ACCURACY FOR CHLORITE AND CHLORINE DIOXIDE
            IN A CHLORINATED SURFACE WATER"
Fortification Level
C1O2
(mg/L)
1.0
not fortified
1.0
2.0
not fortified
C1O2
(mg/L)
not fortified
0.82
0.82
not fortified
1.94
Chlorite
Recovery
(%)
107
not fortified
109
105
not fortified
RSD
(%)
4.2
not fortified
3.7
1.4
not fortified
Chlorine Dioxide
Recovery
(%)
not fortified
100
91.3
not fortified
96.9
RSD
(%)
not fortified
1.9
8.9
not fortified
1.0
      "The drinking water used in these studies had a free available chlorine concentration of 0.9 mg/L.
                                       327.0-26

-------
TABLE 3:   PRECISION AND ACCURACY FOR CHLORITE AND CHLORINE DIOXIDE
            IN A CHLORINATED GROUND WATER3
Fortification Level
C1O2
(mg/L)
1.0
not fortified
1.0
2.0
not fortified
C1O2
(mg/L)
not fortified
0.90
0.90
not fortified
2.0
Chlorite
Recovery
(%)
110
not fortified
107
100
not fortified
RSD
(%)
4.4
not fortified
1.6
2.7
not fortified
Chlorine Dioxide
Recovery
(%)
not fortified
92.0
93.8
not fortified
110
RSD
(%)
not fortified
3.3
3.0
not fortified
1.7
      "The drinking water used in these studies had a free available chlorine concentration of
      0.8 mg/L and a hardness of 325 mg/L (CaCO3).
                                        327.0-27

-------
TABLE 4:   STORAGE STABILITY FOR CHLORITE AND CHLORINE DIOXIDE IN A
             CHLORINATED SURFACE WATER SAMPLE"
Holding
Time
(Hours)
0
2
4
Chlorite Fortified
at 1.0 mg/L
Recovery
(%)
83.3
100
99.3
RSD
(%)
2.5
6.6
2.4
Chlorine Dioxide and
Chlorite Fortified at a Total
Concentration of 1.8 mg/Lb
Recovery
(%)
97.3
98.1
98.1
RSD
(%)
1.4
1.4
2.8
      "The drinking water used in these studies had a free available chlorine concentration of 0.9 mg/L.
      b Chlorine dioxide and chlorite were fortified into the chlorinated drinking water sample using
      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.
                                          327.0-28

-------
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.
                                          327.0-29

-------
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
< 10C 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%.
                                          327.0-30

-------