Environ. Scl. Technol. 1993, 27, 2149-2157
Sensltivity of Cytochrome P450-1A1 Induction in Fish as a Biomarker for Distribution of TCDD and TCDF in the Willamette River, Oregon Lawrence R. Curtls,'~tHlliary M. Carpenter,t Reglna M. Donohoe,t David E. Williams,* Oiaf R. Hedstrom,s Max L. Deinzer,il Michael A. Bellsteln,ll Eugene Foster,I and Richard G a t e d
Department of Fisheries and Wildlife, Department of Food Science, College of Veterinary Medicine, and Department of Agricultural Chemistry, Oregon State University, Corvallis, Oregon 97331, and Oregon Department of Environmental Quallty, Portland, Oregon 97204 The sensitivity of a biomarker for environmental contamination with persistent organochlorines and PAHs (cytochrome P450-1A1 induction in feral fish) was evaluated at six sites along 314 km of the Willamette River, Oregon. Whole-body contamination of Northern squawfish with approximately 2 pg/g of TCDD and 20 pg/g of TCDF in Portland Harbor was significantly higher than upstream sites. Carp muscle TCDF and hepatic biomarker response were also elevated at this site, as were sediment TCDD, TCDF, and PAHs concentrations. In July, cutthroat trout TCDD concentrations above and below a bleached kraft pulp mill were similar. In October cutthroat trout below the mill contained about 2-fold more TCDD than fish upstream. Similar hepatic biomarker profiles occurred in cutthroat trout above and below the mill at both times. Histopathological examination suggested existing organochlorine burdens in adult Willamette River fish were not overtly toxic at any site.
Introduction
Despite efforts to reduce intentional and incidental releases, organochlorines are frequently detected in environmentalsamples. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) occurs as an unwanted byproduct in municipal waste incineration, bleaching of pulp, and synthesis of various chlorinated chemicals, making it a chemical of concern for the forseeable future. 2,3,7,8-Tetrachlorodibenzofuran (TCDF) is a related contaminant which is often produced at higher concentrations in these same processes. Accumulation of TCDD and TCDF by fish is a public and regulatory concern for two principle reasons. First, fish are highly sensitive to the toxicity (1-3) of these contaminants, which can threaten the health of feral fish populations. Second, there is a potential for trophic transfer from contaminated fish to animals which consume them. There is evidence for the transfer of TCDD and TCDF from fish to both wildlife ( 4 ) and humans (5). However, in many cases, the risks associated with this trophic transfer are not clear. Responses of animals which are sensitive to contaminant exposures (biomarkers) can be used as early warning signals for degradation of the environment. Increased cytochrome P450-1A1 expression is a potential sensitive biomarker for exposure of fish to TCDD and TCDF (6). The most *Address correspondence to this author at: Department of Fisheries and Wildlife, Oregon State University, Nash Hall, Room 104, Corvallis, OR 97331-3803. Department of Fisheries and Wildlife. Department of Food Science. I College of Veterinary Medicine. 11 Department of Agricultural Chemistry. Oregon Department of Environmental Quality. 0013-936X/93/0927-2149$04.0010
0 I993 American Chemical Soclety
common techniques for detecting the level of expression of this biomarker involve determination of its enzymatic activity. Ethoxyresorufin-O-deethylase(EROD) activity is probably the most widely accepted of the techniques available (7). It is also possible to measure the specific tissue content of cytochrome P450-1A1 by immunochemistry (i.e., western blots) (8). This can be quite important since high tissue contaminant concentrations can inhibit enzymatic activities (6, 9). Flow-through exposures of rainbow trout to 0.625-10 pg/L fl-naphthoflavoneincrease liver EROD activity and cytochrome P450-1A1 mRNA content in a concentration-dependent manner (9). After 7-day exposure to 50-500 pg/L, EROD induction is inversely related to fl-naphthoflavone concentration while mRNA content increases with concentration (9). Since hepatic content of cytochrome P450-1A1 closely follows expression of its' mRNA (6), inhibition of the protein's catalytic activity (i.e., EROD), not tissue content, is a potential consequence of high inducer concentrations. The objective of this research is to evaluate the sensitivity of a biological response to TCDD and TCDF, by induction of cytochrome P450-1A1, in three species of feral fish collected from sites contaminated to different extents by TCDD and TCDF. Since other agents induce this response, most notably polycyclic aromatic hydrocarbons (PAHs) and coplanar polychlorinated biphenyls (PCBs), fish and sediments were also analyzed for these and a suite of other organic contaminants. Finally, a histopathological assessment of tissues revealed no chemically-induced lesions. Materials and Methods
Sample Collection. Sample sites (Figure 1)impacted by different types of pollution and located within two physically distinct sections of the Willamette River (Table I) were selected. These included a site 2-5 km below a hydroelectric dam (river km 314), a site 1-2 km below a bleached kraft pulp mill discharge (river km 238), a site 1-2 km below a municipal sewage outfall (river km 211)) and a site adjacent to an industrial waste dump contaminated with organochlorines (river km 11). Two additional sites at intermediate locations (river km 258 and 116), which were 10-20 km below sewage outfalls, were sampled. Three fish species were sampled in July and October of 1990: the Northern squawfish (Ptychoceilusoregonensis; all sites), the cutthroat trout (Oncorhynchus clarkii;sites 314,259, and 238 km), and the common carp (Cyprinus carpio; sites 11, 116, and 211 km). Due to the diverse habitat types of the river, not all species were present at each site. Fish were collected by electrofishing and killed by a blow to the head. Immediately thereafter, livers were excised from 1-5 fish of each species (a minimum of three was targeted) and homogenized (10). These samples were Environ. Sci. Technol., Vol. 27, No.
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/COAST FORK
MIDDLE FORK ( R i v e r hm 314; Site 1 )
HARRISBURG HALSEY
(River km 259; Site 2 )
( R i v e r hrn 238; S i t e 3 )
CORVA L L I S
SALEM ( R i v e r k m 116;Site 5 )
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( R i v e r krn 11; Site 6 )
TE
OREGON
Figure 1. Wlllamette River, showing samplesite locatlon and the position of the Wlllamette basin wlthin the state of Oregon (inset).
stored at 4 "C for subsequent laboratory analysis (detailed below). Gills, liver, kidneys, spleen, intestine, and gonads were taken from these same fish and fixed in 10% buffered neutral formalin for histopathology. Squawfish and trout processed in this way ranged from 100to 500 g and common carp ranged from 500 to 2000 g. Additional squawfish and trout (45-200 g) were placed in glass jars for TCDD, TCDF, PCB, and organochlorine pesticide analyses. Carp carcasses were wrapped in aluminum foil. Fish were then placed on ice for transfer to the laboratory. Skinless muscle fillets were prepared from frozen carp for TCDD, TCDF, PCB, PAH, and organochlorine pesticide analyses. Whole squawfish and trout were stored frozen until the time of chemical analyses. Surface sediment from each site was collected in August 1990 with a stainless steel Eckman dredge (0.23 m2), combining 3-5 grabs from areas containing fine-grain material. Subsamples were stored in glass jars at 4 "C. Sediment organic carbon content was determined by the Walkly-Black method (11). Chemical Analysis. Samples and extracts came in contact with only glass, Teflon, and metal prior to GC or GC/MS analysis. All glassware and some metalware were baked at 470 "C for 6 h after being washed and rinsed with distilled water. Teflon and metal that were not baked were rinsed with dichloromethane (DCM) after being rinsed with distilled water. 2150
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Omnisolv-grade2,2,4-trimethylpentane (TMP),hexane, DCM, benzene, methanol, cyclohexane, Tracepur sulfuric acid, and silica gel 60 (70-230 mesh ASTM) were obtained from EM Science, Gibbstown, NJ. Acidic alumina, AG-4, was purchased from Bio-Rad Laboratories, Richmond, CA. B&J brand toluene was obtained from Baxter Healthcare Corp., McGraw Park, IL. Nonane and cesium hydroxide monohydrate (Cs) were obtained from Aldrich Chemical Co., Milwaukee, WI. Potassium hydroxide was obtained from Ashland Chemical Co., Columbus, OH. Technicalgrade anhydrous sodium sulfate was obtained from Kerr McGee, Oklahoma City, OK. Super-A activated carbon (AS-21) was obtained from Anderson Development Co., Adrian MI. Whatman GF/D glass microfiber filters were obtained from W. R. Balston Ltd., England. Sodium sulfate was baked in a muffle furance at 600 "C for 48 h. Silica gel and alumina were washed with methanol and DCM and activated (12) by reaction of silica gel with the respective metal hydroxides. Potassium silicate was stored at 130 "C, and cesium silicate was stored at room temperature in a tightly closed glass jar. Sulfuric acid-silica gel (SA-SG) was prepared by shaking one part concentrated sulfuric acid with 1.5 part activated silica gel until all lumps were broken up. SASG was stored at room temperature in a tightly closed glass jar. Activated carbon purchased as a damp powder was dried for 24 h at 130 "C and then mixed at 2.5% concentration with activated silica gel. The dispersed carbon was stored at 130 "C. Weighed whole fish, muscle, or wet sediment were ground in a Waring blender after mixing with 4-6 weights of anhydrous sodium sulfate. Separate weighed samples of sediment were taken for determination of dry weight and organic carbon content. After being dried, the samplesodium sulfate mixtures were reground and reweighed. Samples of 25 g of fish or wet sediment were added to wintered glass extraction thimbles. Internal standards, 5 ng of [W]-2,3,7,8-TCDD and [13Cl-2,3,7,8-TCDFin 10 pL of nonane (Cambridge Isotope Laboratories, Inc., Cambridge, MA), were added to each sample in the extraction thimble. Samples were extracted in a Soxhlet apparatus with 1:l DCM:cyclohexane. Extracts were applied to a column of (bottom-to-top) 50 g of sodium sulfate, 30 g of silica gel, 30 g of sodium silicate, and 50 g of sodium sulfate. Samples were washed through the column with 1:l DCM:cyclohexane. Sample and wash column eluants were collected as single fractions and concentrated to less than 0.2 mL, reconstituted to 1 mL with hexane, and applied to tandem SA-SG/Cs and activated alumina columns (12). Eluants were concentrated to 0.5 mL. Alumina column sample eluants were applied to dispersed carbon columns (13). The 6-mm (i.d.) columns were packed, bottom-to-top, with glass wool plugs, 5 mm of silica gel, 17 mm of 2.5 % activated carbon in silica gel, and a glass wool plug. Packed columns were washed with 5 mL of DCM, 5 mL of 1:l benzene:DCM, and 5 mL of toluene. The columns were inverted and washed with 10 mL of hexane to remove all toluene. Sample application was followed by three 1-mL washes with cyclohexane, 15 mL of DCM, and 15mL of 3:l benzene:DCM. The columns were inverted, and the dioxinlfuran fractions were eluted with 15mL of toluene. The toluene sample fractions were concentrated to less than 1pL and reconstituted with 1025 pL of nonane.
Table I. General Characteristics and TCDD and TCDF Concentrations of Sediments Taken from Various Sites on the Willamette River in August 1990. 11 kmc 116 kmb 314 kmb 258 kmb 238 kmb 211 kmb physicalcomposition cobbles/gravel 54.7 f 3.0 dry weight ( % ) organic carbon (% ) 0.89 f 0.24 TCDD (pg/g)
