07/21/2003  10:22 FAX                                                                i)002

     '
                                       906R91103
                                                                               20480
                                           NETHOD  200.3
                         SAMPLE PREPARATION  PROCEDURE  FOR SPECTROCHEMICAL
                 DETERMINATION OF TOTAL RECOVERABLE ELEMENTS IN BIOLOGICAL TISSUES
                                         William  HcQaniel
 >'
                                  Environmental Services Division
                                             Region IV
                               U. S. Environmental Protection Agency
 X

"fc
                                           Revision  1.0
                                            April  1991
                                            Adapted by:
                            ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
                                OFFICE OF RESEARCH AND DEVELOPMENT
                               U.  S.  ENVIRONMENTAL PROTECTION AGENCY
                                      CINCINNATI,  OHIO  45268
            S                                  23

            CO
                                         ENVIRONMENTAL PROTECTION AGENCY
                                         WASHINGTON, D.C.2WSO

-------
_07/21/2003 10:30 PAX
                                                                                  Don/oil
                                        METHOD 200.3

               SAMPLE PREPARATION PROCEDURE FOR SPECTROCHEMICAL DETERMINATION
                      OF TOTAL RECOVERABLE ELEMENTS IN BIOLOGICAL TISSUES
       I-   SCOPE AND APPLICATION

            1.1  This method provides sample preparation procedures for the
                 determination of total  recoverable elements in biological  tissue
                 samples.

            1.2  This method is applicable to the- following elements:

                                            Chemical  Abstract Services
                    Analvte                 Registry Numbers (CASRW

                                                  7429-90-5
                                                  7440-36-0
                                                  7440-38-2
                                                  7440-39-3
                                                  7440-41-7
                                                  7440-43-9
                                                  7440-70-2
                                                  7440-47-3
                                                  7440-48-4
                                                  7440-50-8
                                                  7439-89-6
                                                  7439-92-1
                                                  7439-93-2
                                                  7439-95-4
                                                  7439-96-5
                                                  7439-97-6
                                                  7439-98-7
                                                  7440-02-0
                                                  7723-14-0
                                                  7440-09-7
                                                  7782-49-2
                                                  7440-22-4
                                                  7440-23-5
                                                  7440-24-6
                                                  7440-28-0
                                                  7440-29-1
                                                  7440-61-1
                                                  7440-62-2
                                                  7440-66-6

            1.3  Samples  prepared by this  method  can  be  analyzed by inductively
                 coupled  plasma-atomic emission spectrometry (ICP-AES)  Method  200.7,
                 "Determination of Metals  and Trace  Elements by Inductively Coupled
                 Plasma-Atomic Emission  Spectrometry," inductively  coupled  plasma-
                 mass spectrometry (ICP-MS)  Method  200.8,  "Determination  of Metals
Aluminum
Antimony
Arsenic
Barium
Beryl 1 i urn
Cadmium
Calcium
Chromium
Cobalt
Copper
Iron
Lead
Lithium
Magnesium
Manganese
Mercury
Molybdenum
Nickel
Phosphorus
Potassium
Selenium
Silver
Sodium
Strontium
Thallium
Thorium
Uranium
Vanadium
Zinc
(Al)
(Sb)
(As)
(Ba)
(Be)
(Cd)
(Ca)
(Cr)
(Co)
(Cu)
(Fe)
(Pb)
(Li)
(Mg)
(Mn)
(HG)
(Mo)
(Hi)
(P)
(K)
(Se)
(Ag)
(Na)
(Sr)

(Th)
(U)
(V)
(In)
                                             24

-------
07/21/2003 10:22 FAX
                                                                                  g]003
                 and Trace Elements by Inductively Coupled Plasma-Mass Spectrometry,"
                 and stabilized temperature platform graphite furnace atomic
                 absorption (STGFAA), Method 200.9, "Determination of Trace Elements
                 by Stabilized Temperature Graphite Furnace Atomic Absorption
                 Spectrometry".  See analytical methods mentioned for selection of
                 the appropriate method for determination of a specific analyte.

       2.   SUMMARY OF METHOD

            2.1  Up to 5 g of a frozen tissue sample is transferred to a 125 mL
                 flask.  The tissue is digested with nitric acid, hydrogen peroxide
                 and heat.  This digestion results in a clear solution that 1s then
                 analyzed by mass or atomic Spectrometry methods.  The determined
                 metal concentration is reported in microgram/gram (vg/g) wet tissue
                 weight.

       3.   DEFINITIONS

            3.1  TOTAL RECOVERABLE - The concentration of analyte determined to be in
                 either a solid sample or an unfiltered aqueous sample following
  iff              treatment by refluxing with hot dilute mineral acid.
  fo
  fe        3.2  LABORATORY REAGENT BLANK (LRB) - A solution of reagents that is
  [              treated exactly as a sample including exposure to all glassware and
  [.f             equipment that are used with other samples.  The LRB is used to
  $             determine if method analytes or other interferences are present in
  ji!             the laboratory environment, reagents, or apparatus.
   •---•Jt

  K   4-   INTERFERENCES
  m-
            4.1  Chromium contamination of biological  samples from the use of
                 stainless steel has been reported.   Use  of special  cutting
   £j             implements and dissecting board made from materials that are not of
   *y             interest is recommended.  Knife blades made of titanium with Teflon
   -;             handles have been successfully used.

            4.2  In sample preparation, contamination is of prime concern.   The work
                 area, including bench top and fume hood,  should be periodically
                 cleaned in order to eliminate environmental contamination.

            4.3  Chemical interferences are matrix dependent and cannot be predicted.

       5.   SAFETY

            5.1  All  personnel  handling environmental  samples known to contain or to
                 have been in contact with human waste should be immunized against
                 known disease causative agents.

            5.2  Material safety data sheets for all  chemical  reagents should be
                 available to and understood by all personnel  using this method.
                 Concentrated nitric and hydrochloric acids are moderately toxic and
                 extremely irritating to skin and mucus membranes.   Hydrogen peroxide

                                             25

-------
07/21/2003 10:30 FAX                                                               ®OJO/011
                 Is a strong oxidizing reagent.  Use these reagents in a hood
                 whenever possible and if eye or skin contact occurs, flush with
                 large volumes of water.  Always wear safety glasses or a shield for
                 eye protection when working with these reagents.

       6.   APPARATUS AND EQUIPMENT

            6.1  LABWARE - For determination of trace levels of elements,
                 contamination and loss are of prime consideration.  Potential
                 contamination sources include improperly cleaned laboratory
                 apparatus and general contamination within the laboratory
                 environment from dust, etc.  A clean laboratory work area designated
                 for trace element sample handling must be used.  Sample containers
                 can introduce positive and negative errors in the determination of
                 trace elements by contributing contaminants through surface
                 desorption/leaching, or depleting element concentrations through
                 adsorption processes.  All reusable labware (glass, quartz,
                 polyethylene, Teflon, etc.), including the sample container, should
                 be cleaned prior to use or shown to be contaminant free.  Labware
                 should be soaked overnight and thoroughly washed with laboratory-
                 grade detergent and water, rinsed with water, and soaked for four
                 hours in a mixture of dilute nitric and hydrochloric acid (1+2+9),
                 followed by rinsing with water, ASTM type I water and oven drying.

                 NOTE:  Chromic acid must not be used for cleaning glassware.

                 6.1.1  Glassware - Volumetric flasks, graduated cylinders and 125-mL
                        Erlenmeyer flasks.

                 6.1.2  Assorted calibrated pipettes,

                 6.1.3  Wash bottle - One piece stem, Teflon FEP bottle with Tefzel
                        ETFE screw closure, 125-mL capacity,

            6.2  SAMPLE PROCESSING EQUIPMENT

                 6.2.1  Balance - Analytical, capable of accurately weighing to
                        0.1 mg,

                 6.2.2  Hot Plate - (Corning PC100 or equivalent).  An oscillating
                        hot plate will aid in sample digestion.

            6.3  TISSUE DISSECTING EQUIPMENT

                 6.3.1. Dissecting Board: Polyethylene or other inert, nonmetallic
                        material, any non-wetting, easy-to-clean or disposable
                        surface is suitable.  Adhesive backed Teflon or plastic
                        film may be convenient to use.

                 6.3.2  Forceps: Plastic, Teflon or Teflon coated.

-------
                                                                                  i!004
07/21/2003 10:22 FAA
                  6.3.3  Surgical Blades: Disposable stainless steel with stainless
                         steel or plastic handle (Sect. 4.1).

                  6.3.4  Scissors:  Stainless steel.

                  6.3.5  Plastic bags with watertight seal, metal free.

                  6.3.6  Label tape:  Self-adhesive, vinyl coated marking tape,
                         solvent resistant, usable for temperatures from +121'C
                         to -23°C.

                  6.3.7  Polyvinyl chloride or rubber gloves, talc-free.

        7.   REAGENTS AND CONSUMABLE MATERIALS

             7.1  Reagents may contain elemental impurities which might affect
                  analytical data.  High-purity reagents should be used whenever
                  possible.  All acids used for this method must be of ultra high-
                  purity grade.

                  7.1.1  Nitric acid, concentrated (sp.gr. 1.41).

                  7.1.2  Hydrochloric acid, concentrated (sp.gr. 1.19).

                  7.1.3  Hydrogen peroxide (30%)

             7.2  WATER - For all sample preparation and dilutions, ASTM type I water
                  (ASTM D1193) is required.  Suitable water may be prepared by passing
                  distilled water through a mixed bed of anion and cation exchange
                  resins.

        8.   SAMPLE COLLECTION. PRESERVATION AND STORAGE

             8.1  Appropriate individual tissue samples should be taken soon after
                  collection and must be taken prior to freezing2.   If  dissection of
                  the tissue cannot be performed immediately after collection, it
                  should be placed in a plastic bag (Sect. 6.3.5), sealed and placed
                  on ice or refrigerated at approximately 4°C.

             8.2  Prior to dissection, the tissue should be rinsed with metal-free
                  water and blotted dry.  Dissection should be performed within
                  24 hours of collection.  Each individual tissue sample should also
                  be rinsed with metal-free water, blotted dry, and frozen at <-20°C
                  (dry ice).

             8.3  Tissue samples of up to 5 g should be taken using a special
                  implement (Sect. 4.1) and handled with plastic forceps
                  (Sect. 6.3.2)M.

             8.4  A maximum holding time for frozen samples has not been determined.
                                              27

-------
07/21/2003 10:29 FAX
                                                                                  @] 009/011
        9.    CALIBRATION AND STANDARDIZATION

             9.1  Not applicable.   Follow  instructions given  in  the  analytical method
                 selected.

        10.   QUALITY CONTROL

             10.1 Each  laboratory  determining  total  recoverable  elements  is  required
                 to operate a  formal quality  control  (QC) program.  The  minimum
                 requirements  of  a QC  program consist of an  Initial demonstration  of
                 laboratory capability and analysis of  laboratory reagent blanks and
                 fortified blanks and  samples as  a  continuing check on performance.
                 The laboratory is required to maintain performance records that
                 define the quality of data generated.

             10.2 Specific  instructions on accomplishing the  described aspects of the
                 QC program are discussed in  the  analytical  methods.

        11.   PROCEDURE

             11.1 Sample Preparation -  Place up to a 5 g subsample of frozen tissue
                 into  a 125-ml erlenmeyer flask.  Any sample spiking solutions  should
                 be added  at this time and allowed  to be In  contact with the sample
                 prior to  addition of  acid.

             11.2 Add 10 ml of  concentrated nitric acid  and warm on  a hot plate  until
                 the tissue is solubilized.   Gentle swirling the samples or use of an
                 oscillating hot  plate will aid in  this process.

             11.3 Increase  temperature  to  near boiling until  the solution begins to
                 turn  brown.   Cool sample, add an additional 5  ml of concentrated
                 nitric acid and  return to the hot  plate until  the  solution once
                 again begins  to  turn  brown.

             11.4 Cool  sample,  add an additional 2 mL of concentrated nitric acid,
                 return to the hot plate  and  reduce the volume  to 5-10 ml.   Cool
                 sample, add 2 mL of 30%  hydrogen peroxide,  return  sample to the hot
                 plate and reduce the  volume  to 5-10 ml.

             11.5 Repeat Sect.  11.4 until  the  solution is clear  or until  a total of
                 10 mL of  peroxide has been added.  NOTE: A  laboratory reagent  blank
                 is especially critical in this procedure because the procedure
                 concentrates  any reagent contaminants.

             11.6 Cool  the  sample,  add  2 mL of concentrated hydrochloric  acid, return
                 to the hot plate and  reduce  the  volume to 5 mL.

             11.7 Allow the sample to cool and quantitatively transfer to a  100-mL
                 volumetric flask.  Dilute to volume with ASTM  type I water, mix,  and
                 allow any insoluble material  to  separate.   The sample is now ready
                 for analysis  by  either IC"P-AES or  ST6FAA.   For analysis by ICP-MS an
                 additional dilution (1+4) is required,

                                              28

-------
                                                                                  @]005
07/21/2003 10:23 FAX
              11.8  Sample  Analysis  -  Use one of the  analytical  methods  listed in
                   Sect. 1.3.

         12.   CALCULATIONS

              12.1  Not  applicable.  Discussed in analytical  methods listed in Sect.
                   1.3.

         13.   PRECISION AND ACCURACY

              13.1  Not  applicable.  Available data Included  in  analytical  methods
                   listed  in Sect.  1.3.

         14.   REFERENCES

              1.    Versleck, J.,  and  F.  Barbier, "Sample Contamination  as  A Source of
                   Error 1n Trace-Element Analysis of Biological  Samples," Talanta.
                   Vol. 29, pp.  973-984,  1982.

              2.    Ney, J. J.,  and  M.  G.  Martin, "Influences of Prefreezlng on Heavy
                   Metal Concentrations  in Bluegill  Sunfish," Water Res..  Vol. 19,
                   No.  7,  pp.  905-907,  1985.

              3.    "The Pilot  National  Environmental  Specimen Bank," NBS Special
                   Publication 656, U.  S. Department of Commerce,  August,  1983.

              4.    Koirtyohann,  S.  R.,  and H.  C. Hopps,  "Sample Selection, Collection,
                   Preservation and Storage for Data Bank on Trace Elements in Human
                   Tissue," Federation Proceedings,  Vol.  40, No.  8, June,  1981,
                                              29

-------