600-4-85-021
   DETERMINATION OF BENDIOCAR3  IN  INDUSTRIAL
           AND MUNICIPAL WASTEWATERS
    J.S. Warner, T.M.  Engel  and  P.J. Mondron
         Bactelle Columbus Laboratories
              Columbus,  Ohio 43201
            Contract No.  68-03-2956



                Project  Officer

                Thomas Prassley
      Physical and Chemical Methods Branch
Environmental Monitoring  and  Support Laboratory
             Cincinnati,  Ohio  45263
 ENVIRONMENTAL MONITORING AND SUPPORT LABORATORY
        OFFICE OF  RESEARCH AND DEVELOPMENT
       U.S.  ENVIRONMENTAL PROTECTION AGENCY
             CINCINNATI, OHIO 45263

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                                DISCLAIMER
     This report has been reviewed by the Environmental Monitoring and
Support Laboratory, U.S. Environmental Protection Agency, and approved
for publication.  Approval does not signify that the contents necessarily
reflect the view and policies of the U.S. Environmental Protection
Agency, nor does mention of trade names or commercial products constitute
endorsement or recommendation for use.
                                     ii

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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 and Support Laboratory - Cincinnati, conducts
research to:

     •   Develop and evaluate methods to measure the presence and
         concentration of physical, chemical, and radiological
         pollutants in water, wastewater, bottom sediments, and solid
         waste.

     •   Investigate methods for the concentration, recovery, and
         identification of viruses, bacteria and other microbiological
         organisms in water; and, to determine the responses of aquatic
         organisms to water quality.

     •   Develop and operate an Agency-wide quality assurance program
         to assure standardization and quality control of systems  for
         monitoring water and wastewater.

     •   Develop and operate a computerized system for instrument
*        automation leading to improved data collection, analysis,  and
         quality control.

     This report is one of a series that investigates the analytical
behavior of selected pesticides and suggests a suitable  test procedure
for their measurement in wastewater.  The method was modeled after
existing EPA methods being specific yet as simplified as possible.
                      Robert L. Booth, Director
                      Environmental Monitoring and Support
                      Laboratory - Cincinnati
                            ill

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                                ABSTBACT

     A method was developed for the determination of bendiocarb  in
wastewaters.  The method development program consisted_of  a  literature
review; determination of extraction efficiency for the  compound from
water into methyLane chloride; development of a deactivated  Florisil
cleanup procedure; and determination of suitable liquid chromatographic
analysis conditions.

     The final method was applied to a relevant industrial wastewater in
order to determine the precision and accuracy of the method.  The
wastewater was spiked wijrh _the compound at levels of 8.0 ug/L and
80 ug/L.  Recovery for bendiocarb at the 8 ug/L level was 65  ± 24
percent.  Recovery at the 80 ug/L level was 70 ± 4 percent.  The method
detection limit (MDL) for bendiocarb in distilled water was  1.8  ug/L.
The MDL in wastewaters may be higher due to interfering compounds.

     This report was submitted in partial fulfillment of Contract No.
68-03-2956 by Battelle Columbus Laboratories under the sponsorship  of
the U.S. Environmental Protection Agency.  This report covers the
period from February 1, 1982 to April 30, 1982.
                                    iv

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                                 CONTENTS

Foreword
Abstract ...............................     iv
Figures ...............................     vi
Tables  ..............................  •    vii

     1.  Introduction ....... ' .................      1
     2.  Conclusions ......... . ...............      2
              Extraction and Concentration ..............      2
              Cleanup ...... . .................      2
              Chrooatography ............ „ .........      2
              Validation Studies ...................      2
     3 .  Experimental ........ ................      3
              Extraction and Concentration ..............      3
              Cleanup ... .................. ...      3
              Chromatography .....................      3
              Validation Studies ...................      4
     4.  Results and Discussion  ...................      5
              Extraction and Concentration ..............   .   5
              Cleanup ........................      5
              Chromatography .....................      5
              Validation Studies ...................      8

References ..............................      10
Appendix

     A.  Bendiocarb Method 639 ....................      11

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                                 FIGURES




Number                                                            Page




  1     HPLC-UV Chromatogram of 10 ng of Bendiocarb	      6




  2     HPLC-OV Chromatogram of 600 ag of Bendiocarfa	      7




  3     Analytical Curve for Bendiocarb	      9
                                    vi

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                                 TABLES






Number                                                        Page






  1     Analytical Curve Data for Bendiocarb	     8
                                   vii

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                               SECTION 1

                              INTRODUCTION
     Bendiocarb (I) is an insecticide acting by cholinesterase
inhibition and is effective as a contact and stomach poison.  The acute
oral LD50 for rats is 180 rag/kg, which indicates moderate toxicity.
                              (I)

The CAS registry number for bendiocarb is  22781-23-3  and  the  CAS  name
for bendiocarfa is 2,2-dimethyl-l,3-benzodioxol-4-yl methylcarbamate.
It has a melting point of  129-130 C.  A  synonym  used  for  bendiocarb  is
"Ficam".  A GC method of analysis using  a  modified alkali flame
detector has been reported  (L) as well as  gas  chromatographic.(GC)
pyrolysis and GC derivatization methods  (2). It  has been  reported that
bendiocarb is thermally unstable and an  high performance  liquid
chromatographic (HPLC) method has been developed using  reverse phase
chromatography (3).

     Bendiocarb can be extracted from water with methylene chloride.
The selected approach to its determination in  water included  separatory
funnel extraction from water with mehylene chloride,  cleanup  using
Florisil chromatography, and analysis using HPLC with ultraviolet
detection (DVD).  Standard  concentration techniques using
Kuderna-Danish (K-D) equipment were used.  The final  method is included
in Appendix A of this report.

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                                SECTION 2

                               CONCLUSIONS
EXTRACTION AND CONCENTRATION

     Bendiocarb can be extracted  from water  into  methylene  chloride
with greater than 85 percent  recovery using  separatory funnel
techniques.  Use o£ K-D concentration equipment to  perform  extract
concentrations did not significantly  affect  compound  recoveries.

CLEANUP

     Bendiocarb elutes from deactivated  Florisil  with greater  than  80
percent recovery.  This was an effective cleanup  procedure  for a
relevant wasCewater sample.

CHROMATOGRAPHY

     Two HPLC columns, Spherisorb-ODS  and Lichrosorb  RP-2,  were found
to be acceptable for this application.   The  Spherisorb-ODS  column»was
used as the primary column.   The Lichrosorb  RP-2  column was designated
as the alternate column.

VALIDATION STUDIES

     Recoveries of bendiocarb from distilled water  in the 4 to
1000 yg/L concentration range averaged greater than 80 percent.  The
analytical curve constructed  from this data was linear.  The MDL in
distilled water was 1.8 ug/L.  Recoveries of bendiocarb from a relevant
industrial wastewater at the  8 and 80 ug/L levels were 64 i 24 and
70 ± 4 percent, respectively.

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                                SECTION 3

                               EXPERIMENTAL


      Studies  were performed to determine if extractions with separatory
 funnels,  cleanup by Florisil adsorption chromatography, concentration
 using K-D equipment, and analysis using HPLC with UVD would be
 applicable techniques for the determination of bendiocarb in water.

 EXTRACTION AND CONCENTRATION

      Extraction of bendiocarb from water by separatory funnel
 techniques was studied.   One liter of distilled water was used.  The
 distilled water was spiked with bendiocarfa at the 20 Ug/L and 100 ug/L
 levels.   The  samples were adjusted to pH 7 by the addition of 1 N
 suIfuric  acid or 1 £ sodium hydroxide and extracted three times with
 60 mL each of methylene  chloride.  These studies were done in
 duplicate. The extracts were dried by passing them through 10 cm of
 anhydrous granular sodium sulfate and concentrated to two mL.  The
 samples were  solvent exchanged with acetonitrile by concentrating the
 sample to five mL after  the addition of 15 mL of acetonitrile and
 analyzed  by HPLC.

 CLEANUP

      A 10-gram Florisil  column (deactivated with water saturated methylene
 chloride) was  prepared and eluted with 100 mL of 50 percent methylene
 chloride  in petroleum ether.   The bendiocarb,  20 yg dissolved in one mL of
 methylene  chloride,  was  added to  the top of the column.   The column was
 eluted with 50 mL portions of 50  percent methylene chloride in petroleum
 ether  (Fl), methylene chloride (F2),  five percent acetone in methylene
 chloride  (F3),  15  percent  acetone in methylene  chloride  (F4),  and 25
 percent acetone  in  methylene  chloride (F5).  Each fraction was
 concentrated  to  1 mL.  The fractions  were solvent exchanged to
 acetonitrile by reconcentrating  them to  2 mL after the addition of 10 mL
 of acetonitrile.

CHROMATOGRAPHY

     Two  reversed  phase HPLC  columns  were evaluated for  the determination
of bendiocarb:  Spherisorb-ODS  and  Lichrosorb  RP-2.

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VALIDATION STUDIES

     The MDL for bendiocarb was determined by analyzing seven replicate
distilled water samples spiked at the 4 yg/L concentration level.  The
sample extracts were cleaned up using the Florisil cleanup procedure
prior to analysis.  The amounts recovered were determined by external
standard calibration and the MDLs were calculated from these data.

     Distilled water was also spiked in duplicate at the 4, 20, 100,
250, and 1000 yg/L concentration levels and recoveries of the
bendiocarfa were determined as described earlier.  An analytical curve
was generated by plotting the amount spiked into the samples versus the
amount recovered from the samples.

     A relevant industrial wastewater (obtained from a bendiocarb manu-
facturing site) was used for wastewater validation studies.  Seven replicates
of the wastewater were analyzed to determine the background levels.  The
wastewater was spiked with bendiocarfa at the-8 and 80 yg/L concentration
levels, processed and analyzed.  Seven replicate extractions were performed
at each concentration level.

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                                  SECTION 4

                          RESULTS AND DISCUSSION
EXTRACTION AND CONCENTRATION

     Bendiocarb -as extracted  from water with  greater than 35  percent
recovery using separatory funnel  techniques.   Recovery of bendiocarb from
water using separatory funnels were 32  and  96  percent at  20 ug/L and 91
and 93 percent at  100 jig/L.  These data are the  results of duplicate
analyses.

CLEANUP

     Bendiocarb eluted from deactivated Florisil in  the 5 percent acetone
in methylene chloride fraction (F3).  Recovery of 20 yg of bendiocarb  was
82 percent.

CHROMATOGRAPHY

     Both the Spherisorb-ODS and  Lichrosorb RP-2 columns  were  satisfactory
for the determination of bendiocarb.  The Spherisorb-ODS  column was  chosen
as the primary column.  The following conditions were used:

     Column:                   Spherisorb-ODS, 5 micron,
                               250 x 4.6 mm
     Solvent.:                  40 percent acetonitrile/
                               60 percent water
     Flow:                     1 mL/min
     Detector:                 UV (§254  nm
     Injection Volume:         5 uL
     Retention Time:           9.3 min

A chromatogram obtained under  these conditions is shown in Figure 1.

     The Lichrosorb RP-2 column was chosen  as  the alternate column.   The
following conditions were used:

     Column:                   Lichrosorb RP-2,  5 micron,
                               250 x 4.6 mm
     Solvent:                  50 percent acetonitrile/
                               50 percent water
     Flow:                     1 mL/min
     Detector:                 UV (§254  nm
     Injection Volume:         5  uL
     Retention Time:           6.0 min

A chromatogram obtained under  these conditions is shown in Figure 2.
                                5

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•
•H
•
o\
*•>
-

^^A.

2.
I x
1 Y
0 4.0 6.0
Bendlocarb
*

8.0 10.0 12.0 14.0 16.0 18.0 20.0
                    Retention Time,  Min.
Figure 1.   HPLC-UV Chromatogram of 10 ng of Bendiocarb (Column 1)

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            Bendiocarb
"I •-• . -i~|—r-i—i • | --•-
 4.0        6.0        8.0
H.O
16.0
                                  10.0      12.0
                      Retention Time, Min.
Figure 2.  HPLC-UV Chromatogram of 600 ng of Bendiocarb  (Column 2)
'  I  •  '  '  •  1
18.0    20.0

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VALIDATIONS STUDIES

     Recovery of bendiocarb from distilled water at the 4.0 ug/L level
was 3.5 ± 0.6 ug/L.  The MDL in distilled water was calculated to be
1.8 ug/L.  Recoveries of bendiocarb from distilled water at the 4, 20,
100, 250, and 1000 ug/L levexs were 3.5, 17, 82, 200, and 810 yg/L,
respectively.  These data were the averages of duplicate analyses.
Individual data points are given in Table 1.  The resultant analytical
curve is shown in Figure 3.
        TABLE 1.  ANALYTICAL CURVE DATA FOR BENDIOCARB
             Concentration,          Amount  Recovered,
               Ug/L                       ug/L(a)

                   4                       3.3,  3.7

                  20                       16,   18
                 100                       78,   86
                 250                      190,  210

               1000                      770,  850
             (a)  Results of duplicate analyses.
     Recoveries  of bendiocarb  from  a  relevant  wastewater  at  the  8  and
80 ug/L  levels were  65 ±  24  percent and  70 ± 4 percent, respectively.
These  data were  the  averages of  seven replicate analyses.

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             1000 r
              900 -
              800 •
              700
Amount Recovered,
              600 •
              500 -
              400 •
              300 •
             200 •
             100 '
                     100  200   300   400   500   600    700  800   900  1000
                                     Amount Spiked ug/L
                           Figure 3.  Analytical Curve  for Bendiocarb

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                               REFERENCES

1.  Richie, A.R., S.R. Solly, and M.H. Clear.  The Application
    of a Thermionic Detector to the Determination of Nitrogen-
    Containing Compounds on Thin-Layer Chromatograms. J. ChromaEogr.,
    150(2):557-561, 1978.

2.  Whitcoak, R.J., J.B. Reary, and K.C. Overton.  Bendiocarb.
    In: Analytical Methods for Pesticides and Plant Growth Regulators,
    Voll. 10, G. Zweig and J. Sherma, eds. Academic Press, New York,
    San Francisco, and London, 1978. pp. 3-17.

3.  Zehner, J.M., R.A. Simonaitis, and R.E. Bny. High Performance
    Liquid Chromatographic Determination of Bendiocarb on Wool.
    J. Assoc. Off. Anal. Chem.. 63(l):47-48, 1980.
                                   10

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           DETERMINATION OF BENDIOCARB  IN  MUNICIPAL AND  INDUSTRIAL
                    WASTEWATERS  BY  LIQUID  CHROMATOGRAPHY
                                 METHOD 639
1.  Scope and Application
    1.1  This method covers  the  determination  of bendiocarb  pesticide.   The
         following parameter can be determined by this  method:
                   Parameters                          CAS No.
                   Bendiocarb                        22781-23-3
    1.2  This is a high performance liquid chromatographic (HPLC)  method
         applicable to the determination of the compound listed  above in
         municipal and industrial  discharges as provided under 40  CFR 136.1.
         When this method  is used to analyze unfamiliar samples  for the
         compound above, compound identifications should be  supported by at
         least one additional qualitative technique.   This method  describes
         analytical conditions for a second liquid chromatographic column
         that can be used  to confirm measurements made with the  primary
         column.
    1.3  The method detection limit (MDL, defined in  Section 15) for
         bendiocarb is listed in Table 1.  The MDL for a specific
         wastewater may differ from those listed, depending upon the
         nature of interferences in the sample matrix.
                                         11

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    1.4  Any modification of this method beyond those expressly permitted,
         shall be considered a major modification  subject  to  application  and
         approval of alternate test procedures under 40 CFR 136.4 and 136.5.
    1.5  This method is restricted to use by or under the  supervision of
         analysts experienced in the use of liquid chromatography and in
         the interpretation of liquid chromatograms.   Each analyst must
                  •
         demonstrate the ability to generate acceptable results with this
         method using the procedure described in Section 8.2.
2.  Summary of Method
    2.1  A measured volume of sample, approximately 1 liter,  is solvent ex-
         tracted with methyl ene chloride using a separatory funnel.   The
         methylene chloride extract is dried and exchanged to acetonitrile
         during concentration to a volume of 2 ml  or less.  Liquid
         chromatographic conditions are described  which permit the
         separation and measurement of the compounds  in the extract  by
         HPLC-UV. (1)                                                 '
3.  Interferences
    3.1  Method interferences may be caused by contaminants in solvents,
         reagents, glassware, and other sample processing  hardware that lead
         to discrete artifacts or elevated baselines  in liquid
         chromatograms,  ATI of these materials must  be routinely
         demonstrated to be free from interferences under  the  conditions  of
         the analysis by running laboratory reagent blanks as  described in
         Section 8.5.
         3.1.1  Glassware must be scrupulously cleaned. (2)  Clean all
                glassware as soon as possible after use by rinsing with the
                                        12

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                last solvent used in  it.   This  should  be  followed  by
                detergent washing with  hot water and rinses  with tap  and
                disttlled water.  It  should then be drained  dry, and  heated
                in a muffle furnace at  400°C for 15-30 minutes.  Some thermally
                stable materials such as  PCBs may not  be  eliminated by this
                treatment.  Solvent rinses with acetone and  pesticide
                quality hexane may be substituted for  the muffle furnace
                heating.  After drying  and cooling, glassware should  be
                sealed and stored in  a  clean environment  to  prevent any
                accumulation of dust  or other contaminants.   Store inverted
                or capped with aluminum foil.
         3.1.2  The use of high purity  reagents and solvents helps to mini-
                mize interference problems.  Purification of solvents by
                distillation in all-glass systems may  be  required.
    3.2  Matrix interferences may be  caused by  contaminants  that  are  coex-
         tracted from the sample.  The  extent of matrix interferences will
         vary considerably from source  to source, depending  upon  the  nature
         and diversity of the industrial  complex or municipality  being
         sampled.  Unique samples may require additional  cleanup  approaches
         to achieve the MDL listed in Table 1.
4.  Safety
    4.1  The toxicity or carcinogenicity of each reagent  used in  this method
         has not been precisely defined;  however, each chemical  compound
         should be treated as a potential health hazard.   From this
         viewpoint, exposure to these chemicals must  be reduced to the
         lowest possible level by whatever means available.   The  laboratory

                                         13

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         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 data  handling  sheets
         should also be made available to all  personnel involved  in the
         chemical analysis.  Additional  references to  laboratory  safety are
         available and have been identified (3-5)  for  the  information of the
         analyst.
5.  Apparatus and Materials
    5.1  Sampling equipment, for discrete or composite sampling.
         5.1.1  Grab sample bottle - Amber  glass,  1-liter  or 1-quart  volume,
                fitted with screw caps lined with  Teflon.   Foil may be
                substituted for Teflon if the sample  is not  corrosive.   If
                amber bottles are not available,  protect  samples  from light.
                The container and cap liner must  be washed,  rinsed with
                acetone or methylene chloride, and dried  before use to
                minimize contamination.
         5.1.2  Automatic sampler (optional) - Must  incorporate glass sample
                containers for the collection of  a minimum of 250 ml.
                Sample containers must be kept refrigerated at 4°C and
                protected from light during compositing.   If the  sampler
                uses a peristaltic pump, a  minimum length of compressible
                silicone rubber tubing may  be used.   Before use,  however,
                the compressible tubing  should be thoroughly rinsed with
                methanol, followed by repeated rinsings with distilled  water
                to minimize the potential  for contamination of the  sample.
                An integrating flow meter is required to  collect  flow  -
                proportional composites.
                                           14

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5.2  Glassware (All  specifications  are  suggested.   Catalog  numbers  are
     included for illustration  only.)
     5.2.1  Separatory funnel  - 2000-mL,  with Teflon  stopcock.
     5.2.2  Drying column  - Chromatographic  column  400 mm long  x  10 mm
            ID with coarse frit.
     5.2.3  Chromatographic column  - 400  mm  long x  19 mm ID with  250  mL
            reservoir at the top and Teflon  stopcock  (Kontes K-420290 or
            equivalent).
     5.2.4  Concentrator tube,  Kuderna-Danish - 10-mL, graduated  (Kontes
            K-570050-1025 or equivalent).  Calibration must be  checked
            at the volumes employed in  the test.  A ground  glass  stopper
            is used to prevent  evaporation of extracts.
     5.2.5  Evaporative flask,  Kuderna-Danish - 500-mL (Kontes
            K-570001-0500 or equivalent).  Attach to  concentrator tube
            with springs.
     5.2.6  Snyder column, Kuderna-Danish -  three-ball macro (Kontes
            K-503000-0121 or equivalent).
     5.2.7  Snyder column, Kuderna-Danish -  two-ball  micro  (Kontes
            K-569001-0219 or equivalent).
     5.2.8  Vials - Amber glass, 5 to 10 mL  capacity  with Teflon  lined
            screw-cap.
5.3  Boiling chips - approximately 10/40 mesh carborundum.   Heat  to
     400°C for 4 hours or extract in a  Soxhlet extractor with methylene
     chloride.
5.4  Water bath - Heated, capable of temperature control (_+2°C).   The
     bath should be used in a hood.
                                       15

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    5.5  Balance - Analytical,  capable  of  accurately weighing  to  the
         nearest 0.0001  g.
    5.6  Liquid chromatograph - Analytical  system  complete  with liquid
         chromatograph and  all  required accessories including  syringes,
         analytical  columns,  detector and  strip-chart  recorder.   A  data
         system is recommended  for measuring peak  areas.
         5.6.1  Pump -  Isocratic pumping system, constant  flow.
         5.6.2  Column  1 -  Reversed-phase  column,  5 micron  Spherisorb-ODS,
                250 x 4.6 mm  or equivalent.
         5.6.3  Column  2 -  Reversed-phase  column,  5 micron  Lichrosorb  RP-2,
                250 x 4.6 mm  or equivalent.
         5.6.4  Detector -  Ultraviolet  absorbance  detector, 254 nm.
6.  Reagents
    6.1  Reagent water  - Reagent water  is  defined  as  a water in which  an
         interferent is  not observed at the method detection limit  of  each
         parameter of interest.
    6,2  Methylene chloride,  methanol,  acetonitrile,  distilled-in-glass
         quality or equivalent.
    6.3  Sodium sulfate (ACS) Granular, anhydrous; heated  in a muffle
         furnace at 400°C overnight.
    6.4  Sodium hydroxide, 1N_  -  Prepare by  adding 4 g of sodium  hydroxide
         to distilled water  and  diluting to  100 ml.
    6.5  Sulfuric acid,  1N_ - Prepare by adding 2.8 ml of concentrated sulfuric
         acid  to  distilled water and diluting to  100 ml.
    6.6  Florisil -  PR  grade (60/100 mesh).  Purchase activated  at 1250°F
         and store  in a  brown  glass  bottle.  To prepare for use, place 150 g
         in a  wide-mouth jar and heat  overnight at 160-170°C.  Seal tightly
         with  Teflon or aluminum foil-lined  cap and cool to room temperature.
                                          16

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    6.7   Stock  standard  solution  (1.00 yg/uL) - Stock standard solution
         can  be prepared from  pure standard materials or purchased as
         certified  solutions.
         6.7.1   Prepare  stock  standard solutions  by  accurately weighing
                about 0.0100 grams of pure material.  Dissolve the material
                in distilled-in-glass quality methanol  and dilute to volume
                in a 10-mL volumetric flask.   Larger volumes can be  used at
                the convenience of the  analyst.   If compound purity  is
                certified at 96% or  greater,  the  weight can be  used  without
                correction to  calculate  the  concentration of the stock
                standard.  Commercially  prepared  stock  standards can be used
                at any concentration if  they  are  certified by the
                manufacturer or by an independent source.
         6.7.2  Transfer the stock standard  solution into Teflon-sealed
                screw-cap bottles.  Store at  4°C  and protect from light.
                Stock standard solution  should be checked frequently for
                signs of degradation or  evaporation, especially just prior
                to preparing calibration standards from them.
         6.7.3  Stock standard solution  must be replaced after six months
                or sooner if comparison  with quality control check standards
                indicates a problem.
7.  Calibration
    7.1   Establish liquid chromatographic operating parameters  equivalent to
         those indicated in Table 1.  The liquid  chromatographic system can
         be calibrated using the external  standard technique (Section 7.2)
         or the internal standard technique  (Section 7.3).
    7.2   External  standard calibration procedure:
                                        17

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      7.2.1   For  the  compound of  interest, prepare calibration standards
            at a minimum of three concentration levels by adding volumes
            of one or more stock standards to a volumetric flask and
            diluting to volume with acetonitrile.  One of the external
            standards should be at a concentration near, but above, the
            method detection limit.  The other concentrations should
                                                                        »
            correspond to the expected range of concentrations found in
            real samples or should define the working range of the
            detector.
      7.2.2  Using injections of 2 to 5 uL of each calibration standard,
            tabulate peak height or area responses against the mass
            injected.  'The results can be used to prepare a calibration
            curve for  bendiocarb.  Alternatively, the ratio of the
            response to the mass injected, defined as the calibration
            factor (CF), can be calculated at each standard concentration.
            If the relative standard deviation of the calibration factor
            is less than 10% over the working range, linearity through
            the origin can be assumed and the average calibration factor
            can be used in place of a calibration curve.
      7.£.3  The working calibration curve or calibration factor must be
            verified on each working day by the measurement of one or
            more calibration standards.  If the response for
            bendiocarb varies from the predicted response by more
            than ±10%, the test must be repeated using a fresh
            calibration standard.  Alternatively, a new calibration
            curve or calibration  factor must be prepared.
7.3  Internal  standard calibration  procedure.   To use this approach, the
     analyst must select  one  or more internal  standards  similar in
                                     18

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analytical behavior to bendiocarb.   The analyst must further
deomonstrate that the measurement of the internal  standard is
not affected by method or matrix interferences.  Due to these
limitations, no internal standard applicable to all  samples
can be suggested.
7.3.1  Prepare calibration standards at a minimum of three
       concentration levels for bend.iocarb by adding volumes
       of one or more stock standards to a volumetric flask.
       To each calibration standard, add a known constant
       amount of one or more internal standards, and dilute to
       volume with acetonitrile.  One of the standards should be at
       a concentration near, but above, the method detection limit.
       The other concentrations should correspond to the expected
       range of concentrations found in real samples, or should
       define the working range of the detector.
7.3.2  Using injections of 2 to 5 uL of each calibration standard,
       tabulate the peak height or area responses against the
       concentration for bendiocarb and internal standard.
       Calculate response factors (RF) as follows:

                     RF • (AsC1s)/(A1sCs)
       where:
            As  = Response for the compound to be measured.
            A-jS » Response for the internal standard.
            C-jS » Concentration of the internal standard in ug/L.
            Cs  * Concentration of the compound to be measured in
                  ug/L.
                                19

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                If the RF  value over the  working  range  is  constant,  less
                than, 10% relative standard  deviation, the  RF  can  be  assumed
                   *
                to be invariant and the average RF  can  be  used for
                calculations.   Alternatively,  the results  can be  used  to
                plot a calibration curve  of response ratios,  AS/A^S
                against RF.
         7.3.3  The working  calibration curve  or  RF must be verified on each
                working day  by the measurement of one or more calibration
                standards.  If the response for bendiocarb varies from the
                predicted response by more  than _+10%, the test must  be
                repeated using a fresh calibration standard.   Alternatively,
                a new calibration curve must be prepared.
    7.4-  Before using any cleanup procedure, the  analyst must process a
         series of calibration standards  through  the procedure to validate
         elution patterns and the absence of interferences from the
         reagents.
8.  Quality Control
    8.1  Each laboratory using this method  is  required  to operate a formal
         quality control program.  The minimum requirements of this program
         consist of an initial demonstration of laboratory capability and
         the analysis of spiked samples as  a continuing check on
         performance.  The laboratory is  required to maintain performance
         records to define the quality of data that is  generated.
         8.1.1  Before performing any analyses, the analyst must demonstrate
                the ability to generate acceptable accuracy and precision
                                          20

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            with this method.  This ability is  established  as  described
            in Section 8.2.
                *
     8.1.2  In recognition of the rapid advances  occurring  in  chromato-
            graphy, the analyst fs permitted certain  options 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 Section  8.2.
     8.1.3  The laboratory must spike and analyze a minimum of 10% of
            all samples to monitor continuing laboratory performance.
            This procedure is described in Section 8.4.
8.2  To establish the ability to generate acceptable  accuracy  and
     precision, the analyst must perform the following operations.
     8.2.1  Select a representative spike concentration  for each
      *     compound to be measured.  Using stock standards, prepare a
            quality control check sample concentrate  in  methanol 1000
            times more concentrated than the selected concentrations.
     8.2.2  Using a pipet, add 1.00 ml of the check sample  concentrate
            to each of a minimum of four 1000-mL  aliquots of reagent
            water.  A representative wastewater may be used in place of
            the reagent water, but one or more  additional aliquots must
            be analyzed to determine background levels,  and the  spike
            level must exceed twice the background level for the test to
            be valid.  Analyze the aliquots according to the method
            beginning in Section 10.
     8.2.3  Calculate the average percent recovery (R),  and the  standard
            deviation of the percent recovery (s), for the  results.

                                     21

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            Wastewater background corrections must  be made before  R  and
            s caJculations are performed.
     8.2.4  Using the appropriate data from Table 2,  determine  the
            recovery and single operator precision  expected for the
            method, and compare these results to the  values measured in
            Section 8.2.3.  If the data are not comparable, the analyst
            must review potential problem areas and repeat the  test.
8.3  The analyst must calculate method performance  criteria and define
     the performance of the laboratory for each spike concentration  and
     parameter being measured.
     8.3.1  Calculate upper and lower control limits  for method
            performance as follows:
                     Upper Control Limit (UCL) = R  +  3 s
                     Lower Control Limit (LCL) = R  -  3 s
            where R and s are calculated as in Section 8.2.3.   The UCL
            and LCL can be used to construct control  charts (6) that are
            useful in observing trends in performance.
     8.3.2  The laboratory must develop and maintain  separate accuracy
            statements of laboratory performance for  wastewater samples.
            An accuracy statement for the method is defined as  R ^ s.
            The accuracy statement should be developed by the analysis
            of four aliquots of wastewater as described in Section
            8.2.2, followed by the calculation of R and s.  Alternately,
            the analyst may use four wastewater data  points gathered
            through the requirement for continuing  quality control in
                                     22

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            Section 8.4.   The accuracy  statements  should  be  updated
            with,this method.  This  ability  is  established as  described
               *
            regularly. (6)
8.4  The laboratory is required to collect  in duplicate a portion  of
     their samples to monitor spike  recoveries. The frequency of  spiked
     sample analysis must be at least 10% of all samples  or  one sample
     per month, whichever is greater.  One  aliquot of the sample must be
     spiked and analyzed  as described in Section 8.2.   If the  recovery
     for a particular compound does  not fall within the control limits
     for method performance, the results reported  for that compound  in
     all samples  processed as part of the same  set must be qualified  as
     described in Section 13.3.  The laboratory should monitor the
     frequency of data so qualified  to  ensure  that it remains  at or
     below 5%.
8.5  Before processing any samples,  the analyst should demonstrate
     though the analysis  of a 1-liter aliquot  of reagent  water that  all
     glassware and reagents interferences are  under control.  Each time
     a set of samples is  extracted or there is  a change in  reagents,  a
     laboratory reagent blank should be processed  as a safequard against
     laboratory contamination^
8.6  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
                                    23

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         doubt exists  over the  identification  of  a  peak on  the  chromatogram,
         confirmatory  techniques  such  as  liquid chromatography  with  a
                    •
         dissimilar column, must  be used.   Whenever possible, the  laboratory
         should perform analysis  of standard reference materials arrt
         participate in relevant  performance evaluation studies.
9.  Samples Collection. Preservation,  and Handling
    9.1  Grab samples  must be collected in glass  containers.  Conventional
         sampling practices (7) should be followed; however,  the bottle  must
         not be prerinsed with  sample  before collection.   Composite  samples
         should be collected in refrigerated glass  containers in accordance
         with the requirements  of the  program.  Automatic sampling equipment
         must be as free as possible of Tygon  and other potential  sources  of
         contamination.
    9.2  The samples must be iced or refrigerated at 4°C  from the  time of
         collection until extraction.
    9.3  Adjust the pH of the sample to 6 to 8 with IN sodium hydroxide or
         IN sulfuric acid immediately after sampling.
10. Sample Extraction
    10.1 Mark the water meniscus  on the side of  the sample  bottle  for later
         determination of sample  volume.   Pour the  entire sample into a
         2-liter separatory funnel.  Check the pH of the  sample with wide
         range pH paper and adjust to  7 with 1 N  sodium hydroxide  or 1 N
         H2S04.
    10.2 Add 60 ml of  methylene chloride  to the  sample bottle,  seal, and
         shake 30 seconds to rinse the inner walls.  Transfer the  solvent  to
                                          24

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     the separatory funnel  and extract the sample by  shaking  the  funnel
     for 2 mln wath periodic venting to release  excess  pressure.  Allow
                *.
     the organic'layer to separate from the water phase for a minimum  of
     10 min.  If the emulsion interface between  layers  is  more than  one
     third the volume of the solvent layer, the  analyst must  employ
     mechanical techniques  to complete the phase separation.   The
     optimum technique depends upon the sample,  but may include
     stirring, filtration of the emulsion through glass wool,
     centrifugation, or other physical methods.   Collect the  methylene
     chloride extract in a  250 ml Erlenmeyer flask.
10.3 Add a second 60-mL volume of methylene chloride  to the sample
     bottle and repeat the  extraction procedures second time,
     collecting the extract.  Perform a third extraction in the same
     manner and collect the extract.
10.4 Assemble a Kuderna-Danish (K-D) concentrator by  attaching a  10-mL
     concentrator tube to a 250-mL evaporative flask.   Other  concentra-
     tion devices or techniques may be used in place  of the K-D if  the
     requirements of Section 8.2 are met.
10.5 Pour the combined extract through a drying  column  containing about
     10 on of anhydrous sodium sulfate, and collect the extract in  the
     K-D concentrator.  Rinse the Erlenmeyer flask and  column with  20  to
     30 ml of methylene chloride to complete the quantitative transfer.
     Once the flask rinse has passed through the drying column, rinse
     the column with 30 to  40 ml of methylene chloride.
10.6 Add 1 or 2 clean boiling chips to the evaporative  flask  and  attach
     a macro Snyder column.  Prewet the Snyder column by adding
                                    25

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     about  1 ml methylene  chloride to the top.  Place the K-D apparatus
     on  a hot  water bath,  60 to 65°C, so that the concentrator tube is
     partially immersed  in the hot water, and the entire lower rounded
               r
     surface of the flask  is bathed with hot vapor.  Adjust the vertical
     position  of  the  apparatus and the water temperature as required to
     complete  the concentration in 15 to 20 min.  At the proper rate of
     distillation, the balls of the column will actively chatter but the
     chambers  will not flood with condensed solvent.  When the apparent
     volume of liquid reaches  1 ml, remove the K-D apparatus and allow
     it  to  drain.and  cool  for  at  least 10 minutes.
10.7 Remove the macro-Snyder column and  rinse the flask and its lower
     joint  into the concentrator  tube with 1 to 2 ml of methylene
     chloride. A 5-mL syringe  is recommended for this operation.   Add  1
     or  2 clean boiling  chips  and attach a two-ball micro-Snyder column
     to  the concentrator tube.   Prewet the micro-Snyder column with
     methylene chloride  and concentrate  the solvent extract as before.
     When an  apparent volume of  0.5 ml is  reached, or the  solution  stops
     boiling,  remove  the K-D apparatus and allow it to drain and cool
     for 10 minutes.
10.8 Remove the micro-Snyder column and  adjust the volume  of the extract
     to  1.0 ml with methylene  chloride.   Stopper the  concentrator  tube
     and store refrigerated if further processing will not be performed
     immediately.  If the extract is  to  be  stored  longer  than two  days,
     transfer the extract to a screw  capped vial with a Teflon-lined
                                      26

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         cap.   If the sample extract requires  no  further  cleanup,  proceed
         with  solvent exchange to acetonitrile as described beginning  in
         Section  IK5.   If the sample requires cleanup, proceed  to  Section  11
    10.9 Determine the original sample volume  by  refilling the sample  bottle
         to the mark and transferring the water to a 1000-mL graduated
         cylinder.  Record the sample volume to the nearest 5 ml.
11. Cleanup and Separation
    11.1 Cleanup procedures may not be necessary  for a  relatively  clean
         sample matrix.  The cleanup procedure recommended in this method
         has been used for the analysis of various clean  waters  and
         industrial  effluents.  If particular  circumstances demand the use
         of additional  cleanup, the analyst must  demonstrate that  the
         recovery of each compound of interest is no less than 65%..
    11.2 Slurry 10 g of Florisil  in 100 ml of  methylene chloride which *has
         been  saturated with reagent water.  Transfer the slurry to a
         chromatographic column.   Wash the column with  100 ml of
         methylene chloride.  Use a column flow rate of 2 to 2.5 mL/min
         throughout the wash and elution profiles
    11.3 Add the extract from Section 10.8 to  the head  of the column.  Allow
         the solvent to elute from the column  until  the Florisil  is almost
         exposed  to  the air.  Elute the column with 50 mL of methylene
         chloride.  Discard this  fraction.
    11.4 Elute the column with 50 ml of 5% acetone in methylene  chloride.
         Collect  this fraction in a K-D apparatus.   Concentrate  the column
         fraction  to 1  ml as described in  Sections  10.6 and 10.7.
                                         27

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    11.5 Add 10-mL of acetonitrile  to  the  concentrate  along with  1 or  2
         clean  boiling chips.   Attach  a three-ball micro-Snyder column to
               *»
         the concentrator tube.   Prewet the  micro-Snyder  column with
         acetonitrile and concentrate  the  solvent  extract to an apparent
         volume of 1 mL.   Allow the K-D apparatus  to drain and cool for 10
         minutes.
    11.6 Transfer  the liquid  to a 2-mL volumetric  flask and dilute to  the
         mark with acetonitrile.  Mix  thoroughly prior to analysis.   If the
         extracts  will not be analyzed immediately, they  should  be
         transferred to Teflon sealed  screw-cap  vials  and refrigerated.
         Proceed with the liquid chromatographic analysis.
12. Liquid Chromatography
    12.1 Table  1 summarizes the recommended  operating  conditions  for  the
         liquid chromatograph.  Included  in  this table are estimated
         retention times  and  method detection  limits that can  be  achieved by
         this method.  An example of the  separations achieved  by  Column 1
         and Column 2 are shown in  Figures 1 and 2.  Other  columns,
         chromatographic  conditions, or detectors  may  be  used  if  the
         requirements of  Section 8.2 are  met.
    12.2 Calibrate the liquid chromatographic  system daily as  described in
         Section 7.
    12.3 If an  internal standard approach is being used,  the  analyst  must not
         add the internal standard  to  sample extracts  until  immediately before
         injection into the instrument.   Mix thoroughly.
    12.4  Inject 2 to  5 uL of the sample extract by completely filling the
          sample valve  loop.   Record the resulting  peak sizes in area or peak
         height units.
                                      28

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    12.5 The width of the retention time window used to  make  identifications
         should be based upon measurements  of actual  retention time
                    «_
         variations,bf standards over the course of a djy.  Three  times  the
         standard deviation of the retention time for a  compound can  be  used
         to calculate a suggested window size; however,  the experience of
         the analyst should weigh heavily in the interpretation of
         chromatograms.
    12.6 If the response for the peak exceeds the working  range of the
         system, dilute the extract and reanalyze.
    12.7 If the measurement of the peak response is prevented by the
         presence of interferences, further cleanup is required.
13. Calculations
    13.1 Determine the concentration of individual compounds  in the sample.
         13.1.1 If the external  standard calibration procedure is  used,
                calculate the amount of material injected  from the peak
                response using the calibration curve or  calibration factor
                in Section 7.2.2.  The concentration in  the sample can be
                calculated as follows:
                                            (A)(Vt)
                     Concentration, ug/L *
                                            (Vi)(Vs)
                where:
                   A  3 Amount of material  injected, in  nanograms.
                   Vj * Volume of extract injected in uL.   '
                   Y£ =» Volume of total extract in uL.
                   Vs » Volume of water extracted in ml.
                                        29

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         13.1.2 If the internal  standard calibration  procedure was  used,
                calculate the concentration  in  the  sample  using the response
                    *.
                factor (RF)  determined in Section 7.3.2  as follows:
                                              (AS)(IS)
                     Concentration,  ug/L -  (AIS)(RF)(VQ)
                where:
                   As  » Response for the compound  to be measured.
                   A-JS = Response for the internal  standard.
                   Is  = Amount  of internal  standard  added to each
                         extract in  ug.
                   V0  = Volume  of water extracted, in  liters.
    13.2 Report results in micrograms per liter without  correction  for
         recovery data.  When duplicate and  spiked  samples are analyzed,
         report, all data obtained with the sample results.
    13.3 For samples processed as part of a  set where the  laboratory spiked
         sample recovery falls outside of the control limits  in Section 8.3,
         data for the affected compounds must be labeled as suspect.
14. Method Performance
    14.1 The method detection limit  (MDL) is defined  as  the minimum
         concentration of a  substance that can  be measured and reported with
         99% confidence that the value is above zero.(8)  The MDL
         concentrations listed in Table 1 were obtained  using reagent water.
         (1)  Similar results were achieved  using representative
         wastewaters.
    14.2 This method has been tested for linearity  of recovery from spiked
         reagent water and has been  demonstrated to be applicable over the
         concentration range from 10 x MDL to 1000  x  MDL.
                                        30

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14.3 In a single laboratory,  Battelle Columbus Laboratories, using
     spiked wastewater  samples,  the  average  recoveries presented in
     Table 2 were obtained.   Seven replicates of each of two different
     wastewaters were spiked  and analyzed.   The standard deviation of
     the percent recovery  is  also included in Table 2. (1)
                                    31

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                                 REFERENCES
1.  "Development of Methods for Pesticides in Wastewaters," Report for EPA
    Contract 68-03-2956 (In preparation).

2.  ASTM Annual  Book of Standards, Part 31, D3694, "Standard Practice for
    Preparation  of Sample Containers and for Preservation," American Society
    for Testing  and Materials, Philadelphia, PA, p. 679, 1980.

3.  "Carcinogens - Working with Carcinogens," Department of Health, Education,
    and Welfare, Public Health Service, Center for Disease Control, National
    Institute for Occupational Safety and Health, Publication No. 77-206,
    August, 1977.

4.  "OSHA Safety and Health Standards, General Industry," (29 CFR 1910),
    Occupational Safety and Health Administration, OSHA 2206 (Revised,
    January, 1976).

5.  "Safety in Academic Chemistry Laboratories," American Chemical Society
    Publication, Committee on Chemical Safety, 3rd Edition, 1979.

6.  "Handbook for Analytical Quality Control in Water and Wastewater
    Laboratories," EPA-600/4-79-019, U. S. Environmental Protection Agency,
    Environmental Monitoring and Support Laboratory - Cincinnati, Ohio 45268,
    March, 1979.

7.  ASTM Annual  Book of Standards, Part 31, D3370, "Standard Practice for
    Sampling Water," American Society for Testing and Materials, Philadelphia,
    PA, p. 76, 1980.

8.  Glaser,- J. A. et al, "Trace Analysis for Wastewaters," Environmental
    Science and  Technology, 15, 1426 (1981).
                                        32

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      TABLE 1.  CHROMATOGRAPH1C CONDITIONS AND METHOD  DETECTION  LIMITS
                       Retention Time (min.)        Method Detection  Limit
  Parameter            Column 1   Column 2                 (ug/L)


Bendiocarb               9.3        6.0                      1.8
Bendiocarb

Column 1 conditions:  Spherisorb-ODS,  5 micron, 250 x 4.6 mm;  1 mL/min.  flow;
40/60 acetonitrile/water.

Column 2 conditions:  Lichrosorb RP-2, 5 micron, 250 x 4.6 mm;  1 mL/min. flow;
50/50 acetonitrile/water.
                                          33

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           TABLE 2.   SINGLE LABORATORY ACCURACY AND PRECISION(a)



Parameter
Bendiocarb


Average
Percent
Recovery
65
70
Relative
Standard
Deviation,
%

35.6
5.7


Spike
Level
(ug/L)
8
80

Number
of
Analyses
7
7

*
Matrix
Type(b)
1
1
(a)   Column 1 condition-s were used.
(b)   1  = Relevant industrial wastewater.
                                      34

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Ul
                  2.0      4.0       6.0       8.0       10.0       12.0     14.0      16.0      18.0    20.0




                                             Retention Time, Mln.
                             FIGURE 1.   HPLC-UV CHROMATOGRAM OF 10 ng  OF BENDIOCARB  (COLUMN  1)

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         Bendlocarb
                                          14.0
T | r—i -ii \  ' •  •  •  i
 16.0      18.0    20.0
         Retention Time, Mln.
FIGURE 2.   HPLC-UV CHROMATOGRAM OF 600 ug OF BENDIOCARB (COLUMN 2)

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                                    TECHNICAL REPORT DATA
                            (Please read Instructions on the reverse tie fore completing
 1. REPORT NO.
                              2.
                                                            3. RECIPIENT'S ACCESSION NO.
 4. TITLE AND SUBTITLE
                                                             , REPORT DATE
(   Determination  of Bendiocarb in  Industrial  and
   Municipal  Wastewaters
                                                            6. PERFORMING ORGANIZATION COOS
 7. AUTHOH(S)
   J.S. Warner,  T.M. EngeT and P.J.  Mondron
                                                            3. PERFORMING ORGANIZATION REPORT NO,
                                                            10. PROGRAM ELEMENT NO.

                                                               CBEB1C
9. PERFORMING ORGANIZATION NAME AND AOOHSS5
   Battelle Columbus Laboratories
   505 King Avenue
   Columbus, Ohio 43201
                                                            11. CONTRACT/GRANT NO.
                                                               68-03-2956
 12. SPONSORING AGENCY NAME AND ADDRESS
   Environmental Monitoring and Support Laboratory
   Office of Research  and  Development
   U.S. Environmental  Protection Agency
   Cincinnati, Ohio  45268	
                                                            13. TYPE OF REPORT AND PERIOD COVERED
                                                              Final  Report  4/82 - 11/82
                                                            14. SPONSORING AGENCY CODE
                                                               EPA 600/6
 1S. SUPPLEMENTARY NOTES
 16. ABSTRACT
             A oechod was developed  for  the determination  of bendiocarb in
       wastewaters.  The method development program consisted of a literature
       review;  determination of extraction efficiency  for the  compound  from
       water into methylene chloride; development of a deactivated Florisil
       cleanup procedure; and determination of suitable  liquid chromacographic
       analysis conditions.

             The final method was  applied to a relevant industrial wastewater in
       order to determine the precision and accuracy of-the method.  The
       wastewater was spiked with the compound at levels  of 8.0 ug/L and
       80  ug/L.  Recovery for bendiocarb.'at the 3 ug/L  level was 65 * 24
       percent.  Recovery at the  80 ug/L level was 70  ± 4 percent.  The  method
       detection limit (MDL) for  bendiocarb in distilled  'water was 1.3 ug/L.
       The MDL in wastewaters may be higher due to interfering compounds.
 17.
                                 KEY WORDS AND DOCUMENT ANALYSIS
                   DESCRIPTORS
                                               b.lOENTIFIERS/OP6N ENDED TERMS  C.  COSATI 1-icid/CfOUp
 ia. oisrHiauriON STATEMENT
   Distribute  to  Public
                                               19. SECURITY CLASS I IHit Reporti
                                                 Unclassified
21 NO Cf-
     37
                                               20. SECURi TY CLASS i Hit
                                                  Unclassi fied
                                                                           22

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