A r , c h i v e s of

E nvironrnental c ontamination

Arch. Environ. Contam. Toxicol. 20, 56-60 (1991)

9 1991 Springer-Vedag New York lnc~

Polychlorinated Biphenyl Congeners in Blood of Wisconsin Sport Fish Consumers W. S o n z o g n i *,l, L . M a a c k * , T. G i b s o n * , D. D e g e n h a r d t * , H. A n d e r s o n * * , a n d B. F i o r e * * *Laboratory of Hygiene, University of Wisconsin, Madison, Wisconsin 53706, USA and **Division of Health, Wisconsin Department of Health and Social Services, P.O. Box 309, 1 West Wilson, Madison, Wisconsin 53701, USA

Abstract. As part of a study to evaluate the effect of chemical contaminants on Wisconsin sport fish consumers, measurements were made of polychlorinated biphenyl (PCB) congeners in human blood sera. A high resolution gas chromatography procedure was employed, with 13 individual congeners used as standards. Analytical recoveries and precision were >90% and replicates were always within _+30%. Total concentrations, obtained by summing quantified congeners, ranged from 0.6 to 27.1 txg/L. Conventional packed column PCB analysis, conducted on a subset of samples, gave higher total PCB results. On a congener specific basis, congeners 153 (245-245), 138 (234-245), 180 (2345-245) and 118 (245-34) were found most often. These congeners have been reported to be prominent in other matrices. Of the congeners found, 118, 138, and possibly 180 are potentially the most toxic (based on current toxicological information).

Polychlorinated biphenyls (PCBs) are a group of chlorinated hydrocarbons which were produced in the U.S. by the Monsanto Chemical Company under the tradename of Aroclor ~. Aroclor mixtures were used widely by industry prior to being banned in the U.S. in 1977 (Erickson 1986). These Aroctors have been extensively studied, largely because PCBs bioaccumulate in fish and other aquatic organisms, and constitute a health risk to fish consumers (Cordle et al. 1982). A study was initiated in 1985 with the participation of Wisconsin anglers to determine correlations between total PCB body burdens and sport fish consumption, to assess sport fishing and fish consumption habits, and to evaluate comprehension and compliance with the Wisconsin sport fish consumption advisory. A major finding of the study was that increased fish consumption by Wisconsin anglers positively correlated with increased human serum PCB concentrations. This conclusion and other epidemiologically related study results are presented by Fiore et al. (1989). There are several different Aroclor mixtures, each of which contain a unique composition of PCB congeners. Until r e c e n t l y almost all e n v i r o n m e n t a l and biological samples were analyzed by matching the chromatographic PCB pattern to the pattern of pure Aroclors, and results 1 To whom correspondence should be addressed.

were reported as an Aroclor or mixture of Aroclors. Few data have been generated on individual congeners. However, some congeners have been reported to be of much greater toxicological concern than others (Safe 1987; Leece et al. 1985; McKinney and Singh 1981; Goldstein et al. 1977). More information is needed on the levels and types of congeners in various samples in order to provide a better estimate of human health risk or environmental hazard. The purpose of this paper is to complement the Fiore et al. (1989) paper by discussing the analyses of PCB congeners that were conducted on human blood serum samples from Wisconsin fish eaters. Very few data have been published on PCBs in human blood sera, especially on a congener specific basis. This paper presents techniques for measuring PCB congeners in blood, assesses the levels of PCB congeners found in the blood of Wisconsin anglers, compares these levels with PCB congeners in other matrices, and considers the toxicological implications of the individual congeners.

Methods Sample Collection Thirty milliliter (mL) blood samples were collected via antecubital venipuncture from about 200 Wisconsin anglers in the summer and fall of 1986. The blood samples were undisturbed for approximately 40 rain to allow clotting and then centrifuged at 2000 rpm for approximately 10 min. Approximately 5 mL of serum from each 30 mL blood sample was used for chemical analysis. Each sample was drawn off with a glass pipette and placed in a teflon-lined screw-top tube. The samples were refrigerated until delivered to the laboratory and stored frozen at - 10~ until analyzed. Length of storage ranged from one day to two weeks. Of the samples collected from anglers, 173 were analyzed for PCBs.

Extraction and Clean-Up The protocol used in this study for extraction and clean-up is similar to the method reported in Center for Disease Control Laboratory Update 81-108-198 (U.S. Center for Disease Control, undated). PCB and pesticide residues were extracted from 5 mL of human serum with methanol (2 mL) and a l: 1 mix of hexane and ethyl ether (5 mL), followed by rotary mixing (15 min at 60 rpm) and centrifuga-

PCBs in Human Blood

57

Table 1. Concentrations and frequency of occurrence of PCB congeners in human serum (n = 173) Congener No. 153 138 180 118 187 170 28 128 101 70 77 52 44

Structure

Frequency of occurrence (%)

Concentration (Ixg/L) mean

min.

max.

245-245 234-245 2345-245 245-34 2356-245 2345-234 24-4 234-234 245-25 25-34 34-34 25-25 23-25

78 56 42 34 11 5.8 1.2 0.58 0.58 0.58 0.58 0 0

1.46 1.32 1.06 1.12 0.98 0.86 0.80 0.90 0.80 0.70 1.30 ---

0.60 0.60 0.60 0.60 0.60 0.60 0.60 0.90 0.80 0.70 1.30 ---

7.30 6.00 3.50 5.70 2.20 t.40 1.00 0.90 0.80 0.70 1.30 ---

tion (10 min at 1800 rpm). The extraction was repeated twice. The combined extracts were evaporated to approximately 2 mL in isooctane. PCBs were separated from pesticides with Florisil | (8 g) fractionation using a 10 mm i.d. chromatography column and elnted from the column with hexane and ethyl ether. Further clean-up was achieved with silica gel (5 g) fractionation, using a 10 mm i.d. column and eluted with hexane.

Chromatography The extracts containing PCB congeners and p,p'-DDE were analyzed by high resolution gas chromatography, using a Hewlett Packard 5880 gas chromatograph equipped with an autosampler, a splitless injection system, a 60 m by 0.25 mm i.d. fused silica DB-5 (J&W Scientific) coated capillary column with a film thickness of 0.1 ~m and a 63Nickel electron capture detector. The carrier gas was hydrogen and the make-up gas was nitrogen. The temperature program spanned 190~ within 90 minutes. The initial and terminal temperatures were 90 and 280~ respectively. The temperature ramps were as follows: 90-160~ at 20~ 160-200~ at l~ and 200-280 at 2~ Analyses focused on thirteen PCB congeners. The congeners analyzed were (IUPAC number, structure): 153 (245-245), 138 (234-245), 180 (2345-245), 28 (24-4), 128 (234-234), 101 (245-25), 70 (25-34), 77 (34-34), 52 (25-25), 44 (23-25), 170 (2345-234), 187 (2356245), and 118 (245-34). Since congener 77 (34-34) coelutes with congener 110, concentrations reported for 77 actually represent the sum of 110 and 77. However, concentrations of 77 are low relative to 110 (Duinker 1988). The selection of congeners for analysis was based on (1) their expected prevalence as indicated by the literature, (2) preliminary analysis findings, and (3) commercial availability of the standards. Individual pure standards were obtained from Ultra Scientific Inc. Congeners identity for which standards were not available were based on relative retention times. A limit of detection of 0.6 txg/L was statistically determined for all thirteen PCB congeners using a standard statistical approach. Resuits below the limit of detection were reported as not detected (nd). In addition to congener analysis, total PCB data were obtained by conventional packed column gas chromatography using an Aroclor matching pattern technique (Wisconsin Laboratory of Hygiene 1988). Thirty five of the 173 samples were selected for this analysis, which contained relatively high PCB concentrations as determined by congener analyses. Thus, PCBs were all detectable on the packed column. The gas chromatograph was equipped with a 2m by

4mm i.d. packed column coated with 4% SE-30 and 6% OV-210 and operated isothermally at 225~

Quality Control To test accuracy, 22 bovine serum samples (obtained from the U.S. Center for Disease Control, Atlanta, GA) that were free of PCBs were spiked with a mixture (2 ~xg/L) of the PCB congeners. Results were not adjusted for recoveries. Duplicate samples were also analyzed with every ten samples to determine the level of precision.

Results Table 1 presents data on the f r e q u e n c y of o c c u r r e n c e and a b u n d a n c e o f the i n d i v i d u a l c o n g e n e r s . C o n g e n e r s 153 (245-245), 138 (234-245), 180 (2345-245), and 118 (245-34) w e r e found in 78%, 56%, 42%, and 34% of the sera samples, respectively. These congeners were found simultaneously w h e n a b o v e the limit of detection. A standard for congener 118 was not used at the outset of the study (until the 118 c o n g e n e r peak was found to occur in a n u m b e r of samples and c h r o m a t o g r a m s ) . C o n s e q u e n t l y , 118 was q u a n t i f i e d using a standard on less than half of the samples, and the f r e q u e n c y of o c c u r r e n c e of 118 may be biased low. Congeners 153,138, 180, and 118 also had the highest mean concentrations, namely 1.46 tx~L, 1.32 v~g/L, 1.06 ~g/L, and 1.12 Ixg/L, respectively. Congeners which were reported as non-detectable in samples w e r e not included in the determination of the mean. A comparison of these congener concentrations among the samples showed that as congener 153 increased in concentration, the others generally increased within the sample. The nine remaining congeners which were selected for analysis o c c u r r e d in less than 12% of the samples, with m a n y of the congeners in less than 1% of the samples. These congeners had m e a n concentrations less than 1 ~g/L, e x c e p t for one sample that contained c o n g e n e r 110 (and 77) at 1.3

~g/L. There w e r e approximately 15 samples in which additional congeners w e r e o b s e r v e d on their c h r o m a t o g r a m s which did not m a t c h one of the 13 standards. T h e y were tentatively

58

W. Sonzogni et al.

Table 2. Recovery (a) and duplicate (b) results of congener analyses in serum

Table 3. PCB congeners prominent in a variety of human fluid or tissue studies

a. Recovery Results Congener ............. % Recovery ............. no. min. max. mean

No. samples

Sample type (reference)

28 44 52 70 77 101 128 138 153 180 170 187 118

22 22 22 22 19 22 22 22 22 22 15 15 6

68 68 73 83 70 77 69 61 68 72 76 71 71

107 104 108 126 142 126 114 109 109 130 115 114 110

104 90 92 100 99 93 96 95 93 100 103 93 92

b. Duplicate Results

Congener no.

% difference

153 180 138 all others

0-25 0-17 0-28 0

identified based on relative retention times as described in Mullin et al. (1984) and Maack and Sonzogni (1988) or by mass s p e c t r o m e t r y . These congeners includes nos. 74 (245-4), 99 (245-24), 146 (235-245), 156 (2345-34), 183 (2346245), 194 (2345-2345), 201 (2345-2356), and 203 (23456-245). A comparison of total PCB concentrations as determined by congener sums and conventional packed column Aroclor pattern recognition was made for 35 serum samples. Total concentrations by congener sum ranged from zero (no congeners were found above the 0.6 Ixg/L detection limit) to 27.1 Ixg/L. The Aroclor analyses consistently resulted in higher total PCB values. The concentration difference for total PCBs between the two methods ranged from 13% to 75% with an average difference of 48% (s.d. 14%). Possible reasons for the differences include: (1) the Aroclor standards used in the A r o c l o r pattern recognition analyses did not " m a t c h " the PCB distribution in the serum sample and, hence, erroneous results (in this case overestimates) were obtained; (2) the congeners that may have existed at levels undetectable by the current method were treated as zero and thus may have biased total PCB results low; and (3) other congeners were present in the sample which were not quantitated and, therefore, were not included in the total congener sum. Although conventional packed column results were c o n s i s t e n t l y higher than total PCBs c o m p u t e d by adding quantified congeners, the absolute differences were for the most part relatively small. Table 2 presents the quality control results. Acceptable recoveries and precision were regularly obtained. Average recoveries for the congeners ranged from 90% to 107%. Excellent duplication was obtained with the difference in concentration for duplicate runs close to zero for most congeners. Difference between duplicates was greatest for congeners

Blood serum (this study) Blood serum (Bush et al. 1984) Human milk (Safe et al. 1985) (Bush et al. 1985) Human adipose (Focardi and Romei 1987) (Jones 1988) Biota (Jones 1988)

Congener no. 153 138

180

118

x

x

x

x

x

x

x x

x x

x x

x

x x

x x

x x

x

x

x

x

x

153,180, and 138, ranging from 0-25%, O- 17%, and 0-28%, respectively.

Discussion

The PCB congeners found in the blood are also reported to occur in various matrices by other investigators. Bush e t al. (1984) reported congeners 153, 138, 179, and 28/31 as most prominent (38% of the total residue) in maternal blood. The women lived in the vicinity of the Hudson River and Lake Ontario where levels of PCBs and other chlorinated organic compounds in fish are elevated (although data on whether the w o m e n c o n s u m e d fish from the a r e a were not presented). The prominent congeners reported in blood of Wisconsin anglers were also among the congeners detected in other matrices, such as human milk and adipose tissue, fish and other biota. Safe e t al. (1985) reported congeners 28, 74, 99, 118, 138, 153,170, and 180 as the major PCB components in a human milk extract from a woman in Michigan. Bush e t al. (1985) reported that congeners 153, 179, 138, 70, 180, 156, 99, and 105 comprised 52% of the total PCB residue in human milk samples. As in Bush (1984), the samples were collected from women living in New York in the vicinity of Lake Ontario and the Hudson River. It was reported that these women did not consume fresh water fish and few consumed sea fish regularly. Focardi and Romei (1987) reported congeners 99, 138, 153, 170, and 180 as prominent in human adipose tissue. This tissue was obtained from people who were admitted for general surgery at an Italian clinic. Kannan e t al. (1988) reported the occurrence of congeners 77, 169, and 126 in human adipose tissue from a group of Japanese cancer patients. Jones' (1988) review of the literature for PCB congeners in tissue indicated that the most prominent congeners in human tissue were nos. 74, 118, 138, 153, 179, and 180. Jones (1988)also reported that congeners 95, 101, 110, 118, 138, 149, 153,180, and 187 were prevalent in biota, including bivalves, lugworms, shrimp, eel, fish, penguins, porpoises, and whales from various locations in the U.S., Canada, Europe, and Antarctica. Thus, there are a number of con-

PCBs in Human Blood geners that are regularly found in a variety of matrices including 118, t38, 153, and 180 which were found in greatest concentrations in this study. Of the anglers sampled in this study, perch, obtained from both sportfishing and commercial sources, was reported to be eaten most often (Fiore et al. 1989). With respect to sport fish only, chinook salmon (from the Great Lakes) and walleye and perch (from inland waters) comprised a large part of the catch (Fiore et al. 1989). Maack and Sonzogni (1988) performed PCB analyses on fish from various Wisconsin waters including the waters used by anglers sampled in this study. Congeners 138, 153, 180 and 118 were among the most common congeners found in these fish. Concentrations of these congeners were higher in the fish than in the human sofa, although the order of the mean concentrations (153>138>180>118) was the same. Thus, the prominent congeners found in the blood of Wisconsin fish consumers are also conspicuous in a variety of other matrices. Concentrations of the congeners in blood are considerably lower than congener concentrations in other matrices, although the lack of data on congener concentrations in environmental and human samples make it difficult to compare concentrations. Total PCB concentrations in blood, obtained by summing the individual congeners, are generally lower than total PCBs in blood reported by others. Congeners 169, 126, and 77 are the most highly toxic PCBs (Parkinson and Safe 1987). Unfortunately, these congeners tend to co-elute with other congeners and are thus difficult to detect without special techniques. A few investigators have tried special techniques to measure these congeners. Kannan et al. (1988) and Tanabe et al. (1987) have reported the occurrence of these congeners in Japanese residents (adipose tissue), dogs, cats, fish, and marine life. Duinker et al. (1988) detected these three congeners in Aroclor mixtures, but only congener 77 in a seal blubber sample from the Dutch Wadden Sea. Olafsson et al. (1987) did not report the occurrence of these congeners in a snapping turtle from the Upper Hudson River, although it was stated that approximately 10% of the total PCB concentration (in various tissues) contained toxic congeners. Thus, the frequency of occurrence and concentrations of the reportedly highly toxic congeners 77, 126, and 169 are yet to be generally established. With respect to the toxicity of other congeners, it has been reported by Parkinson and Safe (1987) that, in general, the coplanar PCBs are the most toxic, then the mono-ortho analogs, followed by the di-ortho analogs of the coplanar PCBs. The congeners prominent in this study are nos. 153, 138, 180, and 118. Congener 118 is a mono-ortho analog of the coplanar congener 77; congeners 153 and 138 are diortho analogs of the coplanar congener 77; and congener 180 is a di-ortho analog of the coplanar congener 126. Some work has been done on the activity of congeners, as commonly measured by enzyme induction procedures such as cytochrome P-450c, cytochrome P-450d and aryl hydrocarbon hydroxylase (AHH) induction. The activity associated with a biologic (or toxic) response has been determined for various coplanar and mono-ortho analog PCBs (Safe 1987). For example, there was a linear correlation in the rat between body weight loss and thymic atrophy (in vivo) with A H H induction (in vitro) for congener 118. Con-

59 goner 118 induces P-450c and d as well as AHH; congener 138 induces P-450c; congener 153 does not induce P-450c, d, or AHH; and the induction capabilities of congener 180 are not clearly known (Parkinson and Safe 1987). Thus, assuming that the enzyme induction is an indicator or marker of toxicity, congeners 118, 138 and possibly t80 may be of toxicological concern among those identified in the sera of Wisconsin fish eaters. However, assessing the actual human health risk from individual PCB congeners or even total PCBs is difficult. Safe (1987) reported that it is unlikely that environmental exposure to PCBs leads to any adverse human effects. Hayes (1987) reported that "epidemiological studies on human and animal populations exposed to PCBs in the environment have so far not revealed clear evidence for carcinogenicity of PCBs under natural exposure circumstances and much of the evidence for potential carcinogenicity of PCBs in experimental systems leads to a substantial overestimate of the real risks to humans exposed to environmental levels of PCBs". With a contrasting perspective, Tanabe etal. (1987) reported "that coplanar PCBs, especially 3,3',4,4',5 PsCB[no. 126], impose a greater toxic threat than dioxins and furans to humans and probably to wildlife also". Tanabe and coworkers also reported that there is a need for a re-evaluation of PCB toxicity, with particular attention given to toxic congeners. There are questions remaining regarding the toxicological significance of the individual congeners. More data are necessary on levels of PCB congeners, especially those suspected to be of elevated toxicity, in various environmental compartments. Other toxic or biologic endpoints, such as neurotoxicity, need further investigation. Research should be conducted on possible synergistic or antagonistic effects of mixtures of congeners and mixtures of environmental contaminants in general.

Acknowledgments. The authors thank colleagues at the University

of Wisconsin Laboratory of Hygiene, particularly Carol Buelow and Denise Junk, for their help in various aspects of the analyses of PCBs in blood.

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Manuscript received October 15, 1989 and in revised form January 3, 1990.