UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
• SUBJF.CT: tissue Analysis for use in FY78 Regional
         Toxic Data Collection

FROM:    Robert Crim, chief
         Monitoring Branch (WH-553)

TO      Surveillance and Analysis
         .Division Directors
DATE:  2. 5
              The methods included in the enclosed "Sampling and Analysis
          Procedures for Screening of Fisli for Priority Pollutants" should
          pe used for tissue analyses in your FY78 Toxic Data Collection
          Activity.  This program is discussed in Eckardt C. Beck's
          memorandum of June 9, 1977, subject:  FY78 Regional Toxic Data
          Collection.
              Technical questions or comments on these methods should be
         addressed to the Cincinnati Laboratory.

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         UNITED STATES ENVIRONMENTAL PROTECTION AGENCY

                             CINCINNATI. OHIO 40268
                                                ENVIRONMENTAL MONITORING AND
                                               SUPPORT LABORATORY - CINCINNATI

SUBJECT:  Fish Tissue Analysis                 DATE:   August 23,  1977

FROM   :  Dwight G.  Ballinger,
          Environmental  Monitoring and Support
            Laboratory - Cincinnati

TO     :  Surveillance and Analysis Division Directors

               Attached is a copy of "Sampling and Analysis  Procedures
          for Screening of Fish for Priority Pollutants"  for your use
          in collecting and analyzing fish samples in response to the
          State/Regional monitoring operating guidance.   As  with  the
          recent document sent to you on sediment analysis (see memo
          of August 12, 1977), this document should also  be  considered
          as interim guidance.  While we consider it the  Agency's best
          source for the analysis of fish samples for priority pollutants,
          we recognize that changes may be warranted as  a result  of your
          regional experiences on a variety of collected  fish samples.
          As noted in the foreword, we encourage your careful review of
          this protocol and your identification of any problems that may
          arise.

          •Attachment
            As stated above

          cc:  Charles Brunot (w/attachment)

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     SAMPLING Ai-iD ANALYSIS rRCClilHJRES FOR'

   SCREENING OF FISH FOR PRIORITY POLLUTANTS
     U. S. ENVIRONMENTAL  PROTECTION  AGENCY

Environmental Monitoring  and  Support Laboratory
            Cincinnati, Ohio   45263
                August  23,  1977

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                             Foreword






     These guidelines for sample preparation and analysis of fish




have been prepared by the staff of the Environmental Monitoring and



Sqpport Laboratory - Cincinnati, at the request of the Monitoring and




Data Support Division, Office of Water and Hazardous Wastes, with the




cooperation of many EPA Regional Laboratories,  the Food and Drug Admin-




istration, the Southeast Water Research Laboratory,  the Environmental




Research Laboratory - Duluth, and the National  Institute for Occupational




Safety and Health.




     The procedures represent the current state-of-the-art, but improve-



me,its are anticipated as more experience is obtained.  Users of these




methods are encouraged to identify problems to  assist in updating the




test procedures by contacting the Environmental Monitoring and Support




Laboratory, EPA, Cincinnati, Ohio  45268.

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                             CONTENTS

SAMPLE HANDLING .,	
ANALYSIS OF FISH FOR CHLORINATED PESTICIDES AND POI.YCHLORI.NATED
BfPHENYLS	3


ANALYSIS OF FISH FOR GENERAL ORGANICS BY SOLVENT EXTRACTION ......   8

ANALYSIS OF FISH FOR VOLATILE ORGANICS BY HEAD SPACE ANALYSES ....  14


ANALYSIS OF FISH FOR CYANIDE	Vl9


ANALYSIS OF FISH FOR PHENOL	22


ANALYSIS OF FISH FOR MERCURY	  .  26
                                  t

ANALYSIS OF FISH FOR METALS	30
                                                              •
REFERENCES	40

-------
                          LIST OF TABLES

Table I.      Priority Tollutants Analyzed by Pesticide and
              PCB Procedures	7

Table II.     Base - Neutral F.xtractables	11

Table IJI.    Acid Extractables	13

Taole IV.     Characteristic Ions of Volatile Organics	17

Table V.      Method References for Metals	36

-------
               Sampling and Analysis Procedures for
             Screening of. Fish for Priority Pollutants
                          Sample Handling

1.   Collection

          Separate analyses for all priority pollutants are done '

     on the same sample of fish.  A minimum of 2SO grams is required

     for the total protocol.  Small fish must be combined by species

     to obtain this minimum weight.

2.   Preservation

          Field sampling requires an ice chest packed with dry ice.

     Collected samples are wrapped in aluminum foil, labeled with freezer

     tape, and transported in the chest.  The dry ice must be replaced as

     needed until subsamples are obtained for purgeoble organics.

3.   Processing

          To prepare samples for analytical pretrea'cments, wrap and

     weigh each fish.  Combine small fish by site and species until a

     minimum combined weight of 250 grains is obtained.  Chop the sample

     into one-inch chunks using a sharp knife and mallet.  Grind the sample

     witfr a large commercial meat grinder that has been precooled by grinding

     dry ice.  Thoroughly mix the ground material.  Regrind and mix material
       *
     two additional times.  Clean out any material remaining in the grinder;

     add this to the sample and mix well.  Weigh five 10.0 gram portions
                                                             *
 •
     of the sample into separate 125 vials.  Using a crimper, quickly
     U. S. Environmental Protection Agency
     Environmental Monitoring and Support Laboratory
     Cincinnati, Ohio  45263

-------
                                - "2 -  .





     and tightly secure a septum to each bottle with a seal.   Store these




     sample aliquots in a freezer until ready for Volatile Organics analyses.




     Transfer remaining fish samples to a glass container and store in a




     freezer for later subsampling and analysis.




4, -  Special Equipment and Materials




     4.-1  Knife, heavy blade (or meat cleaver)




     4.2  Mallet, plastic faces, 2-3 pounds.




     4.3  Electric meat grinder, 1/2 HP.




     4.4  Ice chest.




     4.5  Dry ice.




     .4.6  Aluminum foil.




     4.7  Freezer tape, for labels.




     4.8  Freezer




     4.9  Vials, 125 ml "Hypo-Vials" (Pierce Chemical Co., #12995,) or




          equivalent.




     4.JO Septa, "Tuf-Bond" (Pierce #12720,) or equivalent.




     4.^1 Seals, aluminum, (Pierce #13214,) or equivalent.




     4.\2 Crimper, hand,  (Pierce #13212,) or equivalent.

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                                - 3 -
Analysis of Fish for Chlorinated Pesticides and Polychlorinntcd Biphenyls







1.   Scope



     1.1  The chlorinated pesticides and polychlorinatcd biphcnyls (PCBs)



          listed in Table I are extracted from fish with either of the two



          procedures described below.  Method A employs a blender, while a



          Tissumizer or equivalent is required for Method B.  Both proce-



          dures result in an extract that can be incorporated directly into


                                                    1        2
          current EPA test procedures for pesticides  or PCBs  as cited



          in the Federal Register .



2.   Special Apparatus and Materials



   .  2.1  Method A only



          2.1.1     Blender, high speed - Waring Blender, Gourdes, Oir.ni-Mixer,



                    or equivalent.  Explosion proof model recommended.  Quart



                    container is suitable size for routine use.



          2.1.2     Buchner funnel - Porcelain, 12 cm.



          2.1.3     Filter paper - 110 mm "Sharkskin" circles.



          2.1.4     Flask, vacuum filtration - 500 ml.



     2.2  Method B only



          2.2.1     Tissumizer SDT-182EN (available from Tckmar Company,



                    P. 0. Box 37202, Cincinnati, Ohio  45222) or  equivalent.



          2.2.2     Centrifuge - capable of handling  100 ml centrifuge tubes.



     2.3  Method A ^ ^



          2.3.1     Kuderna - Danish concentrator  - 500 ml, with  10 ml

-------
                               -4-
                    graduatcd  receiver  and  3-ball Snyder  column.


          2.3.2     Chromatographic  column  - pyrcx, 20 mm ID x  approximately


                    400 mm long,  with coarse fritted plate on bottom.


3.   Procedures


     3.1  METHOD A:   Weigh a 25-50.g portion of  frozen, ground  fish  and add


          to a high  speed blender.   Add 100 g  anhydrous Na^SO.  to combine with


          the water  present and .to  disintegrate  the sample.  Alternately,


          blend and  mix with a spatula  until the sample and sodium sulfate


          are well mixed .  Scrape  down the sides of the  blender jar and break


          up the caked material with the spatula.  Add  150 ml  of hexane and


          blend at high speed for 2 min.

                                                                  H
          3.1.1     Decant the hexane  supernatant through a 12 cm Buchner


                    filter with two shark-skin papers,  into a  500 ml suction


                    flask.  Scrape  down the sides of the  blender jar and break


                    up the caked material with the spatula.  Re-extract  the


                    residue in the  blender jar with two 100 -J. portions  of


                    hexane, blending 3  min. each time.   (After one min.


                    blending, stop  the  blender,  scrape  the material from the


                    sides of the blender jar  and break  up the caked material


                    with the spatula;  continue blending for one  min.) Scrape


                    down the sides  of the blender jar  and break  up  the caked


                    material between extractions.

                                                                 tt
          3.1.2     Decant the hexane supernatants  through the   Buchncr and


                    combine with the first extract.  After the  last blending,
                                                                      I!
                    transfer the residue from the blender jar  to the  Buchner,

                                                                 ti
                    rinsing the blender jar and  material  in t^ie  Buchner with

-------
                           - 5 -


               three 25-50 ml  portions of hcxanc.   Immediately after
                                                        ii
               the last rinse, prcvss the residue in the Buchncr with

               the bottom of a clean beaker to force out the  remaining

               hcxane.

     3.1.3     Pour the combined extracts and rinses through  a column of

               anhydrous Na-,SO., 20 mm x 100 mm long, and collect the

               eluate in a 500 ml Kuderna-Danish concentrator.  Wash- the

               flask and then the column with small portions  of hexane

               and concentrate the extract below 10 ml.

3.2  METHOD B:  Weigh 20.0 grams of frozen, ground fish in a  100 ml

     centrifuge tube.  Add 20 ml of hexane and insert the Tissumizer

     into the sample.  Turn on the Tissumizer and disperse the fish  in

     the solvent for 1 minute.  Centrifuge and decant the solvent

     through a column of anhydrous Na_SO., 20 mm x 100 mm long, and

     collect the eluate in a 500.ml Kuderna-Danish concentrator.  Repeat

     the dispersion twice more using a 20 ml aliquot each time, com-

     bining all dried portions of solvent in the concentrator.  Rinse

     the Tissumizer and the column with small portions of hexane and

     concentrate the extract below 10 ml.

3.3  Cleanup and Analysis:  Unless prior experience would indicate the

     fish species  fat content  is low  (less than 3g/cxtract),  the hexane-

     acetonitrile  clean-up procedures described in the reference methods

     should be followed.  In all cases, Florisil column chromatography

     should be used to clean up  the extracts before  gas chromatography.  ''

     £n electron capture detector is used for final  measurement, and

     results are calculated in micrograms per kilogram.   Identifications

-------
                                - 6 -
          may be confirmed by GC/MS techniques  as  described  in  the  analytical


                                  (41
          protocol for wastewaters    .



     3.4  Quality Control:  Standard quality assurance  protocols  should  be



          employed, including blanks,  duplicates,  and dosed  samples as described



          in the "Analytical Quality Control Handbook"^  .   Dose  fish



          sample aliquots by injecting minimum  amounts  (<20  yl  total)



          of concentrated pesticide or PCB solutions  into the  solid sub-



          sample 10-15 minutes before extraction.



4.   Reporting of Data



     4.1  Report results in ug/kg on a wet tissue  basis.   Report  all quality



          control (QC) data along with the analytical results  for the  samples.

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                                 - .7  -
              TABLE 1 PRIORITY POLLUTANTS ANALYZED BY
                   PESTICIDE AND PCS PROCEDURES
Pesticides

Aldrin

a-BHC

b-BHC

d-BHC

g-BHC

Chlordane

ODD

DDE

DDT

Dieldrin

a-Endosulfai

b-Endosulfan

Endosulfan sulfate

Endrin

Endrin aldehyde

Heptachlor

Heptachlor epoxide

Toxaphene
PCBs

Aroclor 1016

Aroclor 1221

Aroclor 1232

Aroclor 1242

Aroclor 1248

Aroclor 1254

Aroclor 1260

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                                - 8 -
    Analysis of Fish for General  Organic?  by Solvent  Extraction






1.   Scope




     1.1  Thi"s method is designed to determine those  "unambiguous priority



                                                        f4)
          pollutants" associated  with the  Consent Decree    that are solvent



          extractable and amenable to gas  chromatography.   These compounds




          are listed in Tables II and III  of this section.   It is a gas chroma-



          tographic-mass spectrometric method intended for  qualitative and



          semi-quantitative determination  of these compounds.   While this



          approach has not been sufficiently tested through extensive experi-



          mentation, it is based  on laboratory experience  and is presently




          our best analytical approach for these organic materials in fish.



2.   Special Apparatus and Materials

                                                              \


     2.1  Tissumizer SDT-182EN (available  from Tekmar Company, P. 0. Box



          37202, Cincinnati, Ohio  45222)  or equivalent.




     2.2  Centrifuge - capable of handling 100 ml centrifuge tubes.




     2.3  Separatory Funnels - 2  liter with Teflon stopcock.



     2.4  Organic Free Water - prepared by passing distilled water through an



          activated carbon column.




3.  •Procedure



     3.1  Weigh 20.0 grains of ground, homogeneous fish in  a 100 ml




          centrifuge tube.  Add 20 ml acetonitrile and insert the Tissumizer



          into the sample.  Turn  on Tissumizer and disperse the fish into



          the solvent for 1 minute.  Centrifuge and decant the solvent into

-------
                           -9-
     a 2 liter separator/ funnel which contains 1300 ml  of a 2




     percent aqueous solution of sodium sulfate.   Repeat the dis-



     persion twice using a 20 ml aliquot each time and combine the




     acetpnitrile in the separator/ funnel.




     CAUTION^  The dispersion should be carried out in a fume hood to
     avoid exposure to acetonitrile.




3.2  Adjust the pH of the sodium sulfate acetonitrile solution with 6N NaOi-




     to pH 11 or greater.  Use multirange pH paper for the measurement.   E>




     tract the aqueous acetonitrile solution with 60 ml hexane.  Shake the




     separator/ funnel for two minutes.  Drain the aqueous layer into a 2




     liter Erlenmeyer flask and pour the hexane extract through a short




     column of prerinsed anhydrous sodium sulfate.  Collect the dried




     extract in a 500 ml Kuderna-Danish (K-D) flask fitted with a 10 ml




     ampul.  Repeat the extraction and drying steps twice combining the ex




     tracts.  Evaporate the extract to 5-10 ml in a 500 ml K-D apparatus




     fitted with a 3-ball Snyder column and a 10 ml calibrated receiver




     tube.  Allow the K-D to cool to room temperature.  Remove the receive




     and adjust the volume to 10 ml.  Analyze by GC/MS.  If additional




     •sensitivity is required, add fresh boiling chips, attach a two-ball




     micro-Snyder column, and carefully evaporate to 1.0 ml or when




     active distillation ceases.




3.3  Return the aqueous acetonitrile solution to the separatory funnel




     and adjust the pH with 6N HC1 to pH 2 or less.  Extract three




     times with 60 ml hexane each time.  Dry and concentrate as described




     above.  Analyze by GC/MS.

-------
                                - -10 --
          NOTE:   Should the partition used in this  procedure  not  sufficiently




          remove the lipid material,  gel  permeation may  be  employed.   However,




          special expensive equipment is  necessary  for  this procedure    .




     3.4  Quality Control:  Standard  quality assurance  protocols  should  be




          employed,  including blanks, duplicates and dosed  samples  as  described




          in the "Analytical Quality  Control Handbook"    .  Dosing  can be ac-




          complished by injecting 1-20 ul of a standard  solution,  into the




          homogenized tissue contained in a centrifuge  tube.




5.   Reporting of Data




     4.1  Report results in yg/kg on  a wet tissue basis.  Report  all quality




          control (QC) data along with the analytical results for the  samples.

-------
                                                .J.J.
  Ccrr.pc-jr.c Narr.s

'—chloropher.ol
: hsr.ol
;, •-.-Cic'.-.lorophor.ol
I-r.i~rccr.c-r.ol
i-chioro-rn-c-Gsol
1,4, 6-trichlcrcphenol
2.. -;-di-otr.ylphenol
         (2-nitrooher.ol)
 eut-sratcd anthracene (dlO)
0
0
0
1
1
1
• 1
1
1
1
1
0) 1
.63
.66
.96
.00
.05
.14
.32
.34
.42
.43
.64
.68
Limit of
Detection
   fng)

  100 •
  100
  100
  100
  100
  100
  100
  2 pg
  2 yg
  100
  100   .
   40
Characteristic
El ions (Rel. Int.)
128 (100) , .64 (54), 130(31)
94 (100) , 65(17) , 66(19)
162 (100) , 164(58) , 98(61)
133 (ICO)', 65 (35) , 109 (8)
142(100) , 107(80)', 144 (32)
196(100), 193(92), 200(26)
122 (100) , 107(90) , ,121 (55)
184 (100) , 63 (59) , 154 (53)
198 (ICO) , 182(35) , 77 (28)
65(100) , 139 (45) , 109 (72)
266(100), 264(62), 268(63)
188(100), 94(19-)', 80(13)
CI ions
(Methane)
129
95,
163
140
143
197
123
135
199
140
257
189
/ 1-31,
123,
,. 1G5,
/ 168,
, 171,
, 199,
, 131,
, 213,
, 227 ,
/ ies,
, 255,
, 217
1
12
^,
J_
7
.1
_
s
^_
_
__

   ;olu.T.n:   6'  clciss, 2  ITCT i.d.
             Tenax GC
                °
    -  60/80 ^.
- 300°  @  3°/nin.
            180
            He  •?  3C nil/min

-------
Comound NTar.e
(hexachloro-
   benzene)
V
/ — '
• •
- ;.;
"V
is
.-.
?:•:
r •"
.' ^
— ' "'I
- •«-
" r
- -
zc
-*• w
T *""*
— V-
t '^
•^
t •'-
t '-
-«
zx
— ^J

-t
-« * . .
Z- o
J.U
"r
i-
Z:*1
-cich lorcbenzene
-dichlorobenzene
achloroethane
-r.icrlorcber.zene
( 2-chlorciscpropyl)
-'- * N O v-
c.r!-.lor obutadiene
, -, - tr icnlcrobenzene
( "'-cu "; o "oethvl ) ether
r. ch lor ccycl open tadiene
"DLc". ro.r.e

hlcrcr. cphthalene
:-.^"'hthylene
. . -^. ^- * . w • ^ ^ : . i^.
::I-.cron5
crc-r.s
-dinitro toluene
-ci phony Ihydrazine
_ ^ ^' T-» ^ 4- .- A^ J- — . T * T ^ *-v p
_•.—-. .^ U — O *-,wiL*U-i*O
ic.voscdiphenylanune
achlorcbensene
rc.-cphenyl phenyl ether'-
_,_.,i.;-,v-£rj-%
>..vftcer.e
•3 1 h y 1 ~ h t ha 1 a ta
thy ip^. thai axe
ci'cir.tier.e
cnr:
n-butylphthalate
2 id ins
' 0.35
0.36
0.38
0.39

0.47
.0.55
Or- ^
. 55
0.57
0 . 61
0.64
0.64
n £fl
VJ . O O
0.76 '
0.83
0.86
0.07
0.91
0.93
0.96
0.93
0.99
1.00
1.01
1 .09
1.09 •
1.10
1.15
1.23
1.3Q
1.31
1.38
    bir.sylphthalate
    1.46
Limit of
Detection
   (ng)

   40.
   40
   40
  . 40

   40
   40
   40
   40
   40
   40
   40
   40
   40
   40
   40
   40
  . 40
   40
   40*
   40
   40*
   40
   40 '
   40
   40
   40
   40
   40
   40.
   40
   40*
   40
     Characteristic
    El ions  (Rel.  Int.)

146(100), 148(64),  113(12)
146(100),- 148 (64) ,  113(11)
117(100), 199(61),  201(99)
146(100), 140(64),  113(11)
45(100) ,
225 (100,
74 (100),
128 (100)
93 (100) ,
237(100)
77 (100),
93(100) ,
162(ICO)
152(100)
154 (100)
82(100) ,
166 (ICO)
165(100)
77(100) ,
165(100)
169(100)
284 (100)
248 (100)
178 (100)
170 (100)
163(100)
149(100)
202(100)
202(100)
149(100)
184 (100).
149(100)
                                                                 77(19), 79(12)
                                                                 223(63), 227(65)
                                                                 109 (CO) , 145 (52)
                                                                , 127(10), 129(11)
                                                                 63(99), 95(31)
                                                                , 235(63), 272(12)
                                                                 123(50), 65(15)
                                                                 95(32), 123(21)
                                                                , 164 (32) , 127(31)
                                                                , 153(16), 151(17)
                                                                , 153(95), 152(53)
                                                                 95(14) , 138 (10)
                                                                , 165(80), 167(14)
                                                                , 63(72) , 121(23)
                                                                 93(58), 105(28)
                                                                , 63(72), 121(23)
                                                                  168(71),
                                                                  142(30),
                                                                  250 (99) ,
                                                                  179(16)-,
                                                                  179(16),
                                                                  164(10) ,
                                                                  178(25) ,
                                                                  101(23) ,
                                                                  101(26),
                                                                  150(27) -,
                                                   167(50)
                                                   249 (24)
                                                   141(45)
                                                   176(15)
                                                   176(15)
                                                   194(11)
                                                   150(10)
                                                   100(14)
                                                   .100(17)
                                                   104(10)
(
146,
146,
199,
146,
77,
223,
181,
129,
63,
235,
124,
65,
163,
152,
154,
139,
166,
183,
185,
103,
169,
284,
249,
178,
17S,
1C 1
~J JL ,
177,
203,
203,
149,
CI lone '
Methane)
143,
•14-C,
201 ,
I/O
. - '» -j i
135,
225,
133,
157,
107,
237,
152,
107,
191,
153,
155,
167,
1G7,
211,
213,
211,
170,
236,
251,
179,
179,
163,
223,
231,
231,
I- Ub~,
15
15
2C
1!
137
2 '
2C
1C
10?
^ -
^_ 'w
137
2C
1 1
^ \
i >
^. ^.
22
21
2 I
1 c
2 :;
27
2C
2T
1:
25
24
2--
Z/
        ,92(24) ,
        ,,91)50)
                                                                         185(13)
135, 213, 22
149, 2S9, 32
                                                                                   ^ * t.

-------
                               Table II    Base-neutral Extractables (Cor.t'd.)
                          (hexachlcro-
                             benzene)
	~ •.• •; .-. •p /a
   !2-cthylhexyl)phthalate
   :o (a)anthracene
   :oi b)fluoranthene
   :o (k)fluoranthene
   :c (a)pyre.n.e
   • •"•^. ,'T 9 "5 — Tl\*rvfPv^P
   -•»^- \_/*./^/ \^w.y^/
-------
                           Table II
                        - 11 -
                  Base-neutral Extractables
:hlorcbenzene
:r:lorober.zer.G
.oroethar.e
:hlorcber.zcne
:hloroiscpropyl)

Lorobutadiene
-richlcrober.zene
:. lone
::*.loroethyl) ether
Lorccyclopentadiene
     RRT1
(hexachloro-
   benzene)

    " 0.35
     0.36
     0.33
     0.39
~ '•.'. Icroethcxy) methar.e
nitre toluene
pi-.or.yihydrazine
.-.itrc toluene
oscdiher.lanu.ne
    r.yl phenyl ether'
ylphthalate
Iph thai ate
r.thene

utylphthalate
i:ie
bansylphthalate
     0
     0
     0
     0
     0
     0
     0,
     0
     0
     0
47
55
55
57
61
64
  68
  76
  83
0.06
0.07
0.91
0.93
0.96
  93
  99
  00
  01
  09
     0
     0
     1
     I
     1
     1.09
     1.10
1,
1,
1,
1,
T_ _
1.
15
,23
3Q
31
38
46
Limit of
Detection
   (ng)

   40.  . '
   40
   40
  . 40

   40
   40
   40
   40
   40
   40
   40
   40
   40
   40
   40
   40
  . 40
   40
   40*
   40
   40*
 •  40
   40
   40
 .  40
   40
   40
   40
   4Q
   40
   40*
   40
                              Characteristic
                             El ions (Rel. Int.)

                        •146(100),  148(64), 113(12)
                         146(100),- 148 (64) , 113(11)
                         117(100),  199(61), 201(99)
                         146(100) ,  140 (64), 113(11)
45 (100),
225 (100,
74 (100) ,
12S (100) ,
93(100) ,
237(100) ,
77(100) ,
93 (100) ,
162 (100) ,
152(100),
154(100) ,
82 (100) ,
166 (100) ,
165(100) ,
77(100) ,
165(100) ,
169(100),
284 (100) ,
248(100),
178 (100) ,
178 (100),
163(100) ,
149(100) ,
202(100),
202(100),
77(19), 79(12)
223(63), 227(65)
109 (CO) , 145 (52)
 127(10) , 129 (11)
63(99), 95(31)
 235(63) , 272 (12)
123(50) , 65(15)
95(32), 123(21)
 164(32), 127(31)
 153(16), 151(17)
 153(95), 152(53)
95(14) , 138(18)
 165(80) , 167 (14)
 63 (72) , 121(23)
93(58), 105(23)
 63(72), 121(23)
          167(50)
                                   168(71),
                                   142(30),
                                   250(99),
                                   179(16),
                                   179(16),
                                   164(10),
                                   178(25),
                                   101(23),
                                   101(26),
                                   150(27),
                               149(100) ,
                               184 (100)-,92(24)',
                               149(100), 91)50)
          249(24)
          141(45)
          176(15)
          176(15)
          194(11)
          150(10)
          100(14)
          100(17)
          104(10)
                          CI iono
                         (Methane)'

                      146,  143, -150
                      146,  14C, 150
                      199,  201, 203
                      146,  143, 150
77,
223,
151,
129,
63,
235,
124,
65,
163,
152,
154,
139,
166,
183,
185,
103,
169,
284,
249,
178,
17S,
151,
177,
203,
203,
149,
135,
 225
 133
 157
107,
 237
 152
107,
 191
 153
 155
 167
 167
 211
 213
 211
 170
 236
 251
 179
 179
 153
 223
 231
 231,
 137
i  227
,  2C9
,  .169
 109
,  239
,  164
 137
  203
  101
  1C3
  173
  195
  223
  225
  223
  190
  230
  277
  207
  207
  164
  251
  243
  243
  279
                                         185(13)
                     135,  213,  225
                     149,  299,  327

-------
                          Table II
                      -  12 -'

                Base-neutral Extractables  (Cor.t'd.)
:our.d Xarr.e
(he.xachloro-
   benzene)
-r.thyihexyl ) phthalate
\'a) anthracene
ib) f luorantheno
•.';•:) fluoranthene
.'.•' ) ^v"-^ne
,».•/ ,^ V ^ +^ + , \^f
: a",2,3-cd)?yrer.e
.-.D (2, h) anthracene
(c  h i)pcrylene
     1.46
     1 . 50
     1.54
       66
       66
     173
     J* * I fj
     2.07
     2.12
     2.18
                          1
Limit of
Detection
(ng)
40
40
40
40
40
40
100
100
100
Characteristic
El ions (Rcl. Int.)
m
228 (100) , 229(19) , 226(23)
149(100) , 167(31) , 279(26)
228/100) , 229(19) , 226 (19)
252 (100) , 253(23) , 125(15)
252(100) , 253(23) , 125(1G)
252(100) , 253(23) , 125(21)
' 276 (100) , 138 (28) , 277(27)
278 (100) , 139 (24) , 279 (24)
276 (100) , 138 (37) , 277 (25)
                                                                                        CI  iqr.s
                                                                                        (Methane)
223, 229,
149
223, 229,
252, 253,
252, 253,
                                                                                    252,
                                                                                    276,
     253,
     277/
                                                                                    278,  279,
                                                                                    276,  277,
237
281
231
231
30:
3C7
305
-csodirr.ethylanir.e
„• rsccli-n-prcpy la^.ine
:rc-T:her.yl phenyl  ether
.-. r.idchyrle
lichlcrobcr.sidinc
,G-tetrachlcrocibenzo-
L o *•'. in     '
.-;.! oror.ethyl) ether
                                42(100), 74(88),  44(21)
                                130 (22), 42(64) ,  101(12)
                                204 (100) , 206 (34),  141(29)

                                252(100), 254 (66),  126(16)

                                322(100), 320(90),  59(95)
                                45(100) , 49(14) ,  51(5)
 £?-2250 on 100/120  rr.esh Supelcoport in  s.  6'  x 2 mm id glass column;. He @ 30 ml/min;
 ;-rar.:  50° for  4  min',  then 8 /min to  260  and hold for 15 min.
nditicr.ing of column \vith base is required.

-------
                                           . - 13 -
                                   Table III   Acid  Extractables .
 ur
                     (2-nitrooher.ol)
ropr.-2r.ol

j'.-.lorophcr.ol
cpr.C'r.oi
ro-m-c;resol
t rich lor onhcnol
r.troper.o
.-.itrc-o-cresol
•'r.Ioi-cpher.ol
ated  anthracene  (dlO)
0
0
0
1
1
1
• 1
. 1
1
1
1
0) 1
.53
.66
.96 ' .
.00
.05
.14
.32
.34
.42
.43
.64
.63
Limit of .
Detection
(ng)
100
100
100
100
100
100
100
2 yg
2 yg '
100 •'
100 .
40

Characteristic
El ions (Rel. Int.)
128 (100) , .64 (54) , 130(31)
94 (100) , 65(17) , 66(19)
1G2 (100) , 164 (58) , 93 (61)
133(100)', 65(35) , 109(3),
142(100) , 107 (80), 144 (32)
19G (100) , 193 (92) , 200(26)
122 (100) , 107 (90) , 121(55)
184 (100) , 63 (59) , 154 (53)
198 (ICO) , 132(35) , 77(28)
65(100) , 139(45) , 109 (72)
265 (100) , 264 (62) , 268 (63)
188(100), 94(19)', 80(13)
 CI ions•
(Methane)
                                                                                    129, 131
                                                                                    95, 123,
                                                                                    163, 1G5
                                                                                    140,
                                                                                    143,
                                                                                    197,
                                                                                    123,
                                                                                    185,
                                                                                    199,
                                                                                    1-10,
                                                                                    267,
                                                                                    189,
    168
    171
    1?9
    151
    213
    227
    1GS
    255
    217
,  157
 135
,  167
,  122
,  133
,  201
,  153
,  225
,  23 v
,  122
,  26S
.unui:   6'  glass, 2  irm i.d.
             GC - 60/80 r.esh
              300° @ S°/nin.
       100° -
       He  -S 3C nl/.v.in

-------
                                -14-
   Analysis of Fish for Volatile Orgarucs by Head Space Analyses




1.  Scope




     1.1  This method is designed to determine those "unambiguous  priority



                                                        f4")
          pollutants" associated with the Consent Decree    that   are




          amenable to head space analyses.  These compounds  are listed in




          Table IV of this section.   It is a gas chromatographic-mass




          spectromctric (GC-MS)  method intended for qualitative and semi-




          quantitative determination of these compounds.




          The head space analyses and the liquid-liquid extraction methods




          are complementary to one another.  There is an area of overlap




          between the two and some compounds may be recovered by either method.




          The efficiency of recovery depends on the vapor pressure




          and water solubility of the compounds involved.  Generally, the  area




          of overlap may be identified by compounds boiling  between 130°C  and




          150°C with a water solubility of approximately two percent.  When




          compounds are efficiently recovered by both methods, the chromato-




          graphy determines the method of choice.  The gas chromatographic




          conditions selected for the head space method are, generally,  not •

             i


         • suitable for the determination of compounds eluting later than




          chlorobenzene.




     1.2  While the above approach has not been sufficiently tested




          through extensive experimentation, it is based on laboratory ex-




          perience and is presently our best analytical approach for volatile




          organic materials in fish.




2.   Special Apparatus and Materials




     2.1  Sonificr Cell Disrupter W-350 with microprobc  (manufactured by

-------
                                - 15 -
          Brawson Sonic Power Co.,  Manbury,  Connecticut)  or equivalent.


     2.2   Gas-tight syringe - Sec.

     2.3   Organic Free Water - Prepared by passing distilled water through

          an activated carbon column.

   .  2.4   Head Space Standard Solutions - Prepare three standard methanol


          solutions of the compounds listed in Table IV at the following

          concentrations:  50 ng/yg,  150 ng/yl  and 300 ng/yl.   The standard

          solutions should be stored in the freezer at less than 0°C.   Solutions

          should be allowed to warm to room temperature before dosing.  Fresh

          standards should be prepared weekly.  Procedures for preparing  stan-


          dards are outlined in the purge and  trap section of ref. 4.

3.    Procedure


     3.1   Remove four of the sample vials containing 10.0 grams of homo-

          genized fish from the freezer.  Open the vials and add 10 ml  of

          organic free water to each while the fish is still frozen.  Sonify

          the fish for 30 seconds at maximum probe pov.-er.  Immediately rcseal

          the vials!!

     3.2  Dose one sample vial, through the septum, below the water level with

          10 yl of the 50 ng/yl standard solution.  Dose a second vial, with

          10 yl of the 150 ng/yl standard and  a third vial with 10 yl  of  the
       *
          300 ng/yl standard.

     3.3  Place all four sample Vials into a 90°C water bath for 1 hour.

     3.4  K'hile maintaining the sample at 90°C, withdraw 2.0 ml of the head

          gas with a gas tight syringe and analyze by injecting into a GC,

          operating under the conditions recommended in ref. 4.

-------
                                - 16 -



         NOTE:   Specific GC detectors  may be  substituted  for  the MS.


     3.5  Analyze the  undosed  sample first,  followed  by  the  50  ng/yl


         dosed  sample.-  If no compounds  of  interest  are found  in the


         undosed sample  and the  dosed  sample  produces peaks to indicate


         recovery of  the protocol  compounds,  do  not  analyze the remaining


         samples.   Calculate  lower limits of  detection  from the response


         obtained from the dosed sample.  If  compounds  are  observed


         in the undosed  sample,  analyze  the two  remaining dosed • samples


         in exactly the  same  manner.   Subtract the peak areas  of compounds


         found  in the undosed sample from the corresponding compounds  con-


         tained in the dosed  data; quantify the  unknown.   (NOTE:   If the


         calculated sample  concentration is greater  than  the  concentration


         of the dosed standard used in the  dosing  step, it  is  necessary


         to prepare additional standards in order  to "bracket" the unknown.


         Utilize the  remaining sample  in the  freezer for  this  purpose.)


     3.6 Quality Control:   Standard quality assurance protocols should be


         employed, including  blanks,  duplicates, and dosed  samples as  described


         in the "Analytical Quality Control Handbook"^   .


4.   Reporting of Data


     4,1 Report all results  in ug/kg on a wet tissue basis.  Report all
       i

         quality control (QC) data along with the  analytical  results  for


          the samples.

-------
                                 -  17 -
                                 Table  iv
               Characteristic lens  of  Volatile  Organics
 Compound
 chloromathane
 dichlcrodifluororne thane

 bromomethane
 vinyl chloride
 chloroethane
 methylepe chloride

 trichlcrcfluoromethane
 1,1-dicJiloroethylene
 broiriochlorome thane (IS)

 1,1-dichlo^oethane

 trans-1,2-dichloroethylene
 chloroform
 1,2-dichloroethane

 1,1,1-trichloroethane

 carbon tetrachloride
 bromod;Lchlorome thane

 bis-ch|croniethyl ether
•1,2-dichloropropane

 trans-1,3-dichloropropene
 trichloroethylene
 dibromoch lor orr.e thane
                     \
 cis-1, ij-dichloropropene
El Ions  (Relative
   intensity)	
50(100); 52(33)
85(100)
101(13)
87(33);
103(9)
94(100); 96(94)
62(100); 64(33)
64(100); 66(33)
49(100);51(33);
84(86).; 86(55)
101(100); 103(66)
61(100); 96(80); 98(53)
49(100); 130(88);
128(70); 51(33)
63(100); 65(33); 83(13)
85(8); 98(7);  100(4)
61(100); 96(90); 98(57)
83(100); 85(66)
62(100); 64(33);
98(23); 100(15)
98(100); 99(66);
117(17); 119(16)
j.on usod to
 quantify	;
     50

    101
    . 94
     62
     64

     84
    101
     96

    128

     63
   •  96
     83

     98
117(100);  119(96); 121(30)
83(100)
127(13)
85 (66) ;
129(17)
79(100); 81(33)
63(100); 65(33);
112(4); 114(3)
75(100): 77(33)
95(100)
130(90)
97(66) ;
132(85)
129(100); 127(78)
208(13); 206UO)
75(100); 77(33)
     97
    117

    127
     79

    112
     75

    130

    127
     75

-------
                               TABLE IV  Continued
  CcrTpound
  1,1,2-hrichioroethane

  benzene
  2~chloro2thylvinyl ether
  2-bronio-l-chloroprcpane (IS)
  broTr.oform

  1,1,2,2-tetrachlorocthene

  1,1,2,2-tetrachloroethane

  1,4-dichlorobutane(IS)
  toluene
  chlorobenzene
  ethylbenzene
.  acrolein

  acrylonitrile
 El Ions  (Holci
 ^  intensity)
                          Ion  uncd to
                          quantify
 83(95) ; 35 (GC) ; 97(100) ;
. 99(63) ; 132(9) ; 134(8)
 73(100)
 63(95) ; 65(32) ; 105(18)
 77(100) ; 79(33) ;156(5)
 171(50) ;173 (100) ; 175(50)
 250(4) ; 252(11) ; 254(11) ;
 25G(4)
 129(64)
 164(78)
         131(62);
         166(100)
 83(100);  85(66); 131(7)
 133(7); 166(5); 168(6)
          90(30); 92(10)
          92 (78)
55(100);
91(100);
112(100); 114(33)
91(100); 106(33)
26(49); 27(100);
55(64); 56(83)
26(100); 51(32);
52(75); 53(99)
 97
 78
106
 77

173

164

JL68
 55
 92
112
106

 56

' 53

-------
                                    - -19 -
                      Analysis of Fish for Cyanide



1.    Scope and Application



     1.1  This method is to be used for the determination of cyanide in



          fish.  All samples must be distilled prior to the analytical deter-



          mination.  For cyanide levels exceeding 0.2 rr.g/200 ml of absorbing



          liquid, the silver nitrate titrimctric method is to be used.  For cyan-



          ide levels below this value, the colorimetric procedure is to be used.
                                                            4


2.    Samp1c Preparation



     2.1  A 5-gram portion of the frozen ground fish as described under



          "Sample Handling" is used for the analysis.  The sample should be



          thawed before the analysis begins.



3.   Preparation of_ Calibration Curve



     3.1  The calibration curve 13 prepared by using portions of "spiked" fish



          tissue, distilled in the same manner as the tissue sample being



          analyzed.  For preparation of the calibration standards, choose a 50 g



          portion of fish, weigh and blend in a Waring blender (or equivalent)  •



          Vfith 10 ml of 10% NaOH and sufficient deionized distilled water so



          that the volume of the mixture will be 500 ml.
                                                                     f


     3.2  Using a volumetric pipet whicli lias had the tip removed, withdraw

           «


          eight 50 ml portions, and place in a series of 1  liter boiling flasks.



          Seven of the flasks should be spiked with increasing volumes of the



          cyanide standard as given in 3.8  (Ref. 7).  Adjust the final volume



          of  each flask to 500 ml with deionized distilled water.



     3,3  Add 50 ml of a 5% NaOH'solution to the absorbing  tube and dilute,



          if  necessary, with deionizc-d distilled water  to obtain an adequate

-------
                                    - 20 •
          depth of liquid in the absorber. .Connect the boiling flask,




          condenser, absorber and trap in the train as shown in Fig. 1 (Ref.  7).




     3.4  Continue with step 8.2 through 8.7 (Ref.  7).




     3.5  The calibration curve is prepared by plotting the absorbance versus




          the cyanide concentration.  The blank absorbance value must be sub-




          tracted from each value before plotting the curve.




4.   Sample Procedure




     4.1  Place a weighed portion of the ground fish (approximately 5 g) in




          a blender along with 100 ml of deionized distilled water and 1 ml




          of a 5% NaOH solution.




     4.2  Blend until a homogeneous mixture is obtained and transfer to a




          1 liter boiling flask.  Rinse the blender with several portions of




          deionized distilled water totaling 400 ml ond add to the boiling flask.




     4.3  Add 50 ml of a 5% NaOH solution to the absorbing tube and dilute




          if necessary with deionized distilled water to obtain an adequate




          depth of liquid in the absorber.  Connect the boiling flask, condenser,




          absorber and trap in the train and continue with step 8.2 through




          8.7 (Ref. 7).




     4.4  Read the absorbance and determine the cyanide concentration from




          the calibration curve.




5.   Quality Assurance




    •5.1  Initially, demonstrate quantitative recovery with each distillation




           digestion apparatus by comparing distilled aqueous standards




          to non-distilled aqueous standards.  Each day, distill at least




          one standard to confirm distillation efficiency and purity of reagents.

-------
                                    -  21  -
     5.2  At least 15% of the cyanide analysis should consist of duplicate




          and spiked samples.  Quality control Jimits should be established




          and confirmed as described in Chapter 6 of the "Analytical Quality




          Control Handbook" (Ref. 6).




6.    Reporting o_f_ Data




     6.J  Report cyanide concentrations as follows:  less than 1.0 mg/kg,




          nearest 0.01 mg; 1.0 mg/kg and above, two significant figures.




     6.2  Report all quality control (QC) data along with the analytical




          results for the samples.

-------
                       Analysis of Fish for Phenol


1.    Scope and Application


     1.1  This me_thod is to be used for the determination of phenol in fish.


          Phenols are defined as hydroxy derivatives of benzene and its


          condensed nuclei.  The 4-amino-ant.ipyrinc colorimctric method given


          determines phenol, the ortho- and msta-substituted phenols, and


          under proper pH conditions, those para-substituted phenols in which


          the substitution is a carboxyl, halogen, methoxyl or sulfonic


          acid group.  Presumably, the 4-amino-antipyrine method does not


          determine those para-substituted phenols in which the substitution


          is, an alkyl, afyl, nitro, benzoyl, nitroso, or aldehyde group.


     1.2  All samples must be distilled prior to the determination of phenols


          using the procedure given on page 576 (Ref. 8).  Use method 510 B


          for samples that contain less than 1 mg/kg and method 510 C for


          samples that contain more than 1 mg/kg.


2.   Sample Preparation


.;    2.1  A 5-gram portion of the frozen, ground fish as described under


          "Sample Handling" is used for the analysis.  The sample should be


          thawed before the analysis begins.
           <                                 •

3.   Preparation p_f  Calibration Curve


     3.1  The calibration curve is prepared by using portions of "spiked"
     «

          fish tissue, distilled in the same manner as the tissue samples


          being analyzed.  For preparation of the calibration standards,


          choose a 50 g portion of fish, weigh, and blend in a Waring blender

-------
                               - 23 -
     (or equivalent)  with sufficient cleionizcd distilled water so that



     the total volume of the mixture will  be SOU ml.



3.2  Transfer a 50 ml portion of mixture to a beaker using a volumetric



     pipet which has  had the tip removed and determine the volume



     of (1 + 9) H PO   required to lower the pll to 4.0 using either



     methyl orange indicator or a pll meter.  This volume of (1 + 9)



     H,PO. is to be added to each 50 ml portion of fish mixture prior



     to the distillation step which follows.



3.3  Transfer 50 ml portions of the blended fish mixture to the dis-



     tillation apparatus as shown in Fig.  318:1, p.  241 (Ref.  3), adding



     the volume of H..PO. (determined above) to lower the pll to 4.0.
                    34


     Add 5 ml of a 10% CuSO. solution to each distillation flask along



     with appropriate volumes of the standard phenol solution (Ref. 8,



     p. 579, 3C).  A blank and 7 standards should be distilled for



     preparation of the calibration curve.  [NOTE:  The ir.inimujn



     detectable quantity is 1 pg/1 phenol  in a 500 ml distillate.]



     Adjust the volume in the distillation flask to 500 ml.  Use a



     500 ml graduated cylinder as a receiver.



3.4  Begin the distillation and continue until a distillate volume

       i

     of 450 ml is obtained.  Stop the distillation and add 50 ml



     deionized distilled water to 'the distillation flask after



     boiling has ceased.  Continue the distillation until a total of



     500 ml has been collected.  If the distillate is turbid, acidify



     with (1 + 9) H,PO. and repeat the distillation as described.

-------
                                    - 24 •-





     3.5  Continue with the procedure, as j'.ivcn in the chloroform extraction



          method 510 B, p. 577 (Rcf. 8).  Rcr.d the absorbancc of the standards



          against a reagent blank at a wavelength of 460 nm.  Plot absorbancc



          against micrograms of phenol for the calibration curve.



     3.6  Alternatively, follow the direct photometric method (510 C, p. 580,



          Ref. 8), for those samples in which the phenol concentration exceeds



          1 nig/kg.



4.   Sample Procedure



     4,.!  Place a weighed portion of the ground fish (approximately 5 g)



          in a blender along with 100 ml of dcionized distilled water.  Blend



          un,til a homogeneous mixture is obtained and transfer to a 1 liter



          boiling flask.



     4..2  Rinse the blender with several portions of dcionized distilled



          water and add to the distilling flask.  Add a volume of (1 + 9)



          H PO. to bring the pH of the mixture to 4.0 (the same volume as
           O  ^T


          determined for preparation of the calibration standards may be



          used).  'Add 5 ml of a 10% CuSO  solution and adjust the total



          volume to approximately 500 ml.  Use a 500 ml graduated cylinder



          as a receiver.



     4,.3  Begin the distillation and continue as described in 3.4 - 3.6



          above'.  Read the absorbance and determine the ug of phenol from



          the calibration curve.



5.  -Cuality Assurance


     1              ~
     £.1  Demonstrate quantitative recovery with eacli distillation apparatus



          by comparing aqueous distilled standards to non-distilled standards.



          Each day, distill at least one standard to confirm the distillation



          efficiency and purity of reagents.

-------
     5.2  At least 15* of the phenol analyses should consist of duplicate

          and spiked samples.  Quality control limits should be established
                   *
          and confirmed as described in Ref. 6.

6.   Reporting p_f_ Data

     6.1  Report phenol concentrations as follows:

               Method 510 B to the nearest pg/kg

             '  Method 510 C - when less than 1.0 wg/kg to the nearest

               0.01 yg; 1.0 nig/kg and above'to two significant figures.

     6.2  Report all quality control data when reporting results of sample

          analysis.

-------
                 Analysis of Fish for Mercury


Scope and Application


J.I  This Method is. to be used for the determination of total  mercury


     (organic and inorganic) in fish.   A weighed portion of the sample


     is digested with sulfuric and nitric acid at 58°C followed by


     ovornight oxidation with potassium permanganate at room temperature.


     Mercury is subsequently measured by the conventional cold vapor


     technique (Ref. 7, page 118).


1.2  The range of the method is 0.2 to 5 iig/g but may be extended


     above or below the normal range by increasing or decreasing sample


     size or through instrument and recorder control.


Sample Preparation
',                                                    \

2.1  The sample may be prepared as described under "Sample Handling"


     or the special metal procedure may be used.  A 0.2-0.3 g  portion


     should be taken for each analysis.  The sample should not be allowed


     to thaw before weighing.


Freparation of_ Calibration Curve


3.1  The calibration curve is prepared by using portions of "spiked"


     fish tissue, treated in the same manner as the tissue samples


     being analyzed.  For preparation of the calibration standards


     choose a 5 g portion of fish, and blend in a Waring blender.


3.2  Remove six equal and accurately weighed portions (0.2 g)  with a


     spatula and transfer to each of six dry BOD bottles. "Add 4 ml

                                                                     , K*e
     of cone. H,,SQ. and 1 ml of cone.  HNQ_ and place in a water bath^   o


     at f>8°C until the tissue is completely dissolved (30-60 minutes).

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     3.3  Cool,  and transfer 0,  0.5,  1.0,  2.0,  5.0 and 10.0 nil aliquots




          of the working mercury solution  containing 0 to 1.0 pg of. mercury




          to, the BOD bottles.   Cool to 4°C in an ice bath and cautiously




          ad.d IS ml of potassium permanganate solution.   Allow to stand




          overnight at room temperature under oxidizing conditions.




     3.4  Add enough distilled water so that the total volume is approximately




          125 ml.  Add 6 ml of sodium chloride-hydroxylaminc sulfate solution




          to reduce the excess permanganate.




     3,.5  Wait -it least 30 seconds after the addition of the hydroxylamine.




          Treating each bottle individually, add 5 ml of the stannous sulfate




          solution and immediately attach  the bottle to the aeration apparatus.




     5.6  Continue with the procedure as given on page 121 (Ref. 7).  The




          calibration curve is prepared by plotting the peak height versus




          the mercury concentration.   The  peak height of the blank is




          subtracted from each of the other values.




4.    Sampl_c_ Procedure




     4..1  Weigh 0.2-0.3 portions of the sample and place in the bottom




          of a dry BOD bottle.  Care must  be taken that none of the sample




          adheres to the side of the bottle.  Add 4 ml of cone. H?SO  and




          1' nil of cone. UNO  and place in  water bath at 58°C until the tissue




          is completely dissolved  (30-60 minutes).




    •4.2  Cool, to 4°C in an ice bath and cautiously add 5 ml of potassium




          permanganate solution in I ml increments.  Add 10 ml additional




          permanganate, or more if necessary to maintain oxidizing conditions.




          Allow to stand overnight at room temperature (sec NOTE 1).  Continue

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                                    - 28 -
         .as described under (2.4J.   NOTE 1:   As an alternate to the overnight




          digestion,  the solubilization of the tissue may be carried out in a




          water bath  at 80°C for 30  minutes.   The sample is then cooled and




          15 »nl of potassium permanganate solution added cautiously.  At this




          point the sample is returned to the water bath and digested for an




          additional  90 minutes at 80°C (Ref. 0).  If this method is




          followed, the calibration  standards must also be treated in this




          manner.  Continue as described under (3.4).




5.   Calculation




     5.1  Measure the peak height of the unknown from the chart and read the




          mercury value from the standard curve.




     5.2  Calculate the mercury concentration in the sample by the formula





                       ..  ,       yg  Hg in aliquot
                    Vg He/gram = —*—^—n	'—-
                     6  b 6      wt. of aliquot in gms




     5,.3  Report mercury concentrations as follows:




          Below 0.1 yg/gm, <0.1: between 0.1  and 1 yg/gm, to nearest




          0.01 yg; between 1 and 10  yg/gm, to nearest 0.1 yg; above




          10 yg/gm, to nearest yg.




6.   Quality Assurance




     §..l  Standard quality assurance protocols should be employed, including




          blanks, duplicates and spiked samples as described in the "Analytical




          Quality Control Handbook"  (Ref.  6).




    •6.2  Report all  quality control data when reporting results of sample analyses.




7.   Precision and Accuracy




     7.1  The following standard deviations on replicate fish samples were re-




          corded at the indicated levels:   0.19 yg/gm ±0.02, 0.74 yg/gm ±0.05




          and 2.1 yg/gm ±0.06.  The  coefficients of variation at these levels

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                          - 29 -
at these levels,   added as methyl  mercurjc  chloride,  were  112,




93 and 86%, respectively.

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                                    - 30 -
                       Analysis of Fish for Metals .

1.    Scope

     1.1  This method is designed to determine in whole fish those priority

          pollutants listed in the Consent Decree that are classified as

          heavy metals or considered toxic as they exist in their elemental

          form and associated compounds.  Those pollutants are the following:

          antimony, arsenic, beryllium, cadmium, chromium, copper, lead,

          nickel, selenium, silver, thallium, and zinc.

2.    Summary p_f Method

     T..\  The fish is prepared for analysis by first being chopped into

          small pieces, homogenized in a blender with dry ice, and then

          solubilized by either dissolution after dry ashing or a wet

          oxidation digestion.  After sample preparation, atomic absorption,

          either direct aspiration, gaseous hydride, or a flameless technique,

          is used to measure the concentration of the pollutant in the fish.

3.    Preservation and Handling

     £.}  While an aliquot of the ground fish as prepared under "Sample
                 f
          Handling" may be taken for the metals determination, it may be

          more desirable to prepare an individual fish to avoid possible
           i
          metal contamination from the grinder.  Dust in the laboratory

          environment, impurities in reagents and impurities on laboratory

          apparatus which the sample contacts are all sources of potential

          contamination.  All glassware should be thoroughly washed with
                       i
          detergent and tap water, rinsed with 1:1 nitric acid, tap water,

          and finally deionizcd distilled water in that order.

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                                    - .31 -
          NQTli:   Cliromic acid may be useful  to remove organic deposits



          from glassware;  however, the analyst should be cautioned that



          the glassware must be thoroughly rinsed with water to remove



          the last trace of chromium.  This  is especially important if



          chromium is to be included in the  analytical scheme.  A com-



          mercial product--NOCHUOMlX--available from Codax Laboratories,



          6 Yarick Street, New York, NY 10013, may be used in place of



          chromic acid.



4.    Sample Homogenization



     4.1  If a separate fish sample, other than that prepared under "Sample



          Handling," is to be used for metals analyses, unwrap and weigh the



          frozen fish at the time of processing.  Select a fisli that weighs



          between 50 and 300 grams.  If an analysis is required on a larger



          (>300 grams) a 50 gram representative portion must be taken from



          the sample after it has been pretrcatcd as described, in "Sample



          Handling" on page 1 of this document and proceed to step 4.3.



     ^.2  After weighing, the fish should be chopped into approximately



          one-inch chunks or smaller with a meat cleaver or a knife.and



          mallet (2-3 pounds).  Smaller pieces ensure efficient grinding.



     4.3  Place crushed or pelleted dry ice into the blender container.  The



          weight of dry ice should be approximately equal to, or greater
    «          *


          th.an, the weight of the fish.



     4,4  Turn on the blender for 10 seconds to pulverize the ice and



          chill the blender.

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                                   - 32 -
     1.5  Add  the  pieces  of  fish  and  blend  at high  speed until  the



         mixture  is  homogeneous.   This  usually  requires 2-5  minutes.



         Add  more dry  ice.if needed.



     4.6  Pour the homogcnate into a  plastic bag and  close  the  bag with a



         rubber band.   Do not seal the  bag tightly so  that CCL may  escape.



     ^.7  Place the bag in the freezer (-12°C for at  least  16 hours)  until



         ready to proceed with the digestion step.



         NOTE:  If desired,  the  blender blades  can be  modified in order to



         have the leading edge of the blades  (the  sharpened  edge) turned down



         so that, as it rotates,  the blade will throw  the  material  upwards.



         Stainless steel blades  may  be  a possible  source of  nickel  and



         chromium contamination  and  should be noted  if detected.  If a


         •f   \
         ^tantalum blade is  available, it should be substituted for  the

                 )

         stainless steel.



               The hole in the blender lid should bo  enlarged sufficiently



         to allow the  evolved gas to escape  (1/2 inch  - quartsizc,  1 inch -



         gallonsize).   Hold a cloth  or  labwipe  over  this hole  when  blending



         to prevent loss of the  sample  material.  A  glove  should be worn



         to prevent possible freezing of the skin  by escaping  gas.



5.    Reagents



     5.1  Lfeionizcd distilled water:   Prepare by passing distilled water



          through a mixed bed of cation  and anion exchange  resins.   Use



         deionized distilled water for the preparation of-all  rcngents,



         calibration standards,  and as  dilution water.

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                                       -.35 -
       5.2  Nitric acid (cone.)'-  If metaJ impurities arc found to be present,



            distill reagent grade nitric ncid in a borosilicatc glass.dis-



            tillation apparatus.



       5.3  SuIfuric acid, ACS  grade (95.5 - 36.5%).



       5.4  Sulfuric acid - 20% v/v solution.  Carefully add 200 ml concentrated



            H-,SO. to 500 ml water.  Cool and dilute to 1 liter with water.
             2  4


       5.5  Hydrochloric acid,  ACS grade  (37-38%).



       5.6  Hydrogen Peroxide,  50% stabilized ACS grade.



       5.7  Dry ice (frozen carbon dioxide), pellet form preferred.



  6.   Apparatus



       (,.}  Blender, Waring,  two-speed, stainless steel blade or tantalum blade



            if available, glass container capacity 1000 ml, or equivalent



            equipment.



       (.2  Drying oven - Controllable with the range of 100"C to  150°C witli



            less than ±5°C variation.  Check calibration of oven temperature



            control to ensure accurate ashing temperatures.J Furnace must be



'  "  •  vi'    operated in suitable  fume hood.

       -

       (..3  Hot plate, controllable within the range of 80°C to 400°C.



            Hot plate must be operated in fume hood.



  7.   lirocedure



            Except for mercury  which employs a cold vapor technique,  the other



       pollutants can be divided  into two groups  for continued processing.



            GROUP I:   Be, Cd,  Cr, Cu, Pb, Ni, Ag, Tl, and Zn



            GROUP II:  As and Se

-------
Group I is digested by a dry ashing process  (10) with the use of an ashing




aid, whi}e Group II is prepared utilizing a wet ashing procedure.




7. \ Group I_ - Metals




     7.1,1   Remove the homogenized sample from the freezer and weigh




             approximately 10 grams into a tared 100 ml tail form Pyrcx




             beaker.  Subtract the beaker weight from the total and




             record the wet sample weight.




     7.1,2   Add 25 ml of 20% sulfuric acid.  Mix each sample thoroughly




             with a glass stirring rod ensuring all sample material is




             wetted by the acid.  Rinse the  stirring rod with water into




             the ashing vessel and cover the sample with a ribbed




             watch glass.




     7.1.3   Dry the samples in an oven or furnace at 110+ 5°C until




             a charred viscous sulfuric acid/sample residue remains.




             Usually 12 to 16 hours (overnight) is sufficient.  Transfer




             the ashing vessels containing the dried samples to a cold,




             clean muffle furnace which is provided with good external




             ventilation  (fume hood), ensuring that the sample remain




             covered during the transfer.  Initially set the furnace at




             125°C and increase the temperature approximately every hour




             in 50°C increments up to 275°C.  Mold the temperature at




             275°C for 3 hours.  Finally, increase the temperature




             to 450°C (at 50°C per hour) and hold for 12 to 16 hours




             (overnight).  Remove the covered ashing vessels from the




             furnace and allow to cool to room temperature in a clean,




             draft-free area.

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7.1.4   After initial overnight ashing, some residual carbon nwy




        remain in the samples.  Treat each sample asli with 0.5 ml




        of water and 1 ml of concentrated nitric acid (whether or




        not they are already white).   Evaporate carefully just to




        dryncss on a warm hotplate (in a fume hood).   Place the




        ashing vessels (covered with watch glasses) in a cool muffle




        furnace and raise the temperature to 300°C and hold for




        exactly 30 minutes.  Remove eacli covered sample ash from




        the furnace and allow to cool as before.  If residual




        carbon remains, repeat the nitric acid treatment until a




        carbon-free white ash is obtained.  The covered ashing




        vessels containing the ash may be stored in a dessicator




        or in a laminar flow clean hood.  NOTE:  Copious carbon




        residues (i.e., black ashes)  after overnight ashing may




        indicate inefficient or uneven heating within the furnace.




        Routine calibration of the furnace is advised.




7.1.5   Add 0.5 ml of nitric acid and 10 ml uf water to each cool




        ashing vessel, then warm gently on a hotplate at 80-90°C




        for 5 to 10 minutes to effect dissolution of the ash.




        A small amount of insoluble white siliceous-like residue




        may remain undissolved; do not filter the residue because




        of the possibility of contamination.  Quantitatively




        transfer the contents of each ashing vessel into a  100 ml




        volumetric flask, dilute to volume with water, and  shake




        thoroughly.  Allow any residue to settle to the bottom

-------
                         - 30 -
        of the flask (about 2 hrs.).   Do  not  shake  the  sample




        further before taking yn aliquot  for  analysis.   The  sample




        is now ready for analysis.   Note:   The presence of a




        precipitate other than the  insoluble  siliccous-like  material




        may result in low or  eratic results  for ?b.   Precipitate




        formation can result from heating the samples too long or




        at too high a temperature after nitric acid treatment  of




        the ash.  Precipitate formation must  be avoided by main-




        tenance of appropriate ashing temperatures.




7.1,6   The prepared sample should  be analyzed by atomic absorption




        using either direct aspiration or furnace techniques.   For




        a discussion of basic principles,  the method of standard




        addition, the chelation/solvent extraction  procedures,




        general instrumental operating parameters,  and preparation




        of standards and calibration see  the  section on "Atomic




        Absorption Methods," pages  78-91  (Kef. 7) and the individual




        analyses sheets (page numbers listed  in Table V).




                               Table V_




                Element     Methods for Chemical Analysis

AC
Be
Cd
Cr
Cu
Ni
I'b
Sb
Tl
Zn
of Water
P-
P-
P-
P-
P-
P-
P-
P-
P-
P-
and Wastes, 1974
146
99
101
105
108
141
112
94
149
155

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                               -  37  -
     7.1.7    Because  of the adequate sensitivity by conventional  flame




             AA and  the expected concentration  levels  of cadmium,  copper




             and zinc in the sample, these three elements should  be




             analyzed by direct aspiration.   The furnace technique is




             preferred for the analysis of the  other Group I  metals because




             of their expected low concentration.  When using the furnace




             technique, the operating parameters and instructions as-speci-




             fied by the particular instrument  manufacturer should be




             followed.  If the detected concentration  by the  furnace pro-




             cedure  is beyond the working range of the standard curve,  the




             sample  should either be diluted and reanalyzed or analyzed by




             direct  aspiration.  The method of standard additions should be




             employed when needed.  If the sample matrix is so complex




             that sample dilution followed by furnace  analysis cannot  be




             used, or if the use of chclation/solvent  extraction  technique




             for concentration Ag, Ni, Pb and Tl is preferred, the pro-




             cedure  as described starting on page S9 (Rcf. 7). should




             be utilized.




7.2  Group II - Metals




     7.2.1   Remove the homogenized sample from the freezer and weigh  approx-




             imately 5 grams into a tared 125 ml conical beaker.   Subtract




             the beaker weight from the total and record the wet sample weight.




     7.2.2   Add 5 ml of cone. HNOj.  Then slowly add 6 ml cone.  H2S04




             and cover with a watch glass.




     7.2.3   Place beaker on hot plate and warm slightly.  (NOTE:   Remove




             beaker If foaming becomes excessive.)  Continue heating



             until t IT* m i Y 111 t'f bprntn<--c; d n rk o v n no<; ^ i h I r Tfr\\ \c i n o

-------
        condition is evident.   Po not allow the mixture to char.




        Remove beaker from hotplate and allow to cool.




7.2.4   Add an additional 5 ml of cone. HNO_, cover witli a watch




        glass, and return beaker to hot plate.   Repeat  step 7.2.3.




7.2,5   When mixture again turns brown, cool, and slowly add 5 ml




        of 50% hydrogen peroxide.  Cover with watch glass and




        heat gent.ly until the initial reaction has ceased.  If




        the solution becomes dark, repeat the peroxide  addition,




        several times if necessary, and heat to S0_ fumes.  If




        charring occurs, add further 1 ml portions of hydrogen




        peroxide until the fuming sulfuric acid remains colorless




        or very light yellow.  (If at any stage it seems that the




        sulfuric acid may approach dryness, cool, add 2 to 3




        ml of sulfuric acid, and continue.)




7,2.6   Cool, add 40 ml of cone. IIC1 and dilute to 100  ml with




        deionized distilled water.  The sample is now ready for




        analysis.




7,2.7   The Group II metals should be analyzed by atomic absorption




        using the gaseous hydride technique.  The apparatus setup,




        standard preparation and calibration, and analysis procedure




        that is to be followed is given starting on page 159  (Uef. 8)




        From the prepared sample a 25 ml aliquot should be withdrawn




        and the analysis continued as described in section 3.d,




        page 162  (Rcf. 8).

-------
                                    -  39 -
8.    Calculation

     £.1   From the values read off the appropriate calibration  curve

          calculate the concentration of each metal pollutant in the  fish

          as follows:

          If the concentration of standards in the calibration  curve  is

          plotted as mg/1,

                         mg/1 of constituent        volume of prepared
                         in prepared sample    X	sample in ml	
             yg/gram =             weight of wet sample in g

          If the concentration of standards in the calibration curve  is

          plotted as pg/1,

                         pg/1 of constituent
                         in prepared sample    X    volume of prepared
                         	1000	sample in ml
             pg/gram =             weight of wet sample in g

9.   Quality Assurance
     ,-i	

     ?.l  Standard quality assurance protocols should be employed, including

          blanks, duplicates and dosed samples as described in the

          "Analytical Quality Control Handbook" (6).

     S.2  Report all quality control data when reporting results of

          sample analyses.

-------
                                 -  40
                             REFERENCES
1.   "Method for Orcjanochlorir.c  Pectisidar,  in Industrial  Efflucntr.,"
     U. S. Environmental  Protection  Agency,  Environmental Monitoring
     and Support Laboratory,  Cincinnati,  Ohio,   45268 1973.

2.   "Method for Poll/chlorinated Biphenyls  in Industrial  Effluents, "
     Environmental  Protection Agency,  Environmental  Monitoring and
     Support Laboratory,  Cincinnati,  Ohio  45268  1973.

3.   Federal Register,  Volume 41,  number  232, p. 52780, Wednesday,
     December  1, 1976.
    ^
4.   "Sampling and  Analysis Procedures for  Screening of Industrial
     Effluents for  Priority Pollutants, "  Environmental Protection
     Agency, Environmental  Monitoring and Support Laboratory,  rev.
     April, 1977.

5.   Stalling, D.  t..;  Tindle, R.  C.;  Johnson, J. L. ; (1972)  JAOAC.
     55,  32-38.  "Cleanup of Pesticide and  Polychlorinated Bip};enyl
     Residues  in Fish  Extracts by Gel Peiineation Chromatography. "

6.   Handbook  for Analytical Quality Control in l-'ater and Xasteioater
     Laboratories  (1972).   U. S.  Environmental Protection Agency,
     Technology Transfer.

7.   "Methods  for Chemical Analysis  of Water and Wastes (1974), U. S.
     Environmental  Protection Agency, Technology Transfer.

8.   "Standard Methods for the Examination  of Waster and  Wastewater,
     14th  edition  (1975).

9.   Bishop, J. N., "Mercury in Fish," Ontario Water Resources Connn.,
     Toronto,  Ontario,  Canada, 1971.

10.   Jones,  J. W. ;  Gajan, R. J.; Buyer, X.  W.; Fiorino, J. A.; (1977)
     JAOAC_,  60, 826.  "Dry Ash - Volttmnetric Determination of Cadmium,
     Copper, L'ead,  and Zinc in Foods."

-------