EPA/6QQ/R-93/100
                                                   August 1993
       METHODS FOR THE DETERMINATION

         OF INORGANIC SUBSTANCES IN

           ENVIRONMENTAL  SAMPLES
ENVIRONMENTAL MONITORING SYSTEMS  LABORATORY
    OFFICE OF RESEARCH AND DEVELOPMENT
   U\S.  ENVIRONMENTAL PROTECTION AGENCY
          CINCINNATI, OHIO  45268
                                          Printed on Recycled Paper

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                                  DISCLAIMER

     This manual has been reviewed by the Environmental Monitoring Systems
Laboratory - Cincinnati, U.S. Environmental Protection Agency, and approved
for publication.  Mention of trade names or commercial products does not
constitute endorsement or recommendation for use.
                                      n

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                                   FOREWORD


      Environmental  measurements  are  required  to  determine  the  quality  of
 ambient waters  and  the  character of  waste  effluents.  The  Environmental
 Monitoring  Systems  Laboratory  -  Cincinnati  (EMSL-Cincinnati) conducts  research
 to:

      o    Develop and evaluate analytical  methods to  identify  and measure the
          concentration  of  chemical  pollutants in marine and estuarine waters,
          drinking  waters,  surface waters,  groundwaters, wastewaters,
          sediments, sludges,  and solid wastes.

      o    Investigate methods  for the  identification  and measurement of
          viruses,  bacteria and  other  microbiological organisms  in aqueous
          samples and to determine the responses of aquatic organisms  to water
          quality.

      o    Develop and operate  a  quality assurance program  to support the
          achievement of data  quality  objectives in measurements of pollutants
          in marine and estuarine waters, drinking water,  surface water,
          groundwater, wastewater, sediment and  solid waste.

      o    Develop methods and  models to detect and quantify responses  in
          aquatic and terrestrial organisms exposed to environmental  stressors
          and to correlate  the exposure with effects on chemical and
          biological indicators.

     This EMSL-Cincinnati publication, "Methods for the Determination of
 Inorganic Substances in Environmental  Samples," was prepared as the
continuation of an initiative  to gather together a compendium of standardized
laboratory analytical methods  for the  determination of inorganic substances in
water and wastewater.  We are  pleased  to provide this manual and believe that
it will  be of considerable value to many public and private laboratories
involved in inorganic analyses for regulatory or other reasons.
                                        Thomas A.  Clark,  Director
                                        Environmental  Monitoring Systems
                                        Laboratory - Cincinnati

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                                   ABSTRACT

     This manual contains ten updated and revised automated, semi-automated or
methods amenable to automation for the determination of a variety of inorganic
substances in water and wastewater.

     These methods include and address, in an expanded form, information
concerning safety, quality control, pollution prevention, and waste
management.  Methods were selected which minimize the amount of hazardous
reagents required and maximize sample throughput to allow expanded quality
control.

     Automated methods are included for nitrate-nitrite, phosphorus, and
sulfate.  Semi-automated methods cover cyanide, ammonia, total kjeldahl
nitrogen (TKN), chemical oxygen demand (COD) and generic phenolics.  Methods
amenable to automation include turbidity and inorganic anions by ion
chromatography.
                                      IV

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                               TABLE OF CONTENTS
Method
Number               Title	      Revision   Date    Page
                     Disclaimer	    ii
                     Foreword	   lit
                     Abstract	  .	    iv
                     Acknowledgment.  ........  	    vi
                     Introduction.  .......  	  ....     1
180.1                Determination  of Turbidity            2.0       8/93
                     by Nephelometry
300.0                Determination  of Inorganic Anions     2.1       8/93
                     by Ion Chromatography
335.4                Determination  of Total Cyanide        1.0       8/93
                     by Semi-Automated Colorimetry
350.1                Determination  of Ammonia Nitrogen     2.0       8/93
                     by Semi-Automated Coloriimetry
351.2                Determination  of Total Kjeldahl       2.0       8/93
                     Nitrogen by Semi-Automated
                     Colorimetry
353.2                Determination  of Nitrate-Nitrite      2.0       8/93
                     by Automated Colorimetry
365.1                Determination  of Phosphorus           2.0       8/93
                     by Automated Colorimetry
375.2                Determination  of Sulfate              2.0       8/93
                     by Automated Colorimetry
410.4                Determination  of Chemical Oxygen      2.0       8/93
                     Demand by Semi-Automated Colorimetry
420.4                Determination  of Total Recoverable    1.0       8/93
                     Phenolics by Semi-Automated
                     Colorimetry

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                                ACKNOWLEDGMENTS

     This methods manual was prepared and edited by the Inorganic Chemistry
Branch (ICB) of the Chemistry Research Division, Environmental Monitoring
Systems Laboratory - Cincinnati (EMSL-Cincinnati).

     Major contributors from the ICB include John D. Pfaff for anions by ion
chromatography, Billy Potter for cyanide methodology, Theodore Martin and John
Creed for quality control, and Diane Schirmann for manuscript production.
James O'Dell selected the methods that are included from previous versions
published in "Methods for the Chemical Analysis of Water and Wastes," EPA
600/4-79-020, Revised March 1983.  He reorganized the previous versions into a
format approved by the Environmental Monitoring Management Council and added
new sections to address safety, quality control, pollution prevention, and
waste management.  Last but not least, a very special acknowledgement goes to
former ICB member Morris E. Gales who started this project before his
retirement and was responsible for most of the original versions of these
methods.  Thanks again Mo for your years of dedicated service to our
environment, the USEPA, and your fellow employees.
                                      VI

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                                  INTRODUCTION

      The original  version of this manual  was issued in November 1969 by the
 Federal  Water Pollution Control  Administration as "FWPCA Methods for Chemical
 Analysis of Water and Wastes."  With the  creation of the United States
 Environmental Protection Agency  (USEPA) came "Methods for Chemical  Analysis of
 Water and Wastes 1971" Publication No.  16020—07/71.  The second edition was
 issued in 1974 as EPA 625/6-74-003,  and the third edition in 1979 as EPA
 600/4/79-020.  The current version,  an  updated second printing of the third
 edition, was revised and issued  in March  1983.  The methods contained in the
 1983 manual  form the basis for most of  the methodology approved for compliance
 monitoring of inorganic parameters specified under the Clean Water Act (NPDES)
 and contaminants regulated under the Safe Drinking Water Act.
                                            I •
      In  1991, a number of new and revised metals methods were incorporated
 into a new publication entitled, "Methods for the Determination of Metals in
 Environmental Samples."  Concurrently,  the decision was made to revise and
 update selected non-metal  methods to be issued under the name "Methods for the
 Determination of Inorganic Substances in  Environmental  Samples."

      For both the  metals and non-metals manuals,  several  important  features
 were adopted:

•    Consistent use of terminology,  a feature especially helpful  in the
      quality control  sections where  standardized terminology is not yet
      available.   The terms were  carefully selected to be meaningful  without
      extensive definition, and therefore  should be easy to understand and use.

•    New sections  are included with  expanded useful  coverage of safety,
      quality control,  pollution  prevention and waste management.

•    All  methods are presented in the new EPA standard  Environmental
      Monitoring Management Council  (EMMC)  format.


      Although a number of  other  methods included the 1983 edition of USEPA
 "Methods for Chemical  Analysis of Water and Wastes",  Standard  Methods for the
 Examination  of Water and Wastewater,  and  American  Society for  Testing and
Materials Annual Book of Standards  (ASTM)  are acceptable  for compliance
monitoring,  the revised methods  contained  in this  publication  are considered
to  be the most useful  in terms of future  regulatory  requirements.   They
represent a  selection  of air segmented automated,  semi-automated, or amenable
to  automation methodology  that provides the following advantages over their
manual counterparts.

•    Higher  sample  throughput for faster  analysis  and improved  precision.

•    Faster  analysis  allows  more time to  perform the  updated quality  control
      required to insure valid results.

•    Lower per analysis reagent  consumption  to  reduce waste  production  and
      minimize disposal  costs.

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•    The inclusion of multi-laboratory data generated from USEPA performance
     evaluation studies.
The following methods are  included with specific features and improvements:
•    A revised version of  EPA turbidity Method 180.1 that minimizes the direct
     use of hydrazine sulfate.
•    An updated version of EPA Method 300.0 for anions by ion chromatography.
•    A new stand  alone semi-automated revision of EPA cyanide Method 335.2
     that specifies the use of the downsized midi-distillation procedure.
•    A new semi-automated  version of the EPA phenolics Method 420.2.
•    The optional use of a non-mercury catalyst in EPA TKN Method 351.2.
•    All methods  allow the optional use of reduced reagent and distillation-
     digestion volumes.
•    Most of the  methods include the option of limited performance-based
     modifications or improvements.
                James  W.  O'Dell,  John  D.  Pfaff,  William  L.  Budde
                          Chemistry Research Division
                                  August 1993

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                METHOD 180.1

 DETERMINATION  OF TURBIDITY BY NEPHELOMETRY
         Edited by James W. O'Dell
         Inorganic  Chemistry  Branch
        Chemistry Research Division
                Revision  2.0
                August 1993
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
    OFFICE OF RESEARCH AND DEVELOPMENT
   U.S. ENVIRONMENTAL PROTECTION AGENCY
          CINCINNATI, OHIO  45268

                  180.1-1

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                                 METHOD 180.1
                                                                                     H
                  DETERMINATION OF TURBIDITY BY NEPHELOMETRY
1.0  SCOPE AND APPLICATION

     1.1  This method covers the determination of turbidity in drinking,
          ground, surface, and saline waters, domestic and industrial  wastes.

     1.2  The applicable range is 0 to 40 nephelometric turbidity units (NTU).
          Higher values may be obtained with dilution of the sample.

2.0  SUMMARY OF METHOD

     2.1  The method is based upon a comparison of the intensity of light
          scattered by the sample under defined conditions with the intensity
          of light scattered by a standard reference suspension.  The higher
          the intensity of scattered light, the higher the turbidity.
          Readings, in NTU's, are made in a nephelometer designed according to
          specifications given in sections 6.1 and 6.2.  A primary standard
          suspension is used to calibrate the instrument.  A secondary
          standard suspension is used as a daily calibration check and is
          monitored periodically for deterioration using one of the primary
          standards.

          2.1.1  Formazin polymer is used as a primary turbidity suspension
                 for water because it is more reproducible than other types of
                 standards previously used for turbidity analysis.

          2.1.2  A commercially available polymer primary standard is also
                 approved for use for the National Interim Primary Drinking
                 Water Regulations.  This standard is identified as AMCO-AEPA-
                 1, available from Advanced Polymer Systems.

3.0  DEFINITIONS

     3.1  CALIBRATION BLANK  (CB) — A volume of reagent water fortified with
          the same matrix as the calibration standards, but without the
          analytes, internal standards, or surrogates analytes.

     3.2  INSTRUMENT PERFORMANCE CHECK SOLUTION (IPC) — A solution of one or
          more method analytes, surrogates, internal standards, or other test
          substances used to evaluate the performance of the instrument system
          with respect to a defined set of criteria.

     3.3  LABORATORY REAGENT BLANK (LRB) — An aliquot of reagent water or
          other blank matrices that are treated exactly as a sample including
          exposure to all glassware, equipment, solvents, reagents, internal
          standards, and surrogates that are used with other samples.   The LRB
          is used to determine if method analytes or other interferences are


                                    180.1-2

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           present in  the laboratory environment,  the  reagents,  or  the
           apparatus.

      3.4   LINEAR CALIBRATION RANGE  (LCR)  —  The concentration range  over which
           the  instrument response  is linear.

      3.5   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.

      3.6   PRIMARY CALIBRATION STANDARD  (PCAL) —  A suspension prepared from
           the  primary  dilution stock standard suspension.   The  PCAL
           suspensions  are used to calibrate  the instrument  response  with
           respect to  analyte concentration.

      3.7   QUALITY CONTROL SAMPLE (QCS)  — A  solution  of the method analyte of
           known  concentrations that  is  used  to fortify an aliquot  of LRB
           matrix.   The QCS is obtained  from  a source  external to the
           laboratory,  and is used to check laboratory performance.

      3.8   SECONDARY CALIBRATION  STANDARDS (SCAL)  « Commercially prepared,
           stabilized sealed  liquid or gel  turbidity standards calibrated
           against properly prepared  and diluted formazin or styrene
           divinyl benzene  polymers.

      3.9   STOCK  STANDARD  SUSPENSION  (SSS)  — A concentrated suspension
           containing the  analyte prepared in the  laboratory using  assayed
           reference materials  or purchased from a reputable commercial source.
           Stock  standard  suspension  is  used to prepare calibration suspensions
           and  other needed suspensions.

4.0   INTERFERENCES

      4.1   The  presence of floating debris  and coarse  sediments which settle
           out  rapidly will give low  readings.  Finely divided air bubbles can
           cause  high readings.

      4.2  The  presence of true color, that is the color of water which is due
           to dissolved substances that absorb light,  will  cause turbidities to
           be low, although this effect is generally not significant with
          drinking waters.

      4.3   Light  absorbing materials  such as activated carbon in significant
          concentrations can cause low readings.

5.0  SAFETY

     5.1  The toxicity or carcinogenicity of each  reagent  used in this method
          has not been fully established.   Each  chemical  should be regarded as
          a potential  health hazard and exposure  should be as low as
          reasonably achievable.

                                    180.1-3

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     5.2  Each 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 Material
          Safety Data Sheets (MSDS) should be made available to all personnel
          involved in the chemical analysis.  The preparation of a formal
          safety plan is also advisable.

     5.3  Hydrazine Sulfate (7.2.1) is a carcinogen. It is highly toxic and
          may be fatal if inhaled, swallowed, or absorbed through the skin.
          Formazin can contain residual hydrazine sulfate.  Proper protection
          should be employed.

6.0  EQUIPMENT AND SUPPLIES

     6.1  The turbidimeter shall consist of a nephelometer, with light source
          for illuminating the sample, and one or more photo-electric
          detectors with a readout device to indicate the intensity of light
          scattered at right angles to the path of the incident light.  The
          turbidimeter should be designed so that little stray light reaches
          the detector in the absence of turbidity and should be free from
          significant drift after a short warm-up period.

     6.2  Differences in physical design of turbidimeters will cause
          differences in measured values for turbidity, even though the same
          suspension is used for calibration.  To minimize such differences,
          the following design criteria should be observed:

          6.2.1  Light source:  Tungsten lamp operated at a color temperature
                 between 2200-3000°K.

          6.2.2  Distance traversed by incident light and scattered light
                 within the sample tube:  Total not to exceed 10 cm.

          6.2.3  Detector:  Centered at 90° to the incident light path and not
                 to exceed ± 30° from 90°.  The detector, and filter system if
                 used, shall have a spectral peak response between 400 and 600
                 nm.

     6.3  The sensitivity of the instrument should permit detection of a
          turbidity difference of 0.02 NTU or less in waters having
          turbidities less than 1 unit.  The instrument should measure from 0
          to 40 units turbidity.  Several ranges may be necessary to obtain
          both adequate coverage and sufficient sensitivity for low
          turbidities.

     6.4  The sample tubes to be used with the available instrument must be  of
          clear, colorless glass or plastic.  They should be kept scrupulously
          clean, both inside and out, and discarded when they become scratched
          or etched.  A light coating of silicon oil may be used to mask minor
          imperfections in glass tubes.  They must not be handled at all where
          the light strikes them, but should be provided with sufficient extra
          length, or with a protective case, so that they may be handled.

                                    180.1-4

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        ,   Tubes should be checked,  indexed and read at the orientation  that
           produces the lowest background blank value.

      6.5   Balance —  Analytical,  capable of accurately weighing  to  the  nearest
           0.0001  g.

      6.6   Glassware — Class  A volumetric flasks  and pi pets as required.

 7.0   REAGENTS  AND STANDARDS

      7.1   Reagent water,  turbidity-free:   Pass deionized  distilled  water
           through a 0.45/a pore size  membrane filter,  if such filtered water
           shows a lower turbidity than  unfiltered distilled water.

      7.2   Stock standard  suspension  (Formazin):

           7.2.1  Dissolve 1.00 g  hydrazine sulfate,  (WU2.H2S04,  (CASRN 10034-
                  93-2)  in reagent water and dilute to  100 ml in  a volumetric
                  flask.   CAUTION—CARCINOGEN

           7.2.2  Dissolve 10.00 g hexamethylenetetramine  (CASRN  100-97-0)  in
                  reagent  water and dilute to 100  ml  in  a  volumetric flask.  In
                  a 100  ml volumetric flask,  mix 5.0  ml  of each solution (7.2.1
                  + 7.2.2).  Allow to stand 24  hours  at  25 ± 3°C,  then dilute
                  to the mark  with reagent water.

      7.3   Primary calibration standards:   Mix  and  dilute  10.00 ml of stock
           standard suspension (7.2)  to  100 ml  with  reagent  water.  The
           turbidity of  this suspension  is defined  as 40 NTU.  For other
           values,  mix  and dilute  portions of this  suspension  as  required.

           7.3.1   A new  stock  standard suspension  (7.2) should be  prepared each
                  month.   Primary  calibration standards  (7.3)  should be
                  prepared daily by dilution  of the stock  standard suspension.

      7.4   Formazin in commercially prepared  primary  concentrated  stock
           standard suspension  (SSS)  may be  diluted and used  as required.
           Dilute  turbidity standards should  be prepared daily.

      7.5   AMCO-AEPA-1 Styrene  Divinyl benzene polymer primary standards are
           available for specific  instruments and require  no preparation or
           dilution prior  to use.

      7.6   Secondary standards may be acceptable as a daily calibration check,
           but must be monitored on a routine basis for deterioration and
           replaced as required.

8.0  SAMPLE COLLECTION. PRESERVATION AND STORAGE

     8.1  Samples should  be collected in plastic or glass  bottles.  All
           bottles must be thoroughly cleaned and rinsed with turbidity free
          water.  Volume  collected should be sufficient to insure a

                                    180.1-5

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          representative sample, allow for replicate analysis (if required),
          and minimize waste disposal.

     8.2  No chemical preservation is required.  Cool  sample to 4°C.

     8.3  Samples should be analyzed as soon as possible after collection.   If
          storage is required, samples maintained at 4°C may be held  for up to
          48 h.

9.0  QUALITY CONTROL

     9.1  Each laboratory using this method is required to operate a  formal
          quality control (QC) program.  The minimum requirements of  this
          program consist of an initial demonstration of laboratory capability
          and analysis of laboratory reagent blanks and other solutions as a
          continuing check on performance.  The laboratory is required to
          maintain performance records that define the quality of data
          generated.

     9.2  INITIAL DEMONSTRATION OF PERFORMANCE.

          9.2.1  The initial demonstration of performance is used to
                 characterize instrument performance (determination of LCRs
                 and analysis of QCS).

          9.2.2  Linear  Calibration Range  (LCR) -- The LCR must be determined
                 initially and verified every 6 months or whenever a
                 significant change in instrument response is observed or
                 expected.  The initial demonstration of linearity must use
                 sufficient standards to insure that the resulting curve is
                 linear.  The verification of linearity must use a minimum of
                 a blank and three standards.  If any verification data
                 exceeds the initial  values by ± 10%, linearity must be
                 reestablished.   If any portion of the range is shown to be
                 nonlinear, sufficient standards must be used to clearly
                 define  the nonlinear portion.

          9.2.3  Quality Control  Sample (QCS) — When beginning the use of
                 this method, on  a quarterly basis or as required to meet
                 data-quality needs,  verify the calibration  standards and
                 acceptable instrument performance with the  preparation and
                 analysis of  a  QCS.   If the determined concentrations are  not
                 within  ± 10% of  the  stated values, performance of the
                 determinative  step of the method is unacceptable.  The source
                 of  the  problem must  be identified and corrected before
                 continuing with  on-going  analyses.

     9.3  ASSESSING  LABORATORY  PERFORMANCE

          9.3.1  Laboratory Reagent Blank  (LRB) — The laboratory must  analyze
                 at  least one LRB with each batch of samples.  Data produced


                                    180.1-6

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                  are used to assess contamination from the laboratory
                  environment.

           9.3.2  Instrument Performance Check Solution (IPC) — For all
                  determinations,  the laboratory must analyze the IPC (a  mid-
                  range check standard) and a calibration blank immediately
                  following daily  calibration,  after every tenth sample (or
                  more frequently,  if required)  and at the end of the sample
                  run.  Analysis of the IPC solution and calibration blank
                  immediately following calibration must verify that the
                  instrument is within ± 10% of  calibration.   Subsequent
                  analyses of the  IPC solution must verify the calibration is
                  still  within ± 10%.   If the calibration cannot be  verified
                  within the specified limits, reanalyze the  IPC solution.   If
                  the second analysis  of the IPC solution confirms calibration
                  to  be  outside the limits,  sample analysis must be
                  discontinued,  the cause determined and/or in the case of
                  drift  the instrument recalibrated.   All  samples following  the
                  last acceptable  IPC  solution must be reanalyzed.   The
                  analysis data of  the calibration blank and  IPC solution  must
                  be  kept  on file with the sample  analyses data.  NOTE:
                  Secondary calibration standards  (SS)  may also  be used as the
                  1 i U •

          9.3.3   Where  additional  reference  materials  such as  Performance
                  Evaluation samples are  available,  they should  be analyzed  to
                  provide  additional performance data.   The analysis  of
                  reference  samples  is  a  valuable  tool  for demonstrating the
                  ability  to perform the  method  acceptably.

10.0 CALIBRATION  AND STANDARDIZATION

     10.1 Turbidimeter  calibration:  The manufacturer's operating instructions
          should  be  followed.  Measure standards  on the turbidimeter covering
          the range  of  interest.   If the  instrument is  already calibrated in
          standard turbidity units, this procedure will check the accuracy of
          the calibration scales.   At  least one standard should be run in each
          instrument range  to be used.  Some instruments permit adjustments of
          sensitivity so that scale values will  correspond to turbidities.
          Solid standards,  such as those made of lucite blocks, should never
          be used due to potential  calibration  changes caused by surface
          scratches.   If a pre-calibrated scale  is not supplied, calibration
          curves should be prepared for each range of the instrument.

11.0 PROCEDURE

     11.1 Turbidities less than 40 units:  If possible, allow samples to  come
          to room temperature before analysis.   Mix the sample to thoroughly
          disperse the solids.  Wait until air bubbles disappear then pour the
          sample into the turbidimeter tube.  Read the turbidity directly from
          the instrument scale or  from the appropriate calibration curve.


                                   180.1-7

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     11  2 Turbidities  exceeding  40  units:   Dilute the sample with one or more
         volumes  of turbidity- free water  until the turbidity falls below 40
         units    The  turbidity  of  the  original sample  is then computed from
         the  turbidity of the diluted  sample  and the dilution factor.  For
         example   if  5 volumes  of  turbidity- free water were added to 1 volume
         of sample, and the diluted  sample showed a turbidity of 30 units,
         then the turbidity of  the original sample was 180 units,

         11.2.1 Some  turbidimeters are equipped with several separate  scales.
                The higher scales  are  to  be used only  as indicators of
                required dilution  volumes to  reduce readings to less than 40
                NTU.

                NOTE  1:  Comparative work performed in the Environmental
                Monitoring Systems Laboratory -  Cincinnati  (EMSL-Cincinnati)
                 indicates a progressive error on  sample  turbidities  in excess
                 of 40 units.

12.0 DATA ANALYSTS AND CALCULATIONS

     12.1 Multiply sample readings by appropriate dilution  to  obtain  final
          reading.
     12.2 Report results as follows:
           0.0 - 1.0
             1-10
            10-40
            40 - 100
           100 - 400
           400 - 1000
              > 1000

13.0 METHOD PERFORMANCE
                                                    Record to Nearest:

                                                           O-05
                                                           0.1
                                                           5
                                                          1°
                                                          50
                                                         100
     13.1  In a single laboratory  (EMSL-Cincinnati), using surface water
           samples  at levels of  26, 41, 75 and 180 NTU, the standard deviations
           were ± 0.60, ±  0.94,  ±  1.2  and ± 4.7 units, respectively.

     13.2  The interlaboratory precision and  accuracy data in Table 1 were
           developed using a reagent water matrix.  Values are in NTU.

 14.0 POLLUTION PREVENTION

     14.1  Pollution prevention  encompasses any technique that reduces or
           eliminates the  quantity or  toxicity of waste at the point of
           generation.  Numerous opportunities for pollution prevention exist
           in laboratory operation.  The EPA  has established a preferred
           hierarchy of environmental  management techniques that places
           pollution prevention  as the management option of first choice.

                                    180.1-8

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          Whenever feasible, laboratory personnel should use pollution
          prevention techniques to address their waste generation.  When
          wastes cannot be feasibly reduced at the source, the Agency
          recommends recycling as the next best option.

     14.2 The quantity of chemicals purchased should be based on expected
          usage during its shelf life and disposal cost of unused material.
          Actual reagent preparation volumes should reflect anticipated usage
          and reagent stability.

     14.3 For information about pollution prevention that may be applicable to
          laboratories and research institutions, consult "Less is Better:
          Laboratory Chemical Management .for Waste Reduction," available from
          the American Chemical Society's Department of Government Regulations
          and Science Policy, 1155 16th Street N.W., Washington D.C. 20036,
          (202)872-4477.

15.0 WASTE MANAGEMENT

     15.1 The U.S. Environmental Protection Agency requires that laboratory
          waste management practices be conducted consistent with all
          applicable rules and regulations.  Excess reagents, samples and
          method process wastes should be characterized and disposed of in an
          acceptable manner.  The Agency urges laboratories to protect the
          air, water and land by minimizing and controlling all releases from
          hoods, and bench operations, complying with the letter and spirit of
          any waste discharge permit and regulations, and by complying with
          all solid and hazardous waste 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," available from the
          American Chemical  Society at the address listed in Sect. 14.3.

16.0 REFERENCES

     1.   Annual Book of ASTM Standards, Volume 11.01 Water (1), Standard
          D1889-88A, p. 359, (1993).

     2.   Standard Methods for the Examination of Water and Wastewater, 18th
          Edition, pp. 2-9,  Method 2130B, (1992).
                                    180.1-9

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17.0 TABLES, DIAGRAMS, FLOWCHARTS AND VALIDATION DATA
TABLE 1. INTERLABORATORY PRECISION AND ACCURACY DATA
NUMBER OF
VALUES
REPORTED
373
374
289
482
484
489
640
487
288
714
641
TRUE
VALUE
(T)
0.450
0.600
0.65
0.910
0.910
1.00
1.36
3.40
4.8
5.60
5.95
MEAN
(X)
0.4864
0.6026
0.6931
0.9244
0.9919
0.9405
1.3456
3.2616
4.5684
5.6984
5.6026
RESIDUAL
FOR X
0.0027
-0.0244
0.0183
0.0013
0.0688
-0.0686
-0.0074
-0.0401
-0.0706
0.2952
-0.1350
STANDARD
DEVIATION
(S)
0.1071
0.1048
0.1301
0.2512
0.1486
0.1318
0.1894
0.3219
0.3776
0.4411
0.4122
RESIDUAL
FOR S
-0.0078
-0.0211
0.0005
0.1024
-0.0002
-0.0236
0.0075
-0.0103
-0.0577
-0.0531
-0.1078
REGRESSIONS:  X = 0.955T + 0.54, S = 0.074T + 0.082
                                    180.1-10

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                     METHOD 300.0

DETERMINATION OF INORGANIC ANIONS BY ION CHROMATOGRAPHY
                     John D.  Pfaff
               Inorganic Chemistry Branch
              Chemistry Research  Division
                      Revision 2.1
                      August 1993
      ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
          OFFICE OF RESEARCH AND DEVELOPMENT
         U.S. ENVIRONMENTAL PROTECTION AGENCY
                CINCINNATI,  OHIO  45268
                        300.0-1

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                                METHOD 300.0

           DETERMINATION OF INORGANIC ANIONS BY ION CHROMATOGRAPHY
1.0  SCOPE AND APPLICATION

     1.1  This method covers the determination of the following inorganic
          anions:
             PART A.

             Bromide
             Chloride
             Fluoride
             Nitrate

             PART B.

             Bromate
             Chlorate
Nitrite
Ortho-Phosphate-P
Sulfate
Chlorite
     1.2  The matrices applicable to each method are shown below:

          A.   Drinking water, surface water, mixed domestic and industrial
               wastewaters, groundwater, reagent waters, solids (after
               extraction 11.7), leachates (when no acetic acid is used).

          B.   Drinking water and reagent waters

     1.3  The single laboratory Method Detection Limit  (MDL defined in Sect.
          3.2) for the above analytes is listed in Tables 1A and IB.  The MDL
          for a specific matrix may differ from those listed, depending upon
          the nature of the sample.

     1.4  Method A is recommended for drinking and wastewaters.  The
          multilaboratory ranges tested for each anion  are as follows:

                 Analyte

                 Bromide
                 Chloride
                 Fluoride
                 Nitrate-N
                 Nitrite-N
                 Ortho-Phosphate-P
                 Sulfate
0.63
0.78
0.26
0.42
0.36
0.69
2.85
- 21.0
- 26.0
- 8.49
- 14.0
- 12.0
- 23.1
- 95.0
                                    300.0-2

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      1.5  This method is recommended for use only by or under the  supervision
           of analysts experienced in the use of ion  chromatography and  in  the
           interpretation of the resulting ion chromatograms.

      1.6  When this  method is  used to analyze unfamiliar samples for  any of
           the above  anions,  anion identification  should be  supported  by the
           use of a fortified sample matrix covering  the anions of  interest.
           The fortification  procedure is described in Sect.  11.6.

      1.7  Users of the method  data should state the  data-quality objectives
           prior to analysis.   Users of the method must demonstrate the  ability
           to generate acceptable  results with this method,  using the
           procedures described in Sect.  9.0.

2.0   SUMMARY OF METHOD

      2.1   A  small volume  of  sample,  typically 2 to 3  ml,  is  introduced  into
           an  ion  chromatograph.   The anions  of interest  are  separated and
           measured,  using  a  system comprised  of a guard  column, analytical
           column, suppressor device,  and  conductivity detector.

      2.2   The  main differences  between  Parts  A and B  are the  separator columns
           and  guard  columns.   Sections  6.0 ,and  7,0 will  elicit the
           differences.

      2.3   An extraction procedure  must  be performed to use this method for
           solids  (See  11.7).

      2.4   Limited performance-based method modifications may be acceptable
           provided they are fully  documented  and meet or exceed requirements
           expressed  in Sect. 9.0,  Quality Control.

3.0  DEFINITIONS

     3.1  CALIBRATION BLANK (CB) — A volume of reagent water fortified with
          the same matrix as the calibration standards, but without the
          analytes,  internal standards, or surrogate  analytes.

     3.2  CALIBRATION STANDARD  (CAL) — A solution prepared from the primary
          dilution standard solution or stock standard solutions and the
          internal standards and surrogate analytes.   The CAL solutions  are
          used to calibrate the instrument response with respect to analyte
          concentration.

     3.3  FIELD DUPLICATES (FD) — 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  field
          duplicates  indicate the precision associated with  sample  collection
          preservation and storage, as well  as with laboratory procedures.

     3.4  INSTRUMENT  PERFORMANCE CHECK SOLUTION (IPC)  - A solution of one  or
          more method analytes, surrogates,  internal  standards,  or  other test

                                   300.0-3

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     substances used to evaluate the performance of the instrument system
     with respect to a defined set of criteria.

3.5  LABORATORY FORTIFIED BLANK (LFB) — An aliquot of reagent water or
     other blank matrices to which known quantities of the method
     analytes are added in the laboratory.  The LFB is analyzed exactly
     like a sample, and its purpose is to determine whether the
     methodology is in control, and whether the laboratory is capable of
     making accurate and precise measurements.

3.6  LABORATORY FORTIFIED SAMPLE MATRIX (LFM) — An aliquot of an
     environmental sample to which known quantities of the method
     analytes are added in the laboratory.  The LFM 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 LFM
     corrected for background concentrations.

3.7  LABORATORY REAGENT BLANK (LRB) — An aliquot of reagent water or
     other blank matrices that are treated exactly as a sample including
     exposure to all glassware, equipment, solvents, reagents, internal
     standards, and surrogates that are used with other samples.  The LRB
     is used to determine if method analytes or other interferences are
     present in the laboratory environment, the reagents, or the
     apparatus.

3.8  LINEAR CALIBRATION RANGE (LCR) — The concentration range over which
     the instrument response is linear.

3.9  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.

3.10 METHOD DETECTION LIMIT  (MDL) — The minimum concentration of an
     analyte that can be identified, measured  and reported with 99%
     confidence that the analyte concentration is greater than zero.

3.11 PERFORMANCE EVALUATION SAMPLE  (PE) — A solution of method analytes
     distributed by the Quality Assurance Research Division  (QARD),
     Environmental Monitoring Systems Laboratory (EMSL-Cincinnati), U. S.
     Environmental Protection Agency, Cincinnati, Ohio, to multiple
     laboratories for analysis.  A volume of the solution is added to a
     known volume of reagent water and analyzed with procedures used for
     samples.  Results of analyses are used by QARD to determine
     statistically the accuracy and precision  that can be expected when a
     method is performed by  a competent analyst.  Analyte true values are
     unknown to the analyst.

3.12 QUALITY CONTROL SAMPLE  (QCS) — A solution of method analytes of
     known concentrations that  is used to fortify an aliquot of LRB or

                               300.0-4

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          sample matrix.  The QCS is obtained from a source external to the
          laboratory and different from the source of calibration standards.
          It is used to check laboratory performance with externally prepared
          test materials.

     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.

4.0  INTERFERENCES

     4.1  Interferences can be caused by substances with retention times that
          are similar to and overlap those of the anion of interest.  Large
          amounts of an anion can interfere with the peak resolution of an
          adjacent anion.  Sample dilution and/or fortification can be, used to
          solve most interference problems associated with retention times.

     4.2  The water dip or negative peak that elutes near, and can interfere
          with, the fluoride peak can usually be eliminated by the addition of
          the equivalent of 1 mL of concentrated eluent (7.3 100X) to 100 mL
          of each standard and sample.

     4.3  Method interferences may be caused by contaminants in the reagent
          water, reagents, glassware, and other sample processing apparatus
          that lead to discrete artifacts or elevated baseline in ion
          chromatograms.

     4.4  Samples that contain particles larger than 0.45 microns and reagent
          solutions that contain particles "larger than 0.20 microns require
          filtration to prevent damage  to instrument columns and flow systems.

     4.5  Any anion that is not retained by the column or only slightly
          retained will  elute in the area of fluoride and interfere.   Known
          coelution is caused by carbonate and other small  organic anions.   At
          concentrations of fluoride above 1.5 mg/L,  this interference may not
          be significant,  however,  it is the responsibility of the user to
          generate precision and accuracy information in each  sample matrix.

     4.6  The acetate anion elutes  early during the chromatographic run.   The
          retention times of the anions also seem to  differ when large amounts
          of acetate are present.   Therefore,  this method is not recommended
          for leachates  of solid samples when  acetic  acid is used for pH
          adjustment.

     4.7  The quantitation of unretained peaks should be avoided,  such as  low
          molecular weight organic  acids (formate,  acetate,  propionate etc.)
          which are conductive and  coelute with  or near  fluoride and  would
          bias  the fluoride quantitation in  some  drinking and  most  waste
          waters.

     4.8  Any residual  chlorine  dioxide present  in  the sample  will  result  in
          the formation  of additional chlorite prior  to  analysis.   If any

                                   300.0-5

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           concentration of chlorine dioxide is suspected in the sample purge
           the sample with an inert gas (argon or nitrogen) for about five
           minutes or until no chlorine dioxide remains.
5.0  SAFETY

     5.1  The toxicity or carcinogenicity of each reagent used in this method
          have not been fully established.  Each chemical should be regarded
          as a potential health hazard and exposure should be as low as
          reasonably achievable.  Cautions are included for known extremely
          hazardous materials or procedures.

     5.2  Each 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 Material
          Safety Data Sheets (MSDS) should be made available to all personnel
          involved in the chemical analysis.  The preparation of a formal
          safety plan is also advisable.

     5.3  The following chemicals have the potential to be highly toxic or
          hazardous, consult MSDS.

          5.3.1   Sulfuric acid (7.4)

6.0  Equipment and Supplies

     6.1  Balance — Analytical, capable of accurately weighing to the nearest
          0.0001 g.

     6.2  Ion chromatograph — Analytical system complete with ion chromato-
          graph and all required accessories including syringes, analytical
          columns, compressed gasses and detectors.

          6.2.1   Anion guard column: A protector of the separator column.  If
                  omitted from the system the retention times will be shorter.
                  Usually packed with a substrate the same as that in the
                  separator column.

          6.2.2   Anion separator column:  This column produces the separation
                  shown in Figures 1 and 2.

                  6.2.2.1   Anion analytical column (Method A):  The
                            separation shown in Figure 1 was generated using a
                            Dionex AS4A column (P/N 37041).  An optional
                            column may be used if comparable resolution of
                            peaks is obtained, and the requirements of Sect.
                            9.2 can be met.

                  6.2.2.2   Anion analytical column (Method B).  The
                            separation shown in Figure 2 was generated using a
                            Dionex AS9 column (P/N 42025).  An optional column
                            may be used if comparable resolution of peaks  is

                                    300.0-6

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                            obtained and the  requirements of Sect.  9.2  can  be
                            met.

          6.2.3   Anion suppressor device:  The data presented  in this  method
                  were generated using a Dionex anion micro membrane
                  suppressor  (P/N 37106).

          6.2.4   Detector — Conductivity cell: approximately  1.25 /zL
                  internal volume, (Dionex, or equivalent) capable  of
                  providing data as required  in Sect. 9.2.

     6.3  The Dionex AI-450 Data Chromatography Software was used to generate
          all the data in the attached tables.  Systems using a stripchart
          recorder and integrator or other computer based data  system may
          achieve approximately the same MDL's but the user should demonstrate
          this by the procedure outlined in Sect. 9.2.

7.0  Reagents and Standards

     7.1  Sample bottles:   Glass or polyethylene of sufficient volume to
          allow replicate analyses of anions of interest.

     7.2  Reagent water:   Distilled or deionized water,  free of the anions of
          interest.   Water should contain particles no larger than 0.20
          microns.

     7.3  Eluent solution  (Method A and Method B):   Sodium bicarbonate (CASRN
          144-55-8)  1.7 mM,  sodium carbonate (CASRN 497-19-8)  1.8 mM.
          Dissolve 0.2856  g sodium bicarbonate (NaHCO,)  and  0.3816 g of sodium
          carbonate  (Na2C03) in reagent water  (7.2) and dilute to 2  L.

     7.4  Regeneration solution  (micro  membrane suppressor):   Sulfuric acid
          (CASRN-7664-93-9)  0.025N.   Dilute 2.8 ml  cone,  sulfuric acid
          (H2S04) to 4 L with reagent water.

     7.5  Stock standard solutions,  1000  mg/L  (1  mg/mL):   Stock standard
          solutions  may be purchased  as certified solutions  or  prepared from
          ACS reagent  grade  materials  (dried at 105°C  for 30 min)  as listed
          below.

          7.5.1    Bromide  (Br~) 1000 mg/L:  Dissolve 1.2876 g sodium bromide
                  (NaBr, CASRN 7647-15-6)  in reagent water and  dilute  to 1  L.

          7.5.2    Bromate  (Br03')  1000 mg/L:  Dissolve 1.1798g of sodium
                  bromate  (NaBr03, CASRN 7789-38-0) in reagent water and
                  dilute to  1  L.

          7.5.3    Chlorate (C103~) 1000  mg/L:   Dissolve 1.2753g of sodium
                  chlorate (NaC103, CASRN 7775-09-9) in reagent water and
                  dilute to  1  L.
                                   300.0-7

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          7.5.4   Chloride (CL~)  1000 mg/L:   Dissolve 1.6485 g sodium
                  chloride (NaCl, CASRN 7647-14-5) in reagent water and
                  dilute to 1 L.

          7.5.5   Chlorite (C102")  1000  mg/L:   Dissolve  1.3410g  of sodium
                  chlorite (NaC102, CASRN 7758-19-2)  in  reagent water and
                  dilute to 1 L.

          7.5.6   Fluoride (F")  1000 mg/L:  Dissolve  2.2100g sodium fluoride
                  (NaF, CASRN 7681-49-4)  in reagent water and dilute to  1 L.

          7.5.7   Nitrate (NO",-N)  1000  mg/L:   Dissolve  6.0679 g sodium
                  nitrate (NaN03,  CASRN 7631-99-4) in reagent water and
                  dilute to 1 L.

          7.5.8   Nitrite (NO~,-N)  1000  mg/L:   Dissolve  4.9257 g sodium
                  nitrite (NaN02,  CASRN 7632-00-0) in reagent water and
                  dilute to 1 L.

          7.5.9   Phosphate (PO=,-P)  1000 mg/L:   Dissolve 4.3937 g potassium
                  phosphate (KH2PO,,  CASRN 7778-77-0)  in reagent water
                  and dilute to 1 L.

          7.5.10  Sulfate (S04=)  1000 mg/L:   Dissolve 1.8141 g potassium
                  sulfate (K,S04,  CASRN  7778-80-5) in reagent water and
                  dilute to 1 L.

                  NOTE:  Stability of standards:  Stock  standards  (7.5)  are
                         stable for  at  least 1 month when stored  at 4°C.
                         Except for  the  chlorite  standard which  is only  stable
                         for two weeks.   Dilute working  standards  should be
                         prepared weekly, except  those that  contain nitrite
                         and phosphate  should be  prepared fresh  daily.

     7.6  Ethylenediamine preservation  solution:  Dilute  10 mL of
          ethylenediamine (99%) (CASRN  107-15-3)  to 200  mL with  reagent
         water. Use 1 mL of this dilution to each 1 L of sample  taken.

8.0  Sample Collection. Preservation and  Storage

     8.1  Samples should be collected in  plastic  or glass bottles.  All
          bottles must be thoroughly cleaned and  rinsed  with  reagent water.
          Volume collected should be sufficient to insure a  representative
          sample, allow for replicate analysis, if required,  and ;minimize
          waste disposal.

     8.2  Sample preservation and holding times for the  anions  that can  be
          determined by this method  are  as follows:

          Analvte              Preservation              Holding Time

          Bromate              None  required                 28  days

                                    300.0-8

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          Bromide              None required                28 days
          Chlorate             None required                28 days
          Chloride             None required                28 days
          Chlorite             Cool to 4°C                  immediately
          Fluoride             None required                28 days
          Nitrate-N            Cool to 4°C                  48 hours
          Combined             cone. H2SO,                  28 days
      (Nitrate/Nitrite)        to a pH < 2
          Nitrite-N            Cool to 4°C                  48 hours
          0-Phosphate-P        Cool to 4°C                  48 hours
          Sulfate              Cool to 4°C                  28 days

          NOTE: If the determined value for the combined
                nitrate/nitrite exceeds 0.5 mg/L as N",  a resample
                must be analyzed for the individual concentrations
                of nitrate and nitrite.

     8.3  The method of preservation and the holding time for samples
          analyzed by this method are determined by the anions of interest.
          In a given sample, the anion that requires the most preservation
          treatment and the shortest holding time will determine the preser-
          vation treatment.  It is recommended that all  samples be cooled to
          4°C and held for no longer than 28 days for Method A and analyzed
          immediately in Method B.

          NOTE:   If the sample cannot be analyzed for chlorite within < 10
                  minutes, the sample may be preserved by adding 1 ml of the
                  ethylenediamine (EDA) preservation solution (7.6) to 1 L
                  of sample.  This will preserve the concentration of the
                  chlorite for up to 14 days. This addition of EDA has no
                  effect on bromate or chlorate, so they can also be
                  determined in a sample preserved with EDA.   Residual
                  chlorine dioxide should be removed from the sample
                  (per 4.8) prior to the addition of EDA.

9.0  QUALITY CONTROL

     9.1  Each laboratory using this method is required to operate a formal
          quality control (QC) program.  The minimum requirements of this
          program consist of an initial demonstration of laboratory
          capability, and the periodic analysis of laboratory reagent blanks,
          fortified blanks and other laboratory solutions as a continuing
          check on performance.  The laboratory is required to maintain per-
          formance records that define the quality of the data that are
          generated.

     9.2  INITIAL DEMONSTRATION OF PERFORMANCE

          9.2.1   The initial demonstration of performance is used to
                  characterize instrument performance (determination of LCRs
                  and analysis of QCS) and laboratory performance


                                    300.0-9

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             (determination of MDLs) prior to performing analyses by this
             method.

     9.2.2   Linear Calibration Range (LCR) — The LCR must be determined
             initially and verified every 6 months or whenever a
             significant change in instrument response is observed or
             expected.  The initial demonstration of linearity must use
             sufficient standards to insure that the resulting curve is
             linear.  The verification of linearity must use a minimum of
             a blank and three standards.  If any verification data
             exceeds the initial values by ± 10%, linearity must be
             reestablished.  If any portion of the range is shown to be
             nonlinear, sufficient standards must be used to clearly
             define the nonlinear portion.

     9.2.3   Quality Control Sample (QCS) — When beginning the use of
             this method, on a quarterly basis or as required to meet
             data-quality needs, verify the calibration standards and
             acceptable instrument performance with the preparation and
             analyses of a QCS.  If the determined concentrations are not
             within ± 10% of the stated values, performance of the
             determinative step of the method is unacceptable.  The
             source of the problem must be identified and corrected
             before either proceeding with the initial determination of
             MDLs or continuing with on-going analyses.

     9.2.4   Method Detection Limit (MDL) — MDLs must be established for
             all analytes, using reagent water (blank) fortified at a
             concentration of two to three times the estimated instrument
             detection limit.    To determine MDL values, take seven
             replicate aliquots of the fortified reagent water and
             process through the entire analytical method.  Perform all
             calculations defined in the method and report the
             concentration values in the appropriate units.  Calculate
             the MDL as follows:

                                MDL = (t) x (S)

             where, t = Student's t value for a 99% confidence level
                        and a standard deviation estimate with n-1
                        degrees of freedom [t = 3.14 for seven
                        replicates].

                    S = standard deviation of the replicate analyses.

             MDLs should be determined every 6 months, when a new
             operator begins work or whenever there is a significant
             change in the background or instrument response.

9.3  ASSESSING LABORATORY PERFORMANCE
                              300.0-10

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9.3.1   Laboratory Reagent Blank (LRB) — The laboratory must
        analyze at least one LRB with each batch of samples.  Data
        produced are used to assess contamination from the
        laboratory environment.  Values that exceed the MDL indicate
        laboratory or reagent contamination should be suspected and
        corrective actions must be taken before continuing the
        analysis.

9.3.2   Laboratory Fortified Blank (LFB) — The laboratory must
        analyze at least one LFB with each batch of samples.
        Calculate accuracy as percent recovery (Sect. 9.4.2).  If
        the recovery of any analyte falls outside the required
        control limits of 90-110%, that analyte is judged out of
        control, and the source of the problem should be identified
        and resolved before continuing analyses.

9.3.3   The laboratory must use LFB analyses data to assess
        laboratory performance against the required control limits
        of 90-110%.  When sufficient internal performance data
        become available (usually a minimum of 20-30 analyses),
        optional control limits can be developed from the percent
        mean recovery (x) and the standard deviation (S) of the mean
        recovery.  These data can be used to establish the upper and
        lower control limits as follows:

                    UPPER CONTROL LIMIT = x + 3S
                    LOWER CONTROL LIMIT = x - 3S

        The optional control limits must be equal to or better than
        the required control limits of 90-110%.  After each five to
        ten new recovery measurements, new control limits can be
        calculated using only the most recent 20-30 data points.
        Also, the standard deviation (S) data should be used to
        establish an on-going precision statement for the level of
        concentrations included in the LFB.  These data must be kept
        on file and be available for review.

9.3.4   Instrument Performance Check Solution (IPC) — For all
        determinations the laboratory must analyze the IPC (a mid-
        range check standard) and a calibration blank immediately
        following daily calibration, after every tenth sample (or
        more frequently, if required) and at the end of the sample
        run.  Analysis of the IPC solution and calibration blank
        immediately following calibration must verify that the
        instrument is within ± 10% of calibration.  Subsequent
        analyses of the IPC solution must verify the calibration is
        still within ± 10%.  If the calibration cannot be verified
        within the specified limits, reanalyze the IPC solution.  If
        the second analysis of the IPC solution confirms calibration
        to be outside the limits, sample analysis must be
        discontinued, the cause determined and/or in the case of
        drift, the instrument recalibrated.  All  samples following

                         300.0-11

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             the last acceptable IPC solution must be reanalyzed.  The
             analysis data of the calibration blank and IPC solution must
             be kept on file with the sample analyses data.

9.4  ASSESSING ANALYTE RECOVERY AND DATA QUALITY

     9.4.1   Laboratory Fortified Sample Matrix (LFM) — The laboratory
             must add a known amount of analyte to a minimum of 10% of
             the routine samples.  In each case the LFM aliquot must be a
             duplicate of the aliquot used for sample analysis.  The
             analyte concentration must be high enough to be detected
             above the original sample and should not be less than four
             times the MDL.  The added analyte concentration should be
             the same as that used in the laboratory fortified blank.

             9.4.1.1   If the concentration of fortification is less than
                       25% of the background concentration of the matrix
                       the matrix recovery should not be calculated.

     9.4.2   Calculate the percent recovery for each analyte, corrected
             for concentrations measured in the unfortified sample, and
             compare these values to the designated LFM recovery range
             90-110%.  Percent recovery may be calculated using the
             following equation:
                                      II
                               C. - C
X 100
             where,   R  ~  percent recovery.
                      Cs =  fortified  sample concentration.
                      C  -  sample background concentration.
                      s  »  concentration equivalent of analyte added to
                            sample.

     9.4.3   Until sufficient data becomes available (usually a minimum
             of 20 to 30 analysis), assess laboratory performance against
             recovery limits for method A of 80 to 120% and 75 to 125%
             for method B. When sufficient internal performance data
             becomes available develop control limits from percent mean
             recovery and the standard deviation of the mean recovery.

     9.4.4   If the recovery of any analyte falls outside the designated
             LFM recovery range and the laboratory performance for that
             analyte is shown to be in control (Sect. 9.3),  the recovery
             problem encountered with  the LFM is judged to be either
             matrix or solution related, not system related.

     9.4.5   Where reference materials are available, they should be
             analyzed to provide additional performance data.  The
                              300.0-12
                                                                               4»

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                  analysis of reference samples  is  a  valuable  tool  for
                  demonstrating the ability  to perform the  method  acceptably.

          9.4.6   In recognition of the rapid  advances occurring  in chromatog-
                  raphy,  the analyst is permitted certain options,  such  as  the
                  use of  different columns  and/or eluents,  to  improve the
                  separations or lower the  cost  of  measurements.   Each time
           :       such modifications to the  method  are made, the  analyst is
                  required to repeat the procedure  in Sect.  9.2.

          9.4.7   It is recommended that the laboratory adopt  additional
        : .         quality assurance practices  for use with  this method.  The
                  specific practices that are  most  productive  depend upon the
                  needs of the laboratory and  the nature of the samples.
                  Field duplicates may be analyzed  to monitor  the  precision of
                  the sampling technique.  When  doubt exists over  the
                  identification of a peak  in  the chromatogram, confirmatory
                  techniques such as sample  dilution  and fortification,  must
                  be used.  Whenever possible, the  laboratory  should perform
                  analysis of quality control  check samples and participate in
                  relevant performance evaluation sample studies.

          9.4.8   At least quarterly, replicates of LFBs should be analyzed to
                  determine the precision of the laboratory measurements.   Add
                  these results to the on-going  control  charts to  document
                  data quality.

          9.4.9   When using Part B, the analyst should be  aware  of the  purity
                  of the  reagents used to prepare standards.   Allowances must
                  be made when the solid materials  are less than  99% pure.

10.0 Calibration and Standardization

     10.1 Establish ion chromatographic operating parameters equivalent  to
          those indicated in Tables 1A or IB.

   ,10.2 For each analyte of interest, prepare  calibration standards at a
          minimum of three concentration levels  and a blank by adding
      :    accurately measured volumes of one or  more  stock  standards (7.5)  to
          a volumetric flask and diluting to volume with reagent  water.   If
          a sample analyte concentration exceeds the  calibration  range the
          sample may be diluted to fall within the'range.   If  this is not
          possible then three new calibration  concentrations must  be chosen,
          two of which must bracket the concentration of the sample analyte of
          interest.  Each attenuation range of the  instrument  used to analyze
          a sample must be calibrated individually.

     10.3 Using injections of 0.1 to 1.0 ml (determined by  injection loop
          volume) of each calibration standard,  tabulate peak  height or  area
          responses against the concentration.   The results are used to
          prepare a calibration curve for each analyte.  During this pro-
          cedure, retention times must be recorded.

                                   300.0-13

-------
     10.4 The calibration curve must be verified on each working day,  or
          whenever the anion eluent is changed, and after every 20
          samples.  If the response or retention time for any analyte  varies
          from the expected values by more than ± 10%, the test must be
          repeated, using fresh calibration standards.  If the results are
          still more than ± 10%, a new calibration curve must be prepared
          for that analyte.

     10.5 Nonlinear response can result when the separator column capacity is
          exceeded (overloading).  The response of the detector to the sample
          when diluted 1:1, and when not diluted, should be compared.   If the
          calculated responses are the same, samples of this total anionic
          concentration need not be diluted.

11.0 Procedure

     11.1 Tables 1A and IB summarize the recommended operating conditions for
          the ion chromatograph.  Included in these tables are estimated
          retention times that can be achieved by this method.  Other  columns,
          chromatographic conditions, or detectors may be used if the
          requirements of Sect. 9.2 are met.

     11.2 Check system calibration daily and, if required, recalibrate as
          described in Sect. 10.

     11.3 Load and inject a fixed amount of well mixed sample.  Flush
          injection loop thoroughly, using each new sample.   Use the same size
          loop for standards and samples.   Record the resulting peak size in
          area or peak height units.  An automated constant volume injection
          system may also be used.

     11.4 The width of the retention time  window used to make identifications
          should be based upon measurements of actual  retention time varia-
          tions of standards over the course of a day.  Three times the
          standard deviation of a retention time can be used to calculate a
          suggested window size for each analyte.  However,  the experience of
          the analyst should weigh heavily in the interpretation of
          chromatograms.

     11.5 If the response for the peak exceeds the working range of the
          system,  dilute the sample with an appropriate amount of reagent
          water and reanalyze.

     11.6 If the resulting chromatogram fails to produce adequate resolution,
          or if identification of specific anions is questionable,  fortify the
          sample with an appropriate amount of standard and  reanalyze.

          NOTE:    Retention time is inversely proportional  to concentration.
                  Nitrate and sulfate exhibit the greatest amount of change,
                  although all  anions are  affected to  some degree.   In  some
                  cases this peak migration may produce poor resolution or
                  identification.

                                   300.0-14

-------
      11.7 The following extraction should be used for solid materials.  Add an
           amount of reagent water equal to ten times the weight of dry solid
           material taken as a sample.  This slurry is mixed for ten minutes
           using a magnetic stirring device.,  Filter the resulting slurry
           before injecting using a 0.45 /z membrane type filter.  This can be
           the type that attaches directly to the end of the syringe.  Care
           should be taken to show that good recovery and identification of
           peaks is obtained with the user's matrix through the use of
           fortified samples.

      11.8 It has been reported that lower detection limits for bromate
           («7 /jg/L) can be obtained using a. borate based eluent(7).  The use
           of  this eluent or other eluents that improve method performance may
           be considered as a minor modification of the method and as such
           still  are acceptable.

      11.9 Should more complete resolution be needed between peaks the eluent
           (7.3)  can be diluted.   This will  spread  out the  run but will  also
           cause  the later eluting anions  to be  retained longer.   The analyst
           must determine to what extent the eluent is diluted.   This dilution
           should not be considered a  deviation  from the method.

 12.0  DATA ANALYSIS AND CALCULATIONS

      12.1  Prepare  a calibration  curve for each  analyte  by  plotting  instrument
           response against  standard concentration.   Compute sample
           concentration  by  comparing  sample  response  with  the  standard curve
           Multiply answer  by  appropriate  dilution  factor.

      12.2  Report only  those values that fall between  the lowest  and  the
           highest  calibration  standards.  Samples  exceeding  the  highest
           standard  should be diluted  and  reanalyzed.

      12.3  Report results in mg/L.

      12.4  Report  NO ~   as  N
                  NO '   as  N
                  HP04= as  P

13.0 METHODS PERFORMANCE

     13.1 Tables 1A and 2A give the single laboratory (EMSL-Cincinnati) MDL
          for each anion included in the method under the conditions listed.

     13.2 Tables 2A and 2B give the single laboratory (EMSL-Cincinnati)
          standard deviation for each anion included in the method in a
          variety of waters for the listed conditions.

     13.3 Multiple laboratory accuracy and bias  data (St) and estimated single
          operator values (S0)  for reagent,  drinking  and  waste water  using
                                   300.0-15

-------
          method A are given for each anion in Tables 3 through 9.
          19 laboratories were used for this data.
Data from
     13.4 Some of the bias statements, for example chloride and sulfate,  may
          be misleading due to spiking small increments of the anion into
          large naturally occurring concentrations of the same anion.

14.0 POLLUTION PREVENTION

     14.1 Pollution prevention encompasses any technique that reduces  or
          eliminates the quantity or toxicity of waste at the point of
          generation.  Numerous opportunities for pollution prevention exist
          in laboratory operation.  The EPA has established a preferred
          hierarchy of environmental management techniques that places
          pollution prevention as the management option of first choice.
          Whenever feasible, laboratory personnel should use pollution
          prevention techniques to address their waste generation.  When
          wastes cannot be feasibly reduced at the source, the Agency
          recommends recycling as the next best option.

     14.2 Quantity of the chemicals purchased should be based on expected
          usage during its shelf life and disposal cost of unused material.
          Actual reagent preparation volumes should reflect anticipated usage
          and reagent stability.

     14.3 For information about pollution prevention that may be applicable to
          laboratories and research institutions, consult "Less is Better:
          Laboratory Chemical Management for Waste Reduction," available from
          the American Chemical Society's Department of Government
          Regulations and Science Policy, 1155 16th Street N.W., Washington
          D.C. 20036, (202) 872-4477.

15.0 WASTE MANAGEMENT

     15.1 The Environmental Protection Agency requires that laboratory waste
          management practices be conducted consistent with all applicable
          rules and regulations.  Excess reagents, samples and method
          process wastes should be characterized and disposed of in an
          acceptable manner.  The Agency urges laboratories to protect the
          air, water, and land by minimizing and controlling all releases from
          hoods and bench operations, complying with the letter and spirit of
          any waste discharge permit  and regulations,  and by complying with
          all solid and hazardous waste 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," available
          from the American Chemical  Society at the address listed  in Sect.
          14.3.
                                   300.0-16

-------
16.0 REFERENCES                           -f

     1.    "Determination of Inorganic Disinfection By-Products by Ion
          Chromatography", J.  Pfaff, C. Brockhoff.  J.  Am. Water Works Assoc.,
          Vol  82,  No. 4, pg 192.

     2.    Standard Methods for the Examination of Water and Wastewater,
          Method 4110B,  "Anions by Ion Chromatography", 18th Edition of
          Standard Methods (1992).

     3.    Dionex,  System 4000  Operation and Maintenance Manual,  Dionex
          Corp., Sunnyvale, California 94086,  1988.

     4.    Method Detection Limit (MDL) as described  in  "Trace Analyses for
          Wastewater,"  J.  Glaser,  D. Foerst,  G.  McKee,  S.  Quave,  W.  Budde,
          Environmental  Science and Technology,  Vol.  15,  Number  12,  page
          1426,  December,  1981.

     5.    American Society for Testing and Materials. Test Method for Anions
          in Water by Chemically-Suppressed Ion  Chromatography D4327-91.
          Annual Book of Standards,  Vol  11.01  (1993).

     6.    Code  of  Federal  Regulations 40,  Ch.  1,  Pt.  136,  Appendix B.

     7.    Hautman,  D.P.  &  Bolyard,  M.  Analysis of Oxyhalide Disinfection  By-
          products and  other Anions  of Interest  in Drinking Water by Ion
          Chromatography.   Jour, of Chromatog.,  602,  (1992),  65-74.
                                  300.0-17

-------
17.0 TABLES. DIAGRAMS. FLOWCHARTS AND VALIDATION DATA


          TABLE 1A.  CHROMATOGRAPHIC CONDITIONS AND DETECTION LIMITS
                     IN REAGENT WATER (PART A)
            ANALYTE
      *   RETENTION
PEAK #    TIME(MIN)     MDL
                       mg/L
Fluoride
Chloride
Nitrite-N
Bromide
Nitrate-N
o-Phosphate-P
Sulfate
1
2
3
4
5
6
7
1.2
1.7
2.0
2.9
3.2
5.4
6.9
0.01
0.02
0.004
0.01
0.002
0.003
0.02
    Standard Conditions:

    Columns: as specified in 6.2.2.1
    Detector: as specified in 6.2.4
    Eluent: as specified in 7.3
               Pump Rate: 2.0 mL/min.
               Sample Loop: 50 /uL
    MDL calculated from data system using a y-axis selection of
    1000 ns and with a stripchart recorder with an attenuator
    setting of 1 uMHO full scale.

    *  See Figure 1
                                    300.0-18

-------
TABLE IB.  CHROMATOGRAPHIC CONDITIONS AND DETECTION LIMITS
           IN REAGENT WATER (PART B)
ANALYTE
Chlorite
Bromate
Chlorate
*
PEAK #
1
2
4
RETENTION
TIME(MIN)
2.8
3.2
7.1
MDL
mg/L
0.01
0.02
0.003
Standard Conditions:

Column: as specified in 6.2.2.2
Detector: as specified in 6.2.4
Eluent: as specified in 7.3
  * See Figure 2
                                  Pump Rate:  1.0 mL/min,
                                  Sample Loop:  50 #L
                                   Attentuation - 1
                                   y-axis -  500 ns
                        300.0-19

-------
TABLE 2A.  SINGLE-OPERATOR ACCURACY AND BIAS OF STANDARD ANIONS
                             (METHOD A)
ANALYTE
Bromide





Chloride





Fluoride





Nitrate- N





Nitrite- N





o-Phosphate- P



SAMPLE
TYPE
RW
DW
SW
WW
GW
SD
RW
DW
SW
WW
GW
SD
RW
DW
SW
WW
GW
SD
RW
DW
SW
WW
GW
SD
RW
DW
SW
WW
GW
SD
RW
DW
SW
WW
GW
KNOWN NUMBER MEAN
CONC. OF RECOVERY
fma/L} REPLICATES %
5.0
5.0
5.0
5.0
5.0
2.0
20.0
20.0
10.0
20.0
20.0
20.0
2.0
1.0
1.0
1.0
0.4
5.0
10.0
10.0
10.0
10.0
10.0
10.0
10.0
10.0
5.0
5.0
10.0
2.0
10.0
10.0
10.0
10.0
10.0
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
99
105
95
105
92
82
96
108
86
101
114
90
91
92
73
87
95
101
103
104
93
101
97
82
97
121
92
91
96
98
99
99
98
106
95
STANDARD
DEVIATION
Ona/n
0.08
0.10
0.13
0.34
0.34
0.06
0.35
1.19
0.33
5.2
1.3
0.32
0.05
0.06
0.05
0.07
0.07
0.35
0.21
0.27
0.17
0.82
0.47
0.28
0.14
0.25
0.14
0.50
0.35
0.08
0.17
0.26
0.22
0.85
0.33
                                300.0-20

-------
                           TABLE  2A  (CONT'D)
Sulfate            RW     20.0        7       99        0.40
                   DW     50.0        7      105        3.35
                   SW     40.0        7       95        1.7
                   WW     40.0        7      102        6.4
                   GW     40.0        7      112        3.2

  RW = Reagent Water      WW = Mixed Domestic and Industrial Wastewater
  DW = Drinking Water     GW = Groundwater
  SW = Surface Water      SD = USEPA QC Solid (shale)
                              300.0-21

-------
TABLE 2B. SINGLE-OPERATOR ACCURACY AND BIAS OF BY-PRODUCT
                             (PART B)
ANALYTE
                  NUMBER     MEAN   STANDARD
SAMPLE   SPIKE      OF     RECOVERY DEVIATION
 TYPE   (mg/L)  REPLICATES    %      (mg/L)
Bromate RW 5.0
1.0
0.1
0.05
DW 5.0
1.0
0.1
0.05
Chlorate RW 5.0
1.0
0.1
0.05
DW 5.0
1.0
0.1
0.05
Chlorite RW 5.0
1.0
0.1
0.05
DW 5.0
1.0
0.1
0.05
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
103
98
155
122
95
85
98
98
101
97
100
119
101
115
121
110
100
98
86
94
96
100
76
96
0.07
0.04
0.005
0.01
0.04
0.02
0.005
0.005
0.06
0.01
0.01
0.05
0.04
0.01
0.005
0.01
0.04
0.01
0.01
0.01
0.03
0.02
0.00
0.01
  RW s  Reagent  Water
  DW =  Drinking Water
                                300.0-22

-------
TABLE 3.     MULTIPLE LABORATORY (n=19)
             DETERMINATION OF BIAS FOR FLUORIDE
WATER
Reagent





Drinking





Waste





AM'T ADDED
mg/L
0.26
0.34
2.12
2.55
6.79
8.49
0.26
0.34
2.12
2.55
6.79
8.49
0.26
0.34
2.12
2.55
6.79
8.49
AM'T FOUND
mg/L
0.25
0.29
2.12
2.48
6.76
8.46
0.24
0.34
2.09
2.55
6.84
8.37
0.25
0.32
2.13
2.48
6.65
8.27
st
0.08
0.11
0.07
0.14
0.20
0.30
0.08
0.11
0.18
0.16
0.54
0.75
0.15
0.08
0.22
0.16
0.41
0.36
S0
0.11

0.12

0.19

0.05

0.06

0.25

0.06

0.15

0.20

BIAS
%
-3.8
-14.7
0.0
-2.7
-0.4
-0.4
-7.7
0.0
-1.4
0.0
+0.7
-1.4
-3.8
-5.9
+0.5
-2.7
-2.1
-2.6
                 300.0-23

-------
TABLE 4.    MULTIPLE LABORATORY (n=19)
            DETERMINATION OF BIAS FOR CHLORIDE
WATER
Reagent




Drinking




Waste





AM'T ADDED
mg/L
0.78
1.04
6.50
7.80
20.8
26.0
0.78
1.04
6.50
7.80
20.8
26.0
0.78
1.04
6.50
7.80
20.8
26.0
AM'T FOUND
mg/L
0.79
1.12
6.31
7.76
20.7
25.9
0.54
0.51
5.24
6.02
20.0
24.0
0.43
0.65
4.59
5.45
18.3
23.0
st
0.17
0.46
0.27
0.39
0.54
0.58
0.35
0.38
1.35
1.90
2.26
2.65
0.32
0.48
1.82
2.02
2.41
2.50
S0
0.29
0.14

0.62

0.20
1.48

1.14

0.39

0.83

1.57

BIAS
%
+1.3
+7.7
-2.9
-0.5
-0.5
-0.4
-30.8
-51.0
-19.4
-22.8
-3.8
-7.7
-44.9
-37.5
-29.4
-30.1
-11.8
-11.5
                    300.0-24

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TABLE 5. MULTIPLE LABORATORY (n=19)
         DETERMINATION OF BIAS FOR NITRITE - NITROGEN
WATER
Reagent





Drinking





Waste





AM'T ADDED
mg/L
0.36
0.48
3.00
3.60
9.60
12.0
0.36
0.48
3.00
3.60
9.60
12.0
0.36
0.48
3.00
3.60
9.60
12.0
AM'T FOUND
mg/L
0.37
0.48
3.18
3.83
9.84
12.1
0.30
0.40
3.02
3.62
9.59
11.6
0.34
0.46
3.18
3.76
9.74
12.0
V
0.04
0.06
0.12
0.12
0.36
0.27
0.13
0.14
0.23
0.22
0.44
0.59
0.06
0.07
0.13
0.18
0.49
0.56
So
0.04

0.06

0.26

0.03

0.12

0.28

0.04

0.10

0.26

BIAS
%
+2.8
0.0
+6.0
+6.4
+2.5
+0.6
-16.7
-16.7
+0.7
+0.6
-0.1
-3.1
-5.6
-4.2
+6.0
+4.4
+1.5
+0.3
                   300.0-25

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TABLE 6. MULTIPLE LABORATORY (n=19)
         DETERMINATION OF BIAS FOR BROMIDE
WATER
Reagent




Drinking




Waste





AM'T ADDED
mg/L
0.63
0.84
5.24
6.29
16.8
21.0
0.63
0.84
5.24
6.29
16.8
21.0
0.63
0.84
5.24
6.29
16.8
21.0
AM'T FOUND St S0 BIAS
mg/L %
0.69
0.85
5.21
6.17
17.1
21.3
0.63
0.81
5.11
6.18
17.0
20.9
0.63
0.85
5.23
6.27
16.6
21.1
0.11
0.12
0.22
0.35
0.70
0.93
0.13
0.13
0.23
0.30
0.55
0.65
0.15
0.15
0.36
0.46
0.69
0.63
0.05
0.21

0.36

0.04
0.13

0.57

0.09

0.11

0.43

+9.5
+1.2
-0.6
-1.9
+1.6
+1.5
0.0
-3.6
-2.5
-1.7
+0.9
-0.4
0.0
+1.2
-0.2
-0.3
-1.0
+0.3
                      300.0-26

-------
TABLE 7.  MULTIPLE LABORATORY (n=19)
          DETERMINATION OF BIAS FOR NITRATE - NITROGEN
WATER
Reagent





Drinking





Waste





AM'T ADDED
mg/L
0.42
0.56
3.51
4.21
11.2
14.0
0.42
0.56
3.51
4.21
11.2
14.0
0.42
0.56
3.51
4.21
11.2
14.0
AM'T FOUND
mg/L
0.42
0.56
3.34
4.05
11.1
14.4
0.46
0.58
3.45
4.21
11.5
14.2
0.36
0.40
3.19
3.84
10.9
14.1
st
0.04
0.06
0.15
0.28
0.47
0.61
0.08
0.09
0.27
0.38
0.50
0.70
0.07
0.16
0.31
0.28
0.35
0.74
So
0.02

0.08

0.34

0.03

0.10

0.48

0.06

0.07

0.51

BIAS
%
0.0
0.0
-4.8
-3.8
-1.1
+2.6
+9.5
+3.6
-1.7
0.0
+2.3
+1.6
-14.6
-28.6
-9.1
-8.8
-3.0
+0.4
                       300.0-27

-------
TABLE 8.  MULTIPLE LABORATORY (n=19)
          DETERMINATION OF BIAS FOR ORTHO-PHOSPHATE
WATER
Reagent





Drinking





Waste





AM'T ADDED
mg/L
0.69
0.92
5.77
6.92
18.4
23.1
0.69
0.92
5.77
6.92
18.4
23.1
0.68
0.92
5.77
6.92
18.4
23.1
AM'T FOUND
mg/L
0.69
0.98
5.72
6.78
18.8
23.2
0.70
0.96
5.43
6.29
18.0
22.6
0.64
0.82
5.18
6.24
17.6
22.4
st
0.06
0.15
0.36
0.42
1.04
0.35
0.17
0.20
0.52
0.72
0.68
1.07
0.26
0.28
0.66
0.74
2.08
0.87
S0
0.06

0.18

0.63

0.17

0.40

0.59

0.09

0.34

1.27

BIAS
%
0.0
+6.5
-0.9
-2.0
+2.1
+0.4
+1.4
+4.3
-5.9
-9.1
-2.2
-2.0
-7.2
-10.9
-10.2
-9.8
-4.1
-3.0
                        300.0-28

-------
TABLE 9.  MULTIPLE LABORATORY (n=19)
          DETERMINATION OF BIAS FOR SULFATE
WATER
Reagent





Drinking





Waste





AM'T ADDED
mg/L
2.85
3.80
23.8
28.5
76.0
95.0
2.85
3.80
23.8
28.5
76.0
95.0
2.85
3.80
23.8
28.5
76.0
95.0
AM'T FOUND
mg/L
2.83
3.83
24.0
28.5
76.8
95.7
1.12
2.26
21.8
25.9
74.5
92.3
1.89
2.10
20.3
24.5
71.4
90.3
st
0.32
0.92
1.67
1.56
3.42
3.59
0.37
0.97
1.26
2.48
4.63
5.19
0.37
1.25
3.19
3.24
5.65
6.80
S0
0.52

0.68

2.33

0.41

0.51

2.70

0.24

0.58

3.39

BIAS
%
-0.7
+0.8
+0.8
-0.1
+1.1
+0.7
-60.7
-40.3
-8.4
-9.1
-2.0
-2.8
-33.7
-44.7
-14.7
-14.0
-6.1
-5.0
                          300.0-29

-------
                                   Method  A
                                                         Ret. Time
                                                         1.17
                                                         1.73
                                                         2.02
                                                         2.95
                                                         3.20
                                                         5.38
                                                         6.92
02468
                      Minutes
Figure 1. Chromatogram showing separation using the AS4A column
Ion
F-
ci-
N02-
Br
N03-
HP042-
so42-
mg/L
2
20
2
2
10
2
60
Method B
3
1 2
W
Peak Ret. Time Ion mg/L
1 2.75 CI02- 0.1
2 3.23 Br03- 0.1
3 3.63 Cl- 0.1
4 7.08 CI03- 0.1
4
	 A^
i I u w v
I
0 2 46 8
Minutes
Rgure 2. Chromatogram showing separation using the AS9 column
                                        300.0-30

-------
                        METHOD 335.4

DETERMINATION OF TOTAL CYANIDE BY SEMI-AUTOMATED COLORIMETRY
                  Edited  by  James  W.  O'Dell
                 Inorganic Chemistry Branch
                 Chemistry Research  Division
                        Revision 1.0
                        August  1993
        ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
             OFFICE OF RESEARCH AMD DEVELOPMENT
            U.S. ENVIRONMENTAL PROTECTION AGENCY
                  CINCINNATI, OHIO  45268
                          335.4-1

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                                 METHOD 335.4

         DETERMINATION OF TOTAL CYANIDE BY SEMI-AUTOMATED COLORIMETRY
1.0  SCOPE AND APPLICATION

     1.1  This method covers the determination of cyanide in drinking,  ground,
          surface, and saline waters, domestic and industrial wastes.

     1.2  The applicable range is 5 to 500 /zg/L.

2.0  SUMMARY OF METHOD

     2.1  The cyanide as hydrocyanic acid (HCN) is released from cyanide
          complexes by means of a manual reflux-distillation operation  and
          absorbed in a scrubber containing sodium hydroxide solution.   The
          cyanide ion in the absorbing solution is converted to cyanogen
          chloride by reactions with chloramine-T, that subsequently reacts
          with pyridine and barbituric acid to give a red-colored complex.

     2.2  Reduced volume versions of this method that use the same reagents
          and molar ratios are acceptable provided they meet the quality
          control and performance requirements stated in the method.

     2.2  Limited performance-based method modifications may be acceptable
          provided they are fully documented and meet or exceed requirements
          expressed in Sect. 9.0, Quality Control.

3.0  DEFINITIONS

     3.1  CALIBRATION BLANK (CB) — A volume of reagent water fortified with
          the same matrix as the calibration standards, but without the
          analytes, internal standards, or surrogate analytes.

     3.2  CALIBRATION STANDARD (CAL) — A solution prepared from the primary
          dilution standard solution or stock standard solutions and the
          internal standards and surrogate analytes.  The CAL solutions are
          used to calibrate the instrument response with respect to analyte
          concentration.

     3.3  INSTRUMENT PERFORMANCE CHECK SOLUTION (IPC) — A solution of one or
          more method analytes, surrogates, internal standards, or other test
          substances used to evaluate the performance of the instrument system
          with respect to a defined set of criteria.

     3.4  LABORATORY FORTIFIED BLANK (LFB) — An aliquot of reagent water or
          other blank matrices to which known quantities of the method
          analytes are added in the laboratory.  The LFB is analyzed exactly
          like a sample, and its purpose is to determine whether the
                                    335.4-2

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           methodology is In control, and whether the laboratory is capable of
           making accurate and precise measurements.

      3.5  LABORATORY FORTIFIED SAMPLE MATRIX (LFM)  — An aliquot of an
           environmental  sample to which known quantities of the method
           analytes are added in the laboratory.   The LFM 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
           LFM  corrected for background concentrations.

      3.6  LABORATORY REAGENT BLANK (LRB) — An aliquot of reagent water or
           other blank matrices that are treated  exactly as a  sample including
           exposure to all  glassware,  equipment,  solvents,  reagents,  internal
           standards,  and surrogates that are used with other  samples.   The LRB
           is  used to determine if method analytes or other interferences  are
           present in the laboratory environment,  the reagents,  or the
           apparatus.

      3.7  LINEAR CALIBRATION RANGE (LCR)  — The  concentration  range  over  which
           the instrument response is  linear.

      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.

      3.9  METHOD  DETECTION  LIMIT (MDL)  —  The minimum  concentration  of  an
           analyte that can  be  identified,  measured and reported with 99%
           confidence  that  the  analyte concentration  is greater than  zero.

      3.10  QUALITY CONTROL  SAMPLE  (QCS)  -  A  solution of method analytes of
           known concentrations  that is  used  to fortify an aliquot of LRB or
           sample  matrix.  The  QCS  is obtained from a source external to the
           laboratory  and different  from the  source of calibration standards.
           It  is used  to  check  laboratory performance with externally prepared
           test materials.

     3.11  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.

4.0  INTERFERENCES

     4.1  Several interferences are encountered with this method.  Some of the
          known interferences are aldehydes, nitrate-nitrite,  oxidizing
          agents, such as chlorine, thiocyanate,  thiosulfate and sulfide.
          Multiple interferences may require the analysis of a series of
          laboratory fortified sample matrices (LFM)  to verify the suitability
          of the chosen treatment.  Some interferences are eliminated or
          reduced by the  distillation.

                                    335.4-3

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    4.2  Sulfides  adversely  affect the procedure by producing hydrogen
         sulfide during distillation.  If a drop of the sample on lead
         acetate test  paper  indicates the presence of sulfide, treat 25 ml
         more  of the stabilized  sample (pH £  12) than that required for the
         cyanide determination with  powdered  cadmium carbonate.  Yellow
         cadmium sulfide  precipitates if the  sample contains sulfide.  Repeat
         this  operation until a  drop of the treated sample solution does not
         darken the lead  acetate test paper.   Filter the  solution through a
         dry filter paper into a dry beaker,  and from the filtrate, measure
         the sample to be used for analysis.   Avoid a large excess of cadmium
         and a long contact  time in  order to  minimize a loss by cotnplexation
         or occlusion  of  cyanide on  the precipitated material.

    4.3  High  results  may be obtained for samples that contain nitrate and/or
         nitrite.  During the distillation nitrate and nitrite will form
         nitrous  acid  that will  react with some organic compounds to form
         oximes.   These oximes will  decompose under test  conditions to
         generate  HCN. The  interference of nitrate and nitrite  is eliminated
         by pretreatment  with sulfamic acid.

    4.4  Oxidizing agents, such  as  chlorine,  decompose most of the cyanides.
         Test  a  drop  of the  sample  with  potassium  iodide-starch  paper  (KI-
         starch  paper) at time  of collection; a blue  color  indicates the  need
         for  treatment.   Add ascorbic  acid,  a few  crystals  at  a  time,  until  a
         drop  of sample  produces no color  on  the  indicator  paper; then  add  an
          additional  0.06  g of ascorbic  acid  for each  liter  of  sample volume.
         Sodium  arsenite  has also been  employed to remove oxidizing  agents.

    4.5  Other compatible procedures for the  removal  or  suppression  of
          interferences may be employed  provided they  do  not adversely  effect
          the  overall  performance of the  method.

     4.6   Method  interferences may be caused  by contaminants in  the  reagent
          water,  reagents, glassware, and other sample processing apparatus
          that bias analyte response.
5.0  SAFETY
     5.1  The toxicity or carcinogenicity of each reagent used in this method
          has not been fully established.  Each chemical should be regarded as
          a potential health hazard and exposure should be as low as
          reasonably achievable.  Cautions are included for known extremely
          hazardous materials or procedures.

     5.2  Each 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 Material
          Safety Data Sheets (MSDS) should be made available to all personnel
          involved in the chemical analysis.  The preparation of a formal
          safety plan is also advisable.
                                    335.4-4

-------
      5.3  The following chemicals have the potential  to be highly toxic or
           hazardous,  consult MSDS.
           5.3.1    Hydrochloric acid (7.5)
           5.3.2    Silver nitrate (7.9)
           5.3.3    Potassium cyanide (7.10)
           5.3.4    Sulfuric acid (7.14)
      5.4  Because  of  the toxicity of evolved  hydrogen  cyanide  (HCN),
           distillation  should be performed in a  well vented hood.
 6.0   EQUIPMENT AND SUPPLIES
      6.1   Balance  —  Analytical,  capable of accurately weighing to the  nearest
           0.0001 g.
      6.2   Glassware —  Class  A volumetric  flasks and pi pets  as required.
      6.3   Midi reflux distillation  apparatus  including boiling flask
           condenser,  and  absorber as  shown  in  Figure 1.
      6.4   Heating  mantel  or heating  block  as  required.
      6.5   Automated continuous  flow  analysis  equipment designed to deliver and
           react sample  and  reagents  in the  required order and ratios.
           6.5.1    Sampling  device (sampler)
           6.5.2    Multichannel  pump
           6.5.3    Reaction  unit or manifold
           6.5.4    Colorimetric detector
           6.5.5    Data recording device
7.0  REAGENTS AND STANDARDS
     7.1  Reagent water:  Distilled  or deionized water, free of the analyte of
          interest.  ASTM type II or equivalent.
     7.2  Ascorbic acid: Crystal (CASRN-50-81-7)
     7.3  Chloramine-T:   Dissolve 2.0 g of  chloramine-T (CASRN-127-65-1) in
          500 mL  of reagent water.
     7.4  Magnesium Chloride Solution:  Weigh  510 g of  MgCl2.6H20 (CASRN-7786-
          30-3) into a 1000 ml flask, dissolve and  dilute to 1 1 with  reagent
          water.
                                   335.4-5

-------
7.5
7.6
7.7
7.8
          Pyridine Barbituric Acid Reagent:   Place  15  g  of barbituric  acid
          (CASRN-67-52-7)  in a 1  L beaker.   Wash  the sides of the  beaker  with
          about 100 ml of  reagent water.  Add 75  ml of pyridine  (CASRN-110-86-
          1)  and mix.   Add 15 ml  of cone. HC1  (CASRN-7647-01-0)  and  mix.
          Dilute to 900 ml with reagent water and mix  until  all  the  barbituric
          acid has dissolved.  Transfer the  solution to  a 1-L flask  and dilute
          to  the mark.

          Sodium dihydrogenphosphate buffer, 1 M:  Dissolve 138  g  of
          NaH2P04.H20  (CASRN-10049-21-5) in  1 L of reagent water.  Refrigerate
          this solution.

          Sodium Hydroxide Solution, 1.25 N:  Dissolve 50 g of NaOH  (CASRN-
          1310-73-2) in reagent water,  and  dilute to  1 L with reagent  water.

          Sodium Hydroxide, 0.25  N:  Dilute 200 ml of  1.25 N Sodium  hydroxide
          solution (7.7) to 1 L with reagent water.

     7.9  Standard Silver Nitrate Solution,  0.0192 N:   Prepare by  crushing
          approximately 5  g AgNO, (CASRN-7761-88-8) crystals and drying to
          constant weight  at 40°t.  Weigh out 3.2647  g of dried  AgN03,
          dissolve in reagent water, and dilute to 1000  ml (1 mL = 1 mg CN).

     7.10 Stock Cyanide Solution:  Dissolve 2.51 g of KCN (CASRN-151-50-8)  and
          2 g KOH (CASRN-1310-58-3) in 900  mL of reagent water.   Standardize
          with 0.0192 N AgNO, (7.9).  Dilute to appropriate concentration so
          that 1 ml = 1 mg CN.

     7.11 Standard Cyanide Solution, intermediate:   Dilute 10.0  ml of  stock (1
          ml - 1 mg CN) (7.10) to 100.0 with reagent  water (1 ml = 100.0  //g
          CN.

     7.12 Working Standard Cyanide Solution:  Prepare fresh daily by diluting
          20.0 ml of  intermediate cyanide solution (7.11) to 200.0 ml  with
          reagent water and store in a glass stoppered bottle.  1  ml = 10.0 /jg
          CN.

     7.13 Sulfamic Acid:   (CASRN-212-57-3).

     7.14 Sulfuric Acid, 18N:  Slowly add 500 ml of concentrated H2S04 (CASRN-
          5329-14-6)  to 500 ml of reagent water.

8.0  SAMPLE COLLECTION. PRESERVATION AND STORAGE

     8.1  Samples should be collected in plastic or glass bottles.  All
          bottles must  be  thoroughly cleaned and rinsed with reagent water.
          Volume collected should be sufficient to insure a representative
          sample, allow for replicate analysis (if required), and minimize
          waste disposal.
                               335.4-6

-------
     8.2  If the sample contains chlorine or hydrogen sulfide, see Sect. 4.0
          for treatment.

     8.3  Samples must be preserved with sodium hydroxide pH > 12 and cooled
          to 4°C at the time of collection.

     8.4  Samples should be analyzed as soon as possible after collection.   If
          storage is required, preserved samples are maintained at 4°C and  may
          be held for up to 14 days.

9.0  QUALITY CONTROL

     9.1  Each laboratory using this method is required to operate a formal
          quality control  (QC) program.  The minimum requirements of this
          program consist of an initial demonstration of laboratory
          capability,  the periodic analysis of laboratory reagent blanks,
          fortified blanks,  and other laboratory solutions as a continuing
          check on performance.  The laboratory is required to maintain per-
          formance records that define the quality of the data that are
          generated.

     9.2  INITIAL DEMONSTRATION OF PERFORMANCE

          9.2.1   The  initial  demonstration of performance is used to
                  characterize instrument performance (determination of LCRs
                  and  analysis of QCS)  and laboratory performance
                  (determination of MDLs) prior to performing analyses by this
                  method.

          9.2.2   Linear Calibration Range (LCR)  — The LCR must  be determined
                  initially  and verified every 6  months or whenever a
                  significant  change in instrument response is  observed or
                  expected.  The initial  demonstration  of linearity must use
                  sufficient standards  to insure  that the resulting curve is
                  linear.  The verification  of linearity must use a minimum  of
                  a  blank  and  three standards.   If any  verification data
                  exceeds  the  initial values  by ± 10%,  linearity  must be
                  reestablished.   If any portion  of the  range is  shown  to be
                  nonlinear, sufficient standards  must  be used  to clearly
                  define the nonlinear  portion.

          9.2.3    Quality  Control  Sample  (QCS)  —  When  beginning  the  use of
                  this method,  on  a quarterly  basis  or  as required  to meet
                  data-quality needs, verify the  calibration  standards  and
                  acceptable instrument performance  with  the  preparation  and
                  analyses of  a QCS.  If  the determined  concentrations  are not
                 within ± 10% of  the stated values,  performance  of the
                  determinative step of the method  is unacceptable.   The
                  source of the problem must be identified  and corrected
                  before either proceeding on with the  initial determination
                 of MDLs or continuing with on-going analyses.


                                   335.4-7

-------
     9.2.4   Method Detection Limit (MDL) — MDLs must be established for
             all analytes, using reagent water (blank) fortified at a
             concentration of two to three times the estimated instrument
             detection limit/45  To determine MDL values, take seven
             replicate aliquots of the fortified reagent water and
             process through the entire analytical method.  Perform all
             calculations defined in the method and report the
             concentration values in the appropriate units.  Calculate
             the MDL as follows:

                                  MDL = (t) x (S)

             where, t - Student's t value for a 99% confidence level and
                        a standard deviation estimate with n-1 degrees
                        of freedom [t = 3.14 for seven replicates].

                    S = standard deviation of the replicate analyses.

             MDLs should be determined every 6 months, when a new
             operator begins work or whenever there is a significant
             change in the background or instrument response.

9.3  ASSESSING LABORATORY PERFORMANCE

     9.3.1   Laboratory Reagent Blank (LRB) — The laboratory must
             analyze at least one LRB with each batch of samples.  Data
             produced are used to assess contamination from the
             laboratory environment.  Values that exceed the MDL indicate
             laboratory or reagent contamination should be suspected and
             corrective actions must be taken before continuing the
             analysis.

     9.3.2   Laboratory Fortified Blank (LFB) — The laboratory must
             analyze at least one LFB with each batch of samples.
             Calculate accuracy as percent recovery (Sect. 9.4.2).  If
             the recovery of any analyte falls outside the required
             control limits of 90-110%, that analyte is judged out of
             control, and the source of the problem should be identified
             and resolved before continuing analyses.

     9.3.3   The laboratory must use LFB analyses data to assess
             laboratory performance against the required control limits
             of 90-110%.  When sufficient internal performance data
             becomes available (usually a minimum of 20-30 analyses),
             optional control limits can be developed from the percent
             mean recovery (x) and the standard deviation (S) of the mean
             recovery.  These data can be used to establish the upper and
             lower control limits as follows:

                          UPPER CONTROL LIMIT = x + 3S
                          LOWER CONTROL LIMIT = x - 3S
                               335.4-8

-------
              The  optional  control  limits  must  be  equal  to  or  better  than
              the  required  control  limits  of  90-110%.  After each  five  to
              ten  new recovery  measurements,  new control  limits  can be
              calculated  using  only the most  recent  20-30 data points.
              Also,  the standard  deviation (S)  data  should  be  used to
              establish an  on-going precision statement  for the  level of
              concentrations  included  in the  LFB.  These  data  must be kept
              on file and be  available for review.

     9.3.4    Instrument  Performance Check Solution  (IPC) — For all
              determinations, the laboratory must  analyze the  IPC  (a  mid-
              range  check standard)  and a  calibration blank immediately
              following daily calibration,  after every tenth sample (or
              more frequently,  if required), and at  the end of the sample
              run.   Analysis of the  IPC solution and calibration blank
              immediately following calibration must verify that the
              instrument is within ± 10% of calibration.  Subsequent
              analyses of the IPC solution must verify the  calibration is
              still  within ± 10%.   If the  calibration cannot be verified
             within  the specified limits, reanalyze the  IPC solution.  If
             the second analysis of the IPC solution confirms calibration
             to be  outside the limits, sample analysis must be
             discontinued,  the cause determined and/or in the case of
             drift the instrument recalibrated.  All samples following
             the last acceptable IPC solution must be reanalyzed.   The
             analysis data of the calibration blank and  IPC solution must
             be kept on file with the sample analyses data.

9.4  ASSESSING ANALYTE RECOVERY AND DATA QUALITY

     9.4.1   Laboratory Fortified Sample Matrix (LFM)  — The laboratory
             must add a known amount of analyte to a minimum of 10% of
             the routine  samples.  In each case,  the LFM aliquot must be
             a duplicate  of the aliquot used for sample analysis.   The
             analyte concentration  must be high enough  to be detected
             above the original sample and should  not  be less  than four
             times the MDL.  The added analyte  concentration  should  be
             the same as  that used  in  the  laboratory fortified blank.

     9.4.2   Calculate the  percent  recovery for each analyte,  corrected
             for concentrations measured  in the unfortified sample,  and
             compare these  values to the designated  LFM  recovery range
             90-110%.  Percent  recovery may be  calculate  using the
             following equation:


                R  =   GS ' C     x  100
                       s

             where,  R =  percent recovery.
                    Cs =  fortified sample concentration.
                    C =  sample  background concentration.

                              335.4-9

-------
                         s   =  concentration  equivalent  of  analyte  added to
                               sample.

          943   If the recovery of any analyte  falls outside  the  designated
                  LFM recovery range and the  laboratory  performance for that
                  analyte is shown to be in control  (Sect.  9.3),  the recovery
                  problem encountered with the LFM is judged  to be  either
                  matrix or solution related, not system related.

          9.4.4   Where reference materials are available,  they should be
                  analyzed to provide additional  performance  data.   The
                  analysis of reference samples is a valuable tool  for
                  demonstrating the ability to perform the  method acceptably.

10.0 CALIBRATION AND STANDARDIZATION

     10 1 Prepare a series of at least 3 standards,  covering  the desired
          range, and a blank by pipetting suitable volumes  of working standard
          solution (7.12) into 100 ml volumetric flasks.  To  each standard
          (except those to be distilled) add  20 mL of 1.25  N  sodium hydroxide
          and dilute to 100 ml with reagent water.

     10.2 It is not imperative that all standards be distilled in the same
          manner as the samples.  It is recommended that at least two
          standards (a high and low) and a blank be distilled and compared to
          similar values on the standard curve to insure that the distillation
          technique is reliable.  If distilled standards do not agree within
          ± 10% of the undistilled standards the analyst should find the cause
          of the apparent error before proceeding.  Before distillation,
          standards should contain 4 ml 0.25N NaOH  (7.8) per 50 ml.

     10.3 Set  up the manifold  as  shown in Figure 2  in a hood or a well-
          ventilated area.

     10.4 Allow the instrument to warm up as required.  Pump all reagents,
          with 0.25N NaOH  in the  sample line, until a stable baseline is
          achieved.

     10.5 Place appropriate  standards  in  the sampler in order  of decreasing
          concentration  and  perform  analysis.

     10  6 Prepare  standard  curve  by  plotting instrument response against
          concentration  values.   A  calibration curve may be  fitted  to the
          calibration  solutions  concentration/response  data  using  computer or
          calculator  based  regression  curve  fitting techniques.  Acceptance or
          control  limits  should  be  established using the difference between
          the  measured value of  the  calibration  solution and the "true value
          concentration.

     10  7 After the calibration  has  been  established, it must  be verified by
          the  analysis of a suitable QCS.  If measurements exceed  ± 10% of the
          established QCS value,  the analysis should be terminated and the

                                    335.4-10

-------
           instrument recalibrated.   The new calibration must be verified
           before continuing analysis.   Periodic reanalysis of the QCS is
           recommended as a continuing  calibration check.

 11.0 PROCEDURE

      11.1  Pipet  50  ml of sample  or  an  aliquot  diluted  to  50 ml into  the  MIDI
           distillation boiling flask.   Add  boiling chips  as required.  Pipet
           50  ml  of  sodium hydroxide 0.25  N  (7.8)  into  the absorbing  tube.
           Connect the boiling  flask, condenser,  and absorber in the  train  as
           shown  in  Figure 1.

      11.2  Start  a slow stream  of air entering  the boiling flask by adjusting
           the vacuum source to maintain about  3  bubbles per minute.

      11.3  If  samples contain N03 and/or NO., add 0.2 g  of sulfamic acid (7.13)
           after  the  air  rate is  set through the  air inlet tube.  Mix  for 3 min
           prior  to  addition of H2S04.

      11.4  Slowly  add 5 ml  18 N sulfuric acid (7.14)  through  the  air  inlet
           tube.   Rinse the  tube  with distilled water and  allow  the airflow to
           mix the flask  contents for 3 min.  Pour  2  ml of magnesium chloride
           (7.4)  into the  air inlet and wash down with a stream  of water.

      11.5  Heat the solution to boiling.  Reflux for  one and  one  half hours.
           Turn off heat  and continue the airflow for at least 15 min.  After
           cooling the boiling  flask, disconnect absorber  and close off the
           vacuum source  and remove absorber tube.

      11.6  Fill and connect  reagent containers and start system.  Allow the
           instrument to warm up  as required.  Pump all  reagents, with 0.25N
          NaOH in the sample line, until a stable baseline is achieved.

      11.7 Place standards, distilled standards  and unknown samples (ALL in
          0.25N NaOH) in sampler tray.   Calibrate instrument and begin
          analysis.                                       .

12.0 DATA ANALYSIS AND CALCULATIONS

     12.1  Prepare a  calibration curve by plotting instrument response against
          standard concentration.  Compute sample concentration by comparing
          sample  response with  the standard  curve.   Multiply answer  by
          appropriate dilution  factor.

     12.2  Report  only those values that fall  between the lowest and the
          highest calibration standards.   Samples exceeding the highest
          standard should be diluted and reanalyzed.

     12.3  Report  results in mg/L.
                                   335.4-11

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13.0 METHOD PERFORMANCE

     13.1 The inter!aboratory precision and accuracy data in Table 1 were
          developed using a reagent water matrix.  Values are in mg CN/L.

     13.2 Single laboratory precision data can be estimated at 50 to 75% of
          the inter!aboratory precision estimates.

14.0 POLLUTION PREVENTION

     14.1 Pollution prevention encompasses any technique that reduces or
          eliminates the quantity or toxicity of waste at the point of
          generation.  Numerous opportunities for pollution prevention exist
          in laboratory operation.  The USEPA has established a preferred
          hierarchy of environmental management techniques that places
          pollution prevention as the management option of first choice.
          Whenever feasible, laboratory personnel should use pollution
          prevention techniques to address their waste generation.  When
          wastes cannot be feasibly reduced at the source, the Agency
          recommends recycling as the next best option.

     14.2 The quantity of chemicals purchased should be based on expected
          usage during its shelf life and disposal cost of unused material.
          Actual reagent preparation volumes should reflect anticipated  usage
          and reagent stability.

     14.3 For information about pollution prevention that may be applicable to
          laboratories and research institutions, consult "Less is  Better:
          Laboratory Chemical Management for Waste Reduction,"  available from
          the American Chemical Society's Department of Government
          Regulations and Science Policy, 1155 16th Street N.W., Washington
          D.C. 20036, (202)872-4477.

15.0 WASTE MANAGEMENT

     15.1 The U.S.  Environmental  Protection Agency requires that laboratory
          waste management practices conducted be consistent with  all
           applicable rules and regulations.   Excess Reagents, samples,  and
          method process wastes should  be characterized  and disposed  of in an
           acceptable manner.  The Agency  urges laboratories to  protect  the
           air,  water and land by minimizing  and controlling  all releases from
           hoods, and bench operations,  complying with  the letter  and  spirit of
           any waste discharge permit and  regulations,  and by  complying  with
           all solid and  hazardous waste 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,"  available
           from the American  Chemical Society  at  the address listed in Sect.
           14.3.
                                    335.4-12

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16.0 REFERENCES

     1.    Technicon AutoAnalyzer II Methodology, Industrial Method No. 315-74
          WCUV digestion and distillation, Technicon Industrial Systems,
          Tarrytown, NY  10591, (1974).

     2.    Goulden,  P.O., Afghan, B.K. and Brooksbank, P., Anal. 44. 1845
          (1972).

     3.    USEPA Contract Laboratory Program,  Document Number ILMO 1.0, Method
          for Total Cyanide Analysis by MIDI  Distillation #335.2 CLP-M.

     4.    Code of Federal  Regulations 40, Ch. 1, Pt. 136, Appendix B.
                                   335.4-13

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17.  TABLES. DIAGRAMS. FLOWCHARTS. AND VALIDATION DATA
TABLE 1. INTERLABORATORY PRECISION AND ACCURACY DATA
NUMBER OF
VALUES
REPORTED
126
94
158
118
148
92
132
119
148
94
92
158
TRUE
VALUE
(T)
0.020
0.055
0.090
0.110
0.180
0.270
0.530
0.540
0.610
0.700
0.800
0.970
MEAN
(X)
0.0182
0.0501
0.0843
0.1045
0.1683
0.2538
0.5019
0.5262
0.5803
0.6803
0.7726
0.9508
RESIDUAL
FOR X
0.0002
-0.0014
-0.0008
0.0003
-0.0030
-0.0038
-0.0049
0.0098
-0.0032
0.0105
0.0069
0.0222
STANDARD
DEVIATION
(S)
0.0055
0.0092
0.0171
0.0165
0.0236
0.0275
0.0775
0.0679
0.0851
0.1082
0.0880
0.1464
RESIDUAL
FOR S
0.0000
-0.0007
0.0027
-0.0004
-0.0023
-0.0099
0.0069
-0.0039
0.0043
0.0159
-0.0170
0.0197
REGRESSIONS:  X = 0.959T - 0.001, S = 0.128T + 0.003
                                   335.4-14

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             CONDENSER
1
     DISTILLATION
     UNIT
                                     SUCTION
                            $-   ABSORBER
FIGURE 1.  NIDI DISTILLATION APPARATUS
            335.4-15

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                  335.4-16

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                                                                                 1
                          METHOD 350,,1

DETERMINATION OF AMMONIA NITROGEN BY SEMI-AUTOMATED COLORIMETRY
                   Edited by James W. O'Dell
                   Inorganic  Chemistry Branch
                  Chemistry Research Division
                         Revision 2.0
                          August 1993
          ENVIRONMENTAL  MONITORING  SYSTEMS  LABORATORY
              OFFICE OF RESEARCH AND DEVELOPMENT
             U.S. ENVIRONMENTAL PROTECTION AGENCY
                    CINCINNATI,  OHIO   45268
                            350.1-1

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                                 METHOD 350.1

        DETERMINATION OF AMMONIA NITROGEN BY SEMI-AUTOMATED COLORIMETRY
1.0  SCOPE AND APPLICATION

     1.1  This method covers the determination of ammonia in drinking,  ground,
          surface, and saline waters, domestic and industrial wastes.

     1 2  The applicable range is 0.01 to 2.0 mg/L NH3 as N.   Higher
          concentrations can be determined by sample dilution.  Approximately
          60 samples per hour can be analyzed.

     1.3  This method is described for macro glassware; however, micro
          distillation equipment may also be used.

2.0  SUMMARY OF METHOD

     2 1  The sample is buffered at  a pH of 9.5 with a borate buffer in order
          to decrease hydrolysis of  cyanates and organic nitrogen compounds,
          and is  distilled  into a solution of boric acid.  Alkaline phenol and
          hypochlorite react with ammonia to form indophenol blue that is
          proportional to the ammonia concentration.  The blue color formed is
          intensified with  sodium nitroprusside and measured colorimetrically.

     2.3  Reduced volume versions of this method that use the same reagents
          and molar ratios  are acceptable provided they meet the quality
          control and performance requirements stated in the method.

     2 4  Limited performance-based  method modifications may be acceptable
          provided they are fully documented and meet or exceed requirements
          expressed in Sect. 9.0, Quality Control.

3.0  DEFINITIONS

     3.1  CALIBRATION BLANK (CB) --  A volume of reagent  water fortified with
          the same matrix  as the calibration  standards,  but  without the
          analytes, internal standards,  or  surrogate  analytes.

     3.2  CALIBRATION STANDARD  (CAL) —  A solution prepared  from the primary
          dilution standard solution or  stock  standard solutions and the
          internal standards and surrogate  analytes.  The CAL solutions are
          used  to calibrate the  instrument  response with respect to analyte
          concentration.

     3.3   INSTRUMENT  PERFORMANCE CHECK SOLUTION  (IPC)  — A  solution of one  or
          more  method  analytes,  surrogates,  internal  standards, or  other  test
          substances  used  to evaluate the performance of the instrument system
          with  respect  to  a defined  set  of  criteria.
                                    350.1-2

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 3.4  LABORATORY FORTIFIED BLANK (LFB) — An aliquot of reagent water or
      other blank matrices to which known quantities of the method
      analytes are added in the laboratory.  The LFB is analyzed exactly
      like a sample, and its purpose is to determine whether the
      methodology is in control, and whether the laboratory is capable of
      making accurate and precise measurements.

 3.5  LABORATORY FORTIFIED SAMPLE MATRIX (LFM)  — An aliquot of an
      environmental  sample to which known quantities of the method
      analytes are added in the laboratory.  The LFM 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 LFM
      corrected for  background concentrations.

 3.6  LABORATORY REAGENT BLANK (LRB)  — An aliquot of reagent water  or
      other blank matrices  that are treated exactly as  a  sample including
      exposure to all  glassware,  equipment,  solvents,  reagents,  internal
      standards,  and surrogates that  are used with other  samples.  The LRB
      is  used  to determine  if method  analytes or other  interferences  are
      present  in the laboratory environment, the reagents,  or the
      apparatus.

 3.7  LINEAR CALIBRATION RANGE (LCR)  -- The  concentration  range over  which
      the instrument response is  linear.

 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.

 3.9  METHOD DETECTION LIMIT  (MDL)  — The minimum concentration  of an
      analyte  that can be identified, measured and reported with 99%
      confidence  that the analyte concentration  is greater than zero.

 3.10  QUALITY  CONTROL SAMPLE  (QCS)  — A solution of method analytes of
      known concentrations  that is  used to fortify an aliquot of LRB or
      sample matrix.   The QCS  is obtained from a source external to the
      laboratory  and different from the source of calibration standards.
      It  is used  to check laboratory performance with externally prepared
     test materials.

3.11 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.
                              350.1-3

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4.0  INTERFERENCES

     4.1  Cyanate, which may be encountered in certain industrial effluents,
          will hydrolyze to some extent even at the pH of 9.5 at which
          distillation is carried out.

     4 2  Residual chorine must be removed by pretreatment of the sample with
          sodium thiosulfate or other reagents before distillation.

     4 3  Method interferences may be caused by contaminants in the reagent
          water, reagents, glassware, and other sample processing apparatus
          that bias analyte response.

5.0  SAFETY

     5 1  The toxicity or carcinogenicity of each reagent used in this method
          have not been fully established.  Each chemical should be regarded
          as a potential health hazard  and exposure should be as low as
          reasonably  achievable.  Cautions are included for known extremely
          hazardous materials or procedures.

     5 2  Each 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 Material
          Safety Data Sheets  (MSDS)  should be made available to  all personnel
          involved in the chemical analysis.  The preparation of a formal
          safety plan is also advisable.

     5.3  The  following chemicals have  the potential  to be highly toxic  or
          hazardous,  consult MSDS.

          5.3.1    Sulfuric  acid  (7.6)

          5.3.2    Phenol  (7.7)

          5.3.3    Sodium  nitroprusside  (7.10)

 6.0  EQUIPMENT AND SUPPLIES

      6.1  Balance -  Analytical,  capable of accurately weighing  to  the nearest
          0.0001  g.

      6.2  Glassware  - Class A volumetric flasks  and  pipets  as  required.

      6.3  An all-glass  distilling apparatus  with an  800-1000-mL flask.

      6.4  Automated continuous flow analysis equipment  designed to deliver and
           react sample and reagents in the required  order and  ratios.

           6.4.1   Sampling device (sampler)

           6.4.2   Multichannel  pump

                                     350.1-4

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          6.4.3   Reaction unit or manifold

          6.4.4   Colorimetric detector

          6.4.5   Data recording device

7.0  REAGENTS AND STANDARDS

     7.1  Reagent water - Ammonia free:  Such water is best prepared by
          passage through an ion exchange column containing a strongly acidic
          cation exchange resin mixed with a strongly basic anion exchange
          resin.  Regeneration of the column should be carried out according
          to the manufacturer's instructions.

          NOTE 1:  All solutions must be made with ammonia-free water.

     7.2  Boric acid solution (20 g/L):  Dissolve 20 g H3B03  (CASRN  10043-35-
          3) in reagent water and dilute to 1 L.

     7.3  Borate buffer:  Add 88 ml of 0.1 N NaOH (CASRN 1310-73-2) solution
          to 500 ml of 0.025 M sodium tetraborate solution (5.0 g anhydrous
          Na2B40, [CASRN 1330-43-4] or 9.5 g Na2B40/10H20 [CASRN 1303-96-4]  per
          L) ana dilute to 1 L with reagent water.

     7.4  Sodium hydroxide, 1 N:  Dissolve 40 g NaOH in reagent water and
          dilute to 1 L.

     7.5  Dechlorinating reagents:  A number of dechlorinating reagents may be
          used to remove residual chlorine prior to distillation.  These
          include:

          7.5.1   Sodium thiosulfate:  Dissolve 3.5 g Na2S203'5H20 (CASRN
                  10102-17-7) in reagent water and dilute to 1 L.  One ml of
                  this solution will  remove 1 mg/L of residual chlorine in 500
                  ml of sample.

          7.5.2   Sodium sulfite:  Dissolve 0.9 g Na2S03 (CASRN 7757-83-7)  in
                  reagent water and dilute to 1 L. One ml removes 1 mg/L Cl
                  per 500 ml of sample.

     7.6  Sulfuric acid 5 N:  Air scrubber solution.  Carefully add 139 ml of
          cone, sulfuric acid (CASRN 7664-93-9) to approximately 500 ml of
          reagent water.  Cool to room temperature and dilute to 1 L with
          reagent water.

     7.7  Sodium phenolate:  Using a 1-L Erlenmeyer flask, dissolve 83 g
          phenol (CASRN 108-95-2) in 500 ml of distilled water.  In small
          increments, cautiously add with agitation, 32 g of NaOH.
          Periodically cool flask under water faucet.   When cool, dilute to
          1 L with reagent water.
                                   350.1-5

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     7.8  Sodium hypochlorite solution:  Dilute 250 ml of a bleach solution
          containing 5.25% NaOCl  (CASRN 7681-52-9) (such as "Clorox") to 500
          ml with reagent water.  Available chlorine level should approximate
          2% to 3%.  Since "Clorox" is a proprietary product, its formulation
          is subject to change.  The analyst must remain alert to detecting
          any variation in this product significant to its use in this
          procedure.  Due to the  instability of this product, storage over an
          extended period should be avoided.

     7.9  Disodium ethylenediamine-tetraacetate (EDTA) (5%):  Dissolve 50 g of
          EDTA (disodium salt) (CASRN 6381-92-6) and approximately six pellets
          of NaOH in 1 L of reagent water.

     7.10 Sodium nitroprusside (0.05%):  Dissolve 0.5 g of sodium
          nitroprusside (CASRN 14402-89-2) in 1 L of reagent water.

     7.11 Stock solution:  Dissolve 3.819 g of anhydrous ammonium chloride,
          NH4C1  (CASRN 12125-02-9),  dried at 105°C,  in reagent water,  and
          dilute to 1 L.  1.0 ml = 1.0 mg NH3-N.

     7.12 Standard Solution A:  Dilute 10.0 ml of stock solution (7.11) to 1 L
          with reagent water.  1.0 ml = 0.01 mg NH3-N.

     7.13 Standard Solution B:  Dilute 10.0 ml of standard solution A (7.12)
          to 100.0 ml with reagent water.  1.0 mL = 0.001 mg NH3-N.

8.0  SAMPLE COLLECTION. PRESERVATION AND STORAGE

     8.1  Samples should be collected in plastic or glass bottles.   All
          bottles must be thoroughly cleaned and rinsed with reagent water.
          Volume collected should be sufficient to insure a representative
          sample, allow for replicate analysis (if required), and minimize
          waste disposal.

     8.2  Samples must be preserved with H2S04 to  a pH < 2 and cooled to  4°C
          at the time of collection.

     8.3  Samples should be analyzed as soon as possible after collection.  If
          storage is required, preserved samples are maintained at  4°C and may
          be held for up to 28 days.

9.0  QUALITY CONTROL

     9.1  Each laboratory using this method is required to operate  a formal
          quality control (QC) program.  The minimum requirements of this
          program consist of an initial demonstration of laboratory
          capability,  and the periodic analysis of laboratory reagent blanks,
          fortified blanks and other laboratory solutions as a continuing
          check on performance.  The laboratory is required to maintain per-
          formance records that define the quality of the data that are
          generated.
                                    350.1-6

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9.2  INITIAL DEMONSTRATION OF PERFORMANCE

     9.2.1   The initial demonstration of performance is used to
             characterize instrument performance (determination of LCRs
             and analysis of QCS) and laboratory performance
             (determination of MDLs) prior to performing analyses by this
             method.

     9.2.2   Linear Calibration Range (LCR) — The LCR must be determined
             initially and verified every 6 months or whenever a
             significant change in instrument response is observed or
             expected.  The initial demonstration of linearity must use
             sufficient standards to insure that the resulting curve is
             linear.  The verification of linearity must use a minimum of
             a blank and three standards.  If any verification data
             exceeds the initial  values by ± 10%, linearity must be
             reestablished.  If any portion of the range is shown to be
             nonlinear, sufficient standards must be used to clearly
             define the nonlinear portion.

     9.2.3   Quality Control  Sample (QCS) — When beginning the use of
             this method, on  a quarterly basis or as required to meet
             data-quality needs,  verify the calibration standards and
             acceptable instrument performance with the preparation and
             analyses of a QCS.   If the determined concentrations are not
             within ± 10% of  the  stated values,  performance of the
             determinative step of the method is unacceptable.   The
             source of the problem must be identified and corrected
             before either proceeding with the initial  determination of
             MDLs or continuing with on-going analyses.

     9.2.4   Method Detection Limit (MDL) — MDLs must be established for
             all  analytes, using  reagent water (blank)  fortified at a
             concentration of two to three times the estimated  instrument
             detection limit.    To determine MDL values, take seven
             replicate aliquots of the fortified reagent water  and
             process through  the  entire analytical  method.   Perform all
             calculations defined in the method  and report  the
             concentration values in the appropriate units.   Calculate
             the  MDL as follows:

                               MDL = (t) x (S)

             where,  t = Student's t value for a  99% confidence  level
                        and a standard deviation estimate with  n-1
                        degrees of freedom [t =  3.14 for seven
                        replicates].

                    S = standard  deviation of the replicate  analyses.
                              350.1-7

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             MDLs should be determined every 6 months, when a new
             operator begins work or whenever there is a significant
             change in the background or instrument response.

9.3  ASSESSING LABORATORY PERFORMANCE

     9.3.1   Laboratory Reagent Blank (LRB) — The laboratory must
             analyze at least one LRB with each batch of samples.  Data
             produced are used to assess contamination from the
             laboratory environment.  Values that exceed the MDL indicate
             laboratory or reagent contamination should be suspected and
             corrective actions must be taken before continuing the
             analysis.

     9.3.2   Laboratory Fortified Blank (LFB) — The laboratory must
             analyze at least one LFB with each batch of samples.
             Calculate accuracy as percent recovery (Sect. 9.4.2).  If
             the recovery of any analyte falls outside the required
             control limits of 90-110%,  that analyte is judged out of
             control, and the source of the problem should be identified
             and resolved before continuing analyses.

     9.3.3   The laboratory must use LFB analyses data to assess
             laboratory performance against the required control limits
             of 90-110%.  When sufficient internal performance data
             become available (usually a minimum of 20-30 analyses),
             optional control limits can be developed from the percent         Mm
             mean recovery (x) and the standard deviation (S) of the mean
             recovery.  These data can be used to establish the upper and
             lower control  limits as follows:

                         UPPER CONTROL LIMIT = x + 3S
                         LOWER CONTROL LIMIT = x - 3S

             The optional  control limits must be equal to or better than
             the required control limits of 90-110%.   After each five to
             ten new recovery measurements, new control limits can be
             calculated using only the most recent 20-30 data points.
             Also,  the standard deviation (S) data should be used to
             established an on-going precision statement for the level  of
             concentrations included in  the LFB.  These data must be kept
             on file and be available for review.

     9.3.4   Instrument Performance Check Solution (IPC) — For all
             determinations the laboratory must analyze the IPC (a mid-
             range  check standard) and a calibration  blank immediately
             following daily calibration,  after every tenth sample (or
             more frequently, if required)  and at  the end of the sample
             run.  Analysis of the IPC solution and calibration blank
             immediately following calibration must verify that the
             instrument is  within ± 10%  of calibration.  Subsequent
             analyses of the IPC solution  must verify the calibration is

                              350.1-8

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             still within ± 10%.  If the calibration cannot be verified
             within the specified limits, reanalyze the IPC solution.   If
             the second analysis of the IPC solution confirms calibration
             to be outside the limits, sample analysis must be
             discontinued, the cause determined and/or in the case of
             drift, the instrument recalibrated.  All samples following
             the last acceptable IPC solution must be reanalyzed.   The
             analysis data of the calibration blank and IPC solution must
             be kept on file with the sample analyses data.

9.4  ASSESSING ANALYTE RECOVERY AND DATA QUALITY

     9.4.1   Laboratory Fortified Sample Matrix (LFM) — The laboratory
             must add a known amount of analyte to a minimum of 10% of
             the routine samples.  In each case the LFM aliquot must be a
             duplicate of the aliquot used for sample analysis.  The
             analyte concentration must be high enough to be detected
             above the original sample and should not be less than four
             times the MDL.  The added analyte concentration should be
             the same as that used in the laboratory fortified blank.

     9.4.2   Calculate the percent recovery for each analyte, corrected
             for concentrations measured in the unfortified sample, and
             compare these values to the designated LFM recovery range
             90-110%.  Percent recovery may be calculate using the
             following equation:
                R =  Cs   C    x 100
             where,   R  =  percent recovery.
                      Cs -  fortified sample  concentration.
                      C  =  sample background concentration.
                      s  =  concentration equivalent of analyte added to
                            sample.

     9.4.3   If the recovery of any analyte falls outside the designated
             LFM recovery range and the laboratory performance for that
             analyte is shown to be in control (Sect. 9.3),  the recovery
             problem encountered with the LFH is judged to be either
             matrix or solution related, not system related.

     9.4.4   Where reference materials are available, they should be
             analyzed to provide additional performance data.  The
             analysis of reference samples is a valuable tool for
             demonstrating the ability to perform the method acceptably.
                               350.1-9

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10.0 CALIBRATION AND STANDARDIZATION

     10.1 Prepare a series of at least 3 standards, covering the desired
          range, and a blank by diluting suitable volumes of standard
          solutions (7.12, 7.13) to 100 ml with reagent water.

     10.2 Process standards and blanks as described in Sect. 11, Procedure.

     10.3 Set up manifold as shown in Figure 1.

     10.4 Prepare flow system as described in Sect. 11, Procedure.

     10.5 Place appropriate standards in the sampler in order of decreasing
          concentration and perform analysis.

     10.6 Prepare standard curve by plotting instrument response against
          concentration values.  A calibration curve may be fitted to the
          calibration solutions concentration/response data using computer or
          calculator based regression curve fitting techniques.   Acceptance  or
          control limits should be established using the difference between
          the measured value of the calibration solution and the "true value"
          concentration.

     10.7 After the calibration has been established,  it must be verified by
          the analysis of a suitable QCS.  If measurements exceed ± 10% of the
          established QCS value, the analysis should be terminated and the
          instrument recalibrated.  The new calibration must be  verified
          before continuing analysis.  Periodic reanalysis of the QCS is
          recommended as a continuing calibration check.

11.0 PROCEDURE

     11.1 Preparation of equipment:  Add 500 ml of reagent water to an 800-mL
          Kjeldahl  flask.  The addition of boiling chips that have been
          previously treated with dilute NaOH will prevent bumping.  Steam out
          the distillation apparatus until  the distillate shows  no trace of
          ammonia.

     11.2 Sample preparation:   Remove the residual chorine in the sample by
          adding dechlorinating agent (7.5) equivalent to the chlorine
          residual.  To 400 ml of sample add 1 N NaOH  (7.4), until the pH is
          9.5, check the pH during addition with a pH  meter or by use of a
          short range pH paper.

     11.3 Distillation:  Transfer the sample, the pH of which has been
          adjusted  to 9.5, to  an 800-mL Kjeldahl flask and add 25 ml of the
          borate buffer (7.3).  Distill  300 ml at the  rate of 6-10 mL/min.
          into 50 ml of 2% boric acid (7.2) contained  in a 500-mL Erlenmeyer
          flask.

          NOTE 4:  The condenser tip or an  extension of the condenser tip must
          extend below the level of the boric acid solution.

                                   350.1-10

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     11.4 Since the intensity of the color used to  quantify the concentration
          is pH dependent,  the acid concentration of the wash  water and  the
          standard ammonia  solutions should approximate that of the samples.

     11.5 Allow analysis system to warm up as required.  Feed  wash water
          through sample line.

     11.6 Arrange ammonia standards in sampler in order of decreasing
          concentration of nitrogen.  Complete loading of sampler tray with
          unknown samples.

     11.7 Switch sample line from reagent water to  sampler and begin analysis.

12.0 DATA ANALYSIS AND CALCULATIONS

     12.1 Prepare a calibration curve by plotting  instrument response
          against standard concentration.  Compute  sample concentration  by
          comparing sample response with the standard curve.  Multiply answer
          by appropriate dilution factor.

     12.2 Report only those values that fall between the lowest and the
          highest calibration standards.  Samples  exceeding the highest
          standard should be diluted and reanalyzed.

     12.3 Report results in mg NH3-N/L.

13.0 METHOD PERFORMANCE

     13.1 In a single laboratory  (EMSL-Cincinnati), using surface water
          samples at concentrations of 1.41, 0.77,  0.59 and 0.43 mg NH3-N/L,
          the standard deviation was ± 0.005.

     13.2 In a single laboratory  (EMSL-Cincinnati), using surface water
          samples at concentrations of 0.16 and 1.44 mg NH3-N/L, recoveries
          were 107% and 99%, respectively.

     13.3 The inter!aboratory precision and accuracy data in Table 1 were
          developed using a reagent water matrix.   Values are  in mg NH3-N/L.

14.0 POLLUTION PREVENTION

     14.1 Pollution prevention encompasses any technique that  reduces or
          eliminates the quantity  or toxicity of waste at the  point of
          generation.  Numerous opportunities for pollution prevention exist
          in laboratory operation.  The EPA has established a  preferred
          hierarchy of environmental management techniques that places
          pollution prevention as  the management option of first choice.
          Whenever feasible,  laboratory personnel  should use pollution
          prevention techniques to address their waste generation.  When
          wastes  cannot be feasibly reduced at the source, the Agency
          recommends recycling as  the  next best option.


                                    350.1-1.1

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     14.2 The quantity of chemicals purchased should be based on expected
          usage during its shelf life and disposal cost of unused material.
          Actual reagent preparation volumes should reflect anticipated usage
          and reagent stability.

     14.3 For information about pollution prevention that may be applicable to
          laboratories and research institutions, consult "Less is Better:
          Laboratory Chemical Management for Waste Reduction," available from
          the American Chemical Society's Department of Government
          Regulations and Science Policy, 1155 16th Street N.W., Washington
          D.C. 20036, (202)872-4477.

15.0 WASTE MANAGEMENT

     15.1 The U.S. Environmental Protection Agency requires that laboratory
          waste management practices be conducted consistent with all
          applicable rules and regulations.  Excess reagents, samples and
          method process wastes should be characterized and disposed of in an
          acceptable manner.  The Agency urges laboratories to protect the
          air, water and land by minimizing and controlling all releases from
          hoods, and bench operations, complying with the letter and spirit of
          any waste discharge permit and regulations, and by complying with
          all solid and hazardous waste 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," available
          from the American Chemical Society at the address listed in Sect.
          14.3.

16.0 REFERENCES

     1.   Hiller, A., and Van Slyke, D., "Determination of Ammonia in Blood,"
          J. Biol. Chem. 102. p. 499 (1933).

     2.   O'Connor, B., Dobbs, R.,  Villiers, B., and Dean. R., "Laboratory
          Distillation of Municipal  Waste Effluents," JWPCF 39, R 25 (1967).

     3.   Fiore, J.,  and O'Brien, J.E., "Ammonia Determination by Automatic
          Analysis,"  Wastes Engineering 33, p.  352 (1962).

     4.   A Wetting Agent Recommended and Supplied by the Technicon
          Corporation for Use in AutoAnalyzers.

     5.   ASTM "Manual on Industrial Water and Industrial  Waste Water," 2nd
          Ed., 1966 printing, p. 418.

     6.   Booth, R.L., and Lobring.  L.B., "Evaluation of the AutoAnalyzer II:
          A Progress  Report" in Advances in Automated Analysis:  1972
          Technicon International Congress, Vol. 8,  p.  7-10,  Mediad
          Incorporated, Tarrytown,  N.Y., (1973).
                                   350.1-12

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7.   Standards Methods for the Examination of Water and Wastewater, 18th
     Edition, p. 4-77, Methods 4500 NH3 B arid H  (1992).
8.   Annual Book of ASTM Standards, Part 31, "Water," Standard D1426-
     79(C).
9.   Code of Federal Regulations 40, Ch. 1, Pt.  136, Appendix B.
                             350.1-13

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17.0 TABLES. DIAGRAMS. FLOWCHARTS. AND VALIDATION DATA
TABLE 1. INTERLABORATORY PRECISION AND ACCURACY DATA
NUMBER OF
VALUES
REPORTED
134
157
136
195
142
159
156
200
196
156
142
199
TRUE
VALUE
(T)
0.270
0.692
1.20
1.60
3.00
3.50
3.60
4.20
8.76
11.0
13.0
18.0
MEAN
(X)
0.2670
0.6972
1.2008
1.6095
3.0128
3.4991
3.5955
4.2271
8.7257
11.0747
12.9883
17.9727
RESIDUAL
FOR X
-0.0011
0.0059
0.0001
0.0076
0.0069
-0.0083
-0.0122
0.0177
-0.0568
0.0457
-0.0465
-0.0765
STANDARD
DEVIATION
(S)
0.0342
0.0476
0.0698
0.1023
0.1677
0.2168
0.1821
0.2855
0.4606
0.5401
0.6961
1.1635
RESIDUAL
FOR S
0.0015
-0.0070
-0.0112
0.0006
-0.0067
0.0165
-0.0234
0.0488
-0.0127
-0.0495
0.0027
0.2106
REGRESSIONS:  X = 1.003T - 0.003, S = 0.052T + 0.019
                                   350.1-14

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                             METHOD 351.2

DETERMINATION OF TOTAL KJELDAHL NITROGEN BY SEMI-AUTOMATED COLORIMETRY
                       Edited  by James W. O'Dell
                      Inorganic Chemistry Branch
                     Chemistry Research Division
                             Revision  2.0
                              August  1993
             ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
                 OFFICE OF RESEARCH AND DEVELOPMENT
                U.S. ENVIRONMENTAL PROTECTION AGENCY
                       CINCINNATI,  OHIO  45268
                               351.2-1

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                                 METHOD 351.2

     DETERMINATION OF TOTAL KOELDAHL NITROGEN BY SEMI-AUTOMATED COLORIMETRY
1.0  SCOPE AND APPLICATION
     1.1
This method covers the determination of total Kjeldahl nitrogen in
drinking, ground, and surface waters, domestic and industrial
wastes.  The procedure converts nitrogen components of biological
origin such as amino acids, proteins and peptides to ammonia, but
may not convert the nitrogenous compounds of some industrial wastes
such as amines, nitro compounds, hydrazones, oximes, semicarbazones
and some refractory tertiary amines.
     1.2
The applicable range is 0.1 to 20 mg/L TKN.
extended with sample dilution.
                                             The range may be
2.0  SUMMARY OF METHOD
     2.1
     2.2
     2.3
     2.4
     2.5
                                                            for two
The sample is heated in the presence of sulfuric acid, HpSO,  for
and one half hours.  The residue is cooled, diluted to 25 ml and
analyzed for ammonia.  This digested sample may also be used for
phosphorus determination.

Total Kjeldahl nitrogen is the sum of free-ammonia and organic
nitrogen compounds which are converted to ammonium sulfate
(NH4)2S04, under the conditions of digestion described.

Organic Kjeldahl nitrogen is the difference obtained by subtracting
the free-ammonia value from the total Kjeldahl nitrogen value.

Reduced volume versions of this method that use the same reagents
and molar ratios are acceptable provided they meet the quality
control and performance requirements stated in the method.

Limited performance-based method modifications may be acceptable
provided they are fully documented and meet or exceed requirements
expressed in Sect. 9.0, Quality Control.
3.0  DEFINITIONS

     3.1  CALIBRATION BLANK (CB) — A volume of reagent water fortified with
          the same matrix as the calibration standards, but without the
          analytes, internal standards, or surrogate analytes.

     3.2  CALIBRATION STANDARD (CAL) — A solution prepared from the primary
          dilution standard solution or stock standard solutions and the
          internal standards and surrogate analytes.  The CAL solutions are
          used to calibrate the instrument response with respect to analyte
          concentration.

                                    351.2-2

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 3.3   INSTRUMENT PERFORMANCE CHECK SOLUTION  (IPC)  —  A solution  of one  or
      more method analytes,  surrogates,  internal  standards,  or other  test
      substances used to evaluate the  performance  of  the  instrument system
      with respect to a defined set of criteria.

 3.4   LABORATORY FORTIFIED BLANK (LFB)  —  An  aliquot  of reagent  water or
      other blank matrices to which known  quantities  of the  method
      analytes  are added in  the laboratory.   The  LFB  is analyzed exactly
      like a sample,  and its purpose is  to determine  whether the
      methodology is  in control,  and whether  the laboratory  is capable  of
      making accurate and precise measurements.

 3.5   LABORATORY FORTIFIED SAMPLE MATRIX (LFM) —  An  aliquot of  an
      environmental sample to which known  quantities  of the  method
      analytes  are added in  the laboratory.   The LFM  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 LFM
      corrected  for background concentrations.

 3.6   LABORATORY REAGENT BLANK (LRB) —  An aliquot of reagent water or
      other blank matrices that are treated exactly as  a sample  including
      exposure to all  glassware,  equipment, solvents,  reagents,  internal
      standards,  and  surrogates that are used with other samples.  The LRB
      is used to determine if method analytes or other  interferences are
      present in the  laboratory environment,  the reagents, or the
      apparatus.

 3.7   LINEAR CALIBRATION RANGE  (LCR) — The concentration range  over which
      the  instrument  response  is  linear.

 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.

 3.9   METHOD DETECTION  LIMIT. (MDL)  -- The minimum concentration of an
      analyte that can  be identified, measured and reported with  99%
      confidence  that the analyte concentration is greater than zero.

3.10  QUALITY CONTROL SAMPLE  (QCS)  — A solution of method analytes of
      known concentrations that is  used to  fortify an aliquot of  LRB or
      sample matrix.  The QCS is obtained from a source external  to the
      laboratory  and different from the source of calibration standards.
      It is used  to check laboratory performance with externally  prepared
     test materials.

3.11 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.


                               351.2-3

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4.0  INTERFERENCES

     4.1  High nitrate concentrations (10X or more than the TKN level) result
          in low TKN values.  If interference is suspected, samples should be
          diluted and reanalyzed.

     4.2  Method interferences may be caused by contaminants in the reagent
          water, reagents, glassware, and other sample processing apparatus
          that bias analyte response.

5.0  SAFETY

     5.1  The toxicity or carcinogenicity of each reagent used in this method
          have not been fully established.  Each chemical should be regarded
          as a potential health hazard and exposure should be as low as
          reasonably achievable.  Cautions are included for known extremely
          hazardous materials or procedures.

     5.2  Each 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 Material
          Safety Data Sheets (MSDS)  should be made available to all personnel
          involved in the chemical analysis.  The preparation of a formal
          safety plan is also advisable.

     5.3  The following chemicals have the potential to be highly toxic or
          hazardous, consult MSDS.

          5.3.1   Mercury (7.2, 7.3)

          5.3.2   Sulfuric  acid  (7.2, 7.3, 7.4)

          5.3.3   Sodium nitroprusside  (7.9)

6.0  EQUIPMENT AND SUPPLIES

     6.1  Balance - Analytical, capable of accurately weighing to the nearest
          0.0001 g.

     6.2  Glassware - Class A volumetric flasks and pi pets as required.

     6.3  Block digester with tubes.

     6.4  Automated continuous flow  analysis equipment designed to deliver and
          react sample  and  reagents  in  the required order  and ratios.

          6.4.1   Sampling  device  (sampler)

          6.4.2   Multichannel pump

          6.4.3   Reaction  unit  or manifold


                                    351.2-4

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          6.4.4   Colorimetric detector

          6.4.5   Data recording device

7.0  REAGENTS AND STANDARDS

     7.1  Reagent water:  Ammonia free distilled or deionized water, free of
          the analyte of  interest.  ASTM type II or equivalent.

     7.2  Mercuric sulfate:  Dissolve 8 g red mercuric oxide (HgO) (CASRN
          21908-53-2) in 50 mL of 1:4 sulfuric acid (10 ml cone. H2S04:  [CASRN
          7664-93-9] 40 ml reagent water) and dilute to 100 ml with reagent
          water.

     7.3  Digestion solution:  (Sulfuric acid-mercuric sulfate-potassium
          sulfate solution):  Dissolve 133 g of K2SO,  (CASRN  7778-80-5)  in  700
          mL of reagent water and 200 mL of cone. H2S04.   Add 25 mL  of
          mercuric sulfate solution (7.1) and dilute to 1 L.

          NOTE 1:  An alternate mercury-free digestion solution can be
          prepared by dissolving 134 g K2S04  and  7.3 g CuSO,  in 800 mL reagent
          water and then adding 134 mL cone.  H2S04  and diluting to 1 L.  Use
          10 mL solution per 25 mL of sample.

     7.4  Sulfuric Acid solution (4%):  Add 40 ml. of cone, sulfuric acid to
          800 mL of reagent water, cool and dilute to 1 L.

          NOTE 2:  If alternate mercury-free digestion solution is used,
          adjust the above solution to equal  the acid concentration of the
          digested sample (11.6).

     7.5  Stock Sodium Hydroxide (20%):  Dissolve 200 g of sodium hydroxide
          (CASRN 1310-73-2) in 900 mL of reagent water and dilute to 1  L.

     7.6  Stock Sodium Potassium Tartrate solution (20%):  Dissolve 200 g
          sodium potassium tartrate (CASRN 6381-59-5)  in about 800 mL  of
          reagent water and dilute to 1 L.

     7.7  Stock Buffer solution:  Dissolve 134.0 g of sodium phosphate,
          dibasic (Na2HP04)  (CASRN 7558-79-4)  in  about 800 mL of reagent
          water.  Add 20 g of sodium hydroxide and dilute to 1  L.

     7.8  Working Buffer solution:  Combine the reagents in the stated  order,
          add 250 mL of stock sodium potassium tartrate solution (7.6)  to 200
          mL of stock buffer solution (7.7) and mix.   Add xx mL sodium
          hydroxide solution (7.5) and dilute to 1 L.   See concentration
          ranges, Table 2, for composition of working buffer.

     7.9  Sodium Sal icy!ate/Sodium Nitroprusside solution:  Dissolve 150 g of
          sodium salicylate (CASRN 54-21-7) and 0.3 g of sodium nitroprusside
          (CASRN 13755-38-9 or 14402-89-2) in about 600 mL of reagent water
          and dilute to 1 L.

                                   351.2-5

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     7.10 Sodium Hypochlorite solution:  Dilute 6.0 ml sodium hypochlorite
          solution (CASRN 7681-52-9) (Clorox) to 100 ml with reagent water.

     7.11 Ammonium chloride, stock solution:  Dissolve 3.819 g NH4C1  (CASRN
          12125-02-9) in reagent water and bring to volume in a 1 L volumetric
          flask.  1 ml = 1.0 mg NH3-N.

     7.12 Teflon boiling chips.

8.0  SAMPLE COLLECTION. PRESERVATION AND STORAGE

     8.1  Samples should be collected in plastic or glass bottles.  All
          bottles must be thoroughly cleaned and rinsed with reagent water.
          Volume collected should be sufficient to insure a representative
          sample, allow for replicate analysis (if required), and minimize
          waste disposal.

     8.2  Samples must be preserved with H2S04 to a pH <  2  and cooled to 4°C
          at the time of collection.

     8.3  Samples should be analyzed as soon as possible after collection.  If
          storage is required, preserved samples are maintained at 4°C and may
          be held for up to 28 days.

9.0  QUALITY CONTROL

     9.1  Each laboratory using this method is required to operate a formal
          quality control (QC) program.  The minimum requirements of this
          program consist of an initial demonstration of laboratory
          capability, and the periodic analysis of laboratory reagent blanks,
          fortified blanks and other laboratory solutions as a continuing
          check on performance.  The laboratory is required to maintain  per-
          formance records that define the quality of the data that are
          generated.

     9.2  INITIAL DEMONSTRATION OF PERFORMANCE

          9.2.1   The initial demonstration of performance is used to
                  characterize instrument performance (determination of  linear
                  calibration ranges and analysis of QCS) and laboratory
                  performance (determination of MDLs) prior to performing
                  analyses by this method.

          9.2.2   Linear Calibration Range (LCR)  — The LCR must be determined
                  initially and verified every 6 months or whenever a
                  significant change in instrument response is observed  or
                  expected.   The initial demonstration of linearity must use
                  sufficient standards to insure that the resulting curve is
                  linear.  The verification of linearity must use a minimum of
                  a blank and three standards.   If any verification data
                  exceeds the initial values by ± 10%,  linearity must be
                  reestablished.  If any portion of the range is shown to be

                                    351.2-6

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             nonlinear,  sufficient  standards must be used to clearly
             define the  nonlinear portion.

     9.2.3   Quality Control Sample  (QCS) — When beginning the use of
             this method, on a quarterly basis, or as required to meet
             data-quality needs, verify the calibration standards and
             acceptable  instrument  performance with the preparation and
             analyses of a QCS.  If  the determined concentrations are not
             within ± 10% of the stated values, performance of the
             determinative step of the method is unacceptable.  The
             source of the problem must be identified and corrected
             before either proceeding with the initial determination of
             MDLs or continuing with on-going analyses.

     9.2.4   Method Detection Limit  (MDL) -- MDLs must be established for
             all analytes, using reagent water (blank) fortified at a
             concentration of two to three times the estimated instrument
             detection limit.    To  determine MDL values, take seven
             replicate aliquots of the fortified reagent water and
             process through the entire analytical method.  Perform all
             calculations defined in the method and report the
             concentration values in the appropriate units.  Calculate
             the MDL as  follows:

                                 MDL = (t) x (S)

             where, t =  Student's t value for a 99% confidence level  and
                         a standard deviation estimate with n-1 degrees
                         of freedom [t = 3.14 for seven replicates].

                    S =  standard deviation of the replicate analyses.

             MDLs should be determined every six months,  when a new
             operator begins work,  or whenever there is a significant
             change in the background or instrument response.

9.3  ASSESSING LABORATORY PERFORMANCE

     9.3.1   Laboratory Reagent Blank (LRB)  — The laboratory must
             analyze at least one LRB with each batch of  samples.   Data
             produced are used to assess contamination  from the
             laboratory environment.  Values  that exceed  the MDL  indicate
             laboratory or reagent  contamination  should be suspected and
             corrective actions must be taken  before  continuing the
             analysis.

     9.3.2   Laboratory Fortified Blank (LFB)  —  The  laboratory must
             analyze at least one LFB with  each batch of  samples.
             Calculate  accuracy as  percent  recovery  (Sect.  9.4.2).   If
             the recovery of any analyte falls  outside  the required
             control  limits  of 90-110%,  that analyte  is judged  out of


                              351.2-7

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             control, and the source of the problem should be identified
             and resolved before continuing analyses.

     9.3.3   The laboratory must use LFB analyses data to assess
             laboratory performance against the required control limits
             of 90-110%.  When sufficient internal performance data
             become available (usually a minimum of 20-30 analyses),
             optional control limits can be developed  from the percent
             mean recovery (x) and the standard deviation (S) of the  mean
             recovery.  These data can be used to establish the upper and
             lower control limits as follows:

                            UPPER CONTROL LIMIT = x +  3S
                            LOWER CONTROL LIMIT = x -  3S

             The optional control limits must be equal to or better than
             the required control limits of 90-110%.  After each five to
             ten new recovery measurements, new control limits can be
             calculated using only the most recent 20^-30 data points.
             Also, the standard deviation (S) data should be used to
             establish an on-going precision statement for the level  of
             concentrations included in the LFB.  These data must be  kept
             on file and be available for review.

     9.3.4   Instrument Performance Check Solution (IPC) — For all
             determinations the laboratory must analyze the IPC (a mid-
             range check standard) and a calibration blank immediately
             following daily calibration, after every  tenth sample (or
             more frequently, if required), and at the end of the sample
             run.  Analysis of the IPC solution and calibration blank
             immediately following calibration must verify that the
             instrument is within ± 10% of calibration.  Subsequent
             analyses of the IPC solution must verify  the calibration is
             still within ± 10%.  If the calibration cannot be verified
             within the specified limits, reanalyze the IPC solution.  If
             the second analysis of the IPC solution confirms calibration
             to be outside the limits, sample analysis must be
             discontinued, the cause determined and/or in the case of
             drift the instrument recalibrated.  All samples following
             the last acceptable IPC solution must be  reanalyzed.  The
             analysis data of the calibration blank and IPC solution  must
             be kept on file with the sample analyses  data.

9.4  ASSESSING ANALYTE RECOVERY AND DATA QUALITY

     9.4.1   Laboratory Fortified Sample Matrix (LFM)  — The laboratory
             must add a known amount of analyte to a minimum of 10% of
             the routine samples.  In each case the LFM aliquot must  be a
             duplicate of the aliquot used for sample  analysis.  The
             analyte concentration must be high enough to be detected
             above the original sample and should not  be less than four


                               351.2-8

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                   times the MDL.  The added analyte concentration should be
                   the same as that used in the laboratory fortified blank.

           9.4.2   Calculate the percent recovery for each analyte, corrected
                   for concentrations measured in the unfortified sample, and
                   compare these values to the designated LFM recovery range
                   90-110%.  Percent recovery may be calculated using the
                   •Prt 1 1 r\i»i*i nn r-n-iii-»4--irt«.
                   following equation:
                      R =   	l    x 100
                             s

                  where,  R  =   percent recovery.
                         Cs =   fortified sample concentration.
                         C  =   sample background  concentration.
                         s  =   concentration  equivalent  of  analyte  added  to
                               sample.

          9'4'3   ,ILthe  recovery of any  analyte  falls  outside the designated
                  LFM  recovery range and  the 1aboratory performance for  that
                  analyte is shown to  be  in control  (Sect. 9.3), the recovery
                  problem encountered  with the LFM is judged to be either
                  matrix  or solution  related, not system related.

          9.4.4   Where reference materials are available, they should be
                  analyzed to  provide  additional performance data.  The
                  analysis of  reference samples is a valuable tool for
                  demonstrating the  ability to perform the method  acceptably.

10.0 CALIBRATION AND STANDARDI7ATTOM

     10.1 Prepare a series of at least 3 standards,  covering the desired
          range  and a blank by diluting suitable volumes  of standard solution
          (7.11)  with reagent water.

     10.2 Process standards  and blanks as  described  in Sect.  11,  Procedure.

     10.3 Set up  manifold as shown  in Figure  1  and Table 2.

     10.4 Prepare flow system  as  described in Sect.  11,  Procedure.

     10.5 Place appropriate  standards in the  sampler  in  order of decreasina
          concentration and  perform analysis.

     10.6 Prepare standard curve  by plotting  instrument  response against
          concentration values.   A calibration curve  may be fitted  to the
          calibration  solutions concentration/response data using computer or
          calculator based regression curve fitting techniques.  Acceptance or
          control  limits  should be established using  the difference between


                                   351.2-9

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          the measured value of the calibration solution  and  the  "true  value"
          concentration.

     10.7 After the calibration has been established,  it  must be  verified  by
          the analysis of a suitable quality control  sample (QCS).   If
          measurements exceed ± 10% of the established QCS value,  the analysis
          should be terminated and the instrument recalibrated.   The new
          calibration must be verified before continuing  analysis.   Periodic
          reanalysis of the QCS is recommended as a continuing calibration
          check.

11.0 PROCEDURE

     11.1 Pipet 25.0 ml of sample, standard or blank in the digestor tube.

     11.2 Add 5 ml of digestion solution (7.3) and mix with a vortex mixer
          (See Note 1).

     11.3 Add 4-8 Teflon boiling chips (7.12).  CAUTION:   An  excess of  Teflon
          chips may cause the sample to boil over.

     11.4 Place tubes in block digestor preheated to 160°C and maintain
          temperature for 1 h.

     11.5 Reset temperature to 380°C and continue to heat for one and one  half
          hour.
                 (380°C MUST BE MAINTAINED FOR 30 MIN.)

     11.6 Remove digestion tubes, cool and dilute to 25 ml with reagent water.

     11.7 Excluding the salicylate line, place all reagent lines  in their
          respective containers, connect the sample probe to the  sampler and
          start the pump.

     11.8 Flush the sampler wash receptacle with about 25 ml of 4% sulfuric
          acid  (7.4)   (See Note 2).

     11.9 When reagents have been pumping for at least 5 min, place the
          salicylate line in its respective container and allow the system to
          equilibrate.  If a precipitate forms after the addition of
          salicylate, the pH is too low.  Immediately stop the proportioning
          pump and flush the coils with water using a syringe.  Before
          restarting the system, check the concentration of the sulfuric acid
          solutions and/or the working buffer solution.

    11.10 To prevent precipitation of sodium salicylate in the waste tray,
          which can clog the tray outlet, keep the nitrogen flowcell pump  tube
          and the nitrogen Colorimeter "To Waste" tube separate from all other
          lines or keep tap water flowing in the waste tray.

    11.11 After a stable baseline has been obtained, start the sampler  and
          perform analysis.

                                   351.2-10

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 12.0 DATA ANALYSIS AND  CALCULATIONS

     12.1 Prepare a  calibration curve  by plotting  instrument response
          against standard concentration.   Compute sample concentration by
          comparing  sample response with the standard curve.   Multiply answer
          by appropriate dilution factor.

     12.2 Report only those values that fall between the lowest and the
          highest calibration standards.   Samples exceeding the highest
          standard should be diluted and reanalyzed.

     12.3 Report results in mg N/L.

 13.0 METHOD PERFORMANCE

     13.1 In a single laboratory (EMSL-Cincinnati) using sewage samples at
          concentrations of 1.2, 2.6,  and 1.7 mg N/L, the precision was ±
          0.07, ± 0.03, and ± 0.15, respectively.

     13.2 In a single laboratory (EMSL-Cincinnati) using sewage samples at
          concentrations 4.7 and 8.74 mg N/L,  the recoveries were 99% and 99%,
          respectively.

     13.3 The interlaboratory precision and accuracy data in Table 1 were
          developed using a reagent water matrix.  Values are in mg N/L.

14.0 POLLUTION PREVENTION

     14.1 Pollution prevention encompasses any technique that reduces or
          eliminates  the quantity or toxicity  of waste at the point of
          generation.  Numerous  opportunities  for pollution prevention exist
          in laboratory operation.   The EPA has established a preferred
          hierarchy of environmental  management techniques  that places
          pollution  prevention as  the management option  of  first choice.
          Whenever feasible,  laboratory personnel should use  pollution
          prevention  techniques  to  address their waste generation.   When
          wastes  cannot be  feasibly reduced at  the source,  the  Agency
          recommends  recycling as  the next best option.

     14.2 The  quantity of chemicals  purchased  should  be  based on expected
          usage during its  shelf life and  disposal  cost  of  unused material.
          Actual  reagent preparation.volumes should reflect anticipated usage
          and  reagent stability.

     14.3 For  information about  pollution  prevention that may be applicable  to
          laboratories  and  research  institutions,  consult "Less  is  Better:
          Laboratory  Chemical  Management for Waste  Reduction,"  available from
          the American  Chemical  Society's Department of  Government
          Regulations and Science.Policy,  1155  16th Street  N.W., Washington
          D.C.  20036,  (202) 872-4477.
                                  351.2-11

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15.0 WASTE MANAGEMENT

     15.1 The Environmental Protection Agency requires that laboratory waste
          management practices be conducted consistent with all applicable
          rules and regulations.  Excess Reagents and samples and method
          process wastes should be characterized and disposed of in an
          acceptable manner.  The Agency urges laboratories to protect the
          air, water, and land by minimizing and controlling all releases from
          hoods and bench operations, complying with the letter and spirit of
          any waste discharge permit and regulations, and by complying with
          all solid and hazardous waste 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," available from the
          American Chemical Society at the address listed in Sect. 14.3.
16.0 REFERENCES

     1.
     McDaniel, W.H., Hemphill, R.N. and Donaldson, W.T., "Automatic
     Determination of total Kjeldahl Nitrogen in Estuarine Water,"
     Technicon Symposia, pp. 362-367, Vol. 1, 1967.

2.   Gales, M.E. and Booth, R.L., "Evaluation of Organic Nitrogen
     Methods," EPA Office of Research and Monitoring, June, 1972.

3.   Gales, M.E. and Booth, R.L., "Simultaneous and Automated
     Determination of Total Phosphorus and Total Kjeldahl Nitrogen,"
     Methods Development and Quality Assurance Research Laboratory, May
     1974.

4.   Technicon "Total Kjeldahl Nitrogen and Total Phosphorus BD-40
     Digestion Procedure for Water," August 1974.

5.   Gales, M.E., and Booth, R.L.,  "Evaluation of the Technicon Block
     Digester System for the Measurement of Total Kjeldahl Nitrogen and
     Total Phosphorus," EPA-600/4-78-015, Environmental Monitoring and
     Support  Laboratory, Cincinnati, Ohio, 1978.

6.   Code  of  Federal Regulations 40, Ch.  1, Pt. 136, Appendix B.
                                    351.2-12

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17.0 TABLES. DIAGRAMS. FLOWCHARTS. AND VALIDATION DATA
TABLE 1. INTERLABORATORY PRECISION AND ACCURACY DATA
NUMBER OF
VALUES
REPORTED
115
134
127
164
138
115
175
121
120
127
164
175
TRUE
VALUE
(T)
0.380
0.451
1.00
3.10
3.50
5.71
7.00
8.00
15.0
21.0
25.0
26.9
MEAN
(X)
0.3891
0.4807
1.0095
3.0992
3.4765
5.6083
6.9246
7.9991
15.0213
20.4355
24.7157
26.1464
RESIDUAL
FOR X
-0.0091
0.0125
-0.0000
0.0191
0.0020
-0.0452
-0.0008
0.0877
0.2080
-0.2937
0.0426
-0.4000
STANDARD
DEVIATION
(S)
0.0750
0.1181
0.1170
0.2821
0.3973
0.4869
0.6623
0.6283
1.2495
1.7267
2.0147
2.9743
RESIDUAL
FOR S
-0.0135
0.0238
-0.0227
-0.0310
0.0512
-0.0417
0.0272
-0.0894
-0.0462
-0.0644
-0.1067
0.6960
REGRESSIONS:  X - 0.986T + 0.024, S = 0.083T + 0.057
                                   351.2-13

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             TABLE 2.  CONCENTRATION RANGES

  Range               Pump mL/min                  ml NaOH
  mg/LN            Sample   Resample             Buffer (7.7)

 0-1.5              0.80      0.32                    250

 0-5.0              0.16      0.32                    120

0-10.0              0.16      0.16                     80
                        351.2-14

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                              351.2-15

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                           METHOD 353.2

DETERMINATION OF NITRATE-NITRITE NITROGEN BY AUTOMATED COLORIMETRY
                     Edited  by James W. O'Dell
                    Inorganic Chemistry Branch
                   Chemistry Research Division
                           Revision 2.0
                           August  1993
           ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
               OFFICE  OF RESEARCH AND DEVELOPMENT
               U.S.  ENVIRONMENTAL PROTECTION AGENCY
                     CINCINNATI, OHIO  45268
                             353.2-1

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                                 METHOD 353.2

      DETERMINATION  OF NITRATE-NITRITE NITROGEN BY AUTOMATED COLORIMETRY


1.0  SCOPE AND APPLICATION

     1.1  This method covers the determination of nitrite singly,  or nitrite
          and nitrate combined in drinking, ground,  surface, domestic and
          industrial  wastes.

     1.2  The applicable range is 0.05 to 10.0 mg/L nitrate-nitrite nitrogen.
          The range may be extended with sample dilution.

2.0  SUMMARY OF METHOD

     2.1  A filtered sample is passed through a column containing granulated
          copper-cadmium to reduce nitrate to nitrite.  The nitrite (that was
          originally present plus reduced nitrate) is determined by
          diazotizing with sulfanilamide and coupling with N-(l-naphthyl)-
          ethylenediamine dihydrochloride to form a highly colored azo dye
          which is measured colorimetrically.  Separate, rather than combined
          nitrate-nitrite, values are readily obtained by carrying out the
          procedure first with, and then without, the Cu-Cd reduction step.

     2.2  Reduced volume versions of this method that use the same reagents
          and molar ratios are acceptable provided they meet the quality
          control and performance requirements stated in the method.

     2.3  Limited performance-based method modifications may be acceptable
          provided they are fully documented and meet or exceed requirements
          expressed in Sect. 9.0, Quality Control.

3.0  DEFINITIONS

     3.1  CALIBRATION BLANK (CB) — A volume of reagent water fortified with
          the same matrix as the calibration standards, but without the
          analytes, internal standards, or surrogate analytes.

     3.2  CALIBRATION STANDARD (CAL) — A solution prepared from the primary
          dilution standard solution or stock standard solutions and the
          internal standards and surrogate analytes.  The CAL solutions are
          used to calibrate the instrument response with respect to analyte
          concentration.

     3.3  INSTRUMENT PERFORMANCE CHECK SOLUTION (IPC) — A solution of one or
          more method analytes, surrogates,  internal standards, or other test
          substances used to evaluate the performance of the instrument system
          with respect to a defined set of criteria.
                                    353.2-2

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      3.4  LABORATORY FORTIFIED BLANK (LFB)  — An aliquot of reagent water or
           other blank matrices to which known quantities of the method
           analytes are added in the laboratory.   The LFB is analyzed exactly
           like a sample,  and its purpose is to determine whether the
           methodology is  in control,  and whether the laboratory is  capable of
           making accurate and precise measurements.

      3.5  LABORATORY FORTIFIED SAMPLE MATRIX (LFM) — An aliquot of an
           environmental sample to which known quantities of the method
           analytes are added in the laboratory.   The LFM 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 LFM
           corrected for background concentrations.

      3.6  LABORATORY REAGENT BLANK (LRB)  — An aliquot of reagent water  or
           other blank matrices that are treated  exactly as  a sample including
           exposure to all  glassware,  equipment,  solvents,  reagents,  internal
           standards,  and  surrogates that are used with other samples.  The LRB
           is  used  to determine if method analytes or other  interferences  are
           present  in the  laboratory environment,  the reagents,  or the
           apparatus.

      3.7  LINEAR CALIBRATION RANGE (LCR)  — The  concentration  range  over  which
           the  instrument  response is  linear.

      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.

      3.9   METHOD DETECTION  LIMIT (MDL)  — The minimum  concentration  of an
           analyte  that can  be  identified, measured and  reported with 99%
           confidence  that the  analyte concentration  is  greater than  zero.

      3.10  QUALITY  CONTROL SAMPLE  (QCS)— A  solution of method analytes of
           known concentrations that is  used  to fortify  an aliquot of LRB or
           sample matrix.  The  QCS  is obtained from a source external to the
           laboratory  and different  from the  source of calibration standards.
           It is used  to check  laboratory performance with externally prepared
          test materials.

     3.11 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.

4.0   INTERFERENCES

     4.1  Build up of suspended matter in the reduction column  will  restrict
          sample flow.  Since nitrate and nitrite are found in  a soluble
          state, samples may be pre-filtered,

                                    353.2-3

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     4  2   Low results might be obtained for samples  that  contain  high
          concentrations of iron,  copper or other metals.   EDTA is  added  to
          the samples to eliminate this interference.

     4  3   Residual  chlorine can produce a negative interference by  limiting
          reduction efficiency.  Before analysis, samples should  be checked
          and if required,  dechlorinated with sodium thiosulfate.

     4.4   Samples that contain large concentrations  of oil  and grease  will
          coat the surface of the cadmium.  This interference is  eliminated  by
          pre-extracting the sample with an organic  solvent.

     4  5   Method interferences may be caused by contaminants  in the reagent
          water, reagents,  glassware, and other sample processing apparatus
          that bias analyte response.

5.0  SAFETY

     5  1   The toxicity or carcinogenicity of each reagent used in this method
          have not been fully established.  Each chemical should  be regarded
          as a potential health hazard and exposure should be as  low as
          reasonably achievable.  Cautions are included for known extremely
          hazardous materials or procedures.

     5.2  Each 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 Material
          Safety Data Sheets  (MSDS)  should be made  available to all personnel
          involved in the chemical analysis.  The preparation of a formal
          safety plan is also  advisable.

     5.3  The following chemicals have the potential to be highly toxic or
          hazardous,  consult  MSDS.

          5.3.1    Cadmium  (7.1)

          5.3.2    Phosphoric  acid  (7.5)

          5.3.3    Hydrochloric acid  (7.6)

          5.3.4    Sulfuric  acid  (7.8)

          5.3.5    Chloroform  (7.10,  7.11)

 6.0  EQUIPMENT  AND SUPPLIES

     6.1  Balance  — Analytical,  capable of accurately weighing  to  the nearest
          0.0001  g.

     6.2  Glassware  --  Class  A volumetric flasks and  pi pets  as required.



                                     353.2-4

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     6.3  Automated continuous flow analysis equipment designed to deliver and
          react sample and reagents in the required order and ratios.

          6.3.1   Sampling device (sampler)

          6.3.2   Multichannel pump

          6.3.3   Reaction unit or manifold

          6.3.4   Colorimetric detector

          6.3.5   Data recording device

7.0  REAGENTS AND STANDARDS

     7.1  Granulated cadmium:  40-60 mesh (CASRN 7440-43-9).  Other mesh sizes
          may be used.

     7.2  Copper-cadmium:  The cadmium granules (new or used) are cleaned with
          dilute HC1 (7.6) and copperized with 2% solution of copper sulfate
          (7.7) in the following manner:

          7.2.1   Wash the cadmium with HC1 (7.6) and rinse with distilled
                  water.  The color of the cadmium so treated should be
                  silver.

          7.2.2   Swirl 10 g cadmium in 100 ml portions of 2% solution of
                  copper sulfate (7.7) for 5 min or until blue color partially
                  fades, decant and repeat with fresh copper sulfate until a
                  brown colloidal precipitate forms.

          7.2.3   Wash the copper-cadmium with reagent water (at least 10
                  times) to remove all the precipitated copper.  The color of
                  the cadmium so treated should be black.

     7.3  Preparation of reduction column.  The reduction column is a U-
          shaped, 35 cm length, 2 mm I.D. glass tube (Note 1).  Fill the
          reduction column with distilled water to prevent entrapment of air
          bubbles during the filling operations.  Transfer the copper-cadmium
          granules (7.2) to the reduction column and place a glass wool plug
          in each end.  To prevent entrapment of air bubbles in the reduction
          column, be sure that all pump tubes are filled with reagents before
          putting the column into the analytical system.

          NOTE 1:  Other reduction tube configurations, including a 0.081 I.D.
          pump tube, can be used in place of the 2-mm glass tube, if checked
          as in 10.1.

     7.4  Reagent water:  Because of possible contamination, this should be
          prepared by passage through an iori exchange column comprised of a
          mixture of both strongly acidic-cation and strongly basic-anion


                                   353.2-5

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          exchange resins.  The regeneration of the ion exchange column should
          be carried out according to the manufacturer's instructions.

     7.5  Color reagent:  To approximately 800 ml of reagent water, add, while
          stirring, 100 ml cone, phosphoric acid (CASRN 7664-38-2), 40 g
          sulfanilamide (CASRN 63-74-1) and 2 g N-1-naphthylethylenediamine
          dihydrochloride (CASRN 1465-25-4).  Stir until dissolved and dilute
          to 1 L.  Store in brown bottle and keep in the dark when not in use.
          This solution is stable for several months.

     7.6  Dilute hydrochloric acid, 6N:  Add 50 mL of cone. HC1 (CASRN 7647-
          01-0) to reagent water, cool and dilute to 100 ml.

     7.7  Copper sulfate solution, 2%:  Dissolve 20 g of CuS(y5H20 (CASRN
          7758-99-8) in 500 ml of reagent water and dilute to 1 L.

     7.8  Wash solution:  Use reagent water for unpreserved samples.   For
          samples preserved with H2S04,  use  2 ml  H2S04 (CASRN 7764-93-9), per
          liter of wash water.

     7.9  Ammonium chloride-EDTA solution:  Dissolve 85 g of reagent grade
          ammonium chloride (CASRN 12125-02-9) and 0.1 g of disodium
          ethylenediamine tetracetate (CASRN 6381-92-6) in 900 ml of reagent
          water.  Adjust the pH to 9.1 for preserved or 8.5 for non-preserved
          samples with cone, ammonium hydroxide (CASRN 1336-21-6) and dilute
          to 1 L.  Add 0.5 ml Brij-35 (CASRN 9002-92-0).

     7.10 Stock nitrate solution:  Dissolve 7.218 g KN03 (CASRN 7757-79-1)  and
          dilute to 1 L in a volumetric flask with reagent water.  Preserve
          with 2 ml of chloroform (CASRN 67-66-3) per liter.  Solution is
          stable for 6 months.  1 mL = 1.0 mg N03-N.

     7.11 Stock nitrite solution:  Dissolve 6.072 g KN02 in 500 mL of reagent
          water and dilute to 1 L in a volumetric flask.  Preserve with 2 mL
          of chloroform and keep under refrigeration.  1.0 mL = 1.0 mg N02-N.

     7.12 Standard nitrate solution:  Dilute 1.0 mL of stock nitrate solution
          (7.10) to 100 mL.  1.0 mL = 0.01 mg N03-N.   Preserve with .2 mL of
          chloroform.  Solution is stable for 6 months.

     7.13 Standard nitrite solution:  Dilute 10.0 mL of stock nitrite (7.11)
          solution to 1000 mL.  1.0 mL = 0.01 mg N02-N.   Solution is  unstable;
          prepare as required.

8.0  SAMPLE COLLECTION. PRESERVATION AND STORAGE

     8.1  Samples should be collected in plastic or glass bottles.  All
          bottles must be thoroughly cleaned and rinsed with reagent water.
          Volume collected should be sufficient to insure a representative
          sample, allow for replicate analysis (if required), and minimize
          waste disposal.


                                    353.2-6

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     8.2
     8.3
     8.4
Samples must be preserved with H2S04
at the time of collection.
to a pH < 2 and cooled to 4°C
Samples should be analyzed as soon as possible after collection.  If
storage is required, preserved samples are maintained at 4°C and may
be held for up to 28 days.

Samples to be analyzed for nitrate or nitrite only should be cooled
to 4°C and analyzed within 48 hours.
9.0  QUALITY CONTROL

     9.1  Each laboratory using this method is required to operate a formal
          quality control (QC) program.   The minimum requirements of this
          program consist of an initial  demonstration of laboratory capability
          and the periodic analysis of laboratory reagent blanks, fortified
          blanks, and other laboratory solutions as a continuing check on
          performance.  The laboratory is required to maintain performance
          records that define the quality of the data that are generated.

     9.2  INITIAL DEMONSTRATION OF PERFORMANCE

          9.2.1   The initial demonstration of performance is used to
                  characterize instrument performance (determination of LCR
                  and analysis of QCS) and laboratory performance
                  (determination of MDLs) prior to performing analyses by this
                  method.

          9.2.2   Linear Calibration Range (LCR)  — The LCR must be determined
                  initially and verified every 6 months or whenever a
                  significant change in  instrument response is observed or
                  expected.   The initial  demonstration of linearity must use
                  sufficient standards to insure that the resulting curve is
                  linear.  The verification of linearity must use a minimum of
                  a blank and three standards.   If any verification data
                  exceeds the initial  values by ± 10%,  linearity must be
                  reestablished.   If any portion  of the range is shown to be
                  nonlinear,  sufficient  standards must be used to clearly
                  define the nonlinear portion.

          9.2.3   Quality Control  Sample  (QCS)  — When beginning the  use of
                  this method,  on  a quarterly basis or as required  to meet
                  data-quality needs,  verify the  calibration  standards and
                  acceptable instrument  performance with  the  preparation and
                  analyses of a QCS.   If  the determined concentrations are not
                  within ± 10% of  the  stated values,  performance of the
                  determinative step  of  the method is  unacceptable.   The
                  source of  the problem must be  identified  and corrected
                  before either proceeding  with  the initial determination of
                  MDLs or continuing with  on-going analyses.
                                   353.2-7

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     9.2.4   Method Detection Limit (MDL) ~ MDLs must be established for
             all analytes, using reagent water (blank) fortified at a
             concentration of two to three times the estimated instrument
             detection limit.    To determine MDL values, take seven
             replicate aliquots of the fortified reagent water and
             process through the entire analytical method.  Perform all
             calculations defined in the method and report the
             concentration values in the appropriate units.  Calculate
             the MDL as follows:

                                 MDL = (t) x (S)

             where, t = Student's t value for a 99% confidence level and
                        a standard deviation estimate with n-1 degrees
                        of freedom [t =• 3.14 for seven replicates].

                    S = standard deviation of the replicate analyses.

             MDLs should be determined every 6 months, when a new
             operator begins work, or whenever there is a significant
             change in the background or instrument response.

9.3  ASSESSING LABORATORY PERFORMANCE

     9.3.1   Laboratory Reagent Blank (LRB) — The laboratory must
             analyze at least one LRB with each batch of samples.  Data
             produced are used to assess contamination from the
             laboratory environment.   Values that exceed the MDL indicate
             laboratory or reagent contamination should be suspected and
             corrective actions must be taken before continuing the
             analysis.

     9.3.2   Laboratory Fortified Blank (LFB) — The laboratory must
             analyze at least one LFB with each batch of samples.
             Calculate accuracy as percent recovery (Sect. 9.4.2).  If
             the recovery of any analyte falls outside the required
             control limits of 90-110%, that analyte is judged out of
             control, and the source of the problem should be identified
             and resolved before continuing analyses.

     9.3.3   The laboratory must use LFB analyses data to assess
             laboratory performance against the required control  limits
             of 90-110%.   When sufficient internal performance data
             become available (usually a minimum of 20-30 analyses),
             optional control limits  can be developed from the percent
             mean recovery (x) and the standard deviation (S) of the mean
             recovery.  These data can be used to establish the upper and
             lower control  limits as  follows:

                       UPPER CONTROL  LIMIT = x + 3S
                       LOWER CONTROL  LIMIT = x - 3S
                              353.2-8

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             The optional control limits must be equal to or better than
             the required control limits of 90-110%.  After each five to
             ten new recovery measurements, new control limits can be
             calculated using only the most recent 20-30 data points.
             Also, the standard deviation (S) data should be used to
             established an on-going precision statement for the level of
             concentrations included in the LFB.  These data must be kept
             on file and be available for review.

     9.3.4   Instrument Performance Check Solution (IPC) -- For all
             determinations the laboratory must analyze the IPC (a mid-
             range check standard) and a calibration blank immediately
             following daily calibration, after every tenth sample (or
             more frequently, if required), and at the end of the sample
             run.  Analysis of the IPC solution and calibration blank
             immediately following calibration must verify that the
             instrument is within ± 10% of calibration.  Subsequent
             analyses of the IPC solution must verify the calibration is
             still within ± 10%.  If the calibration cannot be verified
             within the specified limits, reanalyze the IPC solution.  If
             the second analysis of the IPC solution confirms calibration
             to be outside the limits, sample analysis must be
             discontinued, the cause determined and/or in the case of
             drift, the instrument recalibrated.  All samples following
             the last acceptable IPC solution must be reanalyzed.   The
             analysis data of the calibration blank and IPC solution must
             be kept on file with the sample analyses data.

9.4  ASSESSING ANALYTE RECOVERY AND DATA QUALITY

     9.4.1   Laboratory Fortified Sample Matrix (LFM) —The laboratory
             must add a known amount of analyte to a minimum of 10% of
             the routine samples.  In each case, the LFM aliquot must be
             a duplicate of the aliquot used for sample analysis.   The
             analyte concentration must be high enough to be detected
             above the original  sample and should not be less than four
             times the MDL.  The added analyte concentration should be
             the same as that used in the laboratory fortified blank.

     9.4,.2   Calculate the percent recovery for each analyte, corrected
             for concentrations measured in the unfortified sample, and
             compare these values to the designated LFM recovery range
             90-110%.  Percent recovery may be calculate using the
             following equation:


                R =  Cs,~  C    x 100
             where, R  =  percent recovery.
                    Cs =  fortified  sample concentration.
                    C  =  sample background  concentration.
                    s  =  concentration equivalent of analyte added to
                          sample.

                              353.2-9

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           9.4.3    If the recovery of any  analyte  falls  outside  the designated
                   LFM recovery range and  the  laboratory performance for that
                   analyte is  shown to be  in control  (Sect.  9.3), the recovery
                   problem encountered with the  LFM  is judged to be either
                   matrix or solution related, not system related.

           9.4.4    Where  reference materials are available,  they should be
                   analyzed to provide additional  performance data.  The
                   analysis of reference samples is  a valuable tool for
                   demonstrating the ability to  perform  the  method acceptably.

10.0 CALIBRATION AND STANDARDIZATION

     10.1  Prepare  a  series of at least 3  standards, covering the desired
           range, and a blank  by diluting  suitable volumes of standard nitrate
           solution (7.12). At least one nitrite standard should be compared to
           a nitrate  standard  at the same  concentration  to verify the
           efficiency of  the reduction  column.

     10.2  Set up manifold  as  shown  in  Figure  1.   Care should be taken not to
           introduce  air  into  the reduction column.

     10.3  Place appropriate standards  in  the  sampler in  order of decreasing
           concentration  and perform analysis.

     10.4  Prepare  standard curve by plotting  instrument  response against
           concentration  values.   A  calibration  curve may be fitted to the
           calibration solutions  concentration/response  data using computer or
           calculator based regression curve fitting techniques.  Acceptance or
           control limits should  be  established  using the difference between
           the measured value  of  the  calibration solution and the "true value"
           concentration.

     10.5 After the  calibration  has  been  established, it must be verified by
           the analysis of  a suitable quality control sample (QCS).   If
          measurements exceed ±  10%  of the established QCS value,  the analysis
           should be  terminated and the instrument recalibrated.  The new
           calibration must be verified before continuing analysis.   Periodic
          reanalysis of the QCS  is recommended  as a continuing calibration
          check.

          NOTE 3:  Condition column  by running  1 mg/L standard for 10 min if a
          new reduction column is being used.   Subsequently wash the column
          with reagents for 20 min.

11.0 PROCEDURE

     11.1 If the pH of the sample is below 5 or above 9, adjust to  between 5
          and 9 with either cone. HC1 or cone. NH4OH.

     11.2 Set up the manifold  as shown in  Figure 1.   Care should be  taken not
          to introduce air into reduction  column.

                                   353.2-10

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     11.3 Allow system to equilibrate as required.  Obtain a stable baseline
          with all reagents, feeding reagent water through the sample line.

     11.4 Place appropriate nitrate and/or nitrite standards in sampler in
          order of decreasing concentration and complete loading of sampler
          tray.

     11.5 Switch sample line to sampler and start analysis.

12.0 DATA ANALYSIS AND CALCULATIONS

     12.1 Prepare a calibration curve by plotting instrument response
          against standard concentration.   Compute sample concentration by
          comparing sample response with the standard curve.   Multiply answer
          by appropriate dilution factor.

     12.2 Report only those values that fall between the lowest and the
          highest calibration standards.   Samples exceeding the highest
          standard should be diluted and reanalyzed.

     12.3 Report results in mg/L as nitrogen.

13.0 METHOD PERFORMANCE

     13.1 Three laboratories participating in an EPA Method Study analyzed
          four natural water samples containing exact increments of inorganic
          nitrate, with the following results:

                                                            Accuracy as	
            Increment as          Precision as
          Nitrate Nitrogen     Standard Deviation       Bias,         Bias,
             mq N/liter            mq N/liter	       %        mq N/liter

                0.29                  0.012            + 5.75       + 0.017
                0.35                  0.092  .         + 18.10       + 0.063
                2.31                  0.318            +4.47       + 0.103
                2.48                  0.176            - 2.69       - 0.067

     13.2 The inter!aboratory precision and accuracy data in Table 1 were
          developed using a reagent water matrix.  Values are in mg N03-N/L.

     13.3 Single laboratory precision data can be estimated at 50% to 75% of
          the inter!aboratory precision estimates.

14.0 POLLUTION PREVENTION

     14.1 Pollution prevention encompasses any technique that reduces or
          eliminates the quantity or toxicity of waste at the point of
          generation.   Numerous opportunities for pollution prevention exist
          in laboratory operation.   The EPA has established a preferred
          hierarchy of environmental  management techniques that places
          pollution prevention as the management option of first choice.

                                   353.2-11

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          Whenever feasible, laboratory personnel should use pollution
          prevention techniques to address their waste generation.  When
          wastes cannot be feasibly reduced at the source, the Agency
          recommends recycling as the next best option.

     14.2 The quantity of chemicals purchased should be based on expected
          usage during its shelf life and disposal cost of unused material.
          Actual reagent preparation volumes should reflect anticipated usage
          and reagent stability.

     14.3 For information about pollution prevention that may be applicable to
          laboratories and research institutions, consult "Less is Better:
          Laboratory Chemical Management for Waste Reduction," available from
          the American Chemical Society's Department of Government
          Regulations and Science Policy, 1155 16th Street N.W., Washington
          D.C. 20036, (202) 872-4477.

15.0 WASTE MANAGEMENT

     15.1 The Environmental Protection Agency requires that laboratory waste
          management practices be conducted consistent with all applicable
          rules and regulations.  Excess reagents, samples, and method
          process wastes should be characterized and disposed of in an
          acceptable manner.  The Agency urges laboratories to protect the
          air, water, and land by minimizing and controlling all releases from
          hoods and bench operations, complying with the letter and spirit of
          any waste discharge permit and regulations, and by complying with
          all solid and hazardous waste 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," available from the
          American Chemical Society at the address listed in Sect. 14.3.

16.0 REFERENCES

     1.   Fiore, 0.,  and O'Brien,  J.E.,  "Automation in Sanitary Chemistry -
          Parts 1 & 2:   Determination of Nitrates and Nitrites," Wastes
          Engineering 33,  128 &238 (1962).

     2.   Armstrong,  F.A., Stearns,  C.R., and Strickland,  J.D., "The
          Measurement of Upwelling and Subsequent Biological  Processes by
          Means of the Technicon AutoAnalyzer and Associated Equipment," Deep
          Sea Research 14, pp.  381-389 (1967).

     3.   Annual Book of ASTM Standards,  Part 31,  "Water," Standard D1254, p.
          366 (1976).

     4.   Standard Methods for the Examination  of Water and Wastewater, 17th
          Edition,  pp.  4-91,  Method  4500-N03 F  (1992).
                                   353.2-12

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Chemical Analyses for Water Quality Manual, Department of the
Interior, FWPCA, R.A. Taft Engineering Center Training Program,
Cincinnati, Ohio 45226 (January, 1966).

Code of Federal Regulations 40, Ch. 1, Pt. 136, Appendix B.
                        353.2-13

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17.0 TABLES. DIAGRAMS. FLOWCHARTS AND VALIDATION DATA
TABLE 1. INTERLABORATORY PRECISION AND ACCURACY DATA
NUMBER OF
VALUES
REPORTED
163
183
213
170
163
172
183
214
172
213
170
214
TRUE
VALUE
(T)
0.250
0.451
0.650
0.950
1.90
2.20
2.41
3.20
6.50
8.00
8.50
10.0
MEAN
(X)
0.2479
0.4441
0.6479
0.9537
1.8987
2.1971
2.3732
3.2042
6.4978
7.9814
8.5135
9.9736
RESIDUAL
FOR X
0.0007
-0.0039
0.0012
0.0074
0.0037
0.0025
-0.0312
0.0109
0.0089
-0.0055
0.0273
-0.0106
STANDARD
DEVIATION
CS)
0.0200
0.0289
0.0398
0.0484
0.0918
0.1164
0.1273
0.1456
0.3156
0.3673
0.3635
0.4353
RESIDUAL
FOR S
-0.0001
-0.0002
0.0017
-0.0031
-0.0024
0.0087
0.0102
-0.0070
0.0148
-0.0008
-0.0271
-0.0227
REGRESSIONS:  X = 0.999T + 0.002, S = 0.045T + 0.009
                                   353.2-14

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                       353.2-15

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                       METHOD  365.1

DETERMINATION OF PHOSPHORUS BY SEMI-AUTOMATED COLORIMETRY
                Edited by James W. O'Dell
                Inorganic  Chemistry Branch
               Chemistry Research Division
                       Revision  2.0
                       August 1993
       ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
            OFFICE OF  RESEARCH AND  DEVELOPMENT
           U.S.  ENVIRONMENTAL PROTECTION AGENCY
                 CINCINNATI, OHIO  45268
                         365.1-1

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                                 METHOD 365.1

             DETERMINATION OF PHOSPHORUS BY AUTOMATED COLORIMETRY


1.0  SCOPE AND APPLICATION

     1.1  This method covers the determination of specified forms of
          phosphorus in drinking, ground, and surface waters, and domestic and
          industrial wastes.

     1.2  The methods are based on reactions that are specific for the
          orthophosphate ion.  Thus, depending on the prescribed pretreatment
          of the sample, the various forms of phosphorus that may be
          determined are defined in Section 3 and given in Figure 1.

          1.2.1   Except for in-depth and detailed studies, the most commonly
                  measured forms are total and dissolved phosphorus, total and
                  dissolved orthophosphate.  Hydrolyzable phosphorus is
                  normally found only in sewage-type samples.  Insoluble forms
                  of phosphorus are determined by calculation.

     1.3  The applicable range is 0.01 to 1.0 mg P/L.  Approximately 20-30
          samples per hour can be analyzed.

2.0  SUMMARY OF METHOD

     2.1  Ammonium molybdate and antimony potassium tartrate react in an acid
          medium with dilute solutions of phosphorus to form an antimony-
          phospho-molybdate complex.  This complex is reduced to an intensely
          blue-colored complex by ascorbic acid.  The color is proportional to
          the phosphorus concentration.

     2.2  Only orthophosphate forms a blue color in this test.  Polyphosphates
          (and some organic phosphorus compounds) may be converted to the
          orthophosphate form by manual sulfuric acid hydrolysis.  Organic
          phosphorus compounds may be converted to the orthophosphate form by
          manual persulfate digestion.5  The developed color is measured
          automatically.

     2.3  Reduced volume versions of this method that use the same reagents
          and molar ratios are acceptable provided they meet the quality
          control and performance requirements stated in the method.

     2.4  Limited performance-based method modifications may be acceptable
          provided they are fully documented and meet or exceed requirements
          expressed in Sect.  9.0, Quality Control.
                                   365.1-2

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3.0  DEFINITIONS

     3.1  CALIBRATION BLANK (CB) — A volume of reagent water fortified with
          the same matrix as the calibration standards, but without the
          analytes, internal standards, or surrogate analytes.

     3.2  CALIBRATION STANDARD (CAL) -- A solution prepared from the primary
          dilution standard solution or stock standard solutions and the
          internal standards and surrogate analytes.  The CAL solutions are
          used to calibrate the instrument response with respect to analyte
          concentration.

     3.3  INSTRUMENT PERFORMANCE CHECK SOLUTION (IPC)  — A solution of one or
          more method analytes, surrogates,  internal standards,  or other test
          substances used to evaluate the performance  of the instrument system
          with respect to a defined set of criteria.

     3.4  LABORATORY FORTIFIED BLANK (LFB) -- An aliquot of reagent water or
          other blank matrices to which known quantities of the  method
          analytes are added in the laboratory.   The LFB is analyzed exactly
          like a sample, and its purpose is  to determine whether the
          methodology is in control, and whether the laboratory  is capable of
          making accurate and precise measurements.

     3.5  LABORATORY FORTIFIED SAMPLE MATRIX (LFM) —  An aliquot of an
          environmental  sample to which known quantities of the  method
          analytes are added in the laboratory.   The LFM 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 LFM
          corrected for  background concentrations.

     3.6  LABORATORY REAGENT BLANK (LRB)  —  An aliquot of reagent water or
          other blank matrices that are treated  exactly as a  sample including
          exposure to all  glassware,  equipment,  solvents,  reagents,  internal
          standards,  and surrogates that are used  with other  samples.   The LRB
          is  used to determine if method analytes  or other interferences  are
          present in the laboratory environment,  the reagents, or the
          apparatus.

     3.7  LINEAR CALIBRATION RANGE (LCR)  —  The  concentration  range  over  which
          the instrument response is  linear.,

     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.

     3.9  METHOD DETECTION  LIMIT  (MDL)  --  The  minimum  concentration  of  an
          analyte that can  be  identified,  measured and  reported with  99%
          confidence  that the  analyte concentration  is  greater than  zero.

                                   365.1-3

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3.10 QUALITY CONTROL SAMPLE (QCS) -- A solution of method analytes of
     known concentrations that is used to fortify an aliquot of LRB or
     sample matrix.  The QCS is obtained from a source external to the
     laboratory and different from the source of calibration standards.
     It is used to check laboratory performance with externally prepared
     test materials.

3.11 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

3.12 TOTAL PHOSPHORUS (P) — All of the phosphorus present in the sample
     regardless of forms, as measured by the persulfate digestion
     procedure.

     3.12.1  TOTAL ORTHOPHOSPHATE (P-ortho) — Inorganic phosphorus
             [(PO*)"3] in the sample as measured by the direct
             colorimetric analysis procedure.

     3.12.2  TOTAL HYDROLYZABLE PHOSPHORUS (P-hydro) — Phosphorus in the
             sample as measured by the sulfuric acid hydrolysis
             procedure, and minus predetermined orthophosphates.  This
             hydrolyzable phosphorus includes polyphosphates [(P207)~ ,
             (P3010)"5,  etc.]  plus some organic phosphorus.

     3.12.3  TOTAL ORGANIC PHOSPHORUS (P-org) -- Phosphorus (inorganic
             plus oxidizable organic) in the sample as measured by the
             persulfate digestion procedure, and minus hydrolyzable
             phosphorus and orthophosphate.

3.13 DISSOLVED PHOSPHORUS (P-D) -- All of the phosphorus present in the
     filtrate of a sample filtered through a phosphorus-free filter of
     0.45 micron pore size and measured by the persulfate digestion
     procedure.

     3.13.1  DISSOLVED ORTHOPHOSPHATE (P-D ortho) ~ As measured by he
             direct colorimetric analysis procedure.

     3.13.2  DISSOLVED HYDROLYZABLE PHOSPHORUS (P-D, hydro) — As
             measured by the sulfuric acid hydrolysis procedure and minus
             predetermined dissolved orthophosphates.

     3.13.3  DISSOLVED ORGANIC PHOSPHORUS (P-D, org) ~ As measured  by
             the persulfate digestion procedure, and minus dissolved
             hydrolyzable phosphorus and orthophosphate.

3.14 The following forms, when sufficient amounts of phosphorus are
     present in the sample to warrant such consideration, may be
     calculated:

     3.14.1  INSOLUBLE PHOSPHORUS (P-I) = (P)-(P-D).
                               365.1-4

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                  3.14.1.1   INSOLUBLE ORTHOPHOSPHATE (P-I, ortho) = (P, ortho)
                             - (P-D, ortho).

                  3.14.1.2   INSOLUBLE HYDROLYZABLE PHOSPHORUS (P-I, hydro) =
                             (P, hydro) - (P-D, hydro).

                  3.14.1.3   INSOLUBLE ORGANIC PHOSPHORUS (P-I, org) = (P,
                             org) -(P-D, org).

     3.15 All phosphorus forms shall be reported as P, mg/L, to the third
          place.

4.0  INTERFERENCES

     4.1  No interference is caused by copper, iron, or silicate at
          concentrations many times greater than their reported concentration
          in seawater.  However, high iron concentrations can cause
          precipitation of, and subsequent loss, of phosphorus.

     4.2  The salt error for samples ranging from 5% to 20% salt content was
          found to be less than 1%.

     4.3  Arsenate is determined similarly to phosphorus and should be
          considered when present in concentrations higher than phosphorus.
          However, at concentrations found in sea water, it does not
          interfere.

     4.4  Sample turbidity must be removed by filtration prior to analysis for
          orthophosphate.  Samples for total or total  hydrolyzable phosphorus
          should be filtered only after digestion.  Sample color that absorbs
          in the photometric range used for analysis will also interfere.

     4.5  Method interferences may be caused by contaminants in the reagent
          water, reagents,  glassware, and other sample processing apparatus
          that bias analyte response.

5.0  SAFETY

     5.1  The toxicity or carcinogenicity of each reagent used in this method
         , have not been fully established.  Each chemical should be regarded
          as a potential health hazard and exposure should be as low as
          reasonably achievable.  Cautions are included for known extremely
          hazardous materials or procedures.

     5.2  Each 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 Material
          Safety Data Sheets (MSDS) should be made available to all  personnel
          involved in the chemical  analysis.  The preparation of a formal
          safety plan is also advisable.
                                   365.1-5

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     5.3  The following chemicals have the potential to be highly toxic or
          hazardous, consult MSDS.

          5.3.1   Sulfuric acid  (7.2, 7.7)

6.0  EQUIPMENT AND SUPPLIES

     6.1  Balance — Analytical, capable of accurately weighing to the nearest
          0.0001 g.

     6.2  Glassware -- Class A volumetric flasks and pi pets as required.

     6.3  Hot plate or autoclave'.

     6.4  Automated continuous flow analysis equipment designed to deliver and
          react sample and reagents in the required order and ratios.

          6.4.1   Sampling device (sampler)

          6.4.2   Multichannel pump

          6.4.3   Reaction unit or manifold

          6.4.4   Colorimetric detector

          6.4.5   Data recording device

     6.5  Acid-washed glassware:  All glassware used in the determination
          should be washed with hot 1:1 HC1 and rinsed with distilled water.
          The acid-washed glassware should be filled with distilled water and
          treated with all the reagents to remove the last traces of
          phosphorus that might be adsorbed on the glassware.  Preferably,
          this glassware should be used only for the determination of
          phosphorus and after use it should be rinsed with distilled water
          and kept covered until needed again.  If this is done, the treatment
          with 1:1 HC1 and reagents is only required occasionally.  Commercial
          detergent should never be used.

7.0  REAGENTS AND STANDARDS

     7.1  Reagent water:  Distilled or deionized water, free of the analyte of
          interest.  ASTM type II or equivalent.

     7.2  Sulfuric acid solution, 5N:  Slowly add 70 ml of cone. H-,SO,  (CASRN
          7664-93-9) to approximately 400 ml of distilled water.  Cool to, room
          temperature and dilute to 500 mL with distilled water.

     7.3  Antimony potassium tartrate solution:  Weight 0.3 g
          K(SbO)C4H,06.l/2H20 (CASRN 28300-74-5), dissolved in 50 ml distilled
          water in 100-mL volumetric flask, dilute to volume.  Store at 4°C in
          a dark, glass-stoppered bottle.


                                   365.1-6

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 7.4  Ammonium molybdate solution:  Dissolve 4 g (NH,),Mo70P,.4H,0  (CASRN
      12027-67-7) in 100 ml reagent water.  Store in a plastic-Wtle at
      T" L »

 7.5  Ascorbic acid, 0.1M:  Dissolve 1.8 g of ascorbic acid (CASRN 50-81-
      7) in 100 ml of reagent water.  The solution is stable for about a
      week if prepared with water containing no more than trace amounts of
      heavy metals and stored at 4°C.

 7.6  Combined reagent:  Mix the above reagents in the following
      proportions for 100 ml of the mixed reagent:   50 ml of 5N H?SO,
      (7.2),  5 ml of antimony potassium tartrate solution (7.3), 15 ml of
      ammonium molybdate solution (7.4), and 30 ml of ascorbic acid
      solution (7.5).  Mix after addition of each reagent.   All  reagents
      must reach room temperature before they are mixed and must be mixed
      in the  order given.  If turbidity forms in the combined  reagent,
      shake and let stand for a few minutes until  the turbidity disappears
      before  processing.   This volume is sufficient for 4 h operation.
      Since the stability of this solution is limited,  it must be  freshly
      prepared for each run.

      NOTE 1:   A stable solution can be prepared by not including  the
      ascorbic acid  in  the combined reagent.   If this is  done,  the mixed
      reagent  (molybdate,  tartrate,  and acid)  is pumped through  the
      distilled water line and the ascorbic acid solution (30  ml of 7.5
      diluted  to 100 ml with  reagent water)  through  the original mixed
      reagent  line.

 7.7   Sulfuric acid  solution,  11  N:   Slowly add  155  ml  cone. HPSO,  to 600
      mL  reagent water.   When  cool,  dilute  to  500 ml.

 7.8   Ammonium persulfate  (CASRN  7727-54-0).

 7.9   Acid  wash  water:  Add 40 mL  of sulfuric  acid solution  (7.7)  to 1 L
      of  reagent  water  and dilute  to 2  L.   (Not  to be used when  only
      orthophosphate  is being  determined).

 7.-10  Phenolphthalein indicator solution  (5 g/L):  Dissolve 0 5  g  of
      phenolphthalein (CASRN 77-09-8) in  a  solution of 50 ml of  isopropyl
      alcohol   (CASRN 67-63-0)  and  50 ml of reagent water.

 7.11  Stock phosphorus solution:  Dissolve 0.4393 g of predried  (105°C for
      1 h)  Potassium phosphate monobasic: KH2PQ,  (CASRN 7778-77-0) in
      reagent water and dilute to  1000 ml.  1.0 ml = 0.1 mg P.

7.12  Standard phosphorus solution:  Dilute 10.0 ml of stock solution
      (7.11) to 100 ml with reagent water.  1.0 ml = 0.01 mg P.

7.13 Standard phosphorus solution:  Dilute 10.0 ml of standard solution
      (7.12) to 100 ml with reagent water.  1.0 mL - 0.001 mg P.
                              365.1-7

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8.0  SAMPLE COLLECTION. PRESERVATION AND STORAGE

     8.1  Samples should be collected in plastic or glass bottles.   All
          bottles must be thoroughly cleaned and rinsed with reagent water.
          Volume collected should be sufficient to insure a representative
          sample, allow for replicate analysis (if required), and minimize
          waste disposal.

     8.2  Samples must be preserved with H2S04  to  a pH  <  2  and cooled to 4°C
          at the time of collection.

     8.3  Samples should be analyzed as soon as possible after collection.  If
          storage is required, preserved samples are maintained at 4°C and may
          be held for up to 28 days.

9.0  QUALITY CONTROL

     9.1  Each laboratory using this method is required to operate a formal
          quality control (QC) program.  The minimum requirements of this
          program consist of an initial demonstration of laboratory
          capability, and the periodic analysis of laboratory reagent blanks,
          fortified blanks and other laboratory solutions as a continuing
          check on performance.  The laboratory is required to maintain per-
          formance records that define the quality of the data that are
          generated.

     9.2  INITIAL DEMONSTRATION OF  PERFORMANCE

          9.2.1   The initial demonstration of performance is used to
                  characterize instrument performance  (determination of LCRs
                  and analysis of QCS) and laboratory performance
                  (determination of MDLs) prior to performing analyses by this
                  method.

          9.2.2   Linear Calibration Range (LCR) — The  LCR must be determined
                  initially and verified every 6 months  or whenever a
                  significant change in instrument response  is observed or
                  expected.  The initial demonstration of linearity must use
                  sufficient standards to insure that the resulting curve is
                  linear.  The verification of linearity must use a minimum of
                  a blank and three standards.  If any verification data
                  exceeds the initial values by ± 10%, linearity must be
                  reestablished.  If any portion of the  range is shown to be
                  nonlinear, sufficient standards must be used to clearly
                  define the nonlinear portion.

          9.2.3   Quality Control Sample (QCS) — When beginning the use of
                  this method, on a quarterly  basis or as required to meet
                  data-quality needs, verify the calibration standards and
                  acceptable instrument performance with the preparation and
                  analyses of a QCS.  If the determined  concentrations are not
                  within ± 10% of the stated values, performance of the

                                    365.1-8

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             determinative step of the method is unacceptable.  The
             source of the problem must be identified and corrected
             before either proceeding with the initial determination of
             MDLs or continuing with on-going analyses.

     9.2.4   Method Detection Limit (MDL) — MDLs must be established for
             all analytes, using reagent water (blank) fortified at a
             concentration of two to three times the estimated instrument
             detection limit.(5)  To determine MDL values, take seven
             replicate aliquots of the fortified reagent water and
             process through the entire analytical method.  Perform all
             calculations defined in the method and report the
             concentration values in the appropriate units.  Calculate
             the MDL as follows:

                              MDL = (t) x (S)

             where, t = Student's t value for a 99% confidence level and
                        a standard deviation estimate with n-1 degrees
                        of freedom [t = 3.14 for seven replicates].

                    S = standard deviation of the replicate analyses.

             MDLs should be determined every 6 months, when a new
             operator begins work,  or whenever there is a significant
             change in the background or instrument response.

9.3  ASSESSING LABORATORY PERFORMANCE

     9.3.1   Laboratory Reagent Blank (LRB)  — The laboratory must
             analyze at least one LRB with each batch of samples.   Data
             produced are used to assess contamination from the
             laboratory environment.   Values that exceed the MDL indicate
             laboratory or reagent  contamination should be suspected and
             corrective actions must  be taken before continuing the
             analysis.

     9.3.2   Laboratory Fortified Blank (LFB) — The laboratory must
             analyze at least one LFB with each batch of samples.
             Calculate accuracy as  percent recovery (Sect.  9.4.2).   If
             the recovery of any analyte falls outside the required
             control  limits of 90-110%,  that analyte is judged out  of
             control,  and the source  of the  problem should be identified
             and resolved before continuing  analyses.

     9.3.3   The laboratory must use  LFB analyses data to assess
             laboratory performance against  the required control  limits
             of 90-110%.   When sufficient  internal  performance data
             become available (usually a minimum of 20-30 analyses),
             optional  control  limits  can be  developed  from the percent
             mean recovery (x)  and the standard  deviation (S)  of the mean


                              365.1-9

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             recovery.   These data can be used to establish  the  upper  and
             lower control  limits as follows:

                        UPPER CONTROL LIMIT  =  x + 3S
                        LOWER CONTROL LIMIT  =  x - 3S

             The optional  control limits must  be equal  to or better than
             the required  control limits of  90-110%.   After  each five  to
             ten new recovery measurements,  new control  limits can be
             calculated using only the most  recent 20-30 data points.
             Also, the standard deviation (S)  data should be used to
             establish an  on-going precision statement for the level of
             concentrations included in the  LFB.  These data must be kept
             on file and be available for review.

     9.3.4   Instrument Performance Check Solution (IPC) —  For all
             determinations the laboratory must analyze the  IPC (a mid-
             range check standard) and a calibration  blank immediately
             following daily calibration, after every tenth  sample (or
             more frequently, if required) and at the end of the sample
             run.  Analysis of the IPC solution and calibration blank
             immediately following calibration must verify that the
             instrument is within ± 10% of calibration.  Subsequent
             analyses of the IPC solution must verify the calibration  is
             still within  ± 10%.  If the calibration  cannot  be verified
             within the specified limits, reanalyze the IPC  solution.   If
             the second analysis of the IPC  solution  confirms calibration
             to be outside the limits, sample analysis must  be
             discontinued, the cause determined and/or in the case of
             drift the instrument recalibrated.  All  samples following
             the last acceptable IPC solution must be reanalyzed.  The
             analysis data of the calibration blank and IPC  solution must
             be kept on file with the sample analyses data.

9.4  ASSESSING ANALYTE RECOVERY AND DATA QUALITY

     9.4.1   Laboratory Fortified Sample Matrix  (LFM) — The laboratory
             must add a known amount of analyte to a minimum of 10% of
             the routine samples.  In each case the LFM aliquot must be a
             duplicate of the aliquot used for sample analysis.  The
             analyte concentration must be high enough to be detected
             above the original sample and should not be less than four
             times the MDL.  The added analyte concentration should be
             the same as that used in the laboratory fortified blank.

     9.4.2   Calculate the percent recovery for each analyte, corrected
             for concentrations measured in the unfortified  sample, and
             compare these values to the designated LFM recovery range
             90-110%. Percent recovery may be calculate using the
             following equation:
                              365.1-10

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                     R =   f	x 100
                            s

                  where, R  =  percent recovery.
                         Cs =  fortified sample concentration.
                         C  =  sample background concentration.
                         s  =  concentration equivalent of analyte added to
                               sample.

          9.4.3   If the recovery of any analyte falls outside the designated
                  LFM recovery range and the laboratory performance for that
                  analyte is shown to be in control (Sect. 9.3), the recovery
                  problem encountered with the LFM is judged to be either
                  matrix or solution related, not system related.

          9.4.4   Where reference materials are available, they should be
                  analyzed to provide additional performance data.  The
                  analysis of reference samples is a valuable tool for
                  demonstrating the ability to perform the method acceptably.

10.0 CALIBRATION AND STANDARDIZATION

     10.1 Prepare a series of at least 3 standards, covering the desired
          range, and a blank by pipetting and diluting suitable volumes of
          working standard solutions (7.12 or 7.13) into 100 ml volumetric
          flasks.  Suggested ranges include 0.00 to 0.10 and 0.20 to 1.00
          mg/L.

     10.2 Process standards and blanks as described in Sect.  11,  Procedure.

     10.3 Set up manifold as shown in Figure 2.

     10.4 Prepare flow system as described in Sect. 11,  Procedure.

     10.5 Place  appropriate standards in the sampler in  order of decreasing
          concentration and perform analysis.

     10.6 Prepare standard curve by plotting instrument  response  against
          concentration values.   A calibration  curve may be  fitted to  the
          calibration  solutions concentration/response data  using computer or
          calculator based regression curve fitting techniques.   Acceptance  or
          control  limits  should be established  using the difference between
          the measured  value of the calibration  solution and  the  "true value"
          concentration.

     10.7 After  the  calibration has been  established,  it must be  verified by
          the analysis  of a suitable quality control  sample  (QCS).   If
          measurements  exceed  ± 10% of the  established QCS value,  the  analysis
          should be  terminated and the instrument  recalibrated.   The new
          calibration must  be  verified before continuing analysis.   Periodic


                                   365.1-11

-------
          reanalysis of the QCS is recommended as a continuing calibration
          check.

11.0 PROCEDURE

     11.1 Phosphorus

          11.1.1  Add 1 ml of sulfuric acid.solution (7.7) to a 50 ml sample
                  and/or standard in a 125-mL Erlenmeyer flask.

          11.1.2  Add 0.4 g of ammonium persulfate (7.8).

          11.1.3  Boil gently on a pre-heated hot plate for approximately 30-
                  40 min or until a final volume of about 10 ml is reached.
                  Do not allow sample to go to dryness.  Alternately, heat for
                  30 min in an autoclave at 121°C (15-20 psi).

          11.1.4  Cool and dilute the sample to 50 ml.  If sample is not clear
                  at this point, filter.

          11.1.5  Determine phosphorus as outlined (11.3.2) with acid wash
                  water (7.9) in wash tubes.

     11.2 Hydrolyzable Phosphorus

          11.2.1. Add 1 mL of sulfuric acid solution (7.7) to a 50 ml sample
                  and/or standard in a 125 ml Erlenmeyer flask.

          11.2.2  Boil gently on a pre-heated hot plate for 30-40 min until  a
                  final volume of about 10 ml is reached.  Do not allow sample
                  to go to dryness.  Alternatively, heat for 30 min in an
                  autoclave at 121°C (15-20 psi).

          11.2.3  Determine phosphorus as outlined (11.3.2) with acid wash
                  water (7.9) in wash tubes.

     11.3 Orthophosphate

          11.3.1  Add 1 drop of phenolphthalein indicator solution (7.10) to
                  approximately 50 ml of sample.  If a red color develops, add
                  sulfuric acid solution (7.7) drop-wise to just discharge the
                  color.  Acid samples must be neutralized with 1 N sodium
                  hydroxide (40 g NaOH/L).

          11.3.2  Set up manifold as shown in Figure 1.

          11.3.3  Allow system to equilibrate as required.  Obtain a stable
                  baseline with all reagents, feeding reagent water through
                  the sample line.

          11.3.4  Place standards in sampler in order of decreasing
                  concentration, and complete filling of sampler tray.

                                   365.1-12

-------
           11.3.5   Switch  sample  line  from  reagent water to  Sampler  and begin
                   analysis.

 12.0 DATA  ANALYSIS AND  CALCULATIONS

     12.1  Prepare  a calibration  curve by plotting instrument response
           against  standard concentration.   Compute sample  concentration by
           comparing sample response with the standard curve.   Multiply answer
           by appropriate  dilution factor.

     12.2  Report only those values that fall between the lotoest and the
           highest  calibration standards.   Samples exceeding the highest
           standard should be diluted  and reanalyzed.  Any sample whose
           computed value  is less than 5% of its immediate predecessor must be
           rerun.

     12.3  Report results  in mg P/L.

 13.0 METHOD PERFORMANCE

     13.1  Six laboratories (using Technicon AAI equipment)  participating in an
           EPA Method Study, analyzed  four natural water samples containing
           exact increments of orthophosphate, with the following results:

           Increment as     Precision as          	Accuracy as	
           Orthophosphate  Standard Deviation      Bias                Bias
           mg P/liter        mo P/liter           _%	            mq P/liter

             0.04               0.019            +16.7                +0.007
             0.04               0.014             -8.3                -0.003
             0.29               0.087            -15.5                -0.05
             0.30               0.066            -12.8                -0.04

     13.2  In a single laboratory (EMSL), using surface water samples at
          concentrations of 0.04, 0.19,  0.35, and 0.84 mg P/L,  standard
          deviations were ±0.005, ±0.000,  ±0.003,  and ±0.000,  respectively.

     13.3  In a single laboratory (EMSL), using surface water samples at
          concentrations of 0.07 and 0.76 mg P/L,  recoveries were 99% and
          100%,  respectively.

     13.4 The inter!aboratory precision  and accuracy data in Table 1 were
          developed using a reagent water matrix.   Values are in mg P04-P/L.

14.0 POLLUTION PREVENTION

     14.1 Pollution prevention encompasses  any technique  that  reduces  or
          eliminates  the quantity or toxicity of waste at the  point of
          generation.   Numerous  opportunities for pollution  prevention exist
          in laboratory operation.   The  EPA has established  a  preferred
          hierarchy of environmental  management techniques that places
          pollution prevention as the management option of first choice.

                                   365.1-13

-------
          Whenever feasible, laboratory personnel should use pollution
          prevention techniques to address their waste generation.  When
          wastes cannot be feasibly reduced at the source, the Agency
          recommends recycling as the next best option.

     14.2 The quantity of chemicals purchased should be based on expected
          usage during its shelf life and disposal cost of unused material.
          Actual reagent preparation volumes should reflect anticipated usage
          and reagent stability.

     14.3 For information about pollution prevention that may be applicable to
          laboratories and research institutions, consult "Less is Better:
          Laboratory Chemical Management for Waste Reduction," available from
          the American Chemical Society's Department of Government Regulations
          and Science Policy, 1155 16th Street N.W., Washington D.C. 20036,
          (202) 872-4477.

15.0 WASTE MANAGEMENT

     15.1 The Environmental Protection Agency requires that laboratory waste
          management practices be conducted consistent with all applicable
          rules and regulations.  Excess reagents, samples and method
          process wastes should be characterized and disposed of in an
          acceptable manner.  The Agency urges laboratories to protect the
          air, water, and land by minimizing and controlling all releases from
          hoods, and bench operations, complying with the letter and spirit of
          any waster discharge permit and regulations, and by complying with
          all solid and hazardous waste 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," available from the
          American Chemical Society at the address listed in Sect. 14.3.

16.0 REFERENCES

     1.   Murphy, J. and Riley, J., "A Modified Single Solution for the
          Determination of Phosphate in Natural Waters."  Anal. Chim. Acta.,
          27, 31 (1962).

     2.   Gales, M., Jr., Julian, E., and Kroner, R., "Method for Quantitative
          Determination of Total Phosphorus in Water."  Jour. AWWA, 58. No.
          10, 1363 (1966).

     3.   Lobring, L.B. and Booth, R.L., "Evaluation of the AutoAnalyzer II; A
          Progress Report,"  Technicon International Symposium, June, 1972,
          New York, N.Y.

     4.   Standard Methods for the Examination of Water and Wastewater, 18th
          Edition, p. 4-116, Method 4500-P F (1992).

     5.   Code of Federal Regulations 40, Ch. 1, Pt. 136, Appendix B.
                                   365.1-14

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17.0 TABLES. DIAGRAMS. FLOWCHARTS. AND VALIDATION DATA
TABLE 1. INTERLABORATORY PRECISION AND ACCURACY DATA
NUMBER OF
VALUES
REPORTED
54
69
88
87
57
69
53
87
64
57
88
63
TRUE
VALUE
(T)
0.150
0.351
0.625
1.80
2.50
2.75
3.50
3,60
4.00
7.01
8.20
9.00
MEAN
(X)
0.1530
0.3670
0.6090
1.7374
2.4867
2.8344
3.5619
3.4957
3.8523
6.9576
8.0995
8.6717
RESIDUAL
FOR X
-0.0017
0.0140
-0.0141
-0.0444
0.0146
0.1158
0.1038
-0.0610
-0.0989
0.0383
0.0068
-0.2099
STANDARD
DEVIATION
(S) i
0.0128
0.0368
0.0413
0.1259
0.1637
0.2019
0.2854
0.2137
0.3158
0.5728
0.5428
0.6770
RESIDUAL
FOR S
-0.0010
0.0084
-0.0069
-0.0072
-0.0200
0.0002
0.0295
-0.0495
0.0237
0.0632
-0.0528
0.0236
REGRESSIONS:  X - 0.986T + 0.007, S = 0.072T + 0.003
                                   365.1-15

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-------

-------
                   METHOD 375.2

DETERMINATION OF SULFATE BY AUTOMATED COLORIMETRY
            Edited by James W. O'De'll
            Inorganic  Chemistry  Branch
           Chemistry Research Division
                  Revision 2.0
                   August 1993
   ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
       OFFICE OF RESEARCH AND DEVELOPMENT
      U.S. ENVIRONMENTAL PROTECTION AGENCY
             CINCINNATI,  OHIO  45268
                     375.2-1

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                                 METHOD 375.2

          DETERMINATION OF SULFATE IN WATER BY AUTOMATED COLORIMETRY
1.0  SCOPE AND APPLICATION

     1.1  This automated method is applicable to drinking, ground and surface
          water, domestic and industrial wastes.

     1.2  The applicable range is 3 to 300 mg S04/L.   The sensitivity of  the
          method can be increased by a minor modification to analyze samples
          in the range of 0.5 to 30 mg S04/L.   Approximately 30  samples per
          hour can be analyzed.

2.0  SUMMARY OF METHOD

     2.1  The sample is first passed through a sodium form cation-exchange
          column to remove multivalent metal ions.  The sample containing
          sulfate is then reacted with an alcohol solution of barium chloride
          and methyl thymol blue (MTB) at a pH of 2.5-3.0 to form barium
          sulfate.  The combined solution is raised to a pH of 12.5-13.0  so
          that excess barium reacts with MTB.  The uncomplexed MTB color  is
          gray; if it is all chelated with barium, the color is blue.
          Initially, the barium and MTB are equimolar and equivalent to 300 mg
          S04/L; thus the amount of uncomplexed MTB is equal to  the sulfate
          present.

     2.2  Reduced volume versions of this method that use the same reagents
          and molar ratios are acceptable provided they meet the quality
          control and performance requirements stated in the method.

     2.3  Limited performance-based method mpdifications may be acceptable
          provided they are fully documented and meet or exceed requirements
          expressed in Sect. 9.0, Quality Control.

3.0  DEFINITIONS

     3.1  CALIBRATION BLANK (CB) — A volume of reagent water fortified with
          the same matrix as the calibration standards, but without the
          analytes, internal standards, or surrogate analytes.

     3.2  CALIBRATION STANDARD  (CAL) — A solution prepared from the primary
          dilution standard solution or stock standard solutions and the
          internal standards and surrogate analytes.  The CAL solutions are
          used to calibrate the instrument response with respect to analyte
          concentration.

     3.3  INSTRUMENT PERFORMANCE CHECK SOLUTION  (IPC) — A  solution of one or
          more method analytes, surrogates, internal standards, or other test
                                    375.2-2

-------
     substances used to evaluate the performance of the instrument system
     with respect to a defined set of criteria.

3.4  LABORATORY FORTIFIED BLANK (LFB) — An aliquot of reagent water or
     other blank matrices to which known quantities of the method
     analytes are added in the laboratory.  The LFB is analyzed exactly
     like a sample, and its purpose is to determine whether the
     methodology is in control, and whether the laboratory is capable of
     making accurate and precise measurements.

3.5  LABORATORY FORTIFIED SAMPLE MATRIX (LFM) — An aliquot of an
     environmental sample to which known quantities of the method
     analytes are added in the laboratory.  The LFM 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 LFM
     corrected for background concentrations.

3.6  LABORATORY REAGENT BLANK (LRB) — An aliquot of reagent water or
     other blank matrices that are treated exactly as a sample including
     exposure to all glassware, equipment, solvents, reagents, internal
     standards, and surrogates that are used with other samples.  The LRB
     is used to determine if method analytes or other interferences are
     present in the laboratory environment, the reagents,  or the
     apparatus.

3.7  LINEAR CALIBRATION RANGE (LCR) — The concentration range over which
     the instrument response is linear.

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.
                             \
3.9  METHOD DETECTION LIMIT (MDL)  -- The minimum concentration of an
     analyte that can be identified,  measured and reported with 99%
     confidence that the analyte concentration is greater than zero.

3.10 QUALITY CONTROL SAMPLE (QCS)  -- A solution of method analytes of
     known concentrations that is  used to fortify an aliquot of LRB or
     sample matrix.  The QCS is obtained from a source external  to the
     laboratory and different from the source of calibration standards.
     It is used to check laboratory performance with externally prepared
     test materials.

3.11 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.
                              375.2-3

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4.0  INTERFERENCES

     4.1  The ion exchange column eliminates interferences from multivalent
          cations.  A mid-scale sulfate standard containing Ca+*  should  be
          analyzed periodically to insure that the column is functioning
          properly.

     4.2  Samples with pH below 2 should be neutralized because high acid
          concentrations elute cations from the ion exchange resin.

     4.3  Turbid samples should be filtered or centrifuged.

     4.4  Method interferences may be caused by contaminants in the reagent
          water, reagents, glassware, and other sample processing apparatus
          that bias analyte response.

5.0  SAFETY

     5.1  The toxicity or carcinogenicity of each reagent used in this method
          have not been fully established.  Each chemical should be regarded
          as a potential health hazard and exposure should be as low as
          reasonably achievable.  Cautions are included for known extremely
          hazardous materials or procedures.

     5.2  Each 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 Material
          Safety Data Sheets (MSDS) should be made available to all personnel
          involved in the chemical analysis.  The preparation of a formal
          safety plan is also advisable.

     5.3  The following chemicals have the potential to be highly toxic or
          hazardous, consult MSDS.

          5.3.1   Barium chloride (7.2)

          5.3.2   Hydrochloric acid (7.3)

6.0  EQUIPMENT AND SUPPLIES

     6.1  Balance — Analytical, capable of accurately weighing to the nearest
          0.0001 g.

     6.2  Glassware — Class A volumetric flasks and pi pets as required.

     6.3  Automated continuous flow analysis equipment designed to deliver and
          react sample and reagents in the required order and ratios.

          6.3.1   Sampling device (sampler)

          6.3.2   Multichannel  pump


                                   375.2-4

-------
          6.3.3   Reaction unit or manifold

          6.3.4   Colorimetric detector

       .   6.3.5   Data recording device

7.0  REAGENTS AND STANDARDS

     7.1   Reagent water:  Distilled or deionized water, free of the analyte of
          interest.  ASTM type II or equivalent.

     7.2   Barium chloride:  Dissolve 0.7630 g of barium chloride dihydrate
          (BaCl2-2H20) (CASRN 10326-27-9) in 250 ml of reagent water and
          dilute to 500 ml.

     7.3   Methyl thymol blue:  Dissolve 0.1182 g of methyl thymol blue (3'3-bis-
          N,N-biscarboxymethyl)-amino methyl thymolsulfone-phthalein
          pentasodium salt) (CASRN 1945-77-3) in 25 ml of barium chloride
          solution (7.2).  Add 4 ml of 1.0 N hydrochloric acid (CASRN 7647-01-
          0) which changes the color to bright orange.  Add 71 ml of reagent
          water and dilute to 500 ml with ethanol.  The pH of this solution is
          2.6.  This reagent should be prepared the day before and stored in a
          brown plastic bottle in the refrigerator.

     7.4   Buffer, pH 10.5 ± 0.5:  Dissolve 6.75 g of ammonium chloride (CASRN
          12125-02-9) in 500 ml of reagent water.  Add 57 ml of concentrated
          ammonium hydroxide (CASRN 1336-21-6) and dilute to 1 L with
          distilled water.

     7.5   Buffered EDTA:  Dissolve 20 g of tetrasodium EDTA (CASRN 64-02-8) in
         J pH 10.5 buffer (7.4), and dilute to 500 ml with buffer.

     7.6   Sodium hydroxide solution (50%):  Dissolve 250 g NaOH (CASRN 1310-
          73-2) in 300 ml of reagent water, cool, and dilute to 500 ml.

     7.7   Sodium hydroxide, 0.18N:  Dilute 7.2 ml of sodium hydroxide solution
          (7.6) to 500 ml.

     7.8   Ion exchange resin:  Bio-Rex 70, 20-50 mesh, sodium form, Bio-Rad
          Laboratories, Richmond, California.  Free from fines by stirring
          with several portions of reagent water and decant the supernate
          before settling is complete.

     7.9   Dilution Water:  Add 0.75 ml of sulfate stock solution (7.10) and 3
          drops of Brij-35 (CASRN 9002-92-0) to 2 L of reagent water.

     7.10 Sulfate stock solution, 1 ml = 1 mg SO,:   Dissolve 1.479. g of dried
          (105°C) Na2S04 (CASRN  7757-82-6)  in  reagent  water  and dilute  to  1  L.

     7.11 Dilute sulfate solution, 1 ml = 0.1 mg S04:   Dilute  50  ml of sulfate
          stock solution (7.10) to 500 ml with reagent water.


                                    375.2-5

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8.0  SAMPLE COLLECTION. PRESERVATION AND STORAGE

     8.1  Samples should be collected in plastic or glass bottles.  All
          bottles must be thoroughly cleansed and rinsed with reagent water.
          Volume collected should be sufficient to insure a representative
          sample, allow for replicate analysis (if required), and minimize
          waste disposal.

     8.2  No chemical preservation required.  Cool sample to 4°C.

     8.3  Samples should be analyzed as soon as possible after collection.  If
          storage is required, samples maintained at 4°C may be held for up
          to 28 days.

9.0  QUALITY CONTROL

     9.1  Each laboratory using this method is required to operate a formal
          quality control  (QC) program.   The minimum requirements of this
          program consist of an initial  demonstration of laboratory
          capability, and the periodic analysis of laboratory reagent blanks,
          fortified blanks and other laboratory solutions as a continuing
          check on performance.  The laboratory is required to maintain per-
          formance records that define the quality of the data that are
          generated.

     9.2  INITIAL DEMONSTRATION OF PERFORMANCE

          9.2.1   The initial  demonstration of performance is used to
                  characterize instrument performance (determination of LCRs
                  and analysis of QCS)  and laboratory performance
                  (determination of MDLs)  prior to performing analyses  by this
                  method.

          9.2.2   Linear Calibration Range (LCR)  —  The LCR must  be determined
                  initially  and verified  every 6  months or whenever a
                  significant  change in  instrument response is  observed or
                  expected.  The initial  demonstration  of linearity must use
                  sufficient standards to  insure  that  the resulting curve is
                  linear.  The verification of linearity must use  a minimum  of
                  a  blank  and  three  standards.   If any  verification data
                  exceeds  the  initial values by ± 10%,  linearity must be
                  reestablished.   If any  portion  of  the range is  shown  to be
                  nonlinear, sufficient standards  must  be used  to  clearly
                  define the nonlinear portion.

          9.2.3    Quality  Control  Sample  (QCS) —  When  beginning the  use of
                  this method,  on  a  quarterly  basis  or  as  required  to meet
                  data-quality needs, verify the  calibration  standards  and
                  acceptable instrument performance with  the  preparation and
                  analyses of  a QCS.  If the determined  concentrations  are not
                 within ± 10% of  the stated values, performance of the
                                   375.2-6

-------
             determinative step of the method is unacceptable.  The
             source of the problem must be identified and corrected
             before either proceeding with the initial determination of
             MDLs or continuing with, on-going analyses.

     9.2.4   Method Detection Limit (MDL) — MDLs must be established for
             all analytes, using reagent water (blank) fortified at a
             concentration of two to three times the estimated instrument
             detection limit.c '  To determine MDL values, take seven
             replicate aliquots of the fortified reagent water and
             process through the entire analytical method.  Perform all
             calculations defined in the method and report the
             concentration values in the appropriate units.  Calculate
             the MDL as follows:

                                  MDL = (t) x (S)

             where, t = Student's t value for a 99% confidence level and
                        a standard deviation estimate with n-1 degrees
                        of freedom [t = 3.14 for seven replicates],

                    S = standard deviation of the replicate analyses.

             MDLs should be determined every 6 months, when a new
             operator begins work, or whenever there is a significant
             change in the background or instrument response.

9.3  ASSESSING LABORATORY PERFORMANCE

     9.3.1   Laboratory Reagent Blank (LRB) -- The laboratory must
             analyze at least one LRB with each batch of samples.  Data
             produced are used to assess contamination from the
             laboratory environment.   Values that exceed the MDL indicate
             laboratory or reagent contamination should be suspected and
             corrective actions must be taken before continuing the
             analysis.

     9.3.2   Laboratory Fortified Blank (LFB) -- The laboratory must
             analyze at least one LFB with each batch of samples.
             Calculate accuracy as percent recovery (Sect. 9.4.2).   If
             the recovery of any analyte falls outside the required
             control limits of 90-110%,  that analyte is judged out of
             control,  and the source of the problem should be identified
             and resolved before continuing analyses.

     9.3.3   The laboratory must use LFB analyses data to assess
             laboratory performance against the required control  limits
             of 90-110%.   When sufficient internal  performance data
             become available (usually a minimum of 20-30 analyses),
             optional  control limits  can be developed from the percent
             mean recovery (x)  and the standard deviation (S)  of the mean


                              375.2-7

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             recovery.  These data can be used to establish the upper and
             lower control limits as follows:

                           UPPER CONTROL LIMIT = x + 3S
                           LOWER CONTROL LIMIT = x - 3S

             The optional control limits must be equal to or better than
             the required control limits of 90-110%.  After each five to
             ten new recovery measurements, new control limits can be
             calculated using only the most recent 20-30 data points.
             Also, the standard deviation (S) data should be used to
             established an on-going precision statement for the level of
             concentrations included in the LFB.  These data must be kept
             on file and be available for review.

     9.3.4   Instrument Performance Check Solution (IPC) — For all
             determinations the laboratory must analyze the IPC (a mid-
             range check standard) and a calibration blank immediately
             following daily calibration, after every tenth sample (or
             more frequently, if required) and at the end of the sample
             run.  Analysis of the IPC solution and calibration blank
             immediately following calibration must verify that the
             instrument is within ± 10% of calibration.  Subsequent
             analyses of the IPC solution must verify the calibration is
             still within ± 10%.  If the calibration cannot be verified
             within the specified limits, reanalyze the IPC solution.  If
             the second analysis of the IPC solution confirms calibration
             to be outside the limits, sample analysis must be
             discontinued, the cause determined and/or in the case of
             drift the instrument recalibrated.  All samples following
             the last acceptable IPC solution must be reanalyzed.   The
             analysis data of the calibration blank and IPC solution must
             be kept on file with the sample analyses data.

9.4  ASSESSING ANALYTE RECOVERY AND DATA QUALITY

     9.4.1   Laboratory Fortified Sample Matrix (LFM) — The laboratory
             must add a known amount of analyte to a minimum of 10% of
             the routine samples.  In each case, the LFM aliquot must be
             a duplicate of the aliquot used for sample analysis.  The
             analyte concentration must be high enough to be detected
             above the original  sample and should not be less than four
             times the MDL.   The added analyte concentration should be
             the same as that used in the laboratory fortified blank.

     9.4.2   Calculate the percent recovery for each analyte,  corrected
             for concentrations measured in the unfortified sample,  and
             compare these values to the designated LFM recovery range
             90-110%. Percent recovery may be calculate using the
             following equation:
                              375.2-8

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                     R =  Cs " C    x 100
                            s

                  where, R  =  percent recovery.
                         Cs =  fortified  sample concentration.
                         C  =  sample background concentration.
                         s  =  concentration equivalent of analyte added to
                               sample.

          9.4.3   If the recovery of any analyte falls outside the designated
                  LFM recovery range and the laboratory performance for that
                  analyte is shown to be in control (Sect. 9.3),  the recovery
                  problem encountered with the LFM is judged to be either
                  matrix or solution related, not system related.

          9.4.4   Where reference materials are available, they should be
                  analyzed to provide additional performance data.  The
                  analysis of reference samples is a valuable tool for
                  demonstrating the ability to perform the method acceptably.

10.0 CALIBRATION AND STANDARDIZATION

     10.1 Set up the manifold for high (0-300 mg S04/L)  or low  (0-30 mg S04/L)
          level samples as described in Figure 1.

          10.1.1  High level working standards, 10-300 mg/L:  As  a minimum,
                  prepare high level working standards by diluting the
                  following volumes of stock standard (7.10) to 100 ml.

                       ml Stock                          mq/L SO^

                           1                                 10
                           5                                 50
                          10                                100
                          15                                150
                          25                                250
                          30                                300

          10.1.2  Low level working standards, 1-30 mg/L:  Prepare at least
                  this number of low level  working standards by diluting the
                  following volumes of dilute sulfate solution  (7.11) to 100
                  mL.
                                   375.2-9

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mL Stock

    1
    5
   10
   15
   25
   30
                                                         mq/L SO,
                                                            1.0
                                                            5.0
                                                          10.0
                                                          15.0
                                                          25.0
                                                          30.0
     10.2 The  ion exchange column  is prepared by pulling a slurry of the resin
          into  a piece of glass tubing 7.5 inches long, 2.0 mm ID and 3.6 mm
          OD.   This  is conveniently done by using a pipet and a loose fitting
          glass wool plug in the tubing.  Care should be taken to avoid
          allowing air bubbles to  enter the column.  If air bubbles become
          trapped, the column should be prepared over again.  The column can
          exchange the equivalent  of 35 mg of calcium.  For the high level
          manifold this corresponds to about 900 samples with 200 mg/L Ca.
          The column should be prepared as often as necessary to assure that
          no more than 50% of its  capacity is used up.
     10.3 Allow the instrument to warm up as required.
          until a stable baseline is achieved.
                                 Pump all  reagents
     10.4 Analyze all working standards in duplicate at the beginning of a run
          to develop a standard curve.  Control standards are analyzed every
          hour to assure that the system remains properly calibrated.  Since
          the chemistry is non-linear, data recording devices should be
          adjusted accordingly.

     10.5 Prepare standard curve by plotting instrument response against
          concentration values.  A calibration curve may be fitted to the
          calibration solutions concentration/response data using computer or
          calculator based on regression curve fitting techniques.  Acceptance
          or control limits should be established using the difference the
          measured value of the calibration solution and the "true value"
          concentration.

     10.6 After the calibration has been established, it must be verified by
          the analysis of a suitable quality control sample (QCS).  If
          measurements exceed ± 10% of the established QCS value, the analysis
          should be terminated and the instrument recalibrated.   The new
          calibration must be verified before continuing analysis.  Periodic
          reanalysis of the QCS is recommended as a continuing calibration
          check.

11.0 PROCEDURE

     11.1 Set up instrument as specified under calibration and standardization
          (10.0).
                                   375.2-10

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     11.2 Fill and connect reagent containers and start system.  Allow the
          system to equilibrate as required.  Pump all reagents until a stable
          baseline is achieved.

     11.3 Place standards and samples in sampler tray.  Calibrate instrument,
          and begin analysis.

     11.4 At the end of each day, the system should be washed with the
          buffered EDTA solution (7.5).  This is done by placing the
          methyl thymol blue line and the sodium hydroxide line in reagent
          water for a few minutes and then in the buffered EDTA solution for
          10 min.  Wash the system with reagent water for 15 min before
          shutting down.

12.0 DATA ANALYSIS AND CALCULATIONS

     12.1 Prepare a calibration curve by plotting instrument response
          against standard concentration.   Compute sample concentration by
          comparing sample response with the standard curve.   Multiply answer
          by appropriate dilution factor.

     12.2 Report only those values that fall between the lowest and the
          highest calibration standards.   Samples exceeding the highest
          standard should be diluted and reanalyzed.

     12.3 Report results in mg/L.

13.0 METHOD PERFORMANCE

     13.1 In a single laboratory the estimated standard deviation, calculated
          from duplicate analyses of 26 surface and wastewaters at a mean
          concentration of 100 mg/L was ±1.6 mg/L.

     13.2 The mean recovery from 24 surface and wastewaters was 102%.

14.0 POLLUTION PREVENTION

     14.1 Pollution prevention encompasses any technique that reduces or
          eliminates the quantity or toxicity of waste at the point of
          generation.  Numerous opportunities for pollution prevention exist
          in laboratory operation.   The EPA has established a preferred
          hierarchy of environmental  management techniques that places
          pollution prevention as the management option of first choice.
          Whenever feasible,  laboratory personnel  should use pollution
          prevention techniques to address their waste generation.  When
          wastes cannot be feasibly reduced at the source, the Agency
          recommends recycling as the next best option.

     14.2 The quantity of chemicals purchased should be based on expected
          usage during its shelf life and disposal  cost of unused material.
          Actual  reagent preparation  volumes should reflect anticipated usage
          and reagent stability.

                                   375.2-11

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     14.3 For information about pollution prevention that may be applicable to
          laboratories and research institutions, consult "Less is Better:
          Laboratory Chemical Management for Waste Reduction," available from
          the American Chemical Society's Department of Government
          Regulations and Science Policy, 1155 16th Street N.W., Washington
          D.C. 20036, (202) 872-4477.

15.0 WASTE MANAGEMENT

     15.1 The Environmental Protection Agency requires that laboratory waste
          management practices be conducted consistent with all applicable
          rules and regulations.  Excess Reagents and samples and method
          process wastes should be characterized and disposed of in an
          acceptable manner.  The Agency urges laboratories to protect the
          air, water, and land by minimizing and controlling all releases from
          hoods and bench operations, complying with the letter and spirit of
          any waster discharge permit and regulations, and by complying with
          all solid and hazardous waste 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," available from the
          American Chemical Society at the address listed in Sect. 14.3.

16.0 REFERENCES

     1.   Lazrus, A.L., Hill, K.C. and Lodge, J.P., "Automation in Analytical
          Chemistry," Technicon Symposia, 1965.

     2.   Coloros, E., Panesar, M.R. and Parry, F.P., "Linearizing the  ,
          Calibration Curve in Determination of Sulfate by the Methyl thymol
          Blue Method," Anal. Chem. 48, 1693 (1976).

     3.   Code of Federal Regulations 40, Ch. 1, Pt. 136, Appendix B.
                                   375.2-12

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17.0 TABLES. DIAGRAMS. FLOWCHARTS.  AND VALIDATION DATA
           »
N WATER
8
8
       LU

       §
                                   Li
                                      e
                                             in
                          G5
                          S
                       2
                            Si
                                             6    8    1
                 in
                                                      .Q
                                                      3
                                                      03


                                                      i
                                                      03
                                                      -3
                                                      §
                                                      .2
                                                                3
                                   375.2-13

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                METHOD 410.4

THE  DETERMINATION  OF  CHEMICAL  OXYGEN  DEMAND
        BY  SEMI-AUTOMATED  COLORIMETRY
          Edited by James W. O'Dell
         Inorganic Chemistry Branch
        Chemistry Research Division
                Revision 2,,0
                 August 1993
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
    OFFICE OF RESEARCH AND DEVELOPMENT
   U.S. ENVIRONMENTAL PROTECTION AGENCY
          CINCINNATI, OHIO  45268
                  410.4-1

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                                 METHOD 410.4

                  THE DETERMINATION OF CHEMICAL OXYGEN DEMAND
                         BY SEMI-AUTOMATED COLORIMETRY
1.0  SCOPE AND APPLICATION

     1.1  This method covers the determination of chemical oxygen demand (COD)
          in ground and surface waters, domestic and industrial wastes.

     1.2  The applicable range is 3-900 mg/L.

2.0  SUMMARY OF METHOD

     2.1  Sample, blanks, and standards in sealed tubes are heated in an oven
          or block digestor in the presence of dichromate at 150°C.  After two
          hours, the tubes are removed from the oven or digester, cooled, and
          measured spectrophotometrically at 600 nm.  The colorimetric
          determination may also be performed manually.

     2.2  Reduced volume versions of this method that use the same reagents
          and molar ratios are acceptable provided they meet the quality
          control and performance requirements stated in the method.

     2.3  Limited performance-based method modifications may be acceptable
          provided they are fully documented and meet or exceed requirements
          expressed in Sect. 9.0, Quality Control.

3.0  DEFINITIONS

     3.1  CALIBRATION BLANK (CB) — A volume of reagent water fortified with
          the same matrix as the calibration standards, but without the
          analytes, internal standards, or surrogate analytes.

     3.2  CALIBRATION STANDARD (CAL) -- A solution prepared from the primary
          dilution standard solution or stock standard solutions and the
          internal standards and surrogate analytes.  The CAL solutions are
          used to calibrate the instrument response with respect to analyte
          concentration.

     3.3  INSTRUMENT PERFORMANCE CHECK SOLUTION (IPC) — A solution of one or
          more method analytes, surrogates, internal standards, or other test
          substances used to evaluate the performance of the instrument system
          with respect to a defined set of criteria.

     3.4  LABORATORY FORTIFIED BLANK (LFB) — An aliquot of reagent water or
          other blank matrices to which known quantities of the method
          analytes are added in the laboratory.  The LFB is analyzed exactly
          like a sample, and its purpose is to determine whether the
                                    410.4-2

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          methodology  is  in control,  and whether the laboratory  is capable of
          making  accurate  and  precise measurements.

     3.5  LABORATORY FORTIFIED SAMPLE MATRIX  (LFM) — An aliquot of an
          environmental sample to which known quantities of the method
          analytes  are  added in the laboratory.  The LFM 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 LFM
          corrected for background concentrations.

     3.6  LABORATORY REAGENT BLANK (LRB) — An aliquot of reagent water or
          other blank matrices that are treated exactly as a sample including
          exposure  to all  glassware,  equipment, solvents, reagents, internal
          standards, and surrogates that are used with other samples.  The LRB
          is used to determine if method analytes or other interferences are
          present in the laboratory environment, the reagents, or the
          apparatus.

     3.7  LINEAR  CALIBRATION RANGE (LCR) —The concentration range over which
          the instrument response is  linear.

     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.

     3.9  METHOD DETECTION LIMIT (MDL) -- The minimum concentration of an
          analyte that can be  identified,  measured and reported with 99%
          confidence that  the  analyte concentration is greater than zero.

     3.10 QUALITY CONTROL  SAMPLE (QCS) -- A solution of method analytes of
          known concentrations that is used to fortify an aliquot of LRB or
          sample matrix.  The QCS is obtained from a source external  to the
          laboratory and different from the source of calibration standards.
          It is used to check laboratory performance with externally prepared
          test materials.

     3.11 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.

4.0  INTERFERENCES

     4.1  Chlorides are quantitatively oxidized by dichromate and represent a
          positive interference.   Mercuric sulfate is added to the digestion
          tubes to complex the chlorides.

     4.2  Method interferences may be caused by contaminants  in the reagent
          water, reagents,  glassware,  and  other sample  processing apparatus
          that bias analyte response.

                                   410.4-3

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5.0  SAFETY
     5.1  The toxicity or carcinogenicity of each reagent used in this method
          has not been fully established.  Each chemical should be regarded as
          a potential health hazard and exposure should be as low as
          reasonably achievable.  Cautions are included for known extremely
          hazardous materials or procedures.
     5.2  Each 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 Material
          Safety Data Sheets (MSDS) should be made available to all personnel
          involved in the chemical analysis.  The preparation of a formal
          safety plan is also advisable.
     5.3  The following chemicals have the potential to be highly toxic or
          hazardous, consult MSDS.
          5.3.1   Mercuric sulfate (7.2)
          5.3.2   Potassium dichromate (7.2)
          5.3.3   Sulfuric acid (7.2, 7.3, 7,4)
6.0  EQUIPMENT AND SUPPLIES
     6.1  Balance — Analytical, capable of accurately weighing to the nearest
          0.0001 g.
     6.2  Glassware — Class A volumetric flasks and pi pets as required.
     6.3  Block digestor or drying oven capable of maintaining 150°C.
     6.4  Muffle furnace capable of 500°C.
     6.5  Culture tube with Teflon-lined screw cap, 16 x 100 mm or 25 x 150
          mm.
     6.6  Automated continuous flow analysis equipment designed to deliver and
          react sample and reagents in the required order and ratios.
          6.6.1   Sampling device (sampler)
          6.6.2   Multichannel pump
          6.6.3   Reaction unit or manifold
          6.6.4   Colorimetric detector
          6.6.5   Data recording device
                                    410.4-4

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7.0  REAGENTS AND STANDARDS

     7.1  Reagent water:  Distilled or deionized water, free of the analyte of
          interest.  ASTM type II or equivalent.

     7.2  Digestion solution:  Add 5.1 g potassium dichromate K2Cr207  (CASRN
          7778-50-9), 84 ml cone, sulfuric acid H,S04  (CASRN  8014-95-7)  and
          16.7 g mercuric sulfate HgS04 (CASRN 7783-35-9)  to  250 ml of reagent
          water, cool and dilute to 500 ml. CAUTION: CAN BE VERY HOT!

     7.3  Catalyst solution:  Add 22 g silver sulfate Ag2S04  (CASRN 10294-26-
          5) to a 4.09 kg bottle of cone. H2S04.   Stir  until  dissolved.

     7.4  Sampler wash solution:  Add 250 mL of cone. H2S04 to  250  ml  of
          reagent water.  CAUTION: PREPARE CAREFULLY, HIGH HEAT GENERATION!

     7.5  Stock potassium hydrogen phthalate standard:  Dissolve 0.425 g KHP
          (CASRN 877-24-7) in 400 mL of reagent water and dilute to 500 mL.
          1 mL = 1 mg COD.

8.0  SAMPLE COLLECTION. PRESERVATION AND STORAGE

     8.1  Samples should be collected in plastic or glass bottles.   All
          bottles must be thoroughly cleansed and rinsed with reagent water.
          Volume collected should be sufficient to insure a representative
          sample, allow for replicate analysis (if required), and minimize
          waste disposal.

     8.2  Samples must be preserved with H2S04 to  a pH  < 2 and  cooled  to 4°C
          at the time of collection.

     8.3  Samples should be analyzed as soon as possible after collection.  If
          storage is required, preserved samples maintained at 4°C may be held
          for up to 28 days.

9.0  QUALITY CONTROL

     9.1  Each laboratory using this method is required to operate a formal
          quality control (QC) program.  The minimum requirements of this
          program consist of an initial demonstration of laboratory
          capability, and the periodic analysis of laboratory reagent blanks,
          fortified blanks,  and other laboratory solutions as a continuing
          check on performance.  The laboratory is required to maintain per-
          formance records that define the quality of the data that are
          generated.

     9.2  INITIAL DEMONSTRATION OF PERFORMANCE

          9.2.1   The initial  demonstration of performance is used  to
                  characterize instrument performance (determination of linear
                  calibration ranges and analysis of QCS)  and laboratory


                                   410.4-5

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             performance (determination of MDLs) prior to performing
             analyses by this method.

     9.2.2   Linear Calibration Range (LCR) — The LCR must be determined
             initially and verified every 6 months or whenever a
             significant change in instrument response is observed or
             expected.  The initial demonstration of linearity must use
             sufficient standards to insure that the resulting curve is
             linear.  The verification of linearity must use a minimum of
             a blank and three standards.  If any verification data
             exceeds the initial values by ± 10%, linearity must be
             reestablished.  If any portion of the range is shown to be
             nonlinear, sufficient standards must be used to clearly
             define the nonlinear portion.

     9.2.3   Quality Control Sample (QCS) — When beginning the use of
             this method, on a quarterly basis or as required to meet
             data-quality needs, verify the calibration standards and
             acceptable instrument performance with the preparation and
             analyses of a QCS.  If the determined concentrations are not
             within ± 10% of the stated values, performance of the
             determinative step of the method is unacceptable.  The
             source of the problem must be identified and corrected
             before either proceeding with the initial determination of
             MDLs or continuing with on-going analyses.

     9.2.4   Method Detection Limit (MDL) -- MDLs must be established for
             all analytes, using reagent water (blank) fortified at a
             concentration of two to three times the estimated instrument
             detection limit.<2)  To determine MDL values, take seven
             replicate aliquots of the fortified reagent water and
             process through the entire analytical method.  Perform all
             calculations defined in the method and report the
             concentration values in the appropriate units.  Calculate
             the MDL as follows:

                                MDL - (t) x (S)

             where, t = Student's t value for a 99% confidence level and
                        a standard deviation estimate with n-1 degrees
                        of freedom [t = 3.14 for seven replicates].

                    S = standard deviation of the replicate analyses.

             MDLs should be determined every 6 months, when a new
             operator begins work, or whenever there is a significant
             change in the background or instrument response.

9.3  ASSESSING LABORATORY PERFORMANCE

     9.3.1   Laboratory Reagent Blank (LRB) — The laboratory must
             analyze at least one LRB with each batch of samples.  Data

                               410.4-6

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        produced  are used to assess contamination from the
        laboratory environment.  Values that exceed the MDL indicate
        laboratory or reagent contamination should be suspected and
        corrective actions must be taken before continuing the
        analysis.

9.3.2   Laboratory Fortified Blank (LFB) ~ The laboratory must
        analyze at least one LFB with each batch of samples.
        Calculate accuracy as percent recovery (Sect. 9.4.2).  If
        the recovery of any analyte falls outside the required
        control limits of 90-110%, that analyte is judged out of
        control, and the source of the problem should be identified
        and resolved before continuing analyses.

9.3.3   The laboratory must use LFB analyses data to assess
        laboratory performance against the required control limits
        of 90-110%.  When sufficient internal performance data
        become available (usually a minimum of 20-30 analyses),
        optional control limits can be developed from the percent
        mean recovery (x) and the standard deviation (S) of the mean
        recovery.  These data can be used to establish the upper and
        lower control limits as follows:

                        UPPER CONTROL LIMIT = x + 3S
                        LOWER CONTROL LIMIT = x - 3S

        The optional  control  limits must be equal  to or better than
        the required control  limits of 90-110%.  After each five to
        ten new recovery measurements,  new control  limits can be
        calculated using only the most recent 20-30 data points.
        Also, the standard deviation (S) data should be used to
        established an on-going precision statement for the level  of
        concentrations included in the LFB.   These data must be kept
        on file and be available for review.

9.3.4   Instrument Performance Check Solution (IPC)  -- For all
        determinations,  the laboratory must analyze the IPC (a mid-
        range check standard)  and a calibration blank immediately
        following daily calibration,  after every tenth sample (or
        more frequently,  if required),  and at the  end of the sample
        run.  Analysis of the  IPC solution and calibration blank
        immediately following  calibration must verify that the
        instrument is within  ± 10% of calibration.   Subsequent
        analyses of the IPC solution  must verify the calibration  is
        still  within  ± 10%.   If the calibration cannot be verified
        within the specified  limits,  reanalyze the  IPC solution.   If
        the second analysis of the IPC  solution confirms  calibration
        to be outside the limits,  sample analysis must be
        discontinued,  the cause determined and/or  in  the  case  of
        drift,  the instrument  recalibrated.   All samples  following
        the last acceptable IPC solution must  be reanalyzed.   The
                         410.4-7

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                  analysis data of the calibration blank and IPC solution must
                  be kept on file with the sample analyses data.

     9.4  ASSESSING ANALYTE RECOVERY AND DATA QUALITY

          9.4.1   Laboratory Fortified Sample Matrix (LFM) — The laboratory
                  must add a known amount of analyte to a minimum of 10% of
                  the routine samples.  In each case, the LFM aliquot must be
                  a duplicate of the aliquot used for sample analysis.  The
                  analyte concentration must be high enough to be detected
                  above the original sample and should not be less than four
                  times the MDL.  The added analyte concentration should be
                  the same as that used in the laboratory fortified blank.

          9.4.2   Calculate the percent recovery for each analyte, corrected
                  for concentrations measured in the unfortified sample, and
                  compare these values to the designated LFM recovery range
                  90-110%.  Percent recovery may be calculated using the
                  following equation:
                     R =   f	_    x 100
                            s

                  where, R  =  percent recovery.
                         Cs =  fortified sample concentration.
                         C  -  sample background concentration.
                         s  =  concentration equivalent of analyte added to
                               sample.

          9.4.3   If the recovery of any analyte falls outside the designated
                  LFM recovery range and the laboratory performance for that
                  analyte is shown to be in control (Sect. 9.3), the recovery
                  problem encountered with the LFM is judged to be either
                  matrix or solution related, not system related.

          9.4.4   Where reference materials are available, they should be
                  analyzed to provide additional performance data.  The
                  analysis of reference samples is a valuable tool for
                  demonstrating the ability to perform the method acceptably.

10.0 CALIBRATION AND STANDARDIZATION

     10.1 Prepare a series of at least 3 standards, covering the desired
          range, by diluting appropriate volumes of the stock standard
          (7.5)and a blank.

     10.2 Process standards and blanks as described under Procedure (11.0).

     10.3 Set up manifold as shown in Figure 1.


                                   410.4-8

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      10.4 Allow  the  instrument  to warm  up  as  required.   Pump  all reagents
          until  a  stable  baseline is  achieved.

      10.5 Place  appropriate  standards in the  sampler  in  order of decreasing
          concentration and  perform analysis.

      10.6 Prepare  a  standard curve by plotting instrument response against
          concentration values.  A calibration curve may be fitted to the
          calibration solutions concentration/response data using computer or
          calculator based regression curve fitting techniques.  Acceptance or
          control  limits  should be established using the difference between
          the measured value of the calibration solution and  the "true value"
          concentration.

     10.7 After the calibration has been established, it must be verified by
          the analysis of a suitable QCS.  If measurements exceed ± 10% of the
          established QCS value, the analysis should be terminated and the
          instrument recalibrated.   The new calibration must be verified
          before continuing analysis.   Periodic reanalysis of the QCS is
          recommended as a continuing calibration check.

11.0 PROCEDURE

     11.1 Wash all  culture tubes and screw caps with 20%  H2SO, before their
          first use to prevent  contamination.   Trace contamination  may be
          removed from the tubes by  igniting them in a muffle  furnace at 500°C
          for 1 h.

     11.2 Pi pet 2.5 mL of  sample, standard  or  blank,  into 16 x 100  mm tubes  or
          10  mL into 25 x  100 mm tubes.

     11.3 Add 1.5 ml of digestion solution  (7.2)  to  the 16 x 100 mm tubes or
          6.0 ml  to the 25 x  150 mm  tubes and  mix.

     11.4 Add 3.5 ml  of catalyst solution  (7.3) carefully down the  side  of the
          16  x 100  mm tubes or  14.0 ml to the  25  x 150 mm tubes.

     11.5 Cap tubes tightly and  shake to mix layer.  CAUTION:  Tubes  are  hot.

     11.6 Place tubes  into a  block digester or oven at 150°C and heat for 2 h.

     11.7 Remove, mix, and cool  tubes.  Allow  any precipitate  to settle.

     11.8 Fill and  connect reagent containers  and start system.  Allow the
         instrument to warm up  as required.,   Pump all reagents until  a stable
         baseline  is achieved.

     11.9 Place standards,  blanks, and samples in sampler tray.  Calibrate
         instrument, and  begin analysis.
                                   410.4-9

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12.0 DATA ANALYSIS AND CALCULATIONS

     12.1 Prepare a calibration curve by plotting instrument response
          against standard concentration.  Compute sample concentration by
          comparing sample response with the standard curve.  Multiply answer
          by appropriate dilution factor.

     12.2 Report only those values that fall between the lowest and the
          highest calibration standards.  Samples exceeding the highest
          standard should be diluted and reanalyzed.

     12.3 Report results in mg/L.

13.0 METHOD PERFORMANCE

     13.1 The inter!aboratory precision and accuracy data in Table 1 were
          developed using a reagent water matrix.  Values are in mg COD/L.

     13.2 Single laboratory precision data can be estimated at 50 to 75% of
          the inter!aboratory precision estimates.

14.0 POLLUTION PREVENTION

     14.1 Pollution prevention encompasses any technique that reduces or
          eliminates the quantity or toxicity of waste at the point of
          generation.  Numerous opportunities for pollution prevention exist
          in laboratory operation.  The EPA has established a preferred
          hierarchy of environmental management techniques that places
          pollution prevention as the management option of first choice.
          Whenever feasible, laboratory personnel should use pollution
          prevention techniques to address their waste generation.  When
          wastes cannot be feasibly reduced at the source, the Agency
          recommends recycling as the next best option.

     14.2 The quantity of chemicals purchased should be based on expected
          usage during its shelf life and disposal cost of unused material.
          Actual reagent preparation volumes should reflect anticipated usage
          and reagent stability.

     14.3 For information about pollution prevention that may be applicable to
          laboratories and research institutions, consult "Less is Better:
          Laboratory Chemical Management for Waste Reduction," available from
          the American Chemical Society's Department of Government
          Regulations and Science Policy, 1155 16th Street N.W., Washington
          D.C. 20036, (202) 872-4477.

15.0 WASTE MANAGEMENT

     15.1 The Environmental Protection Agency requires that laboratory waste
          management practices be conducted consistent with all applicable
          rules and regulations.  Excess reagents, samples, and method
          process wastes should be characterized and disposed of in an

                                   410.4-10

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                                                                                        1
          acceptable manner.  The Agency urges laboratories to protect the
          air, water, and land by minimizing and controlling all releases from
          hoods and bench operations, complying with the letter and spirit of
          any waste discharge permit and regulations, and by complying with
          all solid and hazardous waste 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," available from the
          American Chemical Society at the address listed in Sect. 14.3.

16.0 REFERENCES

     1.    Jirka, A.M., and M.J. Carter, "Micro-Semi-Automated Analysis of
          Surface and Wastewaters for Chemical Oxygen Demand."  Anal. Chem.
          47:1397, (1975).

     2.    Code of Federal Regulations 40,  Ch. 1, Pt. 136, Appendix B.
                                   410.4-11

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17.0 TABLES. DIAGRAMS. FLOWCHARTS. AND VALIDATION DATA
TflRI F 1 . INTERLABORATORY PRECISION AND ACCURACY DATA
NUMBER OF
VALUES
REPORTED
241
144
140
112
261
181
262
182
141
250
144
113
TRUE
VALUE
(T)
18.2
26.3
28.5
43.5
46.6
50.0
65.4
76.2
91.7
121
201
229
MEAN
(X)
18.9398
26.1454
32.7275
42.8360
45.3034
49.4740
63.2876
75.7960
94.0772
117.7424
196.9391
221.8109
RESIDUAL
FOR X
-0.4220
-1.0445
3.4115
-0.9763
-1.5049
-0.6201
-1.6894
0.3816
3.6833
-0.9678
0.9151
-1.2730
STANDARD
DEVIATION
(S)
5.2026
5.6142
6.2230
6.4351
6.7677
7.0494
7.6041
8.4490
7.9289
9.6197
14.6995
17.3403
RESIDUAL
FOR S
-0.0964
-0.0888
0.4103
-0.1257
0.0523
0.1644
-0.0489
0.2573
-1.0358
-0.8063
0.2837
1.5280
REGRESSIONS:   X  -  0.966T  -  1.773,  S  =  0.050T +  4.391
                                    410.4-12

-------
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410.4-13

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-------
                METHOD 420.4

DETERMINATION OF TOTAL RECOVERABLE PHENOLICS
        BY SEMI-AUTOMATED COLORIMETRY
          Edited  by James  W.  O'Dell
         Inorganic Chemistry Branch
         Chemistry Research Division
                Revision 1.0
                 August 1993
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
    OFFICE OF RESEARCH AND DEVELOPMENT
   U.S. ENVIRONMENTAL PROTECTION AGENCY
          CINCINNATI, OHIO  45268
                  420.4-1

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                                 METHOD 420.4

                DETERMINATION OF TOTAL RECOVERABLE PHENOLICS
                         BY SEMI-AUTOMATED  COLORIMETRY


1.0  SCOPE AND APPLICATION

     1.1  This method covers the determination of phenolic materials in
          drinking, ground, surface, and saline waters, and domestic and
          industrial wastes.

     1.2  The applicable range is from 2 to 500 /zg/L.  The working ranges are
          2 to 200 /zg/L and 10 to 500 /zg/L.

2.0  SUMMARY OF METHOD

     2.1  This semi-automated method is based on the distillation of phenol
          and subsequent reaction of the distillate with alkaline ferricyanide
          and 4-aminoantipyrine to form a red complex which is measured at 505
          or 520 nm.

     2.2  Color response of phenolic materials with 4-aminoantipyrine is not
          the same for all compounds.  Because phenolic type wastes usually
          contain a variety of phenols, it is not possible to duplicate a
          mixture of phenols to be used as a standard.  For this reason,
          phenol has been  selected as a standard and any color produced by the
          reaction of other phenolic compounds is reported as phenol.  This
          value will represent the minimum concentration of phenolic compounds
          present in the sample.

     2.3  Reduced volume versions of this method that use the same reagents
          and molar ratios are acceptable provided they meet the quality
          control and performance requirements stated in the method.

     2.4  Limited performance based method modifications may be acceptable
          provided they  are fully documented and meet or exceed requirements
          expressed in Sect. 9.0, Quality Control.

3.0  DEFINITIONS

     3.1  CALIBRATION BLANK (CB) — A volume of reagent water fortified with
          the same matrix  as the calibration standards, but without the
          analytes, internal standards, or surrogate analytes.

     3.2  CALIBRATION STANDARD  (CAL) ~ A  solution prepared from the primary
          dilution standard solution or stock standard solutions and the
          internal standards and surrogate analytes.  The CAL solutions are
          used to calibrate the instrument response with respect to analyte
          concentration.

                                    420.4-2

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 3.3  INSTRUMENT PERFORMANCE CHECK SOLUTION (IPC) — A solution of one or
      more method analytes, surrogates, internal standards, or other test
      substances used to evaluate the performance of the instrument system
      with respect to a defined set of criteria.

 3.4  LABORATORY FORTIFIED BLANK (LFB) -- An aliquot of reagent water or
      other blank matrices to which known quantities of the method
      analytes are added in the laboratory.  The LFB is analyzed exactly
      like a sample, and its purpose is to determine whether the
      methodology is in control, and whether the laboratory is capable of
      making accurate and precise measurements.

 3.5  LABORATORY FORTIFIED SAMPLE MATRIX (LFM)  — An aliquot of an
      environmental  sample to which known quantities of the method
      analytes are added in the laboratory.  The LFM 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 LFM
      corrected for  background concentrations.

 3,6  LABORATORY REAGENT BLANK (LRB)  — An aliquot of reagent water or
      other blank matrices  that are treated exactly as a  sample including
      exposure to all  glassware,  equipment,  solvents,  reagents,  internal
      standards,  and surrogates that are used with other  samples.   The LRB
      is  used  to determine  if method analytes or other interferences  are
      present  in the laboratory environment, the reagents,  or the
      apparatus.

 3.7  LINEAR CALIBRATION RANGE (LCR)  --  The concentration  range  over  which
      the instrument response  is  linear.

 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.

 3.9   METHOD DETECTION LIMIT  (MDL) - The minimum concentration of an
      analyte  that can be identified, measured and reported with 99%
      confidence  that the analyte concentration  is greater than zero.

 3.10  QUALITY  CONTROL SAMPLE (QCS) - A solution of method analytes of
      known concentrations  that is used to fortify an aliquot of LRB or
      sample matrix.  The QCS is obtained from a source external to the
      laboratory and different from the source of calibration standards.
      It  is used to check laboratory performance with externally prepared
     test materials.

3.11 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.


                               420.4-3

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4.0  INTERFERENCES

     4.1  Interferences from sulfur compounds are eliminated by acidifying the
          sample to a pH of 4.0 and aerating briefly by stirring.

     4.2  Oxidizing agents such as chlorine, detected by the liberation of
          iodine upon acidification in the presence of potassium iodide, are
          removed immediately after sampling by the addition of an excess of
          ferrous ammonium sulfate (7.11).  If chlorine is not removed, the
          phenolic compounds may be partially oxidized and the results may be
          low.

     4.3  Background contamination from plastic tubing and sample containers
          is eliminated by filling the wash receptacle by siphon (using Kel-F
          tubing) and using glass tubes for the samples and standards.

     4.4  Method interferences may be caused by contaminants in the reagent
          water, reagents, glassware, and other sample processing apparatus
          that bias analyte response.

5.0  SAFETY

     5.1  The toxicity or carcinogen!city of each reagent used in this method
          have not been fully established.  Each chemical should be regarded
          as a potential health hazard and exposure should be as low as
          reasonably achievable.  Cautions are included for known extremely
          hazardous materials or procedures.

     5.2  Each 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 Material
          Safety Data Sheets (MSDS) should be made available to all personnel
          involved in the chemical analysis.  The preparation of a formal
          safety plan is also advisable.

     5.3  The following chemicals have the potential to be highly toxic or
          hazardous, consult MSDS.

          5.3.1   Potassium ferricyanide (7.2)

          5.3.2   Phenol (7.5)

          5.3.3   Sulfuric acid (7.10)

6.0  EQUIPMENT AND SUPPLIES

     6.1  Balance — Analytical, capable of accurately weighing to the nearest
          0.0001 g.

     6.2  Glassware — Class A volumetric flasks and pipets as required.
                                    420.4-4

-------
     6.3  Distillation apparatus, all glass consisting of a 1-L pyrex
          distilling apparatus with Graham condenser.  Reduced volume
          apparatus also may be used.

     6.4  pH meter with electrodes.

     6.5  Automated continuous flow analysis equipment designed to deliver and
          react sample and reagents in the required order and ratios.

          6.5.1   Sampling device (sampler)

          6.5.2   Multichannel pump

          6.5.3   Reaction unit or manifold

          6.5.4   Colorimetric detector

          6.5.5   Data recording device

7.0  REAGENTS AND STANDARDS

     7.1  Reagent water:   Distilled or deionized water,  free of the analyte of
          interest.   ASTM type II or equivalent.

     7.2  Buffered potassium ferricyanide:  Dissolve 1.0 g potassium
          ferricyanide (CSRN 13746-66-2), 1.55 g boric acid (CASRN 10043-35-
          3),  and 1.875 g potassium chloride (CASRN 7447-40-7)  in 400 ml of
          reagent water.   Adjust to pH of 10.3 with 1  N  sodium  hydroxide
          (CASRN 1310-73-2) (7.3)  and dilute to 500 ml.   Add 0.25 mL of Brij-
          35 (CASRN  9002-92-0).   Prepare fresh weekly.

     7.3  Sodium hydroxide (IN):   Dissolve 20  g NaOH in  250 ml  of reagent
          water,  cool  and dilute to 500  ml.

     7.4  4-Aminoantipyrine:   Dissolve 0.13  g  of 4-aminoantipyrine (CASRN 83-
          07-8)  in 150  ml of reagent water and dilute  to  200 mL.   Prepare
          fresh  each  day.

     7.5  Stock  phenol:   Dissolve  0.50 g phenol  (CASRN 108-95-2)  in  500 ml of
          reagent  water and dilute to  500 ml.   Add  0.25 ml  cone.  H2SO,  (CASRN
          7664-93-9)  as preservative.   1.0 mL  = 1.0 mg phenol.

     7.6  Standard phenol  solution A:  Dilute  1.0 ml of stock phenol  solution
          (7.5)  to 100  ml  with reagent water.   1.0  ml = 0.01 mg phenol.

     7.7  Standard phenol  solution B:  Dilute  10.0  ml of  standard  phenol
          solution A  (7.6)  to  100  ml with reagent water.  1.0 mL = 0.001  mg
          phenol.

     7.8  Standard solution C:  Dilute 10.0 mL  of standard phenol  solution B
          (7.7) to 100  mL with reagent water.   1.0  mL = 0.0001 mg  phenol.


                                   420.4-5

-------
     7.9  Sodium hydroxide, 1+9:  Dilute 10 ml of IN NaOH (7.3)  to 100 mL with
          reagent water.

     7 10 Sulfuric acid, 1+9 :  Slowly add 10 ml cone. H2S04 (CASRN 7764-93-9)
          to 70 ml of reagent water.  Cool and dilute to 100  ml with reagent
          water.

     7.11 Ferrous ammonium sulfate:  Dissolve 0.55 g ferrous  ammonium sulfate
          in 250 ml reagent water containing 0.5 ml H2S04 and dilute to 500 mL
          with freshly boiled and cooled reagent water.

8.0  SAMPLE COLLECTION. PRESERVATION AND STORAGE

     8 1  Samples should be collected in glass bottles only.   All bottles must
          be thoroughly cleansed and rinsed with reagent water. Volume
          collected should be sufficient to insure a representative sample,
          allow for replicate analysis  (if required), and minimize waste
          disposal.

     8.2  Samples must be  preserved at  time of collection with H2S04 to a pH
          of < 2 and cooled to  4°C.

     8.3  Samples should be analyzed as soon as possible after collection.  If
          storage is required,  preserved  samples are maintained at 4°C and may
          be held up to 28 days.

9.0  QUALITY CONTROL
     9.1
     Each laboratory using this method is required to operate a formal
     quality control (QC) program.   The minimum requirements of this
     program consist of an initial  demonstration of laboratory
     capability, and the periodic analysis of laboratory reagent blanks,
     fortified blanks and other laboratory solutions as a continuing
     check on performance.  The laboratory is required to maintain per-
     formance records that define the quality of the data that are
     generated.

9.2  INITIAL DEMONSTRATION OF PERFORMANCE

     9.2.1   The initial demonstration of performance is used to
             characterize instrument performance (determination of LCRs
             and analysis of QCS) and laboratory performance
             (determination of MDLs) prior to performing analyses by this
             method.

     9.2.2   Linear Calibration Range (LCR) — The LCR must be determined
             initially and verified every 6 months or whenever a
             significant change in instrument response is observed or
             expected.  The initial demonstration of linearity must use
             sufficient standards to insure that the resulting curve is
             linear.  The verification of linearity must use a minimum of
             a  blank and three standards.  If any verification data

                               420.4-6

-------
             exceeds the initial values by ± 10%, linearity must be
             reestablished.  If any portion of the range is shown to be
             nonlinear, sufficient standards must be used to clearly
             define the nonlinear portion.

     9.2.3   Quality Control Sample (QCS) — When beginning the use of
             this method, on a quarterly basis or as required to meet
             data-quality needs, verify the calibration standards and
             acceptable instrument performance with the preparation and
             analyses of a QCS.  If the determined concentrations are not
             within ± 10% of the stated values, performance of the
             determinative step of the method is unacceptable.  The
             source of the problem must be identified and corrected
             before either proceeding with the initial determination of
             MDLs or continuing with on-going analyses.

     9.2.4   Method Detection Limit (MDL) — MDLs must be established for
             all analytes, using reagent water (blank) fortified at a
             concentration of two to three times the estimated instrument
             detection limit.(4)  To determine MDL values, take seven
             replicate aliquots of the fortified reagent water and
             process through the entire analytical method.   Perform all
             calculations defined in the method and report the
             concentration values in the appropriate units.  Calculate
             the MDL as follows:

                                MDL = (t) x (S)

             where, t = Student's t value for a 99% confidence level and
                        a standard deviation estimate with n-1 degrees
                        of freedom [t = 3.14 for seven replicates].

                    S = standard deviation of the replicate analyses.

             MDLs should be determined every 6 months, when a new
             operator begins work or whenever there is a significant
             change in the background or instrument response.

9.3  ASSESSING LABORATORY PERFORMANCE

     9.3.1   Laboratory Reagent Blank (LRB) — The laboratory must
             analyze at least one LRB with each batch of samples.  Data
             produced are used to assess contamination from the
             laboratory environment.  Values that exceed the MDL indicate
             laboratory or reagent contamination should be suspected and
             corrective actions must be taken before continuing the
             analysis.

     9.3.2   Laboratory Fortified Blank (LFB) — The laboratory must
             analyze at least one LFB with each batch of samples.
             Calculate accuracy as percent recovery (Sect.  9.4.2).  If
             the recovery of any analyte falls outside the required

                               420.4-7  •

-------
             control  limits of 90-110%,  that analyte is judged out of
             control, and the source of the problem should be identified
             and resolved before continuing analyses.

     9.3.3   The laboratory must use LFB analyses data to assess
             laboratory performance against the required control limits
             of 90-110%.  When sufficient internal  performance data
             become available (usually a minimum of 20-30 analyses),
             optional control limits can be developed  from the percent
             mean recovery (x) and the standard deviation (S) of the  mean
             recovery.  These data can be used to establish the upper and
             lower control limits as follows:

                        UPPER CONTROL LIMIT = x + 3S
                        LOWER CONTROL LIMIT = x - 3S

             The optional control limits must be equal to or better than
             the required control limits of 90-110%.  After each five to
             ten new recovery measurements, new control limits can be
             calculated using only the most recent 20-30 data points.
             Also, the standard deviation (S)  data should be used to
             established an on-going precision statement for the level of
             concentrations included in the LFB.  These data must be  kept
             on file and be available for review.

     9.3.4   Instrument Performance Check Solution (IPC) -- For all
             determinations the laboratory must analyze the IPC (a mid-
             range check standard) and a calibration blank immediately
             following daily calibration, after every tenth sample (or
             more frequently, if required), and at the end of the sample
             run.  Analysis of the IPC solution and calibration blank
             immediately following calibration must verify that the
             instrument is within ± 10% of calibration.  Subsequent
             analyses of the IPC solution must verify the calibration is
             still within ± 10%.  If the calibration cannot be verified
             within the specified limits, reanalyze the IPC solution.  If
             the second analysis of the IPC solution confirms calibration
             to be outside the limits, sample analysis must be
             discontinued, the cause determined and/or in the case of
             drift the instrument recalibrated.  All samples following
             the last acceptable IPC solution must be reanalyzed.  The
             analysis data of the calibration blank and IPC solution  must
             be kept on file with the sample analyses data.

9.4  ASSESSING ANALYTE RECOVERY AND DATA QUALITY

     9.4.1   Laboratory Fortified Sample Matrix (LFM)  -- The laboratory
             must add a known amount of analyte to a minimum of 10% of
             the routine samples.  In each case the LFM aliquot must  be a
             duplicate of the aliquot used for sample analysis.  The
             analyte concentration must be high enough to be detected
             above the original sample and should not be less than four

                             '  420.4-8

-------
                   times the MDL.  The added analyte concentration should be
                   the same as that used in the laboratory fortified blank.

           9.4.2   Calculate the percent recovery for each analyte,  corrected
                   for concentrations measured in the unfortified sample  and
                   compare these values to the designated LFM recovery range
                   90-110%.  Percent recovery may be calculated usinq the
                   following equation:


                      R =  GS  "  C     x  100
                             s

                   where,  R  =  percent recovery.
                          Cs =   fortified sample concentration.
                          C  =  sample  background  concentration.
                          s  =  concentration  equivalent of analyte added to
                                sample.

          9.4.3    If the  recovery of any analyte  falls outside the designated
                   LFM  recovery  range and the  laboratory performance for that
                   analyte  is  shown to  be in control  (Sect. 9.3), the recovery
                   problem  encountered  with the LFM is judged to be either
                  matrix or solution related, not system related.

          9.4.4   Where reference materials are available, they should be
                  analyzed to provide  additional  performance data.  The
                  analysis of reference samples is a valuable tool for
                  demonstrating the ability to perform the method acceptably.

10.0 CALIBRATION AND STANDARnT7flTTnM

     10.1 Prepare a series of at least  3 standards,  covering  the desired
          JolSinS /? blank by pipetting suitable volumes of working standard
          solutions (7.6,  7.7, 7.8)  into 100-mL volumetric flasks.   Suggested
          ranges include 1 to 5, 10  to  100,  and 200  to SOO'jag/L.     iu"ested

     10'2 2«nS  "Ot ™perat1ye thaj  a11 standards  be  distilled  in  the  same
          IX ThiS It s??pl?fi  J*  1s recommenc|ed that at least  one  standard
          and  a  blank  be distilled and  compared to similar values  on the
                   CUTfW'??U!;e !hai the ^Illation technique is
                               proceeding.  Before distillation, standards
                               a pH of 4 with H2S04.

    10.3 Set up the manifold as shown in Figure 1 in a hood or a well-
       '  ventilated area.

    10.4 Allow the instrument to warm up as required.  Pump all reagents
         until a stable baseline is achieved.


                                   420.4-9

-------
     10.5 Place appropriate standards in the sampler in order of decreasing
          concentration and perform analysis.

     10.6 Prepare standard curve by plotting instrument response concentration
          values.  A calibration curve may be fitted to the calibration
          solutions concentration/response data using computer or calculator
          based regression curve fitting techniques.  Acceptance or control
          limits should be established using the difference between the
          measured value of the calibration solution and the "true value"
          concentration.

     10.7 After the calibration has been established, it must be verified by
          the analysis of a suitable quality control sample (QCS).  If
          measurements exceed ± 10% of the established QCS value, the analysis
          should be terminated and the instrument recalibrated.   The new
          calibration must be verified before continuing analysis.  Periodic
          reanalysis of the QCS is recommended as a continuing calibration
          check.

11.0 PROCEDURE

     11.1 Distillation

          11.1.1  Measure 500 ml sample into a beaker.   Adjust the pH to
                  approximately 4 with 1+9 NAOH (7.9) or 1+9 H2S04 (7.10), and
                  transfer to the distillation apparatus.

          11.1.2  Distill  450 ml of sample,  stop the distillation,  and when
                  boiling ceases add 50 ml of warm reagent water to  the flask
                  and resume  distillation until  500 mL  have been collected.

          11.1.3  If the distillate is turbid,  filter through  a  prewashed
                  membrane filter.

     11.2 Set up the manifold as shown in Figure 1.

     11.3 Fill  the wash receptacle  by  siphon with  reagent  water.   Use Kel-F
          tubing with a fast  flow (1  L/h).

     11.4 Allow the instrument to warm up as required.   Run a  baseline with
          all  reagents,  feeding reagent  water through the  sample  line.   Use
          polyethylene  tubing  for sample line.   When new tubing  is used,  about
          2  hours  may be required to obtain  a stable baseline.  This  two  hour
          time  period may  be  necessary to remove the residual  phenol  from the
          tubing.

     11.5 Place appropriate phenol  standards  in  sampler  in  order  of decreasing
          concentration.   Complete  loading of sampler tray  with unknown
          samples,  using glass tubes.

     11.6 Switch sample  line from reagent water  to sampler  and begin  analysis.
                                  420.4-10

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12.0 DATA ANALYSIS AND CALCULATIONS

     12.1 Prepare a calibration curve by plotting instrument response
          against standard concentration.  Compute sample concentration by
          comparing sample response with the standard curve.  Multiply answer
          by appropriate dilution factor.

     12.2 Report only those values that fall between the lowest and the
          highest calibration standards.  Samples exceeding the highest
          standard should be diluted and reanalyzed.

     12.3 Report results in  /jg/L.

13.0 METHOD PERFORMANCE

     13.1 The inter!aboratory precision and accuracy data in Table 1 were
          developed using a reagent water matrix.  Values are in mg Phenol/L.

     13.2 Single laboratory precision data can be estimated at 50 to 75% of
          the inter!aboratory precision estimates.

14.0 POLLUTION PREVENTION

     14.1 Pollution prevention encompasses any technique that reduces or
          eliminates the quantity or toxicity of waste at the point of
          generation. Numerous opportunities for pollution prevention exist
          in laboratory operation. The EPA has established a preferred
          hierarchy of environmental management techniques that places
          pollution prevention as the management option of first choice.
          Whenever feasible, laboratory personnel should use pollution
          prevention techniques to address their waste generation.  When
          wastes cannot be feasibly reduced at the source, the Agency
          recommends recycling as the next best option.

     14.2 The quantity of chemicals purchased should be based on expected
          usage during its shelf life and disposal cost of unused material.
          Actual reagent preparation volumes should reflect anticipated usage
          and reagent stability.

     14.3 For information about pollution prevention that may be applicable to
          laboratories and research institutions, consult "Less is Better:
          Laboratory Chemical  Management for Waste Reduction," available from
          the American Chemical Society's Department of Government
          Regulations and Science Policy, 1155 16th Street N.W.,  Washington
          D.C.  20036, (202) 872-4477.

15.0 WASTE MANAGEMENT

     15.1 The Environmental Protection Agency requires that laboratory waste
          management practices be conducted consistent with all  applicable
          rules and regulations.  Excess Reagents and samples and method
          process wastes should be characterized and disposed of in an

                                   420.4-11

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          acceptable manner.  The Agency urges laboratories to protect the
          air,  water, and land by minimizing and controlling all  releases from
          hoods, and bench operations, complying with the letter and spirit of
          any waste discharge permit and regulations, and by complying with
          all solid and hazardous waste 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," available from the
          American Chemical Society at the address listed in Sect. 14.3.

16.0 REFERENCES

     1.   Technicon AutoAnalyzer II Methodology, Industrial Method No. 127-
          71W, Mil.

     2    Standard Methods for the Examination of Water and Wastewater, 14th
          Edition, p. 574, Method 510 (1975).

     3.   Gales, M.E. and Booth, R.L., "Automated 4 AAP Phenolic Method," AWWA
          68, 540 (1976).

     4.   Code of Federal Regulations 40, Ch. 1, Pt. 136, Appendix B.
                                    420.4-12

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17.0 TABLES. DIAGRAMS. FLOWCHARTS. AND VALIDATION DATA
TABLE 1. INTERLABORATORY PRECISION AND ACCURACY DATA
NUMBER OF
VALUES
REPORTED
99
87
76
110
89
107
86
62
76
89
61
110
TRUE
VALUE
(T)
0.020
0.250
0.400
0.545
0.604
0.660
0.800
0.817
0.970
2.96
4.18
4.54
MEAN
(X)
0.0149
0.1443
0.2352
0.3364
0.3610
0.3959
0.4627
0.4692
0.5680
1.7734
2.3916
2.7150
RESIDUAL
FOR X
0.0000
-0.0052
-0.0021
0.0142
0.0043
0.0064
-0.0087
-0.0122
-0.0029
0.0377
-0.0582
0.0545
STANDARD
DEVIATION
(S)
0.0074
0.0268
0.0422
0.0681
0.0625
0.0894
0.0806
0.0776
0.1017
0.3065
0.4044
0.5382
RESIDUAL
FOR S
0.0000
-0.0038
-0.0036
0.0076
-0.0039
0.0173
-0.0057
-0.0104
-0.0017
0.0018
-0.0237
0.0737
REGRESSIONS:  X = 0.585T + 0.003, S = 0.101T + 0.005
                                   420.4-13

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                                                420.4-14
U.S. GOVERNMENT PRINTING OFFICE 1993—750  -002/80267

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