EPA 560/6-8-007
ANALYSIS OF POLYCHLORIMATED BIPHENYL (PCB)
      IN HUMAN BLOOD SERUM SAMPLES
              OCTOBER 1977
    ENVIRONMENTAL PROTECTION AGENCY
       OFFICE OF TOXIC SUBSTANCES
             WASHINGTON, D.C.

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                   DISCLAIMER

This report has been reviewed by the Office of
Toxic Substances, U.S. Environmental Protection
Agency, and approved for publication.  Mention of
trade names or commercial products does not con-
stitute endorsement or recommendation for use.

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 ANALYSIS OF POLYCHLORINATED BIPHENYL  (PCB)
        IN HUMAN BLOOD SERUM SAMPLES
                Final Report
           Research Request No. 2
            Contract 68-01-3248
                Prepared By:

  Charles L. Stratton and Paul C. Geiszler

ENVIRONMENTAL SCIENCE AND ENGINEERING,  INC,
    P. 0. Box 13454, University Station
        Gainesville, Florida  32604
                    For:

         OFFICE OF TOXIC SUBSTANCES
      ENVIRONMENTAL PROTECTION AGENCY
          Washington, D.C.  20460
               October, 1977

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                                CONTENTS
Figures
Tables

     1.  Introduction                                             1
     2.  Analytical Procedure                                     2
             Quantitation                                         2
             Confirmation                                         9

References                                                       16
Appendices

     A.  Analytical data results
     B.  Analytical procedure
     C.  Sample calculation
     D.  Data for PCB pool samples
     £.  Perchlorination procedure

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                                FIGURES






Number                                                         Page




   1       Typical Chromatogram of a Serum Extract               3




   2       Chromatogram of Aroclor 1242                          4




   3       Chromatogram of Aroclor 1254                          5
                                   11

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                                 TABLES


Number                                                          Page

   1       Inter-Run Statistics for PCB Pool  Samples              7

   2       Inter-Laboratory Comparison of PCB Pool
           Sample Results                                         8

   3       Results of Confirmatory Analysis for PCB              10

   4       Reagent Blank Statistics                              14

   5       Comparison of Pattern versus Perchlorination
           Results for PCB Pool B                                15
                                  111

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INTRODUCTION




A total of 208 human blood serum samples and two mother's milk samples




were analyzed for polychlorinated biphenyl (PCB).  The samples were




supplied to Environmental Science and Engineering, Inc. (ESE), in frozen




condition by the Department of Health, Education, and Welfare, Center




for Disease Control, Atlanta, GA (CDC).  This report includes the




analytical results for these samples and an assessment of the degree of




uncertainty involved in the analysis.  The analytical results, expressed




as Aroclor® 1242 and Aroclor® 1254, are tabulated in Appendix A.

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ANALYTICAL PROCEDURE




The analytical procedure  used  is  described,  in detail,  in Appendix B.




This procedure, which  is  based  on the  procedure used by the Michigan




Department of Health,  was developed by the  cooperation  of CDC and ESE.




Quality control was maintained  during  each  sample  run by:




     1.  Establishing  and documenting  chromatogram resolution by




         analysis of a chlorinated hydrocarbon pesticide  mixture;




     2.  The analysis  of  blind  duplicate  samples;




     3.  The analysis  of  reagent  blank samples;




     4.  The analysis  of  a chlorinated hydrocarbon pesticide mixture




         carried through  the procedure;




     5.  The analysis  of  two serum pool samples.









QUANTITATION




Each chromatogram was  quantitated as Aroclor  1242  and Aroclor 1254 as




described in Appendix  B.  Figure  1 is  a typical  chromatogram of  a serum




sample extract.  Figure 2 is a  standard Aroclor  1242  chromatogram at  the




same gas chromatographic  conditions.   Figure  3  is  a standard Aroclor




1254 chromatogram.  Figure 1 shows the  baseline  used  for  peak height




measurement.  The same convention was  used  for  the  construction  of the




baseline on all samples.  The peak heights were  summed  for  all matching




peaks (within a 4 percent retention time window) for  the  respective




Aroclor pattern.  Appendix C shows the  steps  of  typical sample




quantitation.

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Figure 1.  Typical Chromatogram of a Serum Extract

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Figure 2.  Chromatogram of Aroclor 1242

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Ul
                     Figure 3.  Chromatogram of Aroclor 1254

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Each analytical run contained a PCB Pool A and a PCS Pool B  sample.




These pool samples consisted of serum prepared and provided  by  CDC.




Aroclor 1242 was spiked into PCB Pool B.  Appendix D is a summary  of  CDC




analytical results for these two pool samples.








Table 1 is a summary of the inter-run statistics for the PCB pool




samples as analyzed by ESE.  PCB Pool A contained a mean Aroclor 1242




concentration of 6.2 ng/ml and a mean Aroclor 1254 concentration of 7.4




ng/ral using the quantitation convention described above.  These low PCB




levels are very near the analytical detection limit of the method.  At




these levels, the analytical relative standard deviation is  approxi-




mately 36 percent.  PCB Pool B was spiked by CDC with Aroclor 1242 to a




calculated concentration of 81.1 ng/ml.  The mean Aroclor 1242 concen-




tration reported for the 26 analytical runs is 80.5 ng/ml with a




relative standard deviation of 10.7 percent from run to run.  The back-




ground level of Aroclor 1254 in PCB Pool B is 17.7 ng/ml with about a 30




percent relative standard deviation.









Table 2 summarizes inter-laboratory statistics for the PCB pool samples.




The correspondence between CDC and ESE results for Aroclor 1242 in these




pool samples is good.  It should be noted in Table 2 the standard




deviation of the ESE analyses reflects inter-run variability, while the




standard deviation for the CDC analyses reflects intra-run variability.




For PCB Pool B, the ESE mean result is 99 +_ 10 percent of the Aroclor




1242 spiked amount and 92 +_ 10 percent of the mean CDC result.

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Table 1.  Inter-Run Statistics for PCB Pool Samples
                    Aroclor 1242
                                   Aroclor 1254
PCB Pool A
X  =  6.2 ng/ral




n  =  26 runs




a  =  _+  2.4 ng/ml




a% =  + 38.8%
X  =
                                                  n  -
                               7.4 ng/ml




                               26 runs




                         cr  =  +_ 2.5 ng/ml




                         a% =  +• 33.9%
PCB Pool B
X  =




n  =




0  =




a% =
80.5 ng/ml




26 runs




4-8.5 ng/ml




+ 10.7%
X  =  17.7 ng/ml




n  =  26 runs




0  =  _+ 5.4 ng/ml




a% =  + 30.3%

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Table 2.  Inter-Laboratory Comparison of PCB Pool Sample Results
                                    Mean Cone.
                                   Aroclor 1242      No. of     Standard
                                     (ng/ml)        Analyses   Deviation
PCB Pool A
CDC Analysis,
ESE Analysis,
PCB Pool B

Aroclor 1242*
Aroclor 1242

Spiked Aroclor 1242*
CDC Analysis,
ESE Analysis,
Aroclor 1242*
Aroclor 1242

6.9
6.2

81.1
87.5
80.5

5
26

NA
5
26

+_ 0.6
± 2'4

NA
+_ 5.8
_+ 8.5
* Data from Virlyn W. Burse memo dated May 5,  1977 (Appendix D)
                                8

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CONFIRMATION

Confirmation of PCS was accomplished  by  perchlorination to decachloro-

biphenyl (DCS) using antimony pentachloride  as  the  derivatizing reagent

as described by Armour (1973).  Thirteen percent  of the total number of

samples were subjected to perchlorination.   The perchlorination proce-

dure is described in detail in Appendix  E.   Tests have  shown (ESE, 1977)

that this perchlorination procedure yields 100  _+  4.2  percent recovery of

Aroclor standard solutions over the range of 103 ng to  10  ug.



The results of the confirmatory analyses  along  with the pattern matching

results are tabulated in Table 3.  This  table shows the pattern analyzed

values for Aroclor 1242 and Aroclor 1254, the calculated DCB concentra-

tion based on the pattern matching results,  the analyzed DCB concentra-

tion after perchlorination of the sample, and the percent  correspondence

between the actual DCB value and the  calculated DCB value.   Conversions

between Aroclor and DCB concentrations were  calculated,  using the

factors of Armour (1973).  For example,  the  calculation of DCB

concentration in sample A0102 is as follows:

  15 ng/ml Aroclor 1242 . 2 ng/ml Aroclor 1254  _ 32  _„/_-, „_ DrR
            0.52                   0.65               S



Implicit in the use of these conversion  factors is  the  assumption  that

Aroclor 1242 and Aroclor 1254 are present in  the serum  sample  in unmodi-

fied form.   This is definitely not the case  and, hence,  the  assumption

is a very weak point in comparison of the pattern matching results with

the perchlorination results.

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            Table 3.
 CDC No.     Name/Sample
                                                                      Analyzed
A0102




A0103
B0103
40-01




01-01




11-01




46-01
84-03




14-03




.16-01
         PCB pool B
PCB pool B




Reagent blank




PCB pool B









Reagent: blank




PCB pool B
         Reagent blank




         PCB pool B
Run No.
1
1
1
2
3
3
3
4
4
4
5
7
8
8
8
8
9
10
Sequence
No.
9
2
13
1
12
9
4
9
3
4
4
11
1
3
9
11
10
6
Sample
Vol. (ml)
2.5
4.3
3.6
2.5
5.0
2.5
4.6
5.0
2.5
4.2
3.2
5.0
5.0
5.0
5.0
4.4
3.5
3.2
Aroclor 1242
(ng/ml)
95
15
11
96
0
83
16
4
98
12
9
19
7
0
82
7
11
14
Aroclor 1254
(ng/ml)
21
2
5
17
0
20
8
1
9
9
12
23
37
0
17
24
1
15
DCB
(ng/ml)
215
32
29
211
0
191
43
10
202
37
36
72
70
0
184
50
23
50
DCB
(ng/ml)
166
18
15
133
11
150
30
7
132
26
23
63
50
6
134
50
18
17
• X. J.VJU
DCB Calculated
%
77.2
56.3
51.7
63.0
—
78.5
69.8
—
65.3
70.2
65.1
87.7
71.4
—
72.8
100.0
78.3
34.0

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Analyzed
Calculated  Analyzed  11CB Analyzed

Cue No. Name/Sample
-sss
17-01
81-01
53-01
79-04
Nanograde hexane
20-03
23-02
PCB pool B
78-01
Reagent blank
PCB pool B
67-01
86-01
82-02
80-01
57-02
74-01
76-01

Run No.
11
12
13
13
—
14
15
16
16
16
17
17
18
19
20
21
22
23
Sequence
No.
1
11
3
9
—
5
6
3
7
13
1
9
8
8
10
12
5
6
Sample
Vol. (ml)
5.0
3.8
5.0
4.8
5.0
5.0
4.2
5.0
3.8
5.0
4.8
5.0
5.0
5.0
5.0
5.0
3.1
4.7
Aroclor 1242
(ng/ml)
11
20
12
15
0
34
20
81
58
2
71
14
14
10
14
15
33
161
Aroclor 1254
(ng/ml)
9
24
12
15
0
29
26
25
40
0
19
10
17
15
27
12
23
92
DCB
(ng/ml)
35
75
41
52
0
110
78
194
173
4
166
42
53
42
68
47
99
451
DCB
(ng/ml)
14
25
7
19
0
37
16
108
127
13
119
17
36
24
52
27
35
167
DCB Calculated"
%
40.0
33.3
17.1
36.5
—
33.6
20.5
55.6
73.4
—
71.5
40.5
68.3
57.1
76.5
57.4
35.4
37.0

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Analyzed	  Calculated  Analyzed  OCR Analyzed
CUC No.
13-06
—
—
021337
—
—
64-01
—
021338
-Name/Sample

DCB
PCB pool B

Aroclor 1254
PCB pool B

Reagent blank

Run No.
24
—
25
25
—
26
26
26
26
Sequence
No.
3
—
1
3
—
6
7
1Q
13
Sample
Vol. (ml)
2.4
1.0
5.0
3.1
0.5
5.0
4.3
5.0
5.0
Aroclor 1242
(nR/ml)
5
0
70
10
0
72
18
0
220
Aroclor 1254
(ng/ml)
12
0
18
10
480
11
8
1
63
DCB
(ng/ml)
28
2140
162
35
738
155
47
2
520
DCB
(ng/ml)
25
2100
133
22
666
96
14
2
65
DCB Calculated
7,
89.3
98.1
82.1
62.9
90.2
61.7
30.8
—
12.5

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Reagent blank analyses were conducted during each sample run.  Statisti-

cal analysis of the reported reagent blank data is presented in Table 4

for both the pattern quantitation and perchlorination data.  Reagent

blank values are uniformly low.



Table 5 summarizes all analytical results for PCS Pool B.  The

perchlorination results are 83 +_ 13 percent of the CDC reported Aroclor

1242 spike, and 71 _+ 12 percent of the ESE reported total Aroclor

concentration.



For the 26 serum samples analyzed by both pattern matching and

perchlorination, the mean analyzed DCB value is 51.4 percent of the mean

calculated DCB value.  The correlation coefficient between the analyzed

and calculated DCB values is 0.89.  Linear regression analysis yields

the following empirical relationship.


analyzed   = nT**«/analyzed Aroclor 1242 + analyzed Aroclor 1254V  g^Q
DCB (ng/ml)   ' \        0.52                     0.65        )
                                 13

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Table 4.  Reagent Blank Statistics
Pattern Quantitation




                   Aroclor 1242                   Aroclor  1254




                     n  =  36                       n  =   35




                     I  =  0.6 ng/ml                X  =   0.3 ng/ml




                     a  =  jf 1.0 ng/ml              cr  =   +_ 0.7  ng/ml




Perchlorination




                       DCB




                     n  =  5




                     "X  =  7.8 ng/ml  as DCB




                     a  =  + 4.3 ng/ml as DCB
                                  14

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Table 5.  Comparison of Pattern versus Perchlorination  Results  for PCB
          Pool B
                                            PCB (ng/ml)    DCB  (ng/ml)


Spiked Aroclor 1242*                         81.1           156        (c)

Pattern Analyzed Aroclor 1242 (n=26)         80.5 _+ 8.5     155  +_ 16   (c)

Pattern Analyzed Aroclor 1254 (n=26)         17.7 +_ 5.4      27  +_ 8    (c)

Perchlorination Analyzed PCB Pool B (n=9)    NA             130 _+ 21   (a)
* Data from Virlyn W. Burse memo dated May 5, 1977 (Appendix D)
(a) Analytical result
(c) Calculated DCB concentration using conversion factors of Armour
    (1973)
                                  15

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REFERENCES

Armour, J. A.  1973.  Quantitative Perchlorination of Polychlorinated
     Biphenyls as a Method for Confirmatory Residue Measurement and
     Identification.  Journal of the Association of Official Analytical
     Chemists, 56(4):987-993.

Environmental Science and Engineering, Inc.  1977.  Unpublished data.
                                   16

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    APPENDIX A
ANALYTICAL RESULTS

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CDC
Number
A0101
A0102
A0105
A0106
A0107
A0108
A0111
A0112
A0113
A0114
A0115
A0116
A0117
A0118
A0121
A0122
A0124
A0125
B0101
B0102
B0103
B0204
B0405
B0406
39-02
	
40-01
90-01
85-01
01-02
01-02
01-06
PPM
Birthdate 1242
0.009
0.015
0.008
0.006
0.011
0.005
0.005
0.009
0.011
	 0.021
0.011
0.015
	 0.007
0.008
0.002
0.003
0.007
0.005
0.007
	 0.013
0.016
0.020
0.011
0.014
7/03/38 0.003
5/16/42 0.006
1/02/50 0.012
11/16/39 0.016
12/22/55 0.011
7/16/57 0.020
' 8/19/33 * 0.011
12/29/60 0.008
PPM
1254
0.002
0.002
0.002
0.004
0.007
0.004
0.007
0.006
0.005
0.002
0.005
0.003
0.002
0.003
0.003
0.004
0.003
0.003
0.001
0.005
0.008
0.005
0.017
0.009
0.003
0.008
0.009
0.005
0.006
0.010
0.008
0.009

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CDC
Number
01-05
01-04
01-01
91-01
39-03
05-02
04-01
76-03
02-02
05-01
02-01
07-01
06-01
88-02
08-06
08-01
08-02
08-04
08-07
10-01
50-01
10-02
11-01
79-05
46-01
46-02
11-02
50-02
51-01
46-03
47-02
84-03
Birthdate
8/26/64
8/14/38
8/26/33
2/26/45
3/20/67
10/10/15
5/13/07
8/11/72
8/03/43
7/04/19
10/11/41
4/05/12
2/15/34
4/17/30
8/25/71
8/02/34
6/10/40
3/28/63
6/07/64
10/30/51
12/02/25
11/22/47
5/01/25
9/30/71
1/21/42
1/02/63
11/20/21
3/06/24
10/12/55
10/31/61
2/24/45
1/03/72
PPM
1242
0.011
0.019
0.009
0.011
0.005
0.005
0.010
0.008
0.015
0.004
0.051
0.005
0.006
0.042
0.004
0.010
0.007
0.003
0.002
0.002
0.011
0.004
0.019
0.018
0.007
0.005
0.010
0.031
0.016
0.020
%
0.015
0.007
PPM
1254
0.005
0.002
0.012
0.006
0.003
0.009
0.007
0.005
0.008
0.014
0.020
0.005
0.006
0.010
0.006
0.013
0.009
0.005
0.006
0.007
0.011
0.007
0.023
0.025
0.024
0.019
0.018
0.032
0.023
0.014
0.010
0.024

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CDC
Number
12-02
47-01
12-01
13-03
13-07
13-02
14-03
87-01
13-01
13-04
14-01
16-01
16-02
15-02
15-01
79-03
17-01
17-02
52-02
63-01
55-01
56-01
18-01
88-03
18-02
19-01
59-02
19-03
48-01
48-04
81-01
48-02
Birthdate
1/30/31
3/03/42
6/19/29
6/25/61
10/03/64
4/18/40
2/23/60
7/31/48
1/02/39
2/16/63
9/02/36
2/23/54
8/23/53
4/11/32
12/11/28
7/13/68
1/02/49
1/14/50
11/30/47
6/12/41
2/29/29
8/14/46
7/12/45
8/01/56
2/16/45
10/04/47
5/09/28
9/09/66
6/12/39
6/26/68
11/18/40
4/28/43
PPM
1242
0.007
0.000
0.000
0.018
0.007
0.003
0.011
0.002
0.008
0.015
0.022
0.014
0.018
0.047
0.034
0.044
0.020
0.019
0.007
0.024
0.003
0.004
0.012
0.011
0.023
0.009
0.012
0.009
0.015
0.002
0.020
0.008
PPM
1254
0.021
0.009
0.010
0.010
0.001
0.002
0.001
0.004
0.002
0.004
0.006
0.015
0.009
0.014
0.011
0.023
0.008
O.OOS
0.006
0.009
0.026
0.006
0.005
0.010
0.006
0.011
0.016
0.004
0.010
0.011
0.024
0.013

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CDC
Number
48-03
53-01
59-01
53-02
65-01
79-04
80-02
49-02
61-01
49-01
20-03
92-01
20-04
20-01
88-04
77-01
22-01
21-01
23-01
23-02
24-02
26-02
87-02
83-01
45-01
25-02
27-01
29-01
78-01
29-02
28-02
79-01
Birthdate
11/10/63
4/10/31
1/11/28
5/28/21
8/07/40
1/18/70
6/30/03
11/06/15
6/01/05
9/28/24
3/05/40
11/18/46
7/21/71
8/17/40
9/06/60
1/14/22
1/28/39
12/02/48
1/16/42
4/07/44
4/16/27
9/14/13
5/08/51
4/07/36
9/11/34
9/22/16
9/08/46
2/22/19
3/18/31
9/28/15
9/07/65
1/03/46
PPM
1242
0.012
0.012
0.041
0.004
0.013
0.015
0.020
0.032
0.019
0.014
0.034
0.010
0.009
0.001
0.011
0.009
0.006
0.010
0.011
0.020
0.014
0.040
0.007
0.026
0.009
0.006
0.002
0.006
0.058
0.002
0.010
0.013
PPM
1254
0.008
0.012
0.025
0.011
0.007
0.015
0.022
0.053
0.014
0.011
0.029
0.010
0.008
0.006
0.020
0.014
0.017
0.012
0.012
0.026
0.015
0.016
0.006
0.011
0.041
0.009
0.004
0.013
0.040
0.006
0.014
0.024

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CDC
Number
_«.
73-01
71-01
66-01
61-02
67-01
78-02
88-01
24-01
26-01
30-01
30-02
86-01
30-01
54-01
76-02
28-01
42-01
62-01
01-07
82-02
84-01
74-01
34-01
52-01
33-02
75-01
33-01
35-01
80-01
58-01
Birthdate
8/09/15
7/25/29
5/03/40
9/27/53
5/29/33
3/23/32
11/21/34
9/24/28
6/22/24
8/25/08
6/27/34
4/28/34
5/23/26
11/24/57
11/04/19
6/01/49
1/20/29
6/23/34
9/23/02
10/27/59
10/19/51
3/22/50
12/15/37
8/25/52
5/17/51
10/22/51
9/23/18
9/07/53
8/18/—
12/11/89
5/31/49
PPM
1242
0.008
0.011
0.016
0.017
0.004
0.014
0.008
0.015
0.004
0.025
0.003
0.011
0.014
0.012
0.006
0.011
0.004
0.008
0.011
0.009
0.010
0.008
0.007
0.015
0.003
0.005
0.014
0.007
0.004
0.014
0.005
PPM
1254
0.015
0.015
0.009
0.014
0.005
0.010
0.018
0.010
0.012
0.018
0.014
0.017
0.017
0.014
0.011
0.017
0.018
0.014
0.012
0.012
0.015
0.012
0.011
0.009
0.007
0.012
0.024
0.018
0.010
0.027
0.017

-------
CDC
Number
38-03
44-01
70-01
41-01
36-01
84-02
57-01
57-02
38-02
	
38-01
74-01
68-04
68-02
79-02
68-01
68-03
69-01
43-01
76-01
41-05
60-05
02-03
13-05
13-06
14-05
16-03
17-03
19-04
22-02
22-03
23-03
Birthdate
8/22/59
10/01/26
6/27/26
8/22/45
10/25/58
7/16/50
9/04/15
4/14/20
9/10/35
5/07/50
2/12/43
8/25/48
4/18/24
2/24/21
2/26/47
9/14/51
3/07/45
5/07/21
10/10/51
9/27/48
	
	
	
	
	
	
	
	
	
	
	
	
PPM
1242
0.004
0.002
0.004
0.009
0.008
0.010
0.010
0.015
0.013
0.007
0.012
0.033
0.012
0.014
0.020
0.015
0.003
0.169
0.084
0.161
0.004
0.004
0.005
0.002
0.005
0.003
0.004
0.004
0.004
0.003
0.003
0.003
PPM
1254
0.008
0.010
0.007
0.010
0.011
0.014
0.026
0.012
0.018
0.012
0.011
0.023
0.007
0.032
0.022
0.012
0.009
0.103
0.032
0.092
0.009
0.025
0.004
0.006
0.012
0.006
0.003
0.005
0.008
0.020
0.013
0.007

-------
CDC
Number Birthdate
23-04
38-04
38-06
41-02
41-03
41-04
64-01
60-02
60-03
	 4/30/62
	 in/19/ss
	 8/17/40
021336
021338
B7
B8
B9
021337 1/02/50
PPM
1242
0.003
0.004
0.002
0.003
0.006
0.000
0.018
0.006
0.008
0.003
Onn?
• UU J
0.002
Oflfi9
. \J\Jf.
0.160
0.220
0.012
0.015
0.030
0.010
PPM
1254
0.006
0.013
0.009
0.007
0.008
0.008
0.008
0.007
0.011
0.011
n 01 9
u . u J. £.
0.004
Onns
. uu_?
0.066
0.063
0.007
0.011
0.017
0.010

-------
     APPENDIX B
ANALYTICAL PROCEDURE

-------
                      PROCEDURE FOR

ANALYSES OF POLYCHLORINATED BIPHENYL (PCB) IN BLOOD SERUM
                       Prepared by:

                    Toxicology Branch
               Clinical Chemistry4Division
                  Bureau of Laboratories
                Center for Disease Control
                       Atlanta, GA
                           and
       ENVIRONMENTAL SCIENCE AND ENGINEERING, INC,
           P. 0. Box 13454, University Station
                  Gainesville, FL  32604

-------
I.  ANALYTICAL PROCEDURE




    A.  Extraction




        1.  Pipet 5 ml of serum into a 12.5 cm length culture tube  to




            which was added 4 ml of methanol (all solvents should be of




            pesticide quality and stored in glass or teflon containers),




            attach a teflon-lined screw cap.




        2.  Mix the contents of the tube on a rotary mixer (50-55 rpm)




            for 4 minutes.




        3.  Add 5 ml of hexane-ethyl ether (1:1,  V/V) .




        4.  Attach the teflon-lined screw cap and place on a rotary




            mixer (50-55 rpm) for 15 minutes.




        5.  Centrifuge at 2000 rpm for 2-5 minutes.




        6.  Transfer the upper solvent layer by pipette to a 25 ml




            graduated Kuderna-Danish concentrator tube.




        7.  Carry out steps 3-6 twice more with fresh hexane-ethyl ether




            solutions; the extractions are combined in the concentrator




            tube.




        8.  Concentrate the extract to approximately 0.5 ml under a slow




            stream of dry organic-free nitrogen.









    B.  Saponification




        1.  Add 2 ml of a 2% (V/W methanolic potassium hydroxide




            solution, add an ebbulator.




        2.  Attach a micro-snyder column.




        3.  Using a. Kontes Tube Heater, allow the contents to gently




            boil, reducing the volume to 0.3 ml.

-------
        Note:  If a precipitate has formed, add  a  few  drops  of  2%




        methanolic KOH and warm gently in steam  with swirling until




        the precipitate dissolves.



    4.  After the solution has cooled slightly,  add about 2 ml  of




        methanol-water (1:1).




    5.  Allow solution to reach room temperature and add 2 ml hexane




        into tube.




    6.  Stopper tube and shake vigorously.




    7.  Pipet out top layer.




    8.  Repeat steps 7-9 two additional times.








C.  Column Preparation and Elution




    1.  Activate in a 200°C oven for 24 hours Woelm silica gel




        activity Grade I and granular sodium sulfate (Mallinckrodt




        #8024).  Store in air tight flasks.




    2.  Deactivate the silica gel with 3% water  (W/V) and allow to




        equilibrate at least 5 hours before use.




    3.  Pack a small mat of glass wool into the bottom of a 7 mm




        i.d. x 200 mm chromatography column, which contains a 50 ml




        reservoir and a teflon stopcock.




    4.  Mix 3g of silica gel with approximately 50 ml of hexane and




        pour the slurry into the column.




    5.  Allow the silica gel to settle and then top with a 5-7 g




        layer of sodium sulfate.

-------
 6.  Elute the columns with 20 ml of hexane  and  as  the  last  of




     the hexane enters the sodium sulfate  layer,  add  the




     concentrated, saponified extract.  Place  graduated 25 ml




     receiver under column.  Open stopcock and allow  extract  to




     just enter the sodium sulfate layer before  closing stopcock.




 7.  Rinse the concentrator tube with 1 ml of hexane  and  add  to




     the column.  Open stopcock and allow rinse  to  just enter  the




     sodium sulfate layer before closing stopcock.




 8.  Rinse the micro-Snyder-concentrator tube assembly with 2 ml




     of hexane and add to the column.  Open  stopcock  and  allow




     rinse to just enter sodium sulfate layer before  closing




     stopcock.  Note:   The sodium sulfate layer  should never




     become "dry".




 9.  Carefully add 25 ml hexane to the column (without disturbing




     the sulfate layer).  Open stopcock and  allow column  to elute




     until the 25 ml receiver is filled.




10.  Concentrate eluant under a slow stream  of dry, organic-free




     nitrogen to approximately 1.0 ml.




11.  Rinse the assembly by allowing 1-2 ml of hexane  to run down




     the sides of the column.




12.  The solution is then reduced to exactly 1 ml under nitrogen




     and is now ready for GLC analysis.  Note:  After analyzing




     by GLC it may be necessary to further concentrate or dilute




     this solution in order to re-analyze.

-------
    D.  GLC Analysis




        All analyses will be on a Varian 2700 GLC with electron capture




        detector.  The flow rate which gives at least a 10% peak height




        to valley resolution between p,p'-DDE and dieldrin on the 6 ft x




        1/8 in.  column containing 1.5% OV-17-1.95%QF-1 should be used.




        The injector and detector temperature must be higher than the




        column which is run isothermally at 200°C.  Also the operational




        range for the electrometer should be 10~10 and the attentua-




        tion should be x!6 or more.  All analyses must be run at the




        same electrometer setting; therefore, it might be necessary to




        dilute the sample.









    E.  Confirmation




        A minimum of five percent of the samples will be confirmed by




        perchlorination to decachlorobiphenyl to insure that PCB is




        indeed being analyzed.  The samples of the highest concentration




        will be selected by ESE for this analysis.









II. DESCRIPTION OF A "RUN"
    A.  Unknown samples and controls—to be put through the entire




            analytical procedure




            Eight unknown samples (exception:  the 27 samples from




            Mississippi will be in 3 runs of 9 samples each).




        2.  Two controls (made from human serum at CDC and designated as




            PCB Pool A and PCB Pool B).

-------
    3.  One chlorinated hydrocarbon control containing 1 ml  of  the




        following:  Y and  3 hexachlorocyclohexane;




        heptachlorepoxide; o,p'DDE; dieldrin; p,p'-DDE; o.p'DDT,




        p,p'-DDD and P,p'-DDT with a range of 50-100 ng/ml.  This




        control will be prepared by ESE.  When running the unknown




        samples, any peaks that have the same retention times as the




        control-should not.be included for quantitation.




    4.  Two reagent blanks, the second blank should be composed of




        solvents that will be used in the subsequent run to validate




        purity of solvents (especially useful when new solvent




        bottles or lot numbers are used in the next run).








B.  Standards—to be analyzed by gas liquid chromatography




    1.  Three Aroclor 1242 standards of varying concentrations.  The




        upper concentration should be chosen so that the tallest




        peak is approximately 80% full scale.  Then without




        adjusting the attenuation, the next two concentrations




        should be chosen to encompass the working range.




    2.  Three Aroclor 1254 standards of one concentration.  This




        concentration should be chosen such that it falls in the




        middle of the 1242 working concentration range.

-------
III.  INJECTION ORDER




      Std 1242 (High)                     Std 1254 II




      Sample                              Sample




      Sample                              Sample




      Std 1254 I                          Std 1242 (Medium)




      Sample                              Sample




      Sample                              Sample




      Std 1242 (low)                      Std 1254 III




      Sample                              Sample




      Sample                              Sample









 IV.  QUANTITATION




      A.  As 1242




          Baselines for the chromatograms of the standards and the




          samples will be drawn by following down the sloping solvent




          tail and continuing out until a stable baseline is




          established.  The retention times of all the peaks in the




          three 1242 standards will be measured from the solvent front




          to the nearest tenth of a millimeter.  The three retention




          time values for each specific peak in the 1242 standard (i.e.




          from the three separate injections) are averaged and a four




          percent retention time window is then generated from this




          average retention time value.  The four percent retention time




          window (i.e. plus and minus two percent of the retention time




          distance of each particular peak of the 1242 standard) is then




          applied for matching of sample peaks to standards.

-------
Samples that have peaks within  these  windows that are offscale

will be repeated to obtain  all  quantifiable peaks on scale.

The chlorinated hydrocarbon control is  gas  chromatographed in

the same range as the samples (i.e. if  all  or nearly all

samples are in 1.0 ml and 5 microliters  are being injected

then this will be followed  for  the chlorinated hydrocarbon

control).  Peaks in the chlorinated hydrocarbon control  that

are within the retention time windows of the standard and are

offscale in the control will be disregarded and these particu-

lar peaks will not be quantified in the  standards,  blanks,  or

in the samples since they may be attributable to  pesticides in

the samples and not specific PCB's.  Peaks  in the blank,  which

is analyzed in the same range as the samples,  that  fall  in  the

retention time windows are measured and  summed.   This  value is

used as zero nanograms injected in the   generation  of a

standard curve.  All peaks  in each of the tree  Aroclor 1242

standards that have not been discarded are  measured and  summed

and, together with the blank value, are  used  to generate  the
                              k
standard curve.  The curve  is established by  plotting  the

total peak height as the Y axis versus nanograms  of Aroclor

1242 injected on the X axis.  The height  in millimeters  of  all

sample peaks that have not be discarded  and  are in  the

specific windows are measured and summed  and  the  total peak

height value is then plotted on the Y axis  of  the standard

curve to .give nanograms of Aroclor 1242  injected  for  each

sample.  All sample analysis points on this  curve must be

-------
    between the high standard and  the  origin.   Calculations are




    then carried out to give micrograms Aroclor 1242  per




    milliliter sample.








B.  As 1254




    The retention times of all the peaks in  the Aroclor  1254




    standards will be measured from the solvent front  to the




    nearest tenth of a millimeter.  The three retention  time




    values for each specific peak  in the Aroclor 1254  standards




    (i.e. one for each injection) are  averaged  and a  four  percent




    window is then generated from  this value.   Peaks  in  the




    chlorinated hydrocarbon control that are within the  retention




    time windows of the Aroclor 1254 standards  and are offscale in




    the control will be disregarded as was done  in the Aroclor




    1242 quantitation.  All peaks in each of the three Aroclor




    1254 standards that have not been  discarded  are measured and




    summed.  The total peak height of  the three  separate Aroclor




    1254 standards must agree within +_ 10%.  An  average  total peak




    height response factor is then calculated,  i.e. total peak




    height per nanogram Aroclor 1254 injected.   Sample peaks that




    fall within the retention time windows of the Aroclor 1254




    standard,  and have not been discarded must be onscale and are




    then measured and summed to give a total peak height of




    Aroclor 1254 in the samples.  This value is  then divided by




    the Aroclor 1254 response factor to give nanograms of Aroclor




    1254 injected for each sample.  Calculations are then carried




    out to give micrograms Aroolor 1254 per millimeter sample.

-------
c.   REPORTING OF RESULTS




     The chromatograms will  include  the  following information:  initials




     of operator; date; run  number;  chart  speed;  temperature of




     injector; detector; and  column;  carrier  gas  and  flow rate; standing




     current; percent full scale  deflection at  range  of 10~° and




     attenuation of 32; operating electrometer  settings;  volume of




     sample injected; final  sample volume; and  volume of  serum sample.




     The chromatograms of the standards  (1242 and 1254) will include




     concentration and volume injected.









VI.  STORAGE OF SAMPLES




     All serum samples will  be stored  frozen  until  analysis.  Any unused




     serum sample will be stored  for  at  least 2 months  after notifica-




     tion of results.









VII. PREPARATION AND STORAGE OF STANDARD PCB SOLUTIONS




     Aroclor stock solutions will be  prepared using the Aroclor obtained




     directly from Monsanto Company,  St. Louis, Missouri,  from the




     following lots:




                  Aroclor 1016            Lot KC10-7008




                  Aroclor 1242            Lot KC09-415




                  Aroclor 1254            Lot KD05-613




     Approximately 0.05 ml of the Aroclor is placed directly into  a  tare




     weighed 50 ml volumetric flask.  The flask with  Aroclor is then




     weighed and the mass of Aroclor  is  calculated by difference.  The




     flask is filled to volume with nanograde 2,2,4 trimethylpentane at

-------
20°C.  This solution is shaken for a period  of 4 hours  and allowed




to stand at least one full day to assure complete  solution.   This




stock solution is stored at 4°C in the dark.  Dilutions  of this




stock solution are used as Aroclor standards.  All  standards  are




stored in the dark at 4°C when not in immediate use.  They are




brought to room temperature and mixed before each use.  Working




standards should be prepared from the stock solution at  least once




per week.

-------
    APPENDIX C
SAMPLE CALCULATION

-------
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-------
        APPENDIX D
DATA FOR PCB POOL SAMPLES

-------
                                                May ^, j.977
E:IORAKDUK TO THE RECGE:

XCM:  Virlya-W.-Burse,-jieaearch-Chemist, ..Toxicology Branch^ CC,_BL

U3J:  "Characterization"  of the Third ESE  Pool
he purpose of this mer.orandui?. is  to report data obtained by this laboratory
hen an attempt was made  to  "characterize" the third set of pools prepared
or the ESE PCB project.  These pools are to be used as internal bench controls
y ESE once the analysis  of  unknowns is  commenced.

a the process of deterrining. residue levels of AR 1242, in each of the two
ools, the analyst adhered to (as  much as possible) those aspects of the
rotocol proposed for ESE (See JAL's correspondence to Charles Strattcn
fESE, April 20, 1977),  i.e.  I.D.,  II.B., Ill and IV.A.

he values for the two pools are shown in Table 1,  since the3r are to be
iewed as two separate pools no correction was made for background of AR 1242
Ethe spiked pool.

he standard curve used for  calculations is attached.
                                       Virlyn W.  Burse

Attachment
    Dr. Liddle  «"
    .Dr. Ki mb r ough
    Dr. Needh=r".

-------
"CHARACTERIZATION OF THIRD ESE PCB  POOL
•P""^
Pool I.D. Aroclor Sample I.D.
ESE Pro j eel- 1242 A5
PCD I'ool A

" ; " A4
.
11 | " Al
" i i " -A3
" » A2
ESE Project " B2
PCB Pool- B

" " Bl
i
' " » B4
11 ' " B5
11 , " B3
"Datd generated ur. 'up, proposed protocol approach
i
i
i
!
1 ; '
1 ' ,
1
Level of PCB
in ppm
0.0068


0.0072

0.0068
0.0061
0.0077
0.0948


0.0858

010921
0.0816
0.0831





PCB Added Prelim.
in ppm Stats
None x = 0.0069
G.D. - O.COOu
(Jl)% UCL = 0.0001
Nona gg?0 UCL = O.OOB7
95% LCL = 0.0057
None gg% LCL = 0.0051
None '
None
0.0811 x = 0.0875
S.D. = 0.0050
95% UCL = 0.0991
" 99% UCL = 0.1049
95% LCL = 0.0759
11 99% LCL = 0.0701
ii
it






-------
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                                                                        i
                                                                       1 .C.

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        APPENDIX E
PERCHLORINATION PROCEDURE

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           PERCHLORINATION PROCEDURE FOR CONFIRMATION OF PCB
I.  Principal.  Perchlorination of a PCB mixture using antimony penta-




    chloride (SbCl^) as the active reagent results in conversion of




    all PCB present to DCB which is a single isomeric, fully-substituted




    species.  This species has a high electron capture (EC) detector




    response.  It can also be more readily separated from other compounds




    by GC techniques than can the complex PCB mixture.  Quantitation can




    be accomplished by measurement of a single peak.








II.  Sample Preparation.  The sample extract (1.0 ml) prepared in the




    method described above is quantitatively transferred to a Kuderna-




    Danish apparatus with micro-Snyder column.








III.  Removal of Solvent.  The extracted PCB must be quantitatively




    exchanged from the hexane solvent to chloroform.  All residual hexane




    must be removed from the extract prior to perchlorination.  Even small




    amounts of residual hexane will result in the formation of a black




    solid residue upon the addition of SbCl,..  This severely reduces




    PCB recovery.  The hexane is removed by azeotrophic evaporation from




    the hexane/chloroform mixture.

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il Add 3 ml of nanograde chloroform to the Kuderna-Danish receiver con-
   taining the sample extract in hexane and concentrate, using a micro-
   Synder column,  by slow boiling in a tube heater or water bath to about
   0.2 ml.  Do not allow to evaporate to dryness.


.2 Repeat step  III.l three additional times in order to remove all resi-
   dual hexane.  Rinse the micro-Snyder apparatus with a minimum amount
   of chloroform.   Final volume should be approximately 1.0 ml.


.3 Quantitatively  transfer to a reaction vial  (Figure 1)      using three
   chloroform rinses (Total rinse volume about 2 ml).


.4 Add two micro-Hengar boiling chips and immerse reaction vial upright
   in a 70°C water bath to a depth of 6 _+ 2 cm.


.5 Increase water bath temperature slowly until the solvent begins to
   boil.  Boiling temperature should be 72-76°C.


.6 Concentrate slowly to a volume of approximately 0.1 ml.  Under no cir-
   cumstances should the water bath temperature be permitted to exceed
   76°C or the solvent be evaporated to dryness.  If either of these hap-
   pen, PCB will be lost by volatilization and consequent recoveries will
   be low.  The final volume (0.1 ml) may be determined with sufficient
   accuracy by comparison of solvent level with another reaction vial
   containing 0.1  ml of chloroform.

                    *                      V
.7 When a volume of 0.1 ml is achieved, cap the reaction vial immediately
   and allow to cool.

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                          Teflon-lined cap
Figure 1.  Perchlorination reaction vial.

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IV Sample Perchlorination.




.1 To the concentrated sample extract  in  the  reaction vial add 0.2 ml of




   SbCl5                 and immediately  re-seal  the  vial  tightly




   with the Teflon-lined screw cap.









.2 Place the reaction vial  into a preheated  (160 _+  3°C)  aluminum block




   heater for a period of 15 hours.









.3 After the reaction period, remove the  reaction vial from the aluminum




   block heater and allow to cool to room temperature.   Then cool to 0°C




   in an ice water bath.









.4 Cautiously vent pressure from the vial in  a  fume hood,  directing away




   from the analyst.  Add 1 ml of 6 If HC1 to  the  cool reaction vial,




   replae the cap tightly and shake for 30 seconds.   The HC1 stops the




   perchlorination reaction.  CAUTION:  IF THE  REACTION  VIAL IS NOT COOL,




   THE ADDITION OF HC1 MAY  CAUSE DANGEROUS SPLATTERING OF  THE REAGENTS




   FROM THE CONTAINER.









.5 Add 1 ml hexane, shake vigorously for  30 seconds and  carefully draw




   off the hexane layer with a disposable pipet.









.6 Place this hexane extract on the top of a  6  mm x 12 cm  disposable




   pipet packed with 2 g of anhydrous Na2S04.   This column is prewashed




   with hexane.









.7 Repeat steps  IV.5  and  IV.6  five times  to assure complete




   extraction of all DCS from the reaction vial.

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.8  Pass two 1  ml portions of fresh hexane through the Na2SC>4 column

    and collect all fractions in a 10 ml graduated Kuderna-Danish receiving

    vial •



.9  Connect a modified micro-Snyder column to the Kuderna-Danish apparatus

                      add one Hengar boiling chip, and evaporate in a water

    bath (70°C) to less than 0.5 ml.  Care must be taken to avoid bumping

    and loss of sample.



.10 Cool the apparatus to room temperature and remove the micro-Snyder

    column.  Rinse the micro-Snyder column with sufficient fresh hexane to

    bring the volume up to 1.0 ml as indicated on the graduated Kuderna-

    Danish receiving vial.  Remove the micro-Snyder column and mix by gentle

    swirling.



.11 Transfer the extract immediately (before significant solvent

    evaporation) to a properly-labeled Hypo-vial.



.12 Close the Hypo-vial with a Teflon septum and aluminum cap.

             All PCB present in the sample has been converted to DCB in

    1.0 ml hexane.  The sample is now ready for GC analysis.



 V.  Analysis

.1  Analyze the DCB present in the perchlorinated extract by  GC/EC.   The

    following column conditions have been found to be suitable:

           Glass column:   length 6 ft.,  ID 1/8"
           1.5% OV-17/1.95% QF-1 liquid phase
           Chromosorb W-HP, 80/100 mesh support

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          N2> 35 psig  inlet,  approximately  40 ml/min.
          Column temperature—220°C
          Injection  port  temperature—240°C
          Detector Temperature—200°C

   Under  these  conditions,  DCB  will elute  in approximately 20 minutes,

   Figure 2 is  a typical  chromatogram.
.2  Quantitate as DCB by comparison of the peak area with that of a known

   concentration of pure DCB                 taking into consideration

   all concentration factors.  Care must be taken to assure the sample

   concentration and the standard concentration are near the same value

   so the EC detector is operating in the linear range.  Peak areas of

   the standard and the sample should not differ by more than a factor

   of two.

                          A - B
            ng DCB/ml  =   	S    x C

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I"
bl
                                                                                                      I  1
                                                       !    I
o                    :                      .10         :
               I                          minutes
                                      *

Figure  2.  Typical Chromatogram Showing PCBs After Perchlorination to DCB.
20

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   where  A = area of the DCS peak of the sample
          B - area of the DCB peak of the procedural blank

          S = area of DCB standard peak
          C = concentration of the DCB standard in ng
          V = volume of the sample extract
.3 To convert DCB values to approximate equivalent PCB values, Table 1

   may be employed.

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Table 1.  Factors to Mathematically Convert Decachlorobiphenyl  to  an
          Equivalent Amount of Aroclor  (5)
Aroclor
1221
1232
1242
1016
1248
1254
1260
1262
DCS
Av. No. Cl*
1
2
3
3
4
5
6
7
10
MWt
188.5
223
257.5
257.5
292
326.4
361
395.3
499
X**
0.38
0.45
0.52
0.52
0.59
0.65
0.72
0.79
1.00
*  Average whole number of chlorines calculated from percent chlorine
   substitution for a specific Aroclor

t  Molecular weight of Aroclor based on the average whole number of
   chlorines calculated from percent chlorine substitution

** X = molecular wt Aroclor/molecular wt DCS (499).  To convert ppm
   DCB to ppm of a specific Aroclor, multiply ppm x DCB by X for the
   Aroclor.

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                                   TECHNICAL REPORT DATA
                            (Pleat nod Instructions on the reverse before completing)
\. REPORT NO.
  EPA 560/6-8-007
              3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE
  Analysis of Polychlorinated Biphenyl  (PCB) in
  Human Blood Serum Samples
              8. REPORT DATE
               October 1,  1977
              9. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
  Charles L. Stratton and Paul C. Geiszler
              «. PERFORMING ORGANIZATION REPORT NO
9. PERFORMING ORGANIZATION NAME AND ADDRESS
 Environmental Science and Engineering,  Inc.
 P.O.  Box 13454
 Gainesville, Florida  32604
              10. PROGRAM ELEMENT NO.
              11. CONTRACT/GRANT NO.
               68-01-3248
12. SPONSORING AGENCY NAME AND ADDRESS
  Environmental Protection Agency
  Office of Toxic  Substances
  401 M Street, SW
  Washington, D.C.   20460
              13. TYPE OF REPORT AND PERIOD COVERED
               Final  1/77-10/77
              14. SPONSORING AGENCY CODE
               EPA-OTS
15. SUPPLEMENTARY NOTES
16. ABSTRACT
  A total of 208 human blood serum samples and two mother's milk samples were
  analyzed for polychlorinated biphenyl (PCB).  The samples were supplied  to
  Environmental Science and Engineering,  Inc. (ESE) in  frozen condition by the
  Department of Health, Education, and  Welfare, Center  for Disease Control,
  Atlanta, Georgia.   This report includes the analytical  results for these
  samples and an assessment of the degree of uncertainty  involved in the analysis.
17.
                                KEY WORDS AND DOCUMENT ANALYSIS
                  DESCRIPTORS
b.lOENTIFIERS/OPEN ENDED TERMS  C.  COSATI Field/Group
  Polychlorinated biphenyls, PCB, blood
  serum, analytical methods
IS. DISTRIBUTION STATEMENT
                                               19. SECURITY CLASS (TTia At port/
                                                 unclassified
                            21. NO. OP PAGES
                              59
  Unlimited—release to public
  j_
20. SECURITY CLASS IThii pay/if
  unclassified
                                                                          22. PRICE
CPA Form 222O-1 (S-73)

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