5379
                DETERMINATION OF 8ENSULIDE IN-"INDUSTRIAL AND MUNICIPAL
                        WASTEWATERS BY LIQUID'CHROMATOGRAPHY

                                                                 001D80101
                                     METHOD 636
     1.   Scope and Application
         1.1  This method covers the determination of bensulide pesticide.  The-
             following parameter can be determined by this  method:
                       Parameters                          CAS No.
                       Bensulide             •             741-58-2
         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.
             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.
         1.3  The method detection limit (MDL, defined in Section 14)
             for bensulide compound 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.
                                        U.S. Environmental Protection Agency
                                        Region V, Library
                                        230 South Dearborn Street
                                        Chicago, Illinois  60604

-------
              1.4   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 Sectionr8.2.
              1.5   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.
         .2.   Summary of Method
              2.1   A measured volume of sample, approximately 1 liter, is solvent ex-
                   tracted with methylene 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 chromato-
                   graphic conditions are described which permit the separation and
                   measurement  of the compounds in the extract by HPLC-UV.l
         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.
                   All 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.
U,S. EnvfronrnerrtaJ Protection

-------
         3.1.1  Glassware must be scrupulously cleaned2.  Clean all  glass-
                'ware as soon as possible after use by thoroughly rinsing with
                the last solvent used in it.  Follow by washing with hot water
                and detergent and thorough rinsing with tap and reagent water.
                Drain dry, and heat in an oven or muffle furnace at 400°C for
                15 to 30 min.  Some thermally stable materials such as
                PCBs may not be eliminated by this treatment.  Thorouqh rinsing
                with acetone and pesticide quality hexane may be substituted  for
                the heating.  After drying and cooling, seal and  store glass-
                ware 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.  The cleanup procedure in Section II can be used to
         overcome these interferences, but unique samples may require
         additional  cleanup approaches to achieve the MOL 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 potentialvhealth hazard.   From this  view-
         point, exposure to these chemicals must be reduced  to the lowest
         possible level  by whatever means  available.  The laboratory is
                              "•         .
         responsible for maintaining a current awareness  file  of OSHA
         regulations regarding the safe handling of the chemicals specified
         1n 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 identified3"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 - Borosilicate or flint glass,
                1-liter or 1-quart volume, fitted with screw caps lined
                with Teflon.  Aluminum foil may be substituted for  Teflon
                if the sample is not corrosive.  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 mi'nimum 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.
          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
v
                      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 - 25-mL, graduated (Kontes
                      K-570050-2525 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-Oanish - 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-Oanish - 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
         400eC for 4 hours or extract in a Soxhlet extractor with methylene
         chloride.                       _"'-'.'-'
    5.4  Water bath,- Heated, capable of'temperature control (+2gC).  The
         bath should be used in a hood.
    5.5  Balance - Analytical, capable of accurately weighing to the
         nearest O.Q001 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,  270 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 phosphate, monobasic,  (ACS)  crystal.
    6.5  IN^Sulfuric Acid, slowly add 2.8 ml of cone. H2S04 (94%) to about
         50 ml of distilled water.  Dilute to 100 ml with distilled water.

-------
6.6  IN. Sodium Hydroxide.  Dissolved 4.0,-g.rams of NaOH in TOO ml of
     distilled water.
6.7  Florisil - PR grade (60/100 mesh).  Purchase activated at 1250 F
     and store in brown glass bottle.  Ta 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 screw cap and cool to room
     temperature.
6.8  Stock standard solutions (1.00 ug/yL) - Stock standard solutions
     can be prepared from pure standard materials or purchased as
     certified solutions.
     6.8.1  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.
     6.8.2  Transfer the stock standard solutions into Teflon-sealed
            screw-cap bottles.  Store at 4°C and protect from light.
            Stock standard solutions should be checked frequently for
            signs of degradation  or evaporation, especially just prior
            to preparing calibration standards from them.
     6.8.3  Stock standard solutions must be replaced after six months
            or sooner if comparison with check standards indicates a
            problem.

-------
7.  Calibration             -
    7.1  Establish liquid chromatographic-operating parameters  equivalent  to
         those indicated 1n 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:
         7.2.1  For each 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 each compound.   Alternatively, the ratio of the
                response to the mass injected,  defined as the calibration
                factor (CF), can be calculated  for each compound 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.

-------
     7.2.3  The working calibration curve "or calibration factor must be
                                      " -'" >
            verified on each working shift by the measurement of one or
            more calibration standards.  If the response for any
            compound 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 for that compound.
7.3  Internal  standard calibration procedure.  To use this approach, the
     analyst must select one or more internal standards similar in
     analytical  behavior to the compounds of interest.   The analyst  must
     further demonstrate 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 concen-
            tration levels for each compound of interest  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 W_ of each calibration  standard,
            tabulate the peak  height or area responses  against  the

-------
            concentration for each compound and internal standard.
            Calculate response factors (RF) for each compound as
            follows:
                          RF « (AsC1s)/(AisCs)
            where:
                 As  - Response for the compound to be measured.
                 A^s = Response for the internal standard.
                 C-fS s Concentration of the internal standard in ug/L.
                 Cs  = Concentration of the compound to be measured in
                       ug/L.
            If the RF value over the working range is constant, less
            than 1(3% 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/Ajs
            against RF.
     7.3.3  The working calibration curve or RF must be verified on each
            working shift by the measurement of one or more calibration
            standards.  If the response for any compound varies from the
            predicted response by more than J^10%, the test must be
            repeated using a fresh calibration standard.  Alternatively,
            a new calibration curve must be prepared for that compound.
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                    • —; -
    ^•MMB«B*HMHMMBmWMM^««WMM           „           - __ jC _
    8.1  Each laboratory using this method is required to operate a formal
         quality control program.  The minimum requirements of this program
         consist of ah 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
                with this method.  This ability is establishd as described
                in Section 8.2.
         8.1.2  In recognition of the rapid  advances occurring in chromatog-
                graphy, the analyst is 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 pi pet, 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.
            Wastewater background corrections must be  made before R and
            s calculations 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^ that are
            useful  in observing trends in performance.

-------
     8.3.2  The^laboratory must devel-op-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
            Section 8.4.  The accuracy statements should be updated
            with this method.  This ability is established as described
            regularly6.
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 sane 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 safeguard  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
         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 and
         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? 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 1N_ 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
         the separatory funnel  and extract the sample by shaking the  funnel
         for 2 min with periodic vent-ing 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  procedure a 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  25-mL
         concentrator tube to a 500-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 era 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 three-ball macro-Snyder column.  Prewet the Snyder column by adding
     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
     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.   If the sample  extract
     requires no further cleanup, proceed with solvent exchange  to
     acetonitrile and chromatographic analysis as described in Sections
     11.5 and 12 respectively.  If the sample requires cleanup,  proceed
     to Section 10.7.
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-0 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
         cap.
    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 76%.

-------
11.2 Slurry 10 g of Florisil 1n 100 Si'of methylene chloride which has
               x      .              -•""•'.
     been saturated with reagent water,  transfer the slurry to a
     chromatographic column (Florisil may be retained with a pluo. of qlass
     wool).  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.
11.5 Add 15 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 -    "          - Tt "  -
    12.1 Table 1 summarizes the recommended operating conditions for the
         liquid chrcmatograph.  Included in'this table are estimated
                      -i         ~ ••         .       -
         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 HPLC 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 the sample extracts until
         immediately before injections into the instrument.   Mix thoroughly.
    12.4 Inject 2 to 5 uL of the sample extract into the sample valve
         loop.  Record the resulting peak sizes in area or peak heights
         units.
    12.5 The width of the retention time window used to make identifications
         should be based upon measurements of actual  retention  time
         variations of standards over the course of a day.   Three times  the
         standard deviation of a retention time for a compound  can be used
         to calculate a suggested window size;  however, the  experience of
         the analyst should weigh heavily 1n 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 =  -———>.

                where:
                   A  * Amount of'material injected, in nanograms.
                   V, = Volume of  extract 'injected in UL.
                   Vt - Volume of  total extract in UL.
                   Vs » Volume of  water extracted in ml.
         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)(I$)
                     Concentration, ug/L -

                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.
                               -j               -•
                   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 3.3,
         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
                                                     Q
         99% confidence that the value is above zero.   The MDL concentra-
         tions listed in Tabla 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 sp'iked
         reagent water and has been demonstrated  to be applicable  over the
         concentration range from 10 x MOL to 1QOO x MDL.
    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

-------
                                 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, J.5_t 1426 (1981).

-------
      TABLE 1.  CHROMATOGRAPHIC.CONDITIONS AND METHOD DETECTION LIMITS
                       Retention Time (min.)        Method Detection  Limit
  Parameter            Column 1Column 2                 (ug/L)
Bensulide               14.1        7.2                      1.6
Bensulide

Column 1 conditions:  Spherisorb-ODS,  5 micron,  250 x 4.6 mm;  1  mL/min. flow;
55/45 acetonitrile/water.

Column 2 conditions:  Lichrosorb RP-2,  5 micron, 250 x 4.6 mm; 1 mL/min.  flow;
60/40 acetonitrile/water.

-------
             TABLE 2.  SINGLE LABORATORY ACCURACY AND  PRECISIONa)
Parameter
Bensul Ide
Average
Percent
Recovery.
86
76
Standard .
Deviation,
18
18
Spike
Level
25
250
Number
of
Analyses
7
7
Matrix
Type(b)
1
i \
  (a)  Column 1 conditions were used.

  (b)  1 » Relevant Industrial wastewater,
U.S. Environmental Protection Agency
Region V, Library
230 South Dearborn Street
Chicago,  Iflinofs   60604

-------
                                                           «        t
                                                          5       j
                                                           =     
-------
                                                                                                                            in
                                                    CO
                                                    c
                                                    | i| ji f-^1 -T""i~"t  V
                                                                                                                                                  §

-------