Environ. Sci. Techno/. 1995, 29, 1832-1838
Isomer-Specific Determination and Toxic Evaluation of Polychlorinated Biphenyls, Polychlorinated/brominated Dibenzopdioxins and Dibenzofurans, Polybrominated Biphenyl Ethers, and Extractable Organic Halogen in Carp from the Buffalo River, New York BOMMANNA G. LOGANATHAN,*’ KURUNTHACHALAM KANNAN,’ ISAO WATANABE,* MASAHIDE KAWANO,* KIM IRVINE,” S U B O D H KUMAR,Il A N D H A R I S H C . SIKKA’I Skidaway Institute of Oceanography, University System of Georgia, 10 Ocean Science Circle, Sauannah, Georgia 31411, Osaka Prefectural Institute of Public Health, 1-3-69 Nakamichi, Higashinari-ku, Osaka 537, Japan, Department of Environment Conservation, Ehime University, Tarumi 3-5- 7, Matsuyama 790, Japan, and Centre for Environmental Research and Education, State University of N e w York College at Buffalo, 1300 Elmwood Auenue, Buffalo,N e w York 14222
Concentrations of PCB isomers, polychlorinated/ brominated dibenzo-p-dioxins (PCDDdPBDDs) and dibenzofurans (PCDWPBDFs), polybrominated biphenyl ethers (PBBEs), and total extractable organic halogen (EOX) were determined in carp (Cyprinus carpio) of three age classes collected from the Buffalo River, N e w York. Total PCBs concentrations exceeded the United States Food and Drug Administration’s tolerable level of 2pg/g ( w e t w t ) in all the three age classes. Concentration ranges of total PCDDs and PCDFs were 27-146 and 22-99 pg/g, respectively. Among several of the 2,3,7,8-substituted isomers detected, OCDD and OCDF were the most predominantly noticed congeners. Noticeable concentrations of PBBEs were found with a major contribution from tetra-BBEs. Extractable organochlorines (EOCI) were the highest in the total EOX family followed by organobromines and organoiodines. The known organochlorines such as PCBs and DDT accounted for 13-58% of the EOCI, suggesting the presence of considerable levels of other unknown organochlorines. The 2,3,7,8-TCDD equivalents of coplanar PCBs, dioxins, and furans in carp muscle were between 45 and 108 pglg, with 3848% of the toxicity exerted by mono-orlho-PCBs. The estimated allowable con-
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ENVIRONMENTAL SCIENCE &TECHNOLOGY / VOL. 29, NO. 7,1995
sumption rates of carp based on the risk assessment method were much lowerthan the actual consumption values available for the Great Lakes states. Elevated concentrations of PCB isomers other than dioxins and furans suggest the need to protect humans from the consumption of PCB-contaminated carp from the Buffalo River.
Introduction The Buffalo River, NewYork, is one of 43 “areasof concern” around the Great Lakes that have been identified by the International Joint Commissionas exhibitingenvironmental impairment. The impairments identified for the Buffalo River in a level I Remedial Action Plan by the New York State Department of Environmental Conservation (NYSDEC) included the following: restrictions on fish and wildlife consumption, fish tumors and other deformities, degradation of benthos, restrictions on disposal of dredged sediment, and loss of fish andwildlife habitat (1). The NYSDEC identified multiple possible pollutant sources to the Buffalo River, including combined sewer overflows (CSOs), direct industrial discharges, leaching from inactive hazardous waste sites, water column interaction with historically contaminated bed sediment, and upstream point and nonpoint sources such as municipal wastewater treatment plants and agricultural runoff. Among a wide variety of man-made chemicals, organochlorine compounds such as PCBs, dioxins, and furans are of concern as widespread, persistent, and toxic contaminants (2-4). There is ample evidence that certain organochlorines are at least partly responsible for reproductive and immunological abnormalities observed in some aquatic biota (5). In fish, prevalences of a number of pathological conditions including neoplasms, preneoplastic lesions, hydropic vacuolation, and other necrotic and proliferative lesions were correlated significantly with organochlorine contamination in urban embayments and rivers (6-8). The presence of morphologically abnormal fishes in the Buffalo River in recent years (9) suggests the possibility of extensive contamination by organochlorine compounds. Despite this, not much is known about the levels ofvarious contaminants in biota of the Buffalo River. An earlier investigation revealed that the total PCB concentrations in Buffalo River water exceeded the statetargeted guidelines of 1 ng/L (10). A number of other halogenated compounds, including the polychlorinated/ brominated dibenzo-p-dioxins (PCDDslPBDDs)and dibenzofurans (PCDFs/PBDFs) and polybrominated biphenyl ethers are toxic byproducts formed during the synthesis of the primary industrial halogenated aromatics and during combustion. These compounds are highly lipophilic, persistent, and bioaccumulative. In this study, concentrations of PCB isomers, DDT, PCDDslPCDFs, PBDDdPBDFs, and PBBEs were determined in carp, Cyprinus carpio, ~~~~~~~~~
~
~
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* To whom correspondence should be addressed: Fax: (9121-5982310; e-mail address:
[email protected]. Skidaway Institute of Oceanography. t Osaka Prefectural Institute of Public Health. 5 Ehime University. 11 State University of New York College at Buffalo. +
0013-936X/95/0929-1832$09.00/0
G 1995 American
Chemical Society
this method, p,p'-DDTis converted into p,p'-DDE, and p,p'-
TABLE 1
Details of the BllRslo River Car# age class
sexb
age (yr)
young
F8
4.3
middle
M 6 (4.0-5.0) ND 1 F6 5.9 M6 (5.0-9.0) ND 3
wet standard total weight (kg) length (cm) length (cm) 0.95 (0.57-1.2)
32 (26-37)
40 (34-45)
1.6 (1.3-2.2)
39 (36-42)
47 (43-50)
old
F I O 10 4.4 54 65 M5 (6.0-13) (2.3-8.2) (44-66) (55-80) a Numbers in parentheses indicate the range. Number of male (M) and female (F) individuals in a total of 15samples from each age class is given. ND, not determined.
collected from the Buffalo River to understand their baseline concentrations and toxicity implications and to estimate allowable consumption rates based on risk assessment methods. In addition to known organochlorines such as PCBs and DDT, other unknown organohalogens (introduced into the environment either naturally or anthropogenically)are of current concern. Therefore, the concentrations of total extractable organic halogens (sum of extractable organochlorines, organobromines, and organoiodines; EOX = EOCl EOBr EO11 were also determined in carp muscle to evaluate the contribution of identified organohalogens to total extractable organic halogen (EOX).
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Materials and Methods Collection and Preparation of Carp. Forty-eightcarp were collected from the Buffalo River on July 24, 1991. The collection was done using an electroshocking boat. All temporarily stunned fish were brought onboard with nets and were placed directlyfromthe nets into ice-filled coolers lined with clean aluminium foil. All carp samples were taken to the Great Lakes Center's field station, and preliminary processing was done on the day of sample collection. The carp were sorted into three broad age classes (young,middle-aged,and old) accordingto length and girth. The age of each fish was determined by counting the number of annual growth rings on the sampled scales (11). There were 15 fish in each age class. Age, wet weight, standard length (fish snout to end of torso), and total length (fish snout to end of caudal fin) were recorded (Table 1). Tissues from all 15 fish in each age class were pooled, and an aliquot was subjected to chemical analyses. Chemical Analyses. Muscle tissue from each age class (ca. 200 g) was homogenized and Soxhlet extracted using dichloromethane for 20 h. The extract was dehydrated using anhydrous sodium sulfate and K-D (KudernaDanish) concentrated (to 50 mL) after the addition of n-hexane. Aliquots of subsamples were used separately for the determination of organohalogens. Polychlorinated biphenyls, including non-ortho-coplanar congeners and DDT, were determined by digesting the extract in 1 N KOH-ethanol for 1 h (12,131. The extract was transferred to hexane, K-D concentrated, and cleaned on 1.5g of silica gel Wako gel S- 1)packed on a glass column (10 mm i.d. x 20 cm). The hexane eluate after K-D concentration was treated with 5% fuming sulfuric acid and injected into a gas chromatograph equipped with electron capture detector (GC-ECD) and a gas chromatographlmass spectrometer (GUMS) for the quantification of ortho-chlorine-substituted PCB isomers and DDT. In
DDD is decomposed by alkaline treatment. Therefore, the reported DDT concentration is the sum of the p,p'-DDT and p,p'-DDE concentrations. The remaining hexane extract was passed through a column (5 mm i.d.1 packed with 125 mg of activated carbon for the separation of nonortho-coplanar PCB isomers from other ortho-substituted isomers. An initial eluate of 100mL of 20% dichloromethane in hexane contained ortho-chlorine-substitutedPCBs, other xenobiotics, and biogenic substances. The second fraction eluted with 100 mL of benzene in ethyl acetate (50:50) containing non-ortho-coplanar PCBs was microconcentrated, and residues were dissolved in 5 mL of hexane. The hexane extract was cleaned with 5 mL Of 5%fuming sulfuric acid and rinsed with distilled water for quantification. A Hewlett-Packard 5890 Series I1 gas chromatograph fitted with an electron capture 63Nidetector and a 30 m x 0.25 mm (i.d.1 DB-1 coated (0.25 pm thickness) capillary column with helium as the carrier gas was used. Injection was by the splitless mode, the injection port temperature was kept at 250 "C, and the column oven temperature was programmed from 160 to 240 "C at a rate of 2 "C/min. The detector temperature was kept at 300 "C, and nitrogen was used as the makeup gas. Concentrations of individually resolved peaks of PCB isomers were summed to obtain total PCB concentration. For isomer-specilkdetermination of PCBs, a Hewlett-Packard 5890 gas chromatograph fitted with a 5970 mass selective detector (GUMS) having an electron impact (EI) mode at 70eV was used. Fused silica capillary columns, DB-1 and DB-1701, with the same dimensions and film thickness as mentioned above were used for the determination of PCB isomers and non-orthocoplanar PCB congeners, respectively. The oven temperature was programmed from 160 to 230 "C for the DB-1 column and from 180 to 250 "C for the DB-1701 column at a rate of 2 "Clmin. The injector and ion-source temperatures were kept at 260 and 280 "C, respectively.A Hewlett-Packard 5970C data system was employed for the quantification of PCB isomers and congeners. PCB homologues were determined by selective ion monitoring (SIM) at m/z 256, 292, 326, 360, 394, 430, and 464 for tri-, tetra-, penta-, hexa-, hepta-, octa-, and nonachlorobiphenyls, respectively. An equivalent mixture of Kanechlors 300,400,500, and 600 with known PCB composition and content was used as the standard. For coplanar PCBs determination, M+ and (M 2)+ ions were monitored at m/z 290 and 292 (for IUPAC No. 771,324 and 326 (for IUPAC No. 1261, and 358 and 360 (for IUPAC No. 169). Authentic standards of '98% purity were used. Analytical recoveries of coplanar PCBs through the whole procedure were between 70 and 90% (13). A procedural blank was run simultaneously with samples to check interfering or coeluting peaks. For the determination of chlorinated/brominated dioxins and furans, internal standards ([l3C-2,3,7,81T4BDD, 2.5 ng; [l3C-2,3,7,8]T&DD/T4CDF,2 ng each; O&DD/OsCDF, 2 ng each) were spiked to an aliquot of the extract containing 3 g of lipid in 50 mL of hexane, which was then treated three times with 15 mL of concentrated sulfuric acid to remove lipids. The extract was cleaned using a multi-layer column chromatography (20 mm i.d. x 30 cm) packed with 2 g of 10%AgN03-silica, 3 g of 44% H2S04silica, and 2 g of 2% KOH-silica eluting with 200 mL of 5% dichloromethane in hexane, essentially following the method described elsewhere (14). The eluate was passed
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VOL. 29, NO. 7,1995 I ENVIRONMENTAL SCIENCE &TECHNOLOGY
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through 3 g of activated (220 OC for 12 h) neutral alumina column (10 mm i.d. x 30 cm) for fractionation. The first fraction eluted with 30 mL of 4% dichloromethane in n-hexane-contained PBBEs, andthe second fraction eluted with20mLof50% dichloromethane in n-hexane-contained PBDDdPBDFs and PCDDs/PCDFs. Dioxins and furans were quantified using a highresolution gas chromatographhigh-resolution mass spectrometer (HRGC/HRMS). A Shimadzu 14A gas chromatograph equipped with a DB-5 column (30 m x 0.25 mm i.d.; 0.25 pm filmthickness) was used for the analysis of total PCDDdPCDFs, PBDDdPBDFs, and PBBEs. A DB-dioxin column (60 m x 0.25 mm i.d.; 0.25pm film thickness) was used for isomer-specific determination of 2.3,7,8-substitutedPCDDs/PCDFs. The HRGClHRMS usedwas aKIatoS Concept IS. The SIM conditionswere as follows: ionsource mode, EI; ion source temperature, 290 “ C electron energy, 40 eV; postacceleration detector, 8 kV. The masses monitored for analyte were chosen at two most intensive ions of molecular ion cluster. Concentrations of organic chlorine,bromine, and iodine were determined by Instrumental Neutron Activation Analysis [INAA) (15.16). An aliquot of hexane extIact (ca. 0.5 mL) was sealed in an acid-washed polyethylene vial (vials were washed with distilledwater, stored under hexane overnight, and dried in an oven at 60 “C) and irradiated at a neutron fluxof 4.0 x loL3nlcm2s for 2 min using JRR-4 nuclear reactor of the Japan Atomic Energy Research Institute, Ibaragi, Japan. The irradiated vials were transferred fromreactor tolaboratory, andaliquots were pipetted out into counting vials immediately. The counting time employed was 3 min. The y-energy spectra was recorded withtwo Ge solid-statedetectorswithassociatedelectronics interfaced to EG&G Ortec Model GEM-15180 and Canbera Series 35 Plus 4096 channel for peak area calculations. The analyses were based on y-peaks from 38Cl (tliz= 37 min, Ey = 2167 kev), 80Br(tliz= 17.6 min, E? = 616 kev), and L28Z(tli2 = 25 min, Ey = 443 kev). Ammonium chloride, ammonium bromide, and ammonium iodide of known concentration (dissolvedin hexane) were used as standards. Procedural blanks, containing hexane sealed in a polyethylene vial, were run simultaneously to check impurities and to correct sample values, if necessary.
Results and Discussion Polychlorinated Biphenyls and DDT. Concentrations of total PCBs were high in all three age classes, exceeding the FDA tolerable level of 2 pglg (Table 2). An age-dependent PCB accumulation was evident, with the highest level of 5 pg/g (wet wt) in old carp. Isomer-specific determination of PCBs in middle-aged carp revealed the presence of 80 individual isomers (Figure 1). The compositions of tri-, tetra-, penta-, hexa-, hepta-, and octachlorohiphenyls to the total PCB concentrations were 7,21,23,25,20, and 4%. respectively. Hexachlorobiphenyl of IUPAC No. 153 (2,2’,4,4’,5,51recorded the highest concentration of 307 ng/g followed hy IUPAC Nos. 138 (2,2’,3,4,4’,5’) and 180 (2,2’,3,4,4’,5,57,altogether accountingforaboutZO% ofthe total PCB load. A similar pattern of PCB congener distribution was also noticed in young and old carp. Concentrations of non-, mono-, and di-ortho-PCB congeners are given in Table 2. The concentrations of coplanar PCB congeners (thelaterally substituted PCBs that do not possess ortho-chlorine substituents which would restrict free rotation around the central phenyl-phenyl 1834 m ENVIRONMENTAL SCIENCE & TECHNOLOGVIVOL. 29. NO. 7.1995
TABLE 2
Concentrations of PCB Isomers and DOT (ng/g wet wt) in Muscle of Young, Middle-Aged, and Old Carp from the Buffalo River conpdnUPAC No.
fat (%) DDT
total PCBs non-orthoPCBs 77 126 169 mono-orthoPCBs
60 105 118 156 di-orthoPCBs 128 137 138 153 170 180 194
young 13.6 250 2400 1.6 0.063 0.007 50 36 92 8.0 28 11 153 210 52 184 21
middle 11.8 150 4300
26.6 500 5000
2.3 0.150 0.030 41.4
old
2.4 0.340 0.043
135 20
29 72 182 26
50 17 256 307 75 234 36
37 405 450 81 294 40
60
65
1
Middle-agedcarp
.-5
I I
0.6
E c
$
0.4
.-
0.1
Y
I
2
0
FIGURE 1. PCB isomer and congener composition in carp collected from the Buffalo River. Vertical bars represent concentrations of individual PCB isomen relative to the most abundant isomer WPAC No. 153l.the latter being assigned to a relative concentration01 1.0.
bond) were also age-dependent. Generally, concentrations ofPCBsandDDTweresimilarorhigherthanthosereported for various freshwater fish from the Great Lakes during the late 1980s and the early 1990s (17-20). Great concentrations of total PCBs and the presence of a considerable proportion of lower chlorinated members in Buffalo River carp suggestthe existenceofrecent PCB inputs. Combined sewer overflowswere consideredasoneofthemajor sources ofPCBsintotheBuffaloRiverinrecentyears (10,ZO.There appears to be a significant hut unidentified “upstream” source for total PCBs, i.e., in a typical year the loading of total PCBs to the river from combined sewer overflows was 0.15 kg,while total PCBs coming into the “area of concern” from upstream was 1.11 kg (22). Chlorinated DioxinslFurans. Concentrations of total PCDDs were small in carp muscle. Mean PCDD concentrations for young, middle-aged, and old carp were 27,73, and 146 pg/g (wet wt), respectively. Hexa- and heptachlorodibenzo-p-dioxins were the predominant congeners occupying 77-85% of the total PCDDs (Figure 2). Concentrations of total PCDFs in young, middle-aged, and old carp were 22, 99 and 77 pg/g [wet wt), respectively. The congener distribution of PCDFs in carp did not follow a specific pattern with respect to age class. Tetrachloro-
PCDD1
50 1
PCDF
7
loo h
40
8 w
.-.-*e 0
30
;
20
6
10
n" Young
Middle
Young
Old
Ei TetraEd Hepta-
Middle
Old
W Hexa-
0 PentaW Octa-
FIGURE 2. Composition of PCDD and PCDF congeners in Buflalo River carp.
dibenzofurans were predominant in young carp whereas auniform distributionoftetra- to octachlorodibenzofurans, except for pentachlorodibenzofs, was noticed in middle-aged carp. In old carp, tetra- and pentachlorodibenzofurans were proportionally greater (Figure 2). Among various PCDDlPCDF isomers, the 2,3,7,8substituted analogsare more bioactive and persistent than other isomers (23). Therefore, we examined the isomerspecific concentrations of 2,3,7,8-substituted PCDDs/ PCDFs in carp, and the results are presented in Table 3. The concentrations of 2,3,7,8-substituted PCDDs in carp muscle varied from 4 to 26 pg/g (wet wt), accounting for 15-34% of the total PCDD concentration. Most of the 2,3,7,a-~ubstitutedisomers were belowthelimit ofdetection (Table 3). O&DD was the most prevalent isomer in all the age groups. The predominance of OaCDD in sediments from several Great Lakes regions (24) suggests the reason for its increased proportion in carp, which is a bottomfeeding fish. Moreover, the clearance rates of OaCDD/OHCDF were very slow, and a 336-day depuration period did not decrease their concentrations in carp (25). This may be a plausible explanation for higher levels of 08CDD/OsCDF than the other isomers. The concentrations of2,3,7,8-substiNted PCDFs in carp muscle ranged from less than the limit of detection to 42 pg/g [wet wt). In young and old carp, all the 2,3,7,8substituted PCDF isomerswere below the limit of detection whereas in middle-aged carp, 1,2,3.4,6,7,8-H7CDFand OaCDF were detected at 16 and 26 pg/g, respectively. In general, the composition of 2,3,7,8-substiNted PCDD/PCDF isomers to total PCDDlPCDF concentrations was small, which suggests that other less toxic isomers may be prevalent in carp. The most toxic 2,3,7,8-T4CDDIT4CDF was not detected. Analysis of PCDDs/PCDFs in fish from several of the Great Lakes suggested that the isomer and congener pattern varies with location (26, 27). In Lake Ontario fish extracts, 2,3,7,8-TICDD, 1,2,3.7,8-P5CDD.and 1,2,3,6,7,8-HaCDD, 2,3.7,8-T4CDF, 1,2,3,7,8-P5CDF, and Z,3,4,7,8-PsCDFwere the major congeners (28). Similarly, isomer frequency analysis in carp, with total dioxin levels of 50-385 pg/g, collected from four locations in the Great
TABLE 3
Concentrations of 2.3.7.8-Substituted PCOOs/OFs (pglg wet wt) in Muscle of Young, Middle-Aged, and Old Carp from the Buffalo RiveP compd
young
middle
old
____ 13.0
