Contaminant Exposure, Biochemical, and Histopathological Biomarkers in White Suckers from Contaminated and Reference Sites in the Sheboygan River, Wisconsin

J. Great Lakes Res. 23(2):119-130 Internal. Assoc. Great Lakes Res., 1997

Contaminant Exposure, Biochemical, and Histopathological Biomarkers in White Suckers from Contaminated and Reference Sites in the Sheboygan River, Wisconsin* Candy S. Schrank!, Susan M. Cormier2, and Vicki S. Blazer3 1Department

ofNatural Resources Madison, Wisconsin 53707

2Ecological Exposure Research Division U.S. EPA Cincinnati, Ohio 45268 3National Fish Health Research Laboratory Biological Resources Division, U.S.G.S. Kearneysville, West Virginia 25430

ABSTRACT. Fish populations of the lower Sheboygan River, located in east-central Wisconsin, are considered impaired under the Great Lakes Water Quality Agreement between the United States and Canada. Pollutants in the Sheboygan River system include: polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PARs), and heavy metals. This study compared general health and biochemical parameters, histology of selected organs, and contaminant residues and metabolites in a population of white suckers (Catostomus commersoni) from a contaminated reach of the Sheboygan River and an upstream reference site. Fish from the contaminated site had significantly lower hematocrits, significantly induced ethoxyresorufin-O-deethylase (EROD) activity as a measurement of hepatic mixed-function oxygenase activity, higher biliary metabolites of PARs, and higher tissue concentrations of PCBs and p,p'-DDE relative to an upstream reference population. Analysis of covariance suggests that both PCB and PAR exposure may be influencing EROD activities. Fish from the contaminated site featured more basophilic clusters and developing nephrons in kidney tissue suggesting the presence of a nephrotoxicant. Also, more fish exhibited hepatic lesions including diffuse cellular vacuolation, multifocal coagulative necrosis, bile ductal hyperplasia, and foci of cellular alteration which may be biomarkers for contaminant impacts. This study demonstrates that white suckers residing in the lower reaches of the Sheboygan River absorbed significant amounts of PARs and PCBs and also exhibited hematological, biochemical and histological alterations some of which suggest impaired fish condition. INDEX WORDS:

PCBs, PARs, histopathology, EROD, white suckers, Sheboygan River.

INTRODUCTION

marily of cropland with areas of wetlands, urban and developed land, woodland, and agricultural land. The main stem of the Sheboygan River is 278 km long, drops an average of 1.3 rn/km, and is impounded at six locations. It flows into the Sheboygan Harbor which is sheltered from Lake Michigan by a series of breakwaters at the City of Sheboygan. The lower segment of the Sheboygan River watershed has a history of shipping, industrial and municipal activities, and dredging. Consumption

The Sheboygan River watershed is located in east-central Wisconsin (Fig. 1). The river and its tributaries drain about 1,502 km 2 comprised pri* This document has been reviewed in accordance with U.S. Environmental Protection Agency policy and approved for publication. Approval does not signify that the contents necessarily reflect the views or policies of the Agency nor does mention of trade names or commercial products constitute endorsement or recommendation for use.

119

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Schrank et aI.

HighwayJJ

Lake Michigan

N

A

5km FIG. 1. Map of Sheboygan River indicating approximate location in Wisconsin and location of the study and reference sites.

advisories were first issued in 1978 because some fish from the Sheboygan River exceeded the Food and Drug Administration's polychlorinated biphenyl (PCB) tolerance level for marketable fish. A landfill adjacent to the river was designated as a federal Superfund site in 1984 and the river downstream of the Village of Sheboygan Falls was designated as a federal Superfund site in 1985 because of extensive PCB sediment contamination. The lower river and harbor were also designated by the International Joint Commission (UC) of the United States and Canada as an Area of Concern under the Great Lakes Water Quality Agreement. In addition to the Superfund sites and other municipal, industrial, and nonpoint pollution sources, potential pollution sources to the river include a petroleum storage facility and former coal storage and coal gasification plant sites. Contaminants in the Sheboygan River system include: polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAHs), heavy metals (arsenic, barium, cad-

mium, chromium, copper, lead, mercury, nickel, silver, and zinc), and other pollutants (Wisconsin Department of Natural Resources 1988, 1989, 1995). The highest PCB sediment concentrations (4,500 mg/kg PCBs) were found approximately 15 to 25 km upstream of the mouth prior to removal actions that began in 1989. PCB sediment concentrations decline with distance downstream (Sonzogni et al. 1991, David et al. 1994), and sediment contamination is minimal in the upper segments of the Sheboygan River. Further investigation and cleanup of sediment contamination and sources are ongoing (Wisconsin Department of Natural Resources 1988, 1989, 1995). Although the lower Sheboygan River supports a diverse fish community (Fago 1985), fish populations are considered impaired under the HC designation as an Area of Concern (Wisconsin Department of Natural Resources 1988, 1989, 1995). Since 1975, the Wisconsin Department of Natural Resources and others have monitored con-

Biomarkers in White Suckers from the Sheboygan River taminants in fish from the Sheboygan River (Eggold et at. 1996). Activities to remediate sediments are predicated upon preserving or restoring ecosystem health, but no investigations examining physiological impairments to fish have been conducted to date. A variety of biomarkers have been identified as useful indicators of exposure to or the effects of environmental pollutants some of which do not accumulate in tissues. We chose to investigate hepatic ethoxyresorufin-o-deethylase (EROD) activity, total hepatic glutathione (GSH), bile metabolites, and histopathological alterations because other studies (Jimenez and Stegeman 1990, Niimi 1990, Hinton et af. 1992, Melancon 1995) have shown these to be responsive to PAHs and PCBs. The induction of hepatic mixed-function oxygenase (MFO) activity of fish liver is a useful indicator of exposure to certain types of contaminants such as aromatic hydrocarbons and chlorinated hydrocarbons (Payne et af. 1987; Rattner et aZ. 1989; Addison 1995a, b). Induction of MFO is generally thought to be an adaptive response but with potential adverse consequences. According to Addison (1995a, b), induction of the monooxygenase system allows body burdens of contaminants to be reduced but at the same time may increase susceptibility to damage by enhancing the formation of metabolites which are more reactive and potent toxins than a parent compound. In addition, there is evidence that by some as yet unspecified mechanism, the disruption of the monooxygenase system may be related to disruptions in physiological processes such as the reproductive system. Lastly, there is some support that the monooxygenase measurements may serve as an indicator of more serious whole organism or community response (Addison 1995a, b). Ethoxyresorufin-O-deethylase (EROD) activity is a popular way of measuring MFO activity because of its sensitivity, simplicity, high specificity for P4501A-type enzymes, and low occupational hazard (Munkittrick et at. 1993). The tripeptide glutathione plays an important role in many biological processes including detoxification of xenobiotics and removal of hydroperoxides (Akerboom and Sies 1981). An increase in glutathione is believed to represent a compensatory and protective response to contaminant exposure (Nishimoto et at. 1995). Analysis of tissue residues indicates exposure to and availability of bioaccumulating contaminants and metals, yet many contaminants may exert adverse effects without bioaccumulation. Analysis of

121

bile for metabolites of polycyclic aromatic hydrocarbons indicates exposure to and bioavailability of PAHs to fish (Hellou and Payne 1987, Macubbin et af. 1988, Ariese et aZ. 1993, Lin et aZ. 1994, Lin et aZ. 1996). Specific hepatic lesions have been suggested as biomarkers of exposure to anthropogenic environmental toxicants (Baumann et at. 1996, Hinton and Lauren 1990, Hinton et al. 1992). Developing nephrons and basophillic clusters in the kidney have been proposed as general indicators of environmental stress, but may be better suited for detection of specific nephrotoxicants (Cormier et at. 1995b). The purpose of this study was to compare general health conditions, biochemical parameters, histology of selected organs, and contaminant residues and metabolites in white suckers (Catostomus commersoni) from a contaminated reach of the Sheboygan River relative to a reference site. White suckers were selected as the indicator species because they are in contact with the sediment, are common and numerous in the Sheboygan River (Fago 1985), and have been recommended as an environmental sentinel (Munkittrick and Dixon 1989). MATERIALS AND METHODS Study Sites and Fish Collection White suckers (Catostomus commersoni) were collected from the study site on 6 July 1994 using a pulsed DC boomshocker and from the reference site on 7 July 1994 using a streamshocker. The study site was located approximately 1 to 2 km upstream from the mouth of the river and the reference site was approximately 50 km further upstream. Fish of similar size (200 to 400 mm) were targeted at each site to reduce possible effects of maturity and age on other measured parameters. Fish were transferred to a tub for transportation and held in aerated site water until examined within 1 to 6 hours. General After anesthetizing with MS-222 (Argent Chemical Laboratories, Redmond, WA, USA), a blood sample was taken from the branchial vein for determination of hematocrit, leukocrit, and plasma protein levels. Total body length and weight were measured. General appearance of vital organs was assessed by visual observation using a necropsybased procedure described by Goede (1993) and Goede and Barton (1990). Gonads were examined

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and classified as immature, male, or female. The gallbladder condition was examined for degree of fullness and color of the bile. The age of each fish was estimated by examining scale annuli. Biochemical Parameters and Tissue Metabolites

Liver and bile samples were taken from the first sixteen fish examined from the study and reference sites, then frozen in a liquid nitrogen dry shipper. Microsomes and cytosol were prepared and EROD activity was determined using the methods of Lin et ai. (1989) with modifications as specified in Cormier et ai. (1995a). EROD activity was determined by a kinetic measurement of resorufin formation and results are expressed as pmol/mg protein/minute. Total glutathione (reduced plus oxidized) was measured using methods of Akerboom and Sies (1981) with modifications specified in Cormier et ai. (1995a). Total glutathione was analyzed, after deproteinization, at a wavelength of 415 nm and results are expressed as nmol SH-equivalents/mg protein. Bile samples were analyzed on a Perkin Elmer LS50B spectrofluorometer by fixed fluorescence (FF) according to the methods specified in Lin et ai. (1996). Excitation/emission wavelengths of 290/335 nm were used to measure naphthalene-type metabolites indicative of exposure to petroleum fuel and 380/435 nm for benzo(a)pyrene-type metabolites or five-ring chromophores indicative of exposure to contaminants from combustion sources. Fluorescence of the sample was compared to a standard curve of the fluorescence of B(a)P or naphthalene standards (Lin et ai. 1996). The naphthalene-type and benzo(a)pyrene-type metabolites present in the bile samples are expressed as microgram equivalents/mg protein. Protein concentrations for microsomes, cytosols, and bile were measured using Pierce's micro BCA protein assay reagent kit with an extended incubation of 18 hours. Samples were analyzed on a Dynatech MR5000 micro titer plate reader at a wavelength of 550 nm. Histology Tissue samples were fixed in Bouin's solution (Humason 1979) for 24 to 48 hours and transferred to 70% ethanol. Liver samples were taken from the first sixteen fish and spleen, kidney, and gill samples were taken from the first four fish from each

site. The tissue samples were dehydrated, embedded in paraffin, sectioned at 6 /lm, and stained with hematoxylin and eosin (H & E) and Perl's stain for iron (Humason 1979) and scored for abnormalities blindly as to origin. The number of basophilic clusters and developing nephrons in kidney tissue were determined as described by Cormier et ai. (1995b). Basophilic clusters were identified as a cluster of small, basophilic, crescent-shaped cells. Developing nephrons were identified as organized groups of very basophilic epithelial cells, usually forming a narrow lumen. Five mm 2 of kidney tissue were evaluated using image analysis and then the mean number of developing nephrons and basophilic clusters per mm 2 was calculated. The criteria of Hinton et ai. (1992) were used to evaluate hepatic lesions. Each fish was examined for the presence of seven different types of hepatic lesions recognized as biomarkers of contaminant exposure. The seven hepatic lesions were focal and multifocal coagulative necrosis (in the absence of infectious agents), focal and diffuse cellular vacuolation, bile duct proliferation, foci of cellular alteration, and adenoma. The number of fish exhibiting each of these seven lesions is reported for each site. Tissue Residue

After necropsy, ten fish from each site with intact carcasses and similar size were wrapped in aluminum foil, labeled, and frozen. Whole fish were individually homogenized and subsamples were analyzed for percent lipid, arsenic, mercury, cadmium, chromium, copper, lead, zinc, four isomers of chlordane, dieldrin, dichlorodiphenyltrichloroethane (DDT) and metabolites, alpha- and beta-hexachlorocyclohexane, hexachlorobenzene, heptachlor epoxide, toxaphene, and total polychlorinated biphenyls (PCBs) based on aroclor standards. Analyses were conducted according to methods specified in Wisconsin State Laboratory of Hygiene manuals (Wisconsin State Laboratory of Hygiene 1980, 1994). Statistical Analysis Statistical analyses were conducted using SAS System for Windows (Version 6.08, SAS Institute, Cary, North Carolina). Determination of statistical significance was set at p ~ 0.05 for all tests. Tests included: Shapiro-Wilks statistic to determine normality; two-way analysis of variance to investigate

123

Biomarkers in White Suckers from the Sheboygan River the significance of site and sex as classification factors; analyses of covariance using a general linear model with site as a classification factor to determine the significance of site-covariable interactions and covariables on the variability of normal and normalized parameters; and two sample t-tests (assuming equal and unequal variances), Duncan's multiple range, and nonparametric Wilcoxon tests to determine differences between sites. Two-tailed Fisher's exact test was used to determine if the number of fish with and without specific histopathological lesions depended on site of collection. Parameters which included uncensored values were examined using Uncensor 4.0 (Savannah River Ecology Lab, Aiken, SC) using Helsel's robust method of calculating means, variances, standard deviation, and confidence intervals.

RESULTS

General Thirty white suckers were collected from the west bank of the Sheboygan River about 1 km upstream from the mouth of the river. Twenty-nine fish were collected from the upstream reference site. For fish from the study site, the median value for weight was 385 gm (273 to 654), for length it was 331.5 mm (223 to 385), and for age it was 3+ years (2+ to 4+). For fish from the reference site, the median value for weight was 121 gm (92 to 341), for length it was 233 mm (210 to 315), and for age it was 2+ years (l + to 3+). Although we attempted to collect fish of similar size at each site, the weight, length, and age of the fish collected at the study and reference sites were significantly different (p = 0.0001). Weight, length, and age were highly and significantly correlated with each other (p = 0.0001). All of the fish from the reference site were classified as immature. Nineteen of the 30 study

fish were classified as immature, 8 as females and 3 as males. Percent fat in whole body tissue was not significantly different between sites. The study site fish averaged 5.3% fat (n = 13, CV = 44%) and the reference fish averaged 5.3% fat (n = 14, CV = 24%). Few gross differences in the visual condition of fins, organs, and tissues were observed. A higher percentage of the fish from the study site had pale gills, discolored livers, adhesions in between organs, while a higher percentage of fish from the reference site had pseudobranchs or thymus which appeared inflamed. Analysis of the significance of these observations was not conducted. The majority of fish from both sites had gallbladders which were full and yellow, suggesting that most fish had fed a few to several days prior to capture. Hematocrit values were significantly different (p = 0.001) between sites. Study site fish had a median hematocrit of 41 % (n = 23, 9% to 55%). Reference site fish had a median hematocrit of 48% (n = 29, 27% to 54%). Leukocrits were minimal at both sites. Plasma protein averaged 5.0 g/dl (n = 23, CV = 26%) in the study fish and 3.7 g/dl (n = 29, CV = 15%) in the reference fish. Weight was significant (p = 0.005) as a covariable in describing the variation in protein. Plasma protein adjusted for weight was not significantly different between sites.

Biochemical Parameters and Tissue Metabolites ERaD was significantly higher at the study site (Table 1). Analyses were conducted on ERaD data normalized by log-transformation. Table 1 shows the back-transformed mean ERaD activity for each site. ERaD did not differ between the sex classifications at the study site. Examining only the fish

TABLE 1. EROD, total hepatic glutathione, and bile metabolites in white suckers from the reference and study sites, Sheboygan River, Wisconsin. Study Site

Reference Site Parameter

Mean

CV

n

EROD (pmol/mg protein/min) Total GSH (nmol/mg protein) B(a)P equivalents (Ilg/mg protein) Naphthalene equivalents (Ilg/mg protein)

8.60 0.558 0.136 26.95

21 13 42 26

15 15 15 15

* significantly different from reference site at p < 0.005. ** significantly different from reference site at p = 0.0002. CV = coefficient of variation.

Mean 35.19 0.567 0.354 41.23

** ** *

CV

n

22 33 44 36

16 15 15 15

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Schrank et al.

classified as immature, EROD activities remained significantly different (p < 0.001) between sites. Total hepatic GSH was not different between sites (Table 1) but was found to be different between sex classifications (p = 0.05) with averages for immatures, males and females of 0.57 (n = 23), 0.73 (n = 3), and 0.39 nmol/mg protein (n = 4), respectively. Examining only the fish classified as immature, there was no difference in GSH between sites. Measurement of metabolites of PAHs in the bile indicates that PAHs are bioavailable to both populations (Table 1). However, average B(a)P-type and naphthalene-type metabolite values were significantly higher at the study site compared to the reference site suggesting greater exposure to PAHs from combustion processes and petroleum fuel.

Histology A number of histological differences were noted in kidney, spleen, and liver tissues from white suckers collected at the study site versus the reference site (Table 2). Although kidney tissue of fish from both the study and reference sites contained basophilic clusters, significantly more were found in samples from the study site. Developing nephrons were found in all of the study site fish, but in none of the reference site fish. Often clusters containing multiple cross-sections of tubules were observed (Fig. 2), however these were counted as one observation. Only three of the seven types of hepatic lesions

tallied were observed in fish from the reference site (Table 2). One fish from the reference site exhibited bile duct hyperplasia, two had focal areas of coagulative necrosis, and three had focal areas of cellular vacuolation. Six of the seven hepatic lesions tallied were observed in fish from the study site. Cellular vacuolation was present in livers from five study site fish and was of a more diffuse nature than at the reference site. The vacuoles were believed to be fatty, however this could not be definitively determined with our methods. Eleven of the twelve livers from the study site which showed coagulative necrosis had a more extensive involvement than livers from the reference site. Often this necrosis was accompanied by slight inflammation without any signs of infectious agents and appeared to be due to tissue damage. Nine fish from the study site showed bile ductular/ductal hyperplasia. Foci of cellular alteration (basophilic and eosinophilic foci) and one small hepatic adenoma were also noted in livers from the study site. The adenoma was encapsulated and appeared to be growing, based on observed compression of surrounding hepatocytes. The incidence of the following lesions significantly depended on the site of collection: diffuse cellular vacuolation, multifocal coagulative necrosis, bile ductal hyperplasia, and foci of cellular alteration.

Tissue Residues Analysis of whole body tissues indicates that the fish from the study site were exposed to and accu-

TABLE 2. Mean number of selected lesions per mm 2 of kidney tissue (n = 4 fish) and number of fish (n = 16 fish) with selected hepatic lesions from the reference and study sites, Sheboygan River, Wisconsin. Parameter Kidney Basophilic clusters Developing nephrons

Reference Site Observations/mm 2 ± SD 0.3 ± 0.2

0

Number of Fish Liver 3 Focal cellular vacuolation Diffuse cellular vacuolation o Focal coagulative necrosis 2 Multifocal coagulative necrosis o Bile ductal hyperplasia 1 Foci of cellular alteration o Hepatic adenoma o * significantly different from reference site at p < 0.05. ** the number of fish with lesions is dependent on the site of collection (p < 0.05).

Study Site Observations/mm2 ± SD 0.7 ± 0.2 1.1 ± 0.6

* *

Number of Fish

o

5 ** 1 11 ** 9 ** 5 ** 1

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Biomarkers in White Suckers from the Sheboygan River

FIG. 2. Kidney section of white sucker from Sheboygan River study site. A basophilic cluster (closed arrow) as well as a number of groups of developing nephrons (open arrows) are identified.

mulated PCBs and p,p'-DDE to significantly higher concentrations than at the reference site (Table 3). Over both sites, most fish contained less than detectable residues of: cadmium, lead, chlordane isomers, alpha- and beta-hexachlorocyclohexane, hexachlorobenzene, heptachlor epoxide, toxaphene, p,p'-DDT, p,p'-DDD, o,p'-DDT, o,p'-DDD, and o,p'-DDE. Some parameters were at both detectable and less than detectable concentrations. Using Helsel's robust method for those parameters, p,p'-DDE was

found at significantly higher concentrations in study site fish compared to the reference fish. All ten fish from the reference site contained concentrations of p,p'-DDE at less than the level of detection (0.05 flg/g). The lower confidence limit (0.063 to 0.139 flg/g) of the p,p'-DDE concentrations from the study site does not overlap the level of detection. The confidence intervals for the two sites overlapped for dieldrin, arsenic, and chromium. Mercury concentrations (whole body) were not normally distributed nor significantly different be-

TABLE 3. Contaminant residues in white suckers (whole body) from the reference and study sites, Sheboygan River, Wisconsin. Study Site

Reference Site Parameter Dieldrin (Ilg/g) p,p' -DDE (Ilg/g) Total PCB (Ilg/g) Arsenic (Ilg/g) Chromium (Ilg/g) Copper (Ilg/g) Zinc (Ilg/g)

Mean <0.02 < 0.05 0.094 < 0.1 0.26 0.55 13.6

** significantly different from reference site at p = 0.0001. *** significantly different based on confidence limits.

CV

13 21 20 14

n 10 10 10 9 9 9 9

Mean 0.012 0.09 3.550 0.1 0.25 0.66 12.0

*** **

CV

n

67 74 26 0 24 23 13

10 10 10 10 10 10 10

Schrank et at.

126

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FIG. 3. Hepatic log EROD versus biliary naphthalene-equivalents at reference and study sites.

tween the two sites. Concentrations in fish from the reference site ranged from 0.051 Ilg/g to 0.11 0 Ilg/g with a median value of 0.079 Ilg/g (n = 9). Concentrations in fish from the study site ranged from 0.056 Ilg/g to 0.22 Ilg/g with a median value of 0.065 Ilg/g (n = 10).

Relationship Between EROD and Tissue Residues and Metabolites Analysis of covariance indicated that both site (p = 0.0004) and the level of biliary naphthaleneequivalents (p = 0.02) were significant in explaining the variability of the normalized EROD levels (Fig. 3). However, analysis of variance by site indicated that normalized EROD activity was inversely related to PCB tissue concentration (p = 0.03) at the reference site (Fig. 4) and an interaction term (PCB *naphthalene-equi valents) was significant (p = 0.02) at the study site.

Reference Site

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FIG. 4. Hepatic log EROD versus PCB residues (whole fish) at reference and study sites.

DISCUSSION White suckers from the study area had higher tissue concentrations of PCBs and p,p'-DDE and higher biliary metabolites of PAHs compared to the reference fish. The PCB and PAH levels reflect exposure to contaminants in the sediment at the study site. Four sediment samples taken in 1995 in the vicinity of the study site contained 5.46 to 15.0 mglkg as a total of 17 PAHs. Six sediment samples taken from this lower reach in 1995 also contained PCBs from 1.0 to 2.1 mg/kg. Data from the upstream reaches of the Sheboygan River indicate low levels of sediment contaminants (unpublished data, Wisconsin Department of Natural Resources). For perspective, bile metabolite values found in this study were compared to values found by Cormier et ai. (1996). Bile metabolite values from both the reference and study sites of this study were in the range of the metabolites found in the eastern cornbelt plains ecoregion study. Two-thirds of the study site fish exceeded the reference value for B(a)P-metabolites established for white suckers by the eastern cornbelt plains ecoregion study. Only four study site fish exceeded the naphthalene reference. None of the fish from the reference site exceeded the eastern cornbelt reference values. EROD activity in the study site fish was four times that of reference fish and more than 85% of study site fish exceeded reference values based on 456 white suckers sampled in Ohio from 1991-1993 (Lin et ai. 1996). In two fish from the study site, EROD activities were more than 100 pmol/mg protein/minute and the average at the study site (35 pmol/mg protein/minute) was above the 30 pmol/mg protein/minute level indicative of high induction. Other studies have found elevations in EROD activities in white suckers from polluted waters along with changes in other endpoints (Branchaud et ai. 1995; Gagnon et ai. 1994, 1995; Hodson et ai. 1992; Munkittrick et ai. 1991, 1992, 1994). PCBs, PAHs, and other substances have been shown to induce, inhibit, or both induce and inhibit mixed function oxygenase activity (Bucheli and Fent 1995). Our finding that the level of biliary naphthalene-equivalents was significant as a covariable suggests a relationship between EROD activity and petroleum fuel exposure. However, examination of the relationship between normalized EROD levels and PCBs and PAH metabolites at each site suggests that both pollutants may be influencing the EROD activity. This supports Bucheli and Fent's (1995) observation that a direct linear correlation

Biomarkers in White Suckers from the Sheboygan River between a single contaminant and EROD activity cannot always be expected in field situations because most involve exposure to multiple chemicals. Sex, maturity, and reproductive status are factors known to affect basal and inducible levels of monooxygenase activity (Addison 1995b). Although the study site fish were larger and some fish had gonads that were further developed than the reference site fish, weight and sex were not major factors in the EROD activities. It is not surprising that EROD activities in this study were not significantly affected by these factors because most fish appeared immature and were collected between reproductively active seasons. White suckers generally become reproductively active at ages 3 to 4 and spawn in April to May in Wisconsin (Becker 1983). A number of histological differences were noted in kidney, spleen, and liver tissue of white suckers collected at the study site versus the reference site. Developing nephrons and the basophilic clusters from which they arise have been demonstrated in fish exposed to hexachlorobutadiene (Reimschuessel et ai. 1990), mercuric chloride (Reimschuessel and Gonzalez 1992), and tetrachloroethylene (Reimschuessel et ai. 1994). These structures are thought to be a compensatory response to destruction of tubules from exposure to these substances or substances with similar modes of action (Cormier et al. 1995b). Our findings suggest that nephrotoxic compounds may be present at the study site and support the use of these structures as biomarkers. The hepatic lesions, observed primarily at the study site, have been observed in fish after laboratory exposure to a number of different chemicals (Tetra Tech, Inc. 1987) and many of these lesions have previously been reported in white suckers collected from contaminated sites (Baumann et ai. 1996). Hayes et ai. (1990) found proliferative biliary lesions and foci of cellular alteration in white suckers from polluted sites in western Ontario. Cholangiocytic and hepatocytic tumors have been found at a number of sites in the Great Lakes basin (Cairns and Fitzsimons 1988, Hayes et ai. 1990, Smith et ai. 1995). These types of lesions have most often been associated with chronic exposure of fish to organic contaminants such as PAHs and PCBs (Murchelano and Wolke 1985, Myers et al. 1990, Vogelbein et ai. 1990, Baumann and Harschbarger 1994, Moore and Myers 1994). Strong and consistent correlations between PAHs and the prevalence of liver lesions, including neoplasms, foci of cellular alteration, and necrosis, have been documented (Moore and Myers 1994).

127

Foci of cellular alteration are thought to precede development of hepatic neoplasms and indicate carcinogen exposure (Hinton 1994). A larger sample of fish would need to be examined to determine if the prevalence rate of hepatic neoplasms exceeds 5%, proposed as an indicator of environmental degradation (Baumann et al. 1996). Our attempts to assess general health by hepatic glutathione, plasma protein, and hematocrit detected few differences between the study and reference site fish and interpretation was hindered by limited information on normal values. The GSH levels found in this study are similar to reference values set for white suckers from Ohio (Lin et ai. 1989). The Ohio GSH reference values were arbitrarily set at nominal if below and elevated if above 0.6 nmol/mg protein. Plasma protein concentration is a rough indicator of relative energy stores and food availability but may vary with size, sex, or maturity of the fish. Plasma protein may also be affected by osmotic imbalances, temperature, and many contaminants including PCBs and PAHs (Goede and Barton 1990, Folmar 1993). The level of fat or percent lipid of tissues is also a gross measure of surplus energy deposition (Goede and Barton 1990). The plasma protein, whole body fat, and gallbladder data indicate that the fish from the two sites had similar energy stores and were actively feeding. Mean hematocrit of the study site fish was less than that of the reference site fish. Deviations from normal hematocrit levels may indicate acute stress, disease, gill damage, dehydration, stress, or polycythemia. Many chemicals have been associated with altered hematocrits including PCBs and PAHs (Folmar 1993). However, further interpretation is limited by the lack of information on normal values. This study demonstrates that white suckers residing in the lower reaches of the Sheboygan River absorbed significant amounts of PAHs and PCBs and also exhibited hematological, biochemical, and histological alterations, some of which suggest impaired fish condition. Lesions in the kidney tissues suggest that fish at the study site are exposed to a nephrotoxicant in addition to PAHs and PCBs. Exposure to PCBs and PAHs may be responsible for the elevated mixed-function oxygenase activity and incidence of neoplastic and preneoplastic alterations observed in the livers of these fish. Induction of the mixed-function oxygenase activity indicates a response to reduce body burdens of contaminants and may also be a factor in the incidence of the histopathological alterations through formation of

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more reactive compounds. The histopathological alterations suggest long-term exposure to these compounds while biliary metabolites indicate recent exposure. The significance of these alterations to the white sucker population and other species in the contaminated reach of the Sheboygan River is undetermined. This study may provide a baseline for future studies to evaluate clean-up efforts or the significance of the contamination to the white sucker population and supports the use of EROD, bile metabolites, and histopathology as indicators of exposure and response to PARs and PCBs. ACKNOWLEDGMENTS

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