Environ. Sci. Technol. 1998, 32, 1214-1221
Bioaccumulation and Toxic Potential of Extremely Hydrophobic Polychlorinated Biphenyl Congeners in Biota Collected at a Superfund Site Contaminated with Aroclor 1268 K U R U N T H A C H A L A M K A N N A N , * ,† HARUHIKO NAKATA,‡ ROD STAFFORD,§ GREG R. MASSON,| SHINSUKE TANABE,‡ AND JOHN P. GIESY† 201 Pesticide Research Center, National Food Safety and Toxicology Center, Department of Zoology, Institute of Environmental Toxicology, Michigan State University, East Lansing, Michigan 48824, Department of Environment Conservation, Ehime University, Tarumi 3-5-7, Matsuyama 790, Japan, Georgia Environmental Protection Department, 205 Butler Street SE, Suite 1154, Atlanta, Georgia 30334, and U.S. Fish & Wildlife Service, 4270 Norwich Street, Brunswick, Georgia 31520
Aroclor 1268, a highly chlorinated technical PCB mixture composed of primarily hexa- through decachlorobiphenyls, was used to lubricate high voltage process equipment at a chlor-alkali facility in coastal southeastern Georgia. Disposal of process wastes has resulted in extensive contamination of environmental media at the nearby intertidal marsh. In this study, congener distribution, bioaccumulation properties, and toxic potential of superhydrophobic PCBs were examined in blue crab, fish, terrapin, and birds collected at this site. Lipid-normalized mean concentrations of total PCBs in blue crab, striped mullet, yellow tail, sea trout, diamondback terrapin, red-winged blackbird, boat-tailed grackle, mottled duck, and clapper rail were 197, 283, 203, 56, 14, 385, 76, 135, and 10 µg/g, respectively. Hepta-, octa-, and nonachlorobiphenyls collectively accounted for 85-93% of the total PCB concentrations. The PCB congener profile in biota resembled that of Aroclor 1268, although the relative proportions of nona- and decachlorobiphenyls were less. Bioaccumulation of superhydrophobic congeners in biota, including terrapins and birds, was less than would be predicted from Kow, which supports the hypothesis that these compounds have restricted membrane permeability. Superhydrophobic PCB congeners were less efficiently transferred in the food web. The concentrations of non-ortho coplanar congeners in blue crab were 7-8 orders of magnitude less than the total PCB concentrations. Despite notable concentrations of total PCBs, the 2,3,7,8-TCDD toxic equivalents (TEQs) estimated for non- and mono-orthoPCBs in biota were minimal. The toxic effects of higher chlorinated PCBs, including di-ortho-substituted PCBs, need further investigation.
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Introduction A former chlor-alkali plant in coastal southeastern Georgia (United States) utilized Aroclor 1268, an uncommon, more chlorinated technical polychlorinated biphenyl (PCB) mixture to lubricate graphite electrodes used in process equipment. Disposal of process wastes into holding pits near the top of the tidal marshlands and also directly into adjacent marsh (see Figure 1 of ref 1) for over three decades has resulted in contamination of soils on-site and also of sediments in the coastal marsh. It was estimated that over 37 ton of PCBs and 440 ton of mercury may have seeped into marshes and other nearby areas around the 550-acre site. The site, known as LCP (Linden Chemicals and Plastics), is a PCB- and mercurycontaminated high-priority Superfund site. In our previous studies, spatial distribution and composition of PCBs, polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) were examined in soil and sediments collected at this site (1, 2). Sediments collected at this site revealed a characteristic chlorobiphenyl congener pattern with a greater proportion of hepta- through decachlorobiphenyls, characteristic of the sourcesAroclor 1268 (1). Because of the characteristic composition of PCB congeners, their accumulation in biota would provide information to evaluate the suitability of organisms as bioindicators and to assess Aroclor 1268 as a marker of bioaccumulation of superhydrophobic congeners. In this study, bioaccumulation of highly chlorinated PCBs (Cl g6; also referred to as superhydrophobic congeners) was examined in blue crab, fish (striped mullet, sea trout, and yellow tail), terrapin, and birds (red-winged blackbird, boat-tailed grackle, mottled duck, and clapper rail) collected at this site. Bioaccumulation properties of superhydrophobic PCBs is of interest because these congeners deviate from the lipophilicity model of bioaccumulation in fish and benthic invertebrates (3-6). A few earlier studies were conducted under laboratory conditions using sediments spiked with PCB mixtures or sediments taken from areas that were contaminated with tri- through heptachlorobiphenyls. Field studies reporting bioaccumulation of octathrough decachlorobiphenyls are meager. Moreover, little is known regarding the accumulation of superhydrophobic chlorobiphenyl congeners in biota other than fish and benthic invertebrates. The Turtle River-Brunswick Estuary (see Figure 1 of ref 1) provides an habitat for species such as ducks, clapper rail, and turtles and supports economically important sport and commercial fisheries and crabbing. Due to the contamination by mercury and PCBs, the Georgia State Environmental Protection Division has issued consumption restrictions and a commercial fishing ban along the primary stretch of the Turtle River near the mouth of the Purvis Creek since June 1993. Aroclor 1268 contains small amounts of toxic congeners as compared to the relatively less chlorinated formulations such as Arolcors 1260 and 1254 (1). Congener-specific analysis of toxic chlorobiphenyls is important for assessing the risks of PCBs in biota. Therefore, we estimated 2,3,7,8TCDD equivalents (TEQs) of mono-, di-, and non-ortho coplanar PCBs in various organisms collected at this site. Concentrations of organochlorine pesticides such as HCB (hexachlorobenzene), DDTs [1,1,1-trichloro-2,2-bis(p-chlo* To whom correspondence should be addressed; fax: (517) 3535598; e-mail:
[email protected]. † Michigan State University. ‡ Ehime University. § Georgia Environmental Protection Department. | U.S. Fish & Wildlife Service. S0013-936X(97)00943-7 CCC: $15.00
1998 American Chemical Society Published on Web 03/13/1998
Range. e Available age estimates of sea trout were >2 yr and of terrapins were >5 yr. a
Indicates total length for fish and carapace length for crab and turtle.
b
ND, not determined. c Pooled sample.
d
carcass 55 Aug 27, 1995 1
ND
liver 176-177 Aug 27, 1995
boat-tailed grackle (Quiscalus major) red-winged blackbird (Agelaius phoeniceus)
2c
ND
liver breast muscle 300 1500 Aug 15, 1995 Aug 27, 1995 1 1
ND ND
Aug 15, 1995 1
ND
300
breast muscle
detritivore, benthic feeder carnivore (small fish, crustaceans) detritivore (grass, sediment) carnivore (small fish) omnivore (fiddler crab, snails, worms, marsh plants) omnivore (fiddler crab, snails, worms, clams, insects) omnivore (as above) omnivore (emergent grasses, aquatic plants, Gammarus sp., grains) omnivore (invertebrates such as clam, crayfish, snails, frog, lizard) omnivore (mayflies, damsel flies, caddishflies, beetles) hepatopancreas muscle muscle muscle liver (14.3-18.3) 91 (50-113) 344 (326-362) 640 (430-900) 773 (618-1033) Mar 5, 1997 Mar 5, 1997 Mar 5, 1997 Mar 5, 1997 Jul 21, 1997 5 5 5 5 5
blue crab (Callinectes sapidus) yellow tail (Bairdiella chrysoura) striped mullet (Mugil cephalus) sea trout (Cynoscion nebulosus)e diamondback terrapine (Malaclemys terrapin) clapper rail or marsh hen (Rallus longirostris) clapper rail (Rallus longirostris) mottled duck (Anas fulvigula)
15.7 19 (16-20)d 25 (24-25) 34 (30-38) 17 (16.4-18.4)
tissue analyzed weight (g) lengtha (cm) date of collection
n species
TABLE 1. Characteristics of Biota from the LCP Chemicals Superfund Site, Brunswick, GA
Collection of Samples. Striped mullet (Mugil cephalus), spotted seatrout (Cynoscion nebulosus), and silver perch (also called yellow tail) (Bairdiella chrysoura) were collected by gill-net or by hook and line in Purvis Creek, across the LCP site (Figure 1 of ref 1), on March 5, 1997. The sampling points in Purvis Creek were approximately 1 km from the LCP industrial complex. Blue crabs (Callinectes sapidus) were captured in traps. Whole fish and crab were placed in icefilled portable containers and transported to the laboratory and stored at -20 °C until analysis. Mottled duck (Anas fulvigula), boat-tailed grackle (Quiscalus major), red-winged blackbird (Agelaius phoeniceus), and clapper rail (or marsh hen) (Rallus longirostrus) were collected in Purvis Creek by shooting with steel shot during July-August 1995. Adult female diamondback terrapins (Malaclemys terrapin) were collected by gill-net in Purvis Creek in July 1995. Length and weight of individuals were measured. Muscle tissues from individual fish, hepatopancreas from crabs, liver and breast muscle from clapper rail, breast muscle from duck, liver from boat-tailed grackle, carcass of red-winged blackbird, and liver from terrapin were taken for analysis (Table 1). Chemical Analyses. Organochlorine pesticides and PCBs were analyzed according to methods described in our previous publications (1, 7). The method consisted of extraction of sample tissues (10-20 g) with mixed solvents of diethyl ether (300 mL) and hexane (100 mL) using Soxhlet apparatus for 7 h. Fat content was determined from the K-D (Kuderna-Danish) concentrated aliquots of these extracts. The remaining extracts were then transferred to a glass column packed with 20 g of Florisil (Floridin Co.), followed by elution with a mixture of 150 mL of 80% acetonitrile and 20% hexane-washed water. The eluate from the Florisil column was collected in a separatory funnel containing 100 mL of hexane and 600 mL of hexane-washed water. After partitioning, the hexane layer was concentrated, purified with sulfuric acid, and passed through a 12-g Florisilpacked glass column for separation. The first fraction eluted with hexane contained HCB, PCBs, p,p′-DDE, and transnonachlor. The second fraction eluted with 20% dichloromethane in hexane contained HCH isomers (R-, β-, and γ-HCH), chlordanes (trans-chlordane, cis-chlordane, cisnonachlor, and oxychlordane), and p,p′-DDD and p,p′-DDT. Each fraction was concentrated and injected into a HewlettPackard 5890 series II high-resolution gas chromatograph equipped with a 63Ni electron capture detector (GC-ECD). A fused silica capillary column (30 m × 0.25 mm i.d.) coated with DB-1 (100% dimethyl polysiloxane, J&W Scientific, Folsom, CA) at 0.25 µm film thickness was used for the quantification. The column oven was programmed from an initial temperature of 60 °C (1 min hold) to 160 °C at a rate of 20 °C/min, held for 10 min, and then ramped at a rate of 2 °C/min to 260 °C with a final hold time of 20 min. The injector and detector temperatures were maintained at 260 and 280 °C, respectively. Helium and nitrogen were the carrier and the makeup gases, respectively. Concentrations of individually resolved peaks were summed to obtain the total PCB concentration. Compounds identified and quantified by GC-ECD analysis were confirmed by GC-MSD. A Hewlett-Packard 5890 GC coupled with a 5971 mass selective detector (MSD) equipped with a HPG1034C MS Chem Station and operating at an electron impact energy of 70 eV was used for identification and confirmation of individual PCB isomers. A similar column and temperature program as mentioned above were used. PCB homologues were determined by
food item/feeding habit
Materials and Methods
NDb
rophenyl)ethane], HCHs (hexachlorocyclohexanes), and chlordanes were also determined to evaluate their relative significance in the marsh ecosystem.
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TABLE 2. Concentrations (ng/g Wet Wt) and Relative Composition (in Parentheses; Wt %) of PCB Congeners in Tissues of Blue Crab, Fishes, Terrapin, and Birds As Compared with Aroclor 1268 blue crab (n ) 5)
congener trichlorobiphenyl tetrachlorobiphenyl pentachlorobiphenyl hexachlorobiphenyl heptachlorobiphenyl octachlorobiphenyl nonachlorobiphenyl decachlorobiphenyl total PCBs (wet wt) fat (%) a
From ref 1.
b
yellow tail (n ) 5)
mullet (n ) 5)
9
terrapin (n ) 5)
birds (n ) 5)
24 (0.1)b 64 (0.2) 615 (1.7) 4070 (11) 8570 (24) 17200 (49) 4500 (13) 345 (1)
3 (0.1) 19 (1) 135 (6) 180 (8) 530 (24) 975 (44) 360 (16) 24 (1)
0 (0) 10 (0.3) 42 (1) 350 (10) 605 (18) 1470 (44) 860 (25) 47 (1.5)
1 (0.1) 4 (0.5) 12 (2) 40 (5.1) 165 (21) 370 (46) 190 (24) 11 (1.3)
0 (0) 0 (0) 8 (0.51) 38 (2) 260 (17) 730 (47) 470 (30) 53 (3.4)
0 (0) 0 (0) 4 (0.12) 215 (6) 625 (18) 1710 (49.4) 840 (24.3) 67 (2)
35400 (21700-47600)c 18 (16-20)
2230 (330-7860) 1.1 (0.68-2.4)
3390 (2240-4390) 1.2 (0.62-1.5)
790 (200-1520) 1.4 (0.42-1.8)
1560 (1120-2190) 12 (8.9-17)
3460 (300-11300) 3 (1.3-4.4)
aroclor 1268a (0.06) (0.12) (0.19) (4.4) (10) (45) (35) (4.8)
Composition. c Range.
selective ion monitoring (SIM) at m/z 256 and 258, 290 and 292, 324 and 326, 358 and 360, 392 and 394, 428 and 430, 460 and 462, and 494 and 496 for tri-, tetra-, penta-, hexa-, hepta-, octa-, nona-, and decachlorobiphenyls, respectively. Samples were also analyzed by alkaline alcohol digestion to measure the concentrations of non-ortho coplanar PCB congeners (8, 9). Sample tissues were refluxed in 1 N KOHethanol, followed by reextraction in hexane, concentration, and clean up by silica gel column chromatography, as described elsewhere (8). Extracts were then passed through a glass column (5 mm i.d.) packed with 125 mg of activated carbon (Wako Pure Chemical Industries Ltd., Japan) for the separation of non-ortho PCB congeners; 3,3′,4,4′-T4CB (IUPAC No. 77), 3,3′,4,4′,5-P5CB (IUPAC No. 126), and 3,3′,4,4′,5,5′-H6CB (IUPAC No. 169) from ortho-chlorinesubstituted PCBs. An initial eluate of 100 mL of 20% dichloromethane in hexane (v/v) containing PCBs, orthochlorine-substituted PCB, and biogenic substances that interfere with non-ortho congeners was discarded. A second fraction eluted with 100 mL of benzene:ethyl acetate (1:1) containing non-ortho coplanar PCBs was microconcentrated, and residues were extracted into 5 mL of hexane. This hexane extract was acid treated, rinsed in hexane-washed water, and analyzed by GC-ECD and GC-MS. For the analysis of nonortho PCBs by GC-MSD, M+, and (M + 2)+ cluster ions were monitored at m/z 290 and 292, 324 and 326, and 358 and 360 for IUPAC Nos. 77, 126, and 169, respectively. Recoveries of PCB congeners and organochlorine pesticides were determined in fortified samples of corn oil carried through the analytical procedure involving Soxhlet extraction were between 95 and 103%. An equivalent mixture of Kanechlors 300, 400, 500, and 600 with known PCB composition and content was used as the standard (10). Since the site was primarily contaminated with PCB congeners originating from Aroclor 1268 (1), this PCB preparation was also used as a standard to confirm PCB congener composition. Organochlorine pesticides were quantified from individually resolved peak areas with corresponding peak areas of standards. Detection limits for individual PCB congeners were 0.5 ng/g wet wt, whereas those of non-ortho coplanar PCBs were between 20 and 50 pg/g in tissues of fishes and crab, between 90 and 110 pg/g for terrapin, and between 180 and 360 pg/g for birds and of organochlorine pesticides were 1216
sea trout (n ) 5)
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 32, NO. 9, 1998
typically around 1 ng/g. Chlorobiphenyl congeners are referred by their IUPAC numbers throughout the paper unless specified otherwise. Concentrations are given on a wet weight basis, unless specified otherwise.
Results and Discussion Contamination Status. Concentrations and homologue distributions of PCBs in crab, fish, terrapin, and birds collected from the LCP site are presented in Table 2. Lipid-normalized mean concentrations of total PCBs were greater in striped mullet followed by yellow tail, blue crab, and sea trout containing 283, 203, 197, and 56.4 µg/g, respectively. Since the contaminated marsh sediment is the major source of exposure to PCBs at this site (1, 11), benthic detritivores such as striped mullet and blue crab accumulated greater concentrations. The concentrations of total PCBs varied by an order of magnitude among individual sea trout and yellow tails. Normalization of PCB concentrations to lipid content did not reduce the variability. The large variation in concentrations may be due to the movement of fish along the contamination gradient in the marsh (1). Total PCB concentrations in the muscle of yellow tail and mullet were greater than the Food and Drug Administration tolerance limit of 2 µg/g. The concentration of total PCBs of 7860 ng/g in the muscle of a yellow tail was markedly greater than those in other individuals (330-1050 ng/g). Removal of this value reduced the mean PCB concentration in yellow tail to 640 ng/g wet wt. Concentrations of total PCBs in livers of adult female diamondback terrapins ranged from 1120 to 2190 ng/g wet wt. Among birds, red-winged blackbird contained the greatest PCB concentration (11300 ng/g wet wt or 387 µg/g fat wt) followed by mottled duck (1730 ng/g wet wt or 135 µg/g fat wt) and boat-tailed grackle (3300 ng/g wet wt or 75.5 µg/g fat wt). Concentrations of total PCBs in the muscle and liver of clapper rail were 300 (9.4 µg/g fat wt) and 420 ng/g wet wt (11 µg/g fat wt), respectively. The differences in the concentrations of total PCBs among birds may be related to age, sex, and feeding habits. Concentrations of organochlorine pesticides were at least 2 orders of magnitude less than those of PCBs (Table 3). DDTs were detected in all the samples. The hepatopancreas of blue crab contained mean DDT concentrations of 38 ng/
TABLE 3. Concentrations of Organochlorine Pesticides (ng/g Wet Wt) in Biota from the LCP Superfund Site, Brunswick, GA organochlorine
blue crab
HCB
