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Pattern designation of PCBs in human samples
Chemosphere, Vol.15, No.3, Printed in Great Britain
pp
301-307,
1986
PATTERN DESIGNATION OF PCBS IN ~ Mary S. Wolff, Alf Fischbein,
0045-6535/86 $ 3 . 0 0 + .OO 01986 Pergamon Press Ltd.
SAHPLES
Kenneth D. Rosenman, ~ Stephen H. Levin
Division of Environmental and Occupational Medicine Mount Sinai School of Medicine 1 G u s t a v e L. Levy Place, New York, New York 10029 ~New Jersey Department of Health, Occupational and Environmental Health Service ABSTRACT In order to assess the nature of PCB exposures in humans, statistical measures of PCB patterns in blood serum (as Aroclor 1254 or 1260) were made in 348 cases, representing several exposed and non-exposed groups. Although the cases were not representative of any population, most (252/348) had an Aroclor 1260 pattern, with evidence that PCB congeners in blood serum were usually derived from both Aroclor 1254 and 1260. The method is readily applied to routine packed column gc analysis. INTRODUCTION Absorption of polychlorinated biphenyls (PCBs)by humans in various population groups has been characterized according to the total PCB concentration in adipose tissue or blood. The qualitative nature of these PCB exposures has not been recorded in an objective manner. Assessment of the pattern, or relative concentration, of PCB congeners has been made primarily by visual comparison of gas chromatograms (gc) of human samples with commercial PCB mixtures such as Aroclor 1254 and 1260 (1-3). Of the 209 possible PCB isomers, many are present in commercial PCB mixtures, such as the Aroclots, in varying relative concentrations. The "pattern", or the appearance of PCB components observed as gc peaks, represents an approximation of the proportions of PCB congeners present in samples, compared with those in commercial PCB mixtures which constitute their source. The differential accumulation of specific PCB congeners in humans compared with those in commercial" products has been recognized, along with the potential toxicological implication of selective isomer retention. Since different isomeric PCB structures exert widely different toxic manifestations in animal test systems, the importance of characterizing individual PCBs in humans has received attention in recent years (4-9). Different PCB congeners have markedly different rates of metabolism in animals. The pattern, resulting from metabolism of PCB mixtures, represents a qualitative measure of relative PCB isomer retention. Thus, PCB pattern may be an important parameter in evaluating toxicological effects, as well as providing information about the source of exposure. Such was the case among patients with rice oil disease in Japan and Taiwan (8, 9). However, PCB patterns in human exposures have not been characterized systematically. Computer assisted pattern recognition techniques have recently been applied to high resolution (capillary column) gas chromatographic (gc) analyses of environmental samples, notably those in fish, using the method of principal component analysis (10). However, the necessity of interfacing the laboratory data base with additional computation facilities does not render this method readily available to most analysts. In our experience, most serum resembling Aroclor tical evaluation of 348
non-occupational exposures have shown agc pattern o f PCBs i n b l o o d 1260, rather than Aroclor 1254. In this report, we p r e s e n t a s t a t i s cases from nine study groups, which tends to corroborate our earlier
301
302
visual interpretation, but which is based on an objective measure. The statistical method, reported recently (II), was readily incorporated into our laboratory data management computer using a BASIC program. METHODS
The method for analysis of PCBs in serum, including serum preparation and packed column gc analysis, has been reported previously (12, 13). A Perkin-Elmer Sigma 1 gc and data system were used. The method of calculation and peak assignment was that of Webb and McCall (14). Routine gc reports calculate concentrations for Webb and McCall peaks 70 - 372, calibrated using Aroclor 1260. For purposes of pattern evaluation, six major peaks from 348 cases, representing data collected over several years, were studied using the Statistical Analysis System (SAS, Inc., Cary, NC 27511). The IBM computer system via the City University of New York Computer Center was used for computations. Corresponding peak areas were entered for both Aroclor 1254 and 1260 standards~ which were obtained from the U. S. Environmental Protection Agency standard library. The use of Aroclor 1248 was found applicable to only 5 of 348 cases, and was not investigated further. The visual identification of Aroclor 1248, which in our experience is present in rare cases (other than occupational exposure), may indicate that the inclusion of this standard is necessary. Peaks 70, 84, 125, 146, 174, and 280 were used, since these occur in almost all cases and usually account for more than 90 ~ of the total PCB concentration. Cases with more than one missing (non- detectable) peak were not excluded. According to the method of Lea, BramstonCook, and Tschida (11), the coefficient of variation (CV = standard deviation x lO0/mean) was calculated for each sample for peak area ratios to Aroclor 1254 and 1260 standards. For each sample, the area of each peak was divided by the area of the corresponding standard peak, giving a set of sample: standard ratios for both Aroclor 1254 and 1260 for each sample. The Dixon test (Q test) was used to eliminate outliers, according to the method reported (11, 15). This criterion requires that the sample: standard ratios be reassembled in numerical order, with the ultimate and penultimate values, both high and low, tested against the range of values. The first example shown in Table 2 would represent the set {0.66, 0.68, 0.85, 0.91, 1.0, 26} for Aroclor 1254, and the Dixon criterion (x - x ./x - xl = 0.99) would eliminate n n-i n i the value 26 from the set (for n=6, p=0.05, the criterion is 0.56). The Dixon test was performed using a SAS program, for the values representing x , x^, x q, and x of each sample. Outliers exceeding the criterion were then excluded from t~e c~icu~a~ion of ~be CV. The lesser CV of the two sets for each sample then assigned the "PCB pattern" as either Aroclor 1254 or 1260. Data were obtained non-occupati0nally citor manufacture used Aroclor 1260; pected exposure to
from 1-3 laboratory exposed persons from using Aroclor 1016, 49 persons seen in PCBs.
serum analysis batches from six studies, including 214 New York and New Jersey; 60 workers involved in capa1242, 1254; 12 25 transformer manufacture workers who the Mount Sinai Occupational Clinic, many with sus-
RESULTS AND DISCUSSION
The statistical designation of PCB patterns in non- occupationally exposed persons verified our previous impression that most cases resembled Aroclor 1260 (Table 1). Of 214 cases not occupationally exposed, most were so designated (162/214; 76~). Of the 52 cases designated as like Aroclor 1254, 26 had-been so assigned by visual inspection. Of those 52 cases, one subgroup of 21 persons had known, dietary exposure to Aroclor 1254 contaminated fish (e.g., case SI, Table 2). Aroclor 1260 was assigned as the pattern in another 24 cases from this subgroup.
Among 134 persons with potential occupational exposure, Aroclor 1254 patterns were assigned to 14 of 49 clinic patients (many having been referred with a history of PCB exposure), and 30 of 60 capacitor manufacture workers, with known past exposure to various Aroclors (12). Among 25 who worked where Aroclor 1260 was used in transformer manufacture, all had the Aroclor 1260 pattern (Table 3; case TI, Table 2). Our initial view that most human PCB patterns resembled Aroclor 1260 was based mainly on the height of peak 125 being less than half that of peak 146. Peak 125 in Aroclor 1254, under our gc conditions, has approximately 1.3 times the area of peak 146 (T~ble 4). Therefore, in cases where peak 125 on the strip chart appeared near or "greater in peak height than peak 146, an Aroclor 1254 pattern had been assigned. The contribution of peaks 70 and 84 had not been used for visual inspection. Peak 125 is not only larger in Aroclor 1254, compared with Aroclor 1260, but also contains a greater proportion of the relatively persistent isomer,
303
Table 1 Distribution of the coefficients of variation of PCB peak ratios to Aroclor 1254 or 1260 for human serum and Aroclor standards Pattern ( l e s s e r CV)
Study Group:
CV c a l c u l a t e d A r o c l o r 1260 STD
X
as
NX : STD
. . . . . . . . .
1 16 7
2 6 11 18 20
12 11 6 4
.........
Total n u m b e r :
24 90 .........
Aroclor Aroclor
1 3 18 32 49 37
18 4
X
1254 1254 NX
: CV ( ~ ) :
CV c a l c u l a t e d A r o c l o r 1260
as Aroclor Aroclor
STD
: STD
X
NX
:
. . . . . . . . .
:
---
:
. . . . . . . . .
:
: : : : : : : :
2 14
: : : : : : : :
0 10 20 30 40 50 60 70
: : : : : : : :
7 16 1
: : : : : : : :
: : : : : :
80 90 100
5 16 8 11 3 1
: : : : : : ...............
162 : 16 44 : ...............
7 18 10 11 4 2
110
120 ---
52 : : ---
1 7 9 23 21 11 9 7
2 5 28 53 30 17 22 4
X
1260 1254 NX
. . . . . . . . .
2 5 7 14 8
5 8 11 16 9
: 1 5 : 1 3 : 5 : 8 : 1 : .........
3
: 24 90 162 : 16 44 : ................ : .........
52
: 1 : 0 : 1 : : : ................
1
P a t t e r n d e s i g n a t e d by l e s s e r CV, a s A r o c l o r 1254 o r 1260. STD - s u r r o g a t e s t a n d a r d s ; X - p o t e n t i a l l y occupationally not occupationally exposed.
2,3',4,4'5-pentachlorobiphenyl, a r e more r e a d i l y m e t a b o l i z e d p b e n y l r i n g ( 3 , 9, 16, 1 7 ) .
(CV)
Sample groups: exposed; NX -
t h a n A r o c l o r 1260. The o t h e r m a j o r c o m p o n e n t s i n A r o c l o r 1 2 6 0 , because of the presence of unoccupied 3,4- positions on one b i -
P e a k a r e a s , n o r m a l i z e d t o p e a k 146, a r e g i v e n i n T a b l e s 2 a nd 4 t o s i m u l a t e i n n U m e r i c a l f a s h i o n t h e a p p e a r a n c e o f t h e gc a n a l y s e s . ( P e a k 146 c o n t a i n s m a i n l y t h e p r o t o t y p i c a l persistent PCB i s o m e r , 2 , 2 ' , 4 , 4 ' , 5 , 5 ' hexachlorobiphenyl.) The a v e r a g e r a t i o o f p e a k 125 t o p e a k 146 was i n d e e d h i g h e r among p e r s o n s a s s i g n e d t h e A r o c l o r 1254 p a t t e r n ( T a b l e 4 ) , w i t h 74 o f 96 having ratios greater t h a n 0 . 6 , 82 o f 96 g r e a t e r t h a n 0 . 5 . Among t h o s e w i t h t h e A r o c l o r 1260 pattern ( T a b l e 4 ) , 40 o f 252 h a d r a t i o s g r e a t e r t h a n 0 . 6 , 79 o f 252 g r e a t e r t h a n 0 . 5 . Peaks 70 an d 84 w e r e a l s o r e l a t i v e l y h i g h e r f o r A r o c l o r 1254 a s s i g n e d p a t t e r n s , compared with Aroc l o t 1260 p a t t e r n s , consistent w i t h t h e i r p r e s e n c e i n A r o c l o r 1254 (31% b y w e i g h t ; 1 4 ) . Low concentrations o f p e a k s 70 and 84 w e r e common among t r a n s f o r m e r w o r k e r s w i t h t h e A r o c l o r 1260 pattern (Table 4), similar t o A r o c l o r 1260 s t a n d a r d s . In contrast, other persons with the A r o c l o r 1260 p a t t e r n h a d e v i d e n c e o f s u p e r i m p o s e d a c c u m u l a t i o n o f p e a k s 70 a n d 84 f r o m o t h e r PCBs, p r o b a b l y A r o c l o r 1254. The s t a t i s t i c a l evaluation o f PCB p a t t e r n s i n t h e s e human s e r u m s a m p l e s s u g g e s t s t h a t m o s t p e r s o n s h a v e e z p e r i e n c e d a c o m b i n e d e x p o s u r e , w i t h p e a k s 70 a nd 84 d e r i v e d f r o m A r o c l o r 1254 and l a t e r p e a k s f r o m A r o c l o r 1260. The c o n c e n t r a t i o n o f p e a k s 1 2 5 - 1 7 4 may come f r o m e i t h e r Aroclor, and recent studies, i n c l u d i n g o u r own u n p u b l i s h e d r e s u l t s from our laboratory, sugg e s t t h a t t h e r a t i o o f p e a k 1 2 5 : 1 4 6 i s h i g h a f t e r r e c e n t e x p o s u r e s , b u t t h a t p e a k 125 i s m e t a b o l i z e d more r a p i d l y t h a n t h e o t h e r m a j o r p e a k s , o v e r s e v e r a l y e a r s ( 1 7 ) . Therefore, utilizat i o n o f p e a k 125 a l o n e f o r d e s i g n a t i o n o f PCB p a t t e r n may b e i n a d e q u a t e i n m o s t p e r s o n s . The statistical assessment of "pattern" incorporates p e a k s 70 a nd 8 4 , w h i c h a r e a l s o m a j o r compon e n t s of A r o c l o r 1254, and t h u s s h o u l d be a b e t t e r indicator t h a n m e r e l y p e a k 125. Furthermore, in other studies, we h a v e o b s e r v e d t h a t p e a k 70 i s t h e m o s t p e r s i s t e n t of the three p e a k s 7 0, 8 4 , and 1 2 5 , a n d may t h u s b e a more i m p o r t a n t r e f l e c t i o n o f A r o c l o r 1254 e x p o s u r e , a l t h o u g h i t may a l s o b e d e r i v e d f r o m A r o c l o r 1248 and o t h e r l o w e r c h l o r i n a t e d PCBs. P e a k 174 had similar sample-to-standard peak area ratios for either s t a n d a r d A r o c l o r (1254 or 1260), signifying its similar concentration in these standards, relative t o p e a k 146 ( 1 4 ) .
304
In the 96 cases where an Aroclor 1254 pattern was assigned, peak 280 was inevitably eliminated as an outlier. Peak 280, a major congener in Aroclor 1260, is a minor component (<1%) of Aroclot 1254 (14, 16). Peak 70 was also eliminated in 29 of 30 capacitor workers with the Aroclor 1254 pattern, where this peak was very large due to its derivation from lower chlorinated PCBs (Aroclor 1016 and 1242). On the other hand, peaks 70 and 84 constitute only 7% of Aroclor 1260, but 31% of Aroclor 1254 (14, 16). Thus, in cases where the Aroclor 1260 assignment was made, peak 70 was eliminated as an outlier in 114 of 162 non-exposed, 12 of 35 clinic patients, 29 of 30 capacitor workers. In Aroclor 1260 exposed transformer workers, only 5 of 25 had any outliers deleted (2 for peak 70), while 20 of 25 had no outliers (Table 3). This indicates a better "pattern" fit for Aroclor 1260 among directly exposed persons, than for persons with general environmental exposure which occurs through the dietary route. Data for several cases demonstrate both straightforward and ambigious PCB pattern designation using the described method. Cases with relatively median and low CV for a designated pattern were unequivocal cases (cases SI, CI, NI, T1; table 2). Peak 70 was either low or deleted in Aroclor 1260 patterns, and peak 280 was always deleted in Aroclor 1254 patterns. Cases with high CV for both Aroclor patterns can be considered to be ambiguously assigned (cases F1, N2, table 2). In such cases, the standard ratios appear to cluster in 2 subsets (case N2), or to have a low outlier or 2 high outliers not sensitive to the Dixon or MNR test (FI, N2, table 2). The distribution of CV (Table 1) as well as the average minimum of CV among non-exposed persons (39 ± 13 for Aroclor 1254, n = 52; 36 ± 15 for Aroclor 1260, n = 162), suggest that cases where the minimum CV is greater than 55%, the pattern might better be left unassigned. A difference in two CV's less than 10% (case F1, table 2) should also pre = clude an assignment, since standards vary as much as 10~ (Table 1). By excluding cases with high CV and with CV's different by less than I0~, almost 30% of the cases became unclassified
Table 2 PCB
Case ref.
Standard
70
84
peak area ratios to standards for six typical cases peak 125
number 146
174
280
cv(%)
SI
1254 1260
1.0 (21 )
0.66 ( 7.7 )
0.68 3.4
0.91 2.8
0.85 2.1
(26 ) 1.3
18 38
C1
1254 1260
0.35 ( 6.9 )
0.17 1.8
0.38 1.6
0.82 1.8
0.70 1.8
(18.0 ) 1.4
56 10
N1
1254 1260
0.27 4.1
0.38 4.3
0.42 2.3
0.58 1.7
0.51 1.3
( 5.0 ) 0.51
28 65
T1
1254 1260
0.11 0.51
0.37 0.74
0.41 0.87
1.4 1.2
1.6 1.5
(20 ) 0.76
87 38
F1
1254 1260
0.14 2.3
0.13 1.4
0.16 0.67
0.40 0.81
0.46 0.97
( 3.9 ) 0.35
62 64
N2
1254 1260
0.91 14
0.03 0.32
0.88 3.2
3.4 6.3
3.3 6.9
(60 ) 4.1
90 82
$1 C1 N1 T1 F1 N2
Area of sample PCB peaks relative to sample peak 146 0.70 0.49 0.83 (1) 0.69 0.58 0.32 0.17 0.57 (1) 0.75 1.0 0.30 0.42 0.86 (1) 0.69 0.41 0.05 0.18 0.35 (1) 0.84 0.93 0.29 0.30 0.53 (1) 0.95 0.55 0.24 0.01 0.37 (1) 0.77y 0.79
(Peak area to standard area ratios in parentheses the Dixon criterion.)
were eliminated
as outliers using
305
Table 3 Average sample to standard peak area ratios, p e a k 146, f o r v a r i o u s g r o u p s
Aroclor Standard
Sub-group .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
P a t t e r n = A r o c l o r 1254 (mean r a t i o ) peak number 84 125 146 174
70 .
.
normalized to
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
280 .
.
.
.
.
.
CV (7.) .
.
.
.
.
.
N
.
Non-exposed
1254 1260
0.69 2.6 a
0.50 a 2.0 a
0.55 0.99
(1) (1)
1.1 1.0
0.43
39 55
52
Clinic
1254 1260
0.7~ 2.2
0.5 ~ 1.5
O. 78 1.6
(1) (1)
0.99 0.89
32 56
14
0.36
Capacitor workers
1254 1260
1.5 c
0.68 2.4 c
0.89 1.7
(1) (1)
0.93 0.91
0.31 c
28 64
30
Surrogate standards
1254 1260
1.0 8.1 d
1.0 4.6 d
1.O 2.0
(1) (1)
0.98 1.0
1.0 d 0.07 d
11 103
16
Nonexposed
1254 1260
P a t t e r n = A r o c l o r 1260 (mean r a t i o ) 0.44 0.31 e 0.35 (1) 1.05 1.9 e 1.2 e 0.67 (1) 0.97
61 36
162
0.60
Clinic
1254 1260
0.5~ f 1.3-
0.30~ 0.83 z
0.36 0.82
(I) (I)
1.0 0.93
0.54 f
64 36
35
Capacitor workers
1254 1260
1.6 g 1.5 g
0.34 1.18
0.39 0.81
(I) (I)
1.0 0.94
62 38
30
0.53
Transformer workers
1254 1260
0.16. 0.68 h
0.13~ 0.24 b
0.23. 0.46 n
(1) (1)
1.1h 1.0 b
88 46
25
0.84
Surrogate standards
1254 1260
0.I~ 1.0 z
0.23 1.01
0.49. 0.931
(I) (I)
1.0 0.98
- . 1.01
73 8
24
Values without superscripts had N cases, as indicated. Cases with superscripts had peaks not detected or eliminated as outliers. For (superscript): Standard Aroclor-peak #, cases deleted. (a):1254-pk84,7; 1260-pk70,30; pk84,14; (b):1260-pk70,4; pk84,3; (c):1254-pk70,29; 1260-pk84,2; pk280,1; (d): 1254-pk280,10; 1260-pk70,I; pk84,1; pk280,2; (e):1254-pk84,66; 1260-pk70,I15; pk84,80; (f):1254-pk70,1; pk84,9; 1260-pk70,12; pk84,13; pk280,2; (g):1254-pk70~13; 1260-pk70,29; pk84~5; (h):1254p k 8 4 , 1 6 ; p k 1 7 4 , 1 ; 1260-pk70~2; p k 8 4 , 1 7 ; p k 1 2 5 , 2 ; p k 1 7 4 ~ l ; (i):1260-pk70,4; pk84,1; pk125,2; pk280,2. Peak 280 was e x c l u d e d f r o m a l l CV's c a l c u l a t e d a s A r o c l o r 1254.
(36 o f 134 e x p o s e d , 63 o f 214 n o n - e x p o s e d ) . Not c l a s s i f y i n g j u s t c a s e s where the d i f f e r e n c e i n CV's was l e s s t h a n 10%, l e f t a b o u t 20~ u n a s s i g n e d (23 o f 134 e x p o s e d , 50 o f 214 n o u e x p o s e d ) , w h i c h a l s o i n c l u d e d 26 o f 52 c a s e s w h e r e t h e minimum CV was g r e a t e r t h a n 55~. It might also be a p p r o p r i a t e t o d e n o t e s u c h c a s e s a s " m i x e d " e x p o s u r e s , f o r w h i c h a p p r o p r i a t e c r i t e r i a could be a p p l i e d .
The calculation of sample: standard ratios, for gc analyses, as Aroclor 1254 and 1260 and a and a calculation of the minimum CV for each sample is readily accomplished by a BASIC program in the laboratory computer. Since the Dixon criterion requires redimensioning an array, it is rather laborious to incorporate into the usual sample reporting scheme. However, an alternative can be used, with manual computation in questionable cases. Thus, peak 280 was always deleted in calculation of the Aroclor 1254 CV, and similarly peak 70 for Aroclor 1250, so that these peaks could be eliminated from the corresponding ratio sets in the BASIC program. Since the mean and standard deviation are used to obtain the CV, the MB-R (maximum normalized residual) can be calculated by the BASIC program for each sample (MNR = (x~- x)/sd x ~n-1), as an alternative to the Dixon test (15). If this test suggests an outlier~ the Dixon criterion can then be tested manually.
306
Table 4 Areas of PCB peaks relative to peak 146 according to pattern classification
Sub-group
PCB Pattern Assigned
70
Mean peak areas p e a k n u m b e r 84 125 146 174
Non-occupationally exposed
1254 1260
0.46 0.29
0.37 0.24
Clinic patients Capacitor workers Clinic patients CapacitorTransformerworkers Surrogate standards
1254 1254
0.45 4.2
0.34 0.50
1260 1260 1260
0.37 2.2 0.09
1254 1260
0.69 0.09
0.67 0.44
280
1254
Mean CV % 1260
N
(1) (1)
0.78 0.80
0.59 0.81
39 61
55 36
52 162
0.92 I.I
(1) (I)
0.78 0.72
0.55 0.44
32 28
56 64
14 30
0.16 0.26 0.09
0.43 0.51 0.26
(I) (I) (I)
0.85 0.79 0.80
0.93 0.72 1.3
64 62 88
36 38 46
35 30 25
0.79 0.17
1.3 0.62
(1) (I)
0.80 0.78
0.09 1.3
11 73
103 8
16 24
SUMMARY PCB pattern designation in human samples can be incorporated into routine packed- column gc analysis protocols. PCB pattern is important in recognizing occupational or unusual exposure to PCBs, compared with normal environmental exposure. PCB patterns may also indicate the presence of more toxic PCB isomers, such as those with 3,4- substitution patterns in Aroclor 1254. PCB pattern is not a concentration related phenomenon, so that exposure evaluation must include both concentration of PCBs in blood or tissue as well as PCB pattern. However, since elucidation of PCB exposure in clinical studies has relied mainly on observation of PCB concentrations outside the range of normal, utilization of PCB pattern as an adjunct to concentration can provide evidence of the origin of exposure. ACKNOWLEDGMENT The technical assistance by Ms. Marilyn Rivera and manuscript preparation are greatly appreciated.
by Mr. Sidney Sibel
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13. 14. 15.
16. 17.
Lea RE, Bramston-Cook R, T s c h i d a J . P a t t e r n r e c o g n i t i o n f o r i d e n t i f i c a t i o n and q u a n t i t a t i o n o f complex m i x t u r e s i n c h r o m a t o g r a p h y . Anal Chem 5 5 : 6 2 6 - 6 2 9 , 1983. Wolff MS, F i s c h b e i n A, T h o r n t o n J , Rice C, L i l i s R, S e l i k o f f I J . Body b u r d e n o f p o l y c h l o r i n a t e d b i p h e n y l s among p e r s o n s employed i n c a p a c i t o r m a n u f a c t u r i n g . I n t Arch Occup E n v i r o n H e a l t h 49:199-208, 1982. W o l f f , MS, Anderson HA, S e l i k o f f I J . Human t i s s u e b u r d e n s o f h a l o g e n a t e d a r o m a t i c chemic a l s i n Michigan. J Amer Ned Assoc 247:2112-2116, 1982. Webb RG and McCall AC. Q u a n t i t a t i v e PCB s t a n d a r d s f o r e l e c t r o n c a p t u r e gas chromatography. J Chromat S c i 11:366-373, 1973. S n e d e c o r GW and Cochran WG. S t a t i s t i c a l Methods, 7th e d i t i o n . Iowa S t a t e U n i v e r s i t y P r e s s , Ames, Iowa, 1980, p. 279-280; 489-490. See a l s o Dean RB and Dixon WJ. Anal Chem 23:636-638, 1951. Albro PW, C o r b e t t JT, S c h r o e d e r JL. Q u a n t i t a t i v e c h a r a c t e r i z a t i o n o f p o l y c h l o r i n a t e d b i p h e n y l m i x t u r e s ( A r o c l o r s 1248, 1254, and 1260) by gas chromatography u s i n g c a p i l l a r y columns. J Chrom 205:103-111, 1981. Chen PH, Luo ML, Wong CK, CHen CJ. Comparative r a t e s o f e l i m i n a t i o n o f some i n d i v i d u a l p o l y c h l o r i n a t e d b i p h e n y l s from t h e b l o o d o f PCB p o i s o n e d p a t i e n t s i n Taiwan. Food Chem Toxic 20:417-425,1982.
(Received in Germany 2 January i~86)
pp
301-307,
1986
PATTERN DESIGNATION OF PCBS IN ~ Mary S. Wolff, Alf Fischbein,
0045-6535/86 $ 3 . 0 0 + .OO 01986 Pergamon Press Ltd.
SAHPLES
Kenneth D. Rosenman, ~ Stephen H. Levin
Division of Environmental and Occupational Medicine Mount Sinai School of Medicine 1 G u s t a v e L. Levy Place, New York, New York 10029 ~New Jersey Department of Health, Occupational and Environmental Health Service ABSTRACT In order to assess the nature of PCB exposures in humans, statistical measures of PCB patterns in blood serum (as Aroclor 1254 or 1260) were made in 348 cases, representing several exposed and non-exposed groups. Although the cases were not representative of any population, most (252/348) had an Aroclor 1260 pattern, with evidence that PCB congeners in blood serum were usually derived from both Aroclor 1254 and 1260. The method is readily applied to routine packed column gc analysis. INTRODUCTION Absorption of polychlorinated biphenyls (PCBs)by humans in various population groups has been characterized according to the total PCB concentration in adipose tissue or blood. The qualitative nature of these PCB exposures has not been recorded in an objective manner. Assessment of the pattern, or relative concentration, of PCB congeners has been made primarily by visual comparison of gas chromatograms (gc) of human samples with commercial PCB mixtures such as Aroclor 1254 and 1260 (1-3). Of the 209 possible PCB isomers, many are present in commercial PCB mixtures, such as the Aroclots, in varying relative concentrations. The "pattern", or the appearance of PCB components observed as gc peaks, represents an approximation of the proportions of PCB congeners present in samples, compared with those in commercial PCB mixtures which constitute their source. The differential accumulation of specific PCB congeners in humans compared with those in commercial" products has been recognized, along with the potential toxicological implication of selective isomer retention. Since different isomeric PCB structures exert widely different toxic manifestations in animal test systems, the importance of characterizing individual PCBs in humans has received attention in recent years (4-9). Different PCB congeners have markedly different rates of metabolism in animals. The pattern, resulting from metabolism of PCB mixtures, represents a qualitative measure of relative PCB isomer retention. Thus, PCB pattern may be an important parameter in evaluating toxicological effects, as well as providing information about the source of exposure. Such was the case among patients with rice oil disease in Japan and Taiwan (8, 9). However, PCB patterns in human exposures have not been characterized systematically. Computer assisted pattern recognition techniques have recently been applied to high resolution (capillary column) gas chromatographic (gc) analyses of environmental samples, notably those in fish, using the method of principal component analysis (10). However, the necessity of interfacing the laboratory data base with additional computation facilities does not render this method readily available to most analysts. In our experience, most serum resembling Aroclor tical evaluation of 348
non-occupational exposures have shown agc pattern o f PCBs i n b l o o d 1260, rather than Aroclor 1254. In this report, we p r e s e n t a s t a t i s cases from nine study groups, which tends to corroborate our earlier
301
302
visual interpretation, but which is based on an objective measure. The statistical method, reported recently (II), was readily incorporated into our laboratory data management computer using a BASIC program. METHODS
The method for analysis of PCBs in serum, including serum preparation and packed column gc analysis, has been reported previously (12, 13). A Perkin-Elmer Sigma 1 gc and data system were used. The method of calculation and peak assignment was that of Webb and McCall (14). Routine gc reports calculate concentrations for Webb and McCall peaks 70 - 372, calibrated using Aroclor 1260. For purposes of pattern evaluation, six major peaks from 348 cases, representing data collected over several years, were studied using the Statistical Analysis System (SAS, Inc., Cary, NC 27511). The IBM computer system via the City University of New York Computer Center was used for computations. Corresponding peak areas were entered for both Aroclor 1254 and 1260 standards~ which were obtained from the U. S. Environmental Protection Agency standard library. The use of Aroclor 1248 was found applicable to only 5 of 348 cases, and was not investigated further. The visual identification of Aroclor 1248, which in our experience is present in rare cases (other than occupational exposure), may indicate that the inclusion of this standard is necessary. Peaks 70, 84, 125, 146, 174, and 280 were used, since these occur in almost all cases and usually account for more than 90 ~ of the total PCB concentration. Cases with more than one missing (non- detectable) peak were not excluded. According to the method of Lea, BramstonCook, and Tschida (11), the coefficient of variation (CV = standard deviation x lO0/mean) was calculated for each sample for peak area ratios to Aroclor 1254 and 1260 standards. For each sample, the area of each peak was divided by the area of the corresponding standard peak, giving a set of sample: standard ratios for both Aroclor 1254 and 1260 for each sample. The Dixon test (Q test) was used to eliminate outliers, according to the method reported (11, 15). This criterion requires that the sample: standard ratios be reassembled in numerical order, with the ultimate and penultimate values, both high and low, tested against the range of values. The first example shown in Table 2 would represent the set {0.66, 0.68, 0.85, 0.91, 1.0, 26} for Aroclor 1254, and the Dixon criterion (x - x ./x - xl = 0.99) would eliminate n n-i n i the value 26 from the set (for n=6, p=0.05, the criterion is 0.56). The Dixon test was performed using a SAS program, for the values representing x , x^, x q, and x of each sample. Outliers exceeding the criterion were then excluded from t~e c~icu~a~ion of ~be CV. The lesser CV of the two sets for each sample then assigned the "PCB pattern" as either Aroclor 1254 or 1260. Data were obtained non-occupati0nally citor manufacture used Aroclor 1260; pected exposure to
from 1-3 laboratory exposed persons from using Aroclor 1016, 49 persons seen in PCBs.
serum analysis batches from six studies, including 214 New York and New Jersey; 60 workers involved in capa1242, 1254; 12 25 transformer manufacture workers who the Mount Sinai Occupational Clinic, many with sus-
RESULTS AND DISCUSSION
The statistical designation of PCB patterns in non- occupationally exposed persons verified our previous impression that most cases resembled Aroclor 1260 (Table 1). Of 214 cases not occupationally exposed, most were so designated (162/214; 76~). Of the 52 cases designated as like Aroclor 1254, 26 had-been so assigned by visual inspection. Of those 52 cases, one subgroup of 21 persons had known, dietary exposure to Aroclor 1254 contaminated fish (e.g., case SI, Table 2). Aroclor 1260 was assigned as the pattern in another 24 cases from this subgroup.
Among 134 persons with potential occupational exposure, Aroclor 1254 patterns were assigned to 14 of 49 clinic patients (many having been referred with a history of PCB exposure), and 30 of 60 capacitor manufacture workers, with known past exposure to various Aroclors (12). Among 25 who worked where Aroclor 1260 was used in transformer manufacture, all had the Aroclor 1260 pattern (Table 3; case TI, Table 2). Our initial view that most human PCB patterns resembled Aroclor 1260 was based mainly on the height of peak 125 being less than half that of peak 146. Peak 125 in Aroclor 1254, under our gc conditions, has approximately 1.3 times the area of peak 146 (T~ble 4). Therefore, in cases where peak 125 on the strip chart appeared near or "greater in peak height than peak 146, an Aroclor 1254 pattern had been assigned. The contribution of peaks 70 and 84 had not been used for visual inspection. Peak 125 is not only larger in Aroclor 1254, compared with Aroclor 1260, but also contains a greater proportion of the relatively persistent isomer,
303
Table 1 Distribution of the coefficients of variation of PCB peak ratios to Aroclor 1254 or 1260 for human serum and Aroclor standards Pattern ( l e s s e r CV)
Study Group:
CV c a l c u l a t e d A r o c l o r 1260 STD
X
as
NX : STD
. . . . . . . . .
1 16 7
2 6 11 18 20
12 11 6 4
.........
Total n u m b e r :
24 90 .........
Aroclor Aroclor
1 3 18 32 49 37
18 4
X
1254 1254 NX
: CV ( ~ ) :
CV c a l c u l a t e d A r o c l o r 1260
as Aroclor Aroclor
STD
: STD
X
NX
:
. . . . . . . . .
:
---
:
. . . . . . . . .
:
: : : : : : : :
2 14
: : : : : : : :
0 10 20 30 40 50 60 70
: : : : : : : :
7 16 1
: : : : : : : :
: : : : : :
80 90 100
5 16 8 11 3 1
: : : : : : ...............
162 : 16 44 : ...............
7 18 10 11 4 2
110
120 ---
52 : : ---
1 7 9 23 21 11 9 7
2 5 28 53 30 17 22 4
X
1260 1254 NX
. . . . . . . . .
2 5 7 14 8
5 8 11 16 9
: 1 5 : 1 3 : 5 : 8 : 1 : .........
3
: 24 90 162 : 16 44 : ................ : .........
52
: 1 : 0 : 1 : : : ................
1
P a t t e r n d e s i g n a t e d by l e s s e r CV, a s A r o c l o r 1254 o r 1260. STD - s u r r o g a t e s t a n d a r d s ; X - p o t e n t i a l l y occupationally not occupationally exposed.
2,3',4,4'5-pentachlorobiphenyl, a r e more r e a d i l y m e t a b o l i z e d p b e n y l r i n g ( 3 , 9, 16, 1 7 ) .
(CV)
Sample groups: exposed; NX -
t h a n A r o c l o r 1260. The o t h e r m a j o r c o m p o n e n t s i n A r o c l o r 1 2 6 0 , because of the presence of unoccupied 3,4- positions on one b i -
P e a k a r e a s , n o r m a l i z e d t o p e a k 146, a r e g i v e n i n T a b l e s 2 a nd 4 t o s i m u l a t e i n n U m e r i c a l f a s h i o n t h e a p p e a r a n c e o f t h e gc a n a l y s e s . ( P e a k 146 c o n t a i n s m a i n l y t h e p r o t o t y p i c a l persistent PCB i s o m e r , 2 , 2 ' , 4 , 4 ' , 5 , 5 ' hexachlorobiphenyl.) The a v e r a g e r a t i o o f p e a k 125 t o p e a k 146 was i n d e e d h i g h e r among p e r s o n s a s s i g n e d t h e A r o c l o r 1254 p a t t e r n ( T a b l e 4 ) , w i t h 74 o f 96 having ratios greater t h a n 0 . 6 , 82 o f 96 g r e a t e r t h a n 0 . 5 . Among t h o s e w i t h t h e A r o c l o r 1260 pattern ( T a b l e 4 ) , 40 o f 252 h a d r a t i o s g r e a t e r t h a n 0 . 6 , 79 o f 252 g r e a t e r t h a n 0 . 5 . Peaks 70 an d 84 w e r e a l s o r e l a t i v e l y h i g h e r f o r A r o c l o r 1254 a s s i g n e d p a t t e r n s , compared with Aroc l o t 1260 p a t t e r n s , consistent w i t h t h e i r p r e s e n c e i n A r o c l o r 1254 (31% b y w e i g h t ; 1 4 ) . Low concentrations o f p e a k s 70 and 84 w e r e common among t r a n s f o r m e r w o r k e r s w i t h t h e A r o c l o r 1260 pattern (Table 4), similar t o A r o c l o r 1260 s t a n d a r d s . In contrast, other persons with the A r o c l o r 1260 p a t t e r n h a d e v i d e n c e o f s u p e r i m p o s e d a c c u m u l a t i o n o f p e a k s 70 a n d 84 f r o m o t h e r PCBs, p r o b a b l y A r o c l o r 1254. The s t a t i s t i c a l evaluation o f PCB p a t t e r n s i n t h e s e human s e r u m s a m p l e s s u g g e s t s t h a t m o s t p e r s o n s h a v e e z p e r i e n c e d a c o m b i n e d e x p o s u r e , w i t h p e a k s 70 a nd 84 d e r i v e d f r o m A r o c l o r 1254 and l a t e r p e a k s f r o m A r o c l o r 1260. The c o n c e n t r a t i o n o f p e a k s 1 2 5 - 1 7 4 may come f r o m e i t h e r Aroclor, and recent studies, i n c l u d i n g o u r own u n p u b l i s h e d r e s u l t s from our laboratory, sugg e s t t h a t t h e r a t i o o f p e a k 1 2 5 : 1 4 6 i s h i g h a f t e r r e c e n t e x p o s u r e s , b u t t h a t p e a k 125 i s m e t a b o l i z e d more r a p i d l y t h a n t h e o t h e r m a j o r p e a k s , o v e r s e v e r a l y e a r s ( 1 7 ) . Therefore, utilizat i o n o f p e a k 125 a l o n e f o r d e s i g n a t i o n o f PCB p a t t e r n may b e i n a d e q u a t e i n m o s t p e r s o n s . The statistical assessment of "pattern" incorporates p e a k s 70 a nd 8 4 , w h i c h a r e a l s o m a j o r compon e n t s of A r o c l o r 1254, and t h u s s h o u l d be a b e t t e r indicator t h a n m e r e l y p e a k 125. Furthermore, in other studies, we h a v e o b s e r v e d t h a t p e a k 70 i s t h e m o s t p e r s i s t e n t of the three p e a k s 7 0, 8 4 , and 1 2 5 , a n d may t h u s b e a more i m p o r t a n t r e f l e c t i o n o f A r o c l o r 1254 e x p o s u r e , a l t h o u g h i t may a l s o b e d e r i v e d f r o m A r o c l o r 1248 and o t h e r l o w e r c h l o r i n a t e d PCBs. P e a k 174 had similar sample-to-standard peak area ratios for either s t a n d a r d A r o c l o r (1254 or 1260), signifying its similar concentration in these standards, relative t o p e a k 146 ( 1 4 ) .
304
In the 96 cases where an Aroclor 1254 pattern was assigned, peak 280 was inevitably eliminated as an outlier. Peak 280, a major congener in Aroclor 1260, is a minor component (<1%) of Aroclot 1254 (14, 16). Peak 70 was also eliminated in 29 of 30 capacitor workers with the Aroclor 1254 pattern, where this peak was very large due to its derivation from lower chlorinated PCBs (Aroclor 1016 and 1242). On the other hand, peaks 70 and 84 constitute only 7% of Aroclor 1260, but 31% of Aroclor 1254 (14, 16). Thus, in cases where the Aroclor 1260 assignment was made, peak 70 was eliminated as an outlier in 114 of 162 non-exposed, 12 of 35 clinic patients, 29 of 30 capacitor workers. In Aroclor 1260 exposed transformer workers, only 5 of 25 had any outliers deleted (2 for peak 70), while 20 of 25 had no outliers (Table 3). This indicates a better "pattern" fit for Aroclor 1260 among directly exposed persons, than for persons with general environmental exposure which occurs through the dietary route. Data for several cases demonstrate both straightforward and ambigious PCB pattern designation using the described method. Cases with relatively median and low CV for a designated pattern were unequivocal cases (cases SI, CI, NI, T1; table 2). Peak 70 was either low or deleted in Aroclor 1260 patterns, and peak 280 was always deleted in Aroclor 1254 patterns. Cases with high CV for both Aroclor patterns can be considered to be ambiguously assigned (cases F1, N2, table 2). In such cases, the standard ratios appear to cluster in 2 subsets (case N2), or to have a low outlier or 2 high outliers not sensitive to the Dixon or MNR test (FI, N2, table 2). The distribution of CV (Table 1) as well as the average minimum of CV among non-exposed persons (39 ± 13 for Aroclor 1254, n = 52; 36 ± 15 for Aroclor 1260, n = 162), suggest that cases where the minimum CV is greater than 55%, the pattern might better be left unassigned. A difference in two CV's less than 10% (case F1, table 2) should also pre = clude an assignment, since standards vary as much as 10~ (Table 1). By excluding cases with high CV and with CV's different by less than I0~, almost 30% of the cases became unclassified
Table 2 PCB
Case ref.
Standard
70
84
peak area ratios to standards for six typical cases peak 125
number 146
174
280
cv(%)
SI
1254 1260
1.0 (21 )
0.66 ( 7.7 )
0.68 3.4
0.91 2.8
0.85 2.1
(26 ) 1.3
18 38
C1
1254 1260
0.35 ( 6.9 )
0.17 1.8
0.38 1.6
0.82 1.8
0.70 1.8
(18.0 ) 1.4
56 10
N1
1254 1260
0.27 4.1
0.38 4.3
0.42 2.3
0.58 1.7
0.51 1.3
( 5.0 ) 0.51
28 65
T1
1254 1260
0.11 0.51
0.37 0.74
0.41 0.87
1.4 1.2
1.6 1.5
(20 ) 0.76
87 38
F1
1254 1260
0.14 2.3
0.13 1.4
0.16 0.67
0.40 0.81
0.46 0.97
( 3.9 ) 0.35
62 64
N2
1254 1260
0.91 14
0.03 0.32
0.88 3.2
3.4 6.3
3.3 6.9
(60 ) 4.1
90 82
$1 C1 N1 T1 F1 N2
Area of sample PCB peaks relative to sample peak 146 0.70 0.49 0.83 (1) 0.69 0.58 0.32 0.17 0.57 (1) 0.75 1.0 0.30 0.42 0.86 (1) 0.69 0.41 0.05 0.18 0.35 (1) 0.84 0.93 0.29 0.30 0.53 (1) 0.95 0.55 0.24 0.01 0.37 (1) 0.77y 0.79
(Peak area to standard area ratios in parentheses the Dixon criterion.)
were eliminated
as outliers using
305
Table 3 Average sample to standard peak area ratios, p e a k 146, f o r v a r i o u s g r o u p s
Aroclor Standard
Sub-group .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
P a t t e r n = A r o c l o r 1254 (mean r a t i o ) peak number 84 125 146 174
70 .
.
normalized to
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
280 .
.
.
.
.
.
CV (7.) .
.
.
.
.
.
N
.
Non-exposed
1254 1260
0.69 2.6 a
0.50 a 2.0 a
0.55 0.99
(1) (1)
1.1 1.0
0.43
39 55
52
Clinic
1254 1260
0.7~ 2.2
0.5 ~ 1.5
O. 78 1.6
(1) (1)
0.99 0.89
32 56
14
0.36
Capacitor workers
1254 1260
1.5 c
0.68 2.4 c
0.89 1.7
(1) (1)
0.93 0.91
0.31 c
28 64
30
Surrogate standards
1254 1260
1.0 8.1 d
1.0 4.6 d
1.O 2.0
(1) (1)
0.98 1.0
1.0 d 0.07 d
11 103
16
Nonexposed
1254 1260
P a t t e r n = A r o c l o r 1260 (mean r a t i o ) 0.44 0.31 e 0.35 (1) 1.05 1.9 e 1.2 e 0.67 (1) 0.97
61 36
162
0.60
Clinic
1254 1260
0.5~ f 1.3-
0.30~ 0.83 z
0.36 0.82
(I) (I)
1.0 0.93
0.54 f
64 36
35
Capacitor workers
1254 1260
1.6 g 1.5 g
0.34 1.18
0.39 0.81
(I) (I)
1.0 0.94
62 38
30
0.53
Transformer workers
1254 1260
0.16. 0.68 h
0.13~ 0.24 b
0.23. 0.46 n
(1) (1)
1.1h 1.0 b
88 46
25
0.84
Surrogate standards
1254 1260
0.I~ 1.0 z
0.23 1.01
0.49. 0.931
(I) (I)
1.0 0.98
- . 1.01
73 8
24
Values without superscripts had N cases, as indicated. Cases with superscripts had peaks not detected or eliminated as outliers. For (superscript): Standard Aroclor-peak #, cases deleted. (a):1254-pk84,7; 1260-pk70,30; pk84,14; (b):1260-pk70,4; pk84,3; (c):1254-pk70,29; 1260-pk84,2; pk280,1; (d): 1254-pk280,10; 1260-pk70,I; pk84,1; pk280,2; (e):1254-pk84,66; 1260-pk70,I15; pk84,80; (f):1254-pk70,1; pk84,9; 1260-pk70,12; pk84,13; pk280,2; (g):1254-pk70~13; 1260-pk70,29; pk84~5; (h):1254p k 8 4 , 1 6 ; p k 1 7 4 , 1 ; 1260-pk70~2; p k 8 4 , 1 7 ; p k 1 2 5 , 2 ; p k 1 7 4 ~ l ; (i):1260-pk70,4; pk84,1; pk125,2; pk280,2. Peak 280 was e x c l u d e d f r o m a l l CV's c a l c u l a t e d a s A r o c l o r 1254.
(36 o f 134 e x p o s e d , 63 o f 214 n o n - e x p o s e d ) . Not c l a s s i f y i n g j u s t c a s e s where the d i f f e r e n c e i n CV's was l e s s t h a n 10%, l e f t a b o u t 20~ u n a s s i g n e d (23 o f 134 e x p o s e d , 50 o f 214 n o u e x p o s e d ) , w h i c h a l s o i n c l u d e d 26 o f 52 c a s e s w h e r e t h e minimum CV was g r e a t e r t h a n 55~. It might also be a p p r o p r i a t e t o d e n o t e s u c h c a s e s a s " m i x e d " e x p o s u r e s , f o r w h i c h a p p r o p r i a t e c r i t e r i a could be a p p l i e d .
The calculation of sample: standard ratios, for gc analyses, as Aroclor 1254 and 1260 and a and a calculation of the minimum CV for each sample is readily accomplished by a BASIC program in the laboratory computer. Since the Dixon criterion requires redimensioning an array, it is rather laborious to incorporate into the usual sample reporting scheme. However, an alternative can be used, with manual computation in questionable cases. Thus, peak 280 was always deleted in calculation of the Aroclor 1254 CV, and similarly peak 70 for Aroclor 1250, so that these peaks could be eliminated from the corresponding ratio sets in the BASIC program. Since the mean and standard deviation are used to obtain the CV, the MB-R (maximum normalized residual) can be calculated by the BASIC program for each sample (MNR = (x~- x)/sd x ~n-1), as an alternative to the Dixon test (15). If this test suggests an outlier~ the Dixon criterion can then be tested manually.
306
Table 4 Areas of PCB peaks relative to peak 146 according to pattern classification
Sub-group
PCB Pattern Assigned
70
Mean peak areas p e a k n u m b e r 84 125 146 174
Non-occupationally exposed
1254 1260
0.46 0.29
0.37 0.24
Clinic patients Capacitor workers Clinic patients CapacitorTransformerworkers Surrogate standards
1254 1254
0.45 4.2
0.34 0.50
1260 1260 1260
0.37 2.2 0.09
1254 1260
0.69 0.09
0.67 0.44
280
1254
Mean CV % 1260
N
(1) (1)
0.78 0.80
0.59 0.81
39 61
55 36
52 162
0.92 I.I
(1) (I)
0.78 0.72
0.55 0.44
32 28
56 64
14 30
0.16 0.26 0.09
0.43 0.51 0.26
(I) (I) (I)
0.85 0.79 0.80
0.93 0.72 1.3
64 62 88
36 38 46
35 30 25
0.79 0.17
1.3 0.62
(1) (I)
0.80 0.78
0.09 1.3
11 73
103 8
16 24
SUMMARY PCB pattern designation in human samples can be incorporated into routine packed- column gc analysis protocols. PCB pattern is important in recognizing occupational or unusual exposure to PCBs, compared with normal environmental exposure. PCB patterns may also indicate the presence of more toxic PCB isomers, such as those with 3,4- substitution patterns in Aroclor 1254. PCB pattern is not a concentration related phenomenon, so that exposure evaluation must include both concentration of PCBs in blood or tissue as well as PCB pattern. However, since elucidation of PCB exposure in clinical studies has relied mainly on observation of PCB concentrations outside the range of normal, utilization of PCB pattern as an adjunct to concentration can provide evidence of the origin of exposure. ACKNOWLEDGMENT The technical assistance by Ms. Marilyn Rivera and manuscript preparation are greatly appreciated.
by Mr. Sidney Sibel
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(Received in Germany 2 January i~86)