Environ. Sci. Technol. 2000, 34, 4327-4334
Characteristics of Ligands for the Ah Receptor and Sex Steroid Receptors in Hepatic Tissues of Fish Exposed to Bleached Kraft Mill Effluent L . M A R K H E W I T T , * ,† JOANNE L. PARROTT,† KELLY L. WELLS,‡ M. KATHRYN CALP,‡ SUSAN BIDDISCOMBE,‡ MARK E. MCMASTER,† KELLY R. MUNKITTRICK,§ AND GLEN J. VAN DER KRAAK‡ Aquatic Ecosystem Conservation Branch, National Water Research Institute, Environment Canada, Burlington, Ontario, Canada, Department of Zoology, University of Guelph, Guelph, Ontario, Canada, Aquatic Ecosystem Impacts Branch, National Water Research Institute, Environment Canada, Saskatoon, Saskatchewan, Canada, and Department of Biology, University of New Brunswick, Fredericton, New Brunswick, Canada
Male white sucker (Catostomus commersoni) from a population exposed to effluent from a bleached kraft mill and from a reference site were held in effluent or clean water for 4 d. To investigate bioavailable bioactive compounds, hepatic tissues were extracted and fractionated according to octanol-water partition coefficient (Kow) using reverse phase HPLC. Fractions were tested in vitro for the presence of compounds functioning as ligands for (i) the aryl hydrocarbon receptor (AhR) using mixed function oxygenase (MFO) induction in H4IIE cells, (ii) the estrogen receptor (ER) isolated from rainbow trout (Oncorhynchus mykiss) liver, (iii) the androgen receptor (AR) isolated from goldfish (Carassius auratus) testes, and (iv) sex steroid binding protein (SSBP) isolated from goldfish plasma. Polychlorinated dioxins and furans accounted for MFO activity of liver contaminants of log Kow > 5 but multiple nondioxin AhR ligands of log Kow 2 to 5 also caused significant induction. Compounds of log Kow 2 to 5 in livers of exposed fish exhibited significant competition for the AR, ER, and SSBP, indicating potential effects on hormone signaling and transport. The absence of most ligands for the AhR and sex steroid receptors in tissues of preexposed fish held in clean water demonstrates clearance of these compounds after short-term removal from effluent. This study demonstrates the utility of using wild fish tissue burdens to study the lipophilicities and the pharmacokinetic properties of bioavailable compounds functioning as ligands for the AhR and sex steroid hormone receptors.
* Corresponding author phone: (905)319-6924; fax: (905)336-6430; e-mail:
[email protected]. † Environment Canada, Burlington. ‡ University of Guelph. § Environment Canada, Saskatoon, and University of New Brunswick. 10.1021/es0011212 CCC: $19.00 Published on Web 09/09/2000
2000 American Chemical Society
Introduction The effects of pulp mill effluents on wild fish have been well documented. For studies conducted at the pulp mill releasing effluent into Jackfish Bay Lake Superior, the most consistent responses have been induction of hepatic mixed function oxygenase (MFO) activity and effects on reproductive fitness that include reduced gonad size, reduced expression of secondary sexual characteristics, and depressions in circulating levels of gonadal sex steroids (1, 2). Improvements in MFO induction and reproductive performance in white sucker (Catostomus commersoni) have been observed following mill process changes which include installation of secondary treatment, improved spill control, and increased substitution of chlorine dioxide for molecular chlorine (3). At this time it is not possible to directly relate process changes to improvements in fish responses since the responsible chemicals were not identified prior to modifications. The persistence of MFO induction and reproductive alterations in wild fish at Jackfish Bay and other pulp mills may be related to decreased discharges of the responsible compounds or may be associated with chemicals released as a result of these modifications. Characterizing compounds associated with present-day responses is the first step in addressing these issues and would allow evaluations of future process changes. Previous speculation held that polychlorinated dibenzodioxins and dibenzofurans (PCDD/DFs) present in final effluents and biota from receiving environments were solely responsible for MFO induction, but there is evidence that additional nondioxin classes of chemicals interacting with the aryl hydrocarbon receptor (AhR) are involved (4-6). Additional studies have shown that black liquor from bleached kraft mills can elevate MFO activity (7, 8), juvabione was associated with induction in steam condensates at a thermomechanical mill (9), and a chlorinated stilbene has been associated with MFO activity in treated bleached kraft effluent (10). It is important to note that in these investigations the total MFO activity of the effluents has not been accounted for and additional AhR ligands remain unidentified. Although compounds associated with reproductive dysfunction in wild fish have not been examined systematically, certain wood extractives present in final effluents have individually been found to affect endocrine function in fish. β-Sitosterol, the dominant plant sterol consistently measured in final effluents, exhibits estrogenic activity in vitro and can induce vitellogenin production and reduce plasma sex steroids in vivo (11, 12). Abietic acid, pinosylvin, and betulin have also shown the potential to exhibit estrogenic activity (13). The responses measured at Jackfish Bay and other pulp mills have shown effects on gonad size, depressed levels of circulating steroids (3), perturbations in the sex steroid biosynthesis pathway (14), and effects on gonadotropin production and peripheral sex steroid metabolism (15), indicating multiple mechanisms and chemicals are involved. The recent development and characterization of fish-specific sex steroid receptor assays (16, 17) now affords the opportunity to test whether chemicals interacting at the level of the receptor are associated with these responses. Bioassay-directed effluent fractionations have been employed to isolate and characterize compounds associated with MFO induction in effluents (4, 8, 9, 18). Difficulties encountered with fractionation studies of final effluents include the following: (i) fractionation experiments conducted on “grab” samples of effluent do not reflect temporal fluctuations in active chemicals, (ii) toxicological potencies VOL. 34, NO. 20, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. Locations of fish caging sites relative to the bleached kraft mill located in Terrace Bay, ON. Preexposed fish and fish from the reference site at Mountain Bay (not shown) were caged in effluent at the third crossing of Blackbird Creek with Hwy 17 (3X) and in clean water at Sawmill Creek. of effluent samples were influenced by sample handling and storage conditions, (iii) the large amount of high molecular weight lignin material proved a significant interference when investigating low molecular weight extractives, (iv) the complexity of low molecular weight effluent extractives, and (v) uncertainties regarding the bioavailability of identified bioactive components. Our approach to address these concerns was to characterize ligands for the Ah receptor and sex steroid receptors present in tissues of fish exposed to effluent. Previous studies with effluent from the mill located in Terrace Bay have shown MFO activity can be rapidly induced (19) and quickly disappears during a mill shutdown (5). Reductions in gonadal production of sex steroids after 4 and 8 d effluent exposures have also been observed (20). In this study we utilized shortterm effluent exposures to naı¨ve fish from a reference site to investigate ligands associated with rapid uptake. Fish historically exposed to effluent were held in effluent for comparison, as well as in clean water to evaluate the persistence of accumulated ligands. In these experiments we sought to relate the lipophilicities of ligands for the AhR and sex steroid receptors in hepatic tissues to the different regimes of exposure and depuration.
Experimental Section Chemicals and Equipment. Unless otherwise stated, all solvents were distilled in glass (Caledon Laboratories, Georgetown, ON, Canada), and HPLC instrumentation was from the same manufacturer (Waters, Mississauga, ON, Canada). Fish Caging Experiments. The bleached kraft mill chosen for this study is located near the town of Terrace Bay, ON, Canada. Effluent from this mill has consistently been associated with induction of hepatic MFO activity and reproductive dysfunction in wild white sucker since 1988 (3). At the time of fish exposures, the mill was operating at 60% ClO2 substitution for elemental chlorine on hardwood and softwood lines (hardwood DCEDED; softwood DCPEopDED; R. Ferguson, Kimberly Clark, Terrace Bay, ON, Canada, personal communication). Water usage approximates 100,000 m3d-1, effluent passes through a primary clarifier into a 375,000 m3 settling basin followed by a 1.1 million m3 three-cell aerated stabilization basin with a hydraulic retention time averaging 10 d. Effluent is released into Blackbird Creek (48°49′ N - 87°00′ W), approximately 15 km upstream of the mouth in Jackfish Bay, Lake Superior (Figure 1). There are no other known inputs to Blackbird 4328
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Creek, the third crossing with highway 17 was used as the effluent caging site, approximately 6 km downstream of the effluent discharge (3X, Figure 1). Since females can reduce body burdens of accumulated contaminants through maternal transfer (21), males were utilized for caging experiments. Wild male white sucker (39.8 ( 1.7 cm; 826 ( 113 g; mean ( SD) that normally reside in the effluent discharge area (preexposed) were collected during their spawning run from an uncontaminated tributary of Jackfish Lake (Sawmill Creek) in May 1996 using trap nets (Figure 1). Reference males (41.5 ( 1.7 cm; 927 ( 117 g) were collected during the spawning run from the Little Gravel River (48°55′ N - 87°47′ W), which drains into an uninhabited bay on Lake Superior approximately 100 km west of Terrace Bay. Both sites have been utilized for wild fish collections to examine the effects on effluent from the Terrace Bay mill since 1988 (3). For caging studies, 20 reference and 20 preexposed fish were then held simultaneously for 4 d in Sawmill Creek and Blackbird Creek. After exposure, fish were sacrificed by concussion and spinal severance, and measurements of fork lengths, body weights, and liver weights were recorded. Approximately 1 g of liver from each fish was frozen in liquid nitrogen for determination of MFO activity. The balance of liver tissues were pooled, wrapped in hexane rinsed foil, and immediately frozen at -20 °C until extraction and fractionation. Extraction and Fractionation of Liver Tissues. Livers were thawed, patted dry with tissues, weighed, homogenized to a fine slurry using a tissue homogenizer, and pooled. Tissue homogenates were combined with equal portions of sodium sulfate in Soxhlet thimbles and extracted with dichloromethane overnight. Dichloromethane was employed as the extraction solvent since previous work has shown AhR ligands were recovered from dichloromethane extracts of hepatic tissues (22). Extracts were brought up in dichloromethane/hexane (50:50 v/v), and most lipids were separated on a calibrated preparative gel permeation chromatography system (GPC; ABC Laboratories, Columbia, MO). Removal of residual lipids was accomplished using a preparative HPLC GPC column (21.2 × 350 mm Envirosep ABC, Phenomenex, CA). Lipid fractions were cytotoxic to H4IIE cells (data not shown), and all subsequent work focused on lipid-free crude extracts. Compounds in lipid-free liver extracts from each exposure type were fractionated according to their octanol/water partition coefficients (Kow) by reverse phase HPLC. A C18
FIGURE 2. Relationship between retention factor (k′) and log Kow of various pesticides, PAHs, and phenolics used to calibrate HPLC elution conditions for fractionation of liver extracts. column (4.6 × 250 mm Bakerbond 5, 5µm 300 Å, Phenomenex, CA) was eluted with a mobile phase that was initially 60:40 methanol:water (fortified with 10% methanol) and step programmed by 5% methanol every 12 min to 100% and held for 15 min. The unretained retention time for the column (t0) was determined using formamide (23). The retention factors (k′; k′ ) [ti - t0]/t0) were then determined for several phenolics, pesticides, phthalates, and PAHs and plotted against their literature Kow values (24) to determine retention times for fraction collections (Figure 2). HPLC eluents were monitored using a photodiode array detector scanning 210 to 400 nm. This technique has been used to determine Kow values for a variety of environmentally relevant organic contaminants (23, 25). The good correlation between k′ and Kow (R2 ) 0.9538; Figure 2) enabled the collection of fractions of liver extracts according to increasing lipophilicity. MFO Induction in Caged Fish and in Liver Fractions using H4IIE Cells. Hepatic MFO activity in wild white sucker was determined following established protocols (22). HPLC fractions of white sucker liver extracts were reduced to dryness under a gentle stream of nitrogen and brought up in methanol for assessment of MFO activity in H4IIE cells. H4IIE cell culture conditions, dosing regimens and ethoxyresorufinO-deethylase (EROD) methodology can be found elsewhere (22). H4IIE cells were used from passages 5, 6, 15, and 16. Initial dose-response experiments were conducted to determine appropriate doses for fractionation studies. Throughout, doses are based on equivalents of wet weights of liver tissue extracted. Binding Studies with Rainbow Trout Hepatic Estrogen Receptors, Goldfish Testicular Androgen Receptors, and Sex Steroid Binding Protein. Liver fractions were tested for their potential to interact with fish sex steroid receptors using established protocols for rainbow trout hepatic estrogen receptors (ER (26, 27)), goldfish testicular androgen receptors (AR (16)), and goldfish plasma sex steroid binding protein (SSBP (17)). Liver fractions were reduced to just dryness at 35 °C under a gentle stream of nitrogen and dissolved in methanol. Serial dilutions of liver fractions and sex steroid standards contained the same concentration of methanol (1% v/v), and this concentration did not affect hormone binding in the three assays. For each assay, the specific binding of liver fractions was converted into hormone equivalents (pg/g liver) using the corresponding hormone standard curve. For each liver fraction, triplicate (AR) or duplicate (ER, SSBP) competition experiments were conducted on different receptor preparations. Radioimmunoassays of Sex Steroids. The amounts of testosterone and 17β-estradiol in liver fractions with log Kow 3 to 4 were quantified by radioimmunoassay (RIA). The presence of both hormones was predicted in the log Kow 3 to 4 fraction from literature values (testosterone: 3.32, 17βestradiol: 4.01 (24)) and was confirmed with authentic standards. RIAs were conducted on these fractions following established protocols (28).
FIGURE 3. Hepatic EROD activity in male white sucker from exposed and reference locations held for 4 d in effluent or clean water. Reference sucker were held in Sawmill Creek (REF in clean water) and in effluent (REF in effluent) and preexposed sucker were held in Sawmill Creek (EXP in clean water) and in effluent (EXP in effluent). Bars represent means ( SE (n ) 20). Labels above bars denote groups that were not significantly different from each other (p < 0.05). GC-MS Analyses. Analysis conditions for PCDD/DF congeners in the log Kow > 5 fractions by high-resolution GC-MS (GC-HRMS) are reported elsewhere (29). Aliquots of each fraction were reduced to just dryness under nitrogen and dissolved in toluene. PCDD/DF concentrations were determined manually against external standards. 2,3,7,8Tetraachlorodibenzo-p-dioxin (2378-TCDD) toxic equivalents (TEQ) of confirmed isomers were calculated using H4IIE toxic equivalency factors (TEFs) (30). TEQs for non-2,3,7,8substituted congeners were obtained using TEFs that were 2 orders of magnitude lower than those for the highest 2,3,7,8substituted isomer within the appropriate congener series. Predicted H4IIE MFO activities associated with PCDD/DFs were calculated by interpolating TEQs derived from chemical analysis with the 2378-TCDD dose response curve conducted with each H4IIE assay. Low resolution full scan GC-MS analysis of log Kow > 5 fractions were obtained with a HP 5973 MSD. Statistics. MFO data from caged fish and H4IIE cells were log transformed prior to one way ANOVA using statistical software (31). Hormone equivalents generated from binding studies were analyzed using nonparametric ANOVA for effects (Kruskal-Wallis) and pairwise comparisons to determine differences (Tukey). Slopes and intercepts of displacement curves for fractions demonstrating affinity to steroid hormone receptors were compared using ANCOVA.
Results MFO Induction in Caged Fish. Significant hepatic EROD induction was observed in both reference and preexposed male white sucker caged for 4 d in bleached kraft effluent (p < 0.001) (Figure 3). MFO activity for preexposed fish and reference fish held in effluent were similar (p > 0.05), and MFO activity for preexposed fish held in clean water was not different than reference (p > 0.05). Significant EROD induction in caged fish demonstrates rapid ( 5 Fractions. VOL. 34, NO. 20, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 4. HPLC elution profiles of lipid-free liver extracts for each exposure type. Maximum absorbances as a function of retention time (maxplot) are shown for pooled extracts (19-33 g liver equivalents) of each exposure type. Dashed lines depict fraction collections made according to Kow calibration. Significant induction of MFO activity was observed in H4IIE cells incubated with fractions of hepatic tissue from all fish caged in effluent (p < 0.001; Figure 5). For each exposure type, the highest levels of induction were associated with the
most hydrophobic fraction (log Kow > 5) and can be attributed to PCDD/DFs. A comparison between H4IIE MFO activities measured for the log Kow > 5 fractions and MFO activities derived from PCDD/DFs demonstrate that TEQs derived from PCDD/DFs account for the MFO induction associated with these fractions (Inset, Figure 5). Differences associated with the contributions of individual PCDD/DF congeners to total TEQ can be related to exposure and fish origin (Table 1). In all fish preexposed to effluent, >80% TEQs are derived from TCDD. By comparison, TCDD was not detected in reference fish held in clean water or effluent ( 5 fraction. Significant MFO induction associated with compounds of log Kow 2-5 present in livers of reference and preexposed fish held in effluent (p e 0.006; Figure 5) indicates accumulation of multiple nondioxin AhR ligands from effluent within 4 d. The similar level of induction between reference and preexposed fish held in effluent suggests elimination rates of these AhR ligands are not influenced by preexposure to effluent. The lack of induction in the same fractions of preexposed fish held in clean water indicates that these materials were cleared during short-term depuration. Binding of Liver Fractions to Rainbow Trout Hepatic ER. Significant binding to rainbow trout hepatic ER was caused by components with log Kow 3 to 4 present in livers of reference fish held in effluent (p ) 0.038; Figure 6); binding
FIGURE 5. EROD activity in H4IIE cells incubated with fractions of liver extracts from each exposure type. Background activities for each fraction have been subtracted. Asterisks denote significant induction above reference for that fraction (p < 0.05). Bars are means of 3 replicates ( SE. Inset compares EROD activity for log Kow > 5 fractions with activity predicted from PCDD/DF TEQs derived from GC-HRMS analysis (checkered bars).
TABLE 1. PCDD/DF TEQs Derived from GC-HRMS Analysis of log Kow > 5 Fractions of Hepatic Tissues of Sucker from Each Exposure Typea reference fish in clean water
reference fish in effluent
preexposed fish in clean water
preexposed fish in effluent
congener
TEQ (pg/g)
%
TEQ (pg/g)
%
TEQ (pg/g)
%
TEQ (pg/g)
%
2378-TCDF other TCDFs 2378-TCDD 12378-PCDF 1234678-HCDD others
0.125 0.028 ND ND 0.175 0.002
37.7 8.7
0.478 0.069 ND 0.816 0.528 0.022
25.0 3.6
0.078 0.071 87.63 2.045 0.465 0.022
0.1 0.1 97.0 2.3 0.5 0.0
0.110 0.061 6.71 0.926 0.427 0.013
1.3 0.7 81.4 11.2 5.2 0.2
a
52.8 0.7
42.6 27.6 1.2
Contributions of each congener are given as a percentage of the total TEQ. ND < 0.17 pg/g.
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FIGURE 6. 17β-Estradiol equivalents associated with binding of liver fractions to rainbow trout hepatic ER. Asterisks denote significant induction above reference (p < 0.05). Background activities for each fraction have been subtracted. Bars are means of 2 replicates ( SE.
FIGURE 7. Testosterone equivalents associated with binding of liver fractions to goldfish testicular AR. Asterisks denote significant induction above reference for that fraction (p < 0.05). Background activities for each fraction have been subtracted. Bars are means of 3 replicates ( SE. Inset compares testosterone equivalents for log Kow 3 to 4 fractions with binding equivalents predicted from testosterone RIA (checkered bars). of compounds with log Kow 4 to 5 was also elevated but not significant (p ) 0.058). Conversely, these fractions of preexposed fish held in effluent did not bind to the ER (p > 0.05), which was also the case for preexposed fish held in clean water. These results suggest that preexposed fish metabolize ER ligands with log Kow 3 to 4 (and possibly 4 to 5) at rates that are g uptake rates. Regardless of holding conditions, liver fractions from all fish with log Kow > 5 exhibited high levels of affinity for trout ER (p > 0.05; Figure 6). Some insight into the estrogenic activity of these fractions was provided by full scan GC-MS. A phthalate ester was tentatively identified in each fraction at approximately the same concentration. The established affinity of phthalate esters for the ER would account for at least part of, if not all, of the observed activity (32). The source of a phthalate ester in this fraction is suspected to originate from solvent concentration during liver extraction and lipid separation, as indicated by laboratory blanks (data not shown). Displacement curves for all fractions which exhibited significant binding to the ER were examined for parallelism
with the 17β-estradiol standard using ANCOVA. Since fractions are mixtures of unidentified compounds and displacement curves cannot be plotted against any known concentration, binding values were plotted against dilution factors. These comparisons showed that slopes and intercepts were not different (p > 0.05; data not shown), indicating all fractions interacted with a common binding site on the ER with equal affinity. Contributions of endogenous 17β-estradiol to the observed estrogenic activities of the log Kow 3 to 4 fractions were assessed using RIA. 17β-Estradiol was not detected ( 5 that exhibited significant binding (p ) 0.003). Metabolism of AR ligands is demonstrated by the fact that fractions of preexposed fish held in clean water showed no affinity for the AR (p > 0.05; Figure 7). Testosterone was detected using RIA in the log Kow 3 to 4 fractions in fish from each exposure type, ranging from 39 to 109 pg/g. When compared with testosterone equivalents derived from AR binding assays, endogenous hormone accounted for the androgenic activity in these fractions except for reference fish exposed to effluent where additional ligands were present (Inset, Figure 7). Binding of Liver Fractions to Goldfish SSBP. To investigate whether compounds accumulated from effluent could affect the transport of hormones on carrier proteins, liver fractions were assessed for their potential to interact with goldfish SSBP. Relative to reference, significant binding to SSBP was associated with components with log Kow 2 to 4 present in tissues of all fish held in effluent (p < 0.006; Figure 8). In contrast, no significant binding was observed in all fractions of preexposed fish held in clean water, demonstrating that materials interacting with SSBP are accumulated from effluent within 4 d and are metabolized by preexposed fish during short term removal from effluent. Comparison of displacement curves of using ANCOVA showed parallel slopes (p > 0.05) but significantly different intercepts (p < 0.001). This suggests that compounds present in these fractions of hepatic tissues are interacting with the same binding site on SSBP but with different affinities.
Discussion Through the use of tissue burdens, this study has demonstrated that at a site where wild fish have historically exhibited MFO induction and reproductive dysfunction, chemicals with the ability to interact with the AhR and sex steroid receptors are present in fish hepatic tissues after short-term waterborne exposure. Most ligands with log Kow < 5 for all receptors were not present in tissues of preexposed fish removed from effluent short term, demonstrating metabolism of these materials. It was determined that PCDD/DFs present in hepatic tissues accounted for AhR ligands with log Kow > 5. The results of this study also suggest that metabolic rates of ligands for the ER are influenced by historical exposure. MFO Induction in Caged Fish and in H4IIE Cells Incubated with Liver Fractions. All fish exposed to effluent in this study accumulated AhR ligands within 4 d, as evidenced by significant MFO activity (Figure 3). Preexposed 4332
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fish held in clean water showed no significant induction, indicating clearance of AhR ligands. It is estimated that preexposed fish collected during the spawning run in Sawmill Creek would have been removed from exposure for a minimum of 7 d based on a shutdown (5) and caging experiments (19, 33) at this mill. The lack of induction in preexposed fish held in clean water contrasts with the elevated induction in H4IIE cells observed with their log Kow > 5 fraction (Figure 5). A contributing factor may be in the biological systems used in MFO determinations; in vivo fish vs in vitro rat. Another explanation may be that hydrophobic compounds such as PCDD/DFs are normally sequestered in hepatic lipids in vivo and were rendered available to H4IIE cells through homogenation and solvent extraction. Less hydrophobic compounds would be expected to be bioavailable in vivo, which is shown by the metabolism of ligands with log Kow < 5 by preexposed fish held in clean water. TEQs derived from GC-HRMS analysis of PCDD/DFs in the log Kow > 5 fractions showed that PCDD/DFs account for the MFO induction in H4IIE cells (Inset, Figure 5). Although it appears that preexposed fish have accumulated PCDD/ DFs during their lifetimes (Table 1), the correlation of accumulated 12378-PCDF with MFO induction in the log Kow > 5 fraction of reference fish held in effluent indicates fish were exposed during this study (Table 1). This implies that PCDD/DFs were originating from the mill, leaching from historical contamination of sediments in Blackbird Creek, or both. Low levels (2-8 pg/L) of 2378-TCDD and 2,3,7,8tetrachlorodibenzofuran (2378-TCDF) detected in effluent leaving secondary treatment the month before this study (34) indicate releases may have occurred at the time of this study. Other evidence for accumulation of PCDD/DFs is provided by mussels (Elliptio complanatae) caged in Blackbird Creek in 1993 (35). Pentachlorodibenzofurans, including 12378-PCDF, were detected in all mussels caged at 7 sites in Blackbird Creek for 21 d (2.6-6.7 pg/g). Similar levels of MFO induction were associated with less hydrophobic (log Kow 2-5) components present in all fish held in effluent and are related to exposure to multiple nondioxin AhR ligands. The presence of multiple AhR ligands of lower Kow may explain MFO activity not accounted for in previous studies (8, 9, 18). The sesquiterpenoids juvabione and dehydrojuvabione have been identified as natural balsam fir (Abies balsamea) components partially responsible for MFO induction from a TMP mill, but since these compounds do not survive kraft cooking or secondary treatment they are
not expected in the present study (9). Relative to PCDD/DFs, nondioxin AhR ligands in the range of log Kow 2 to 5 would be accumulated primarily across gill membranes, metabolized, and excreted rapidly. This is reflected in the absence of induction associated with liver fractions of log Kow 2 to 5 of preexposed fish held in clean water. It also parallels the lack of induction observed in these fish in vivo during this study (Figure 3), the rapid decline of induction in wild fish after shutdowns at this mill (5) and other mills (33). Although ligands in hepatic tissues likely arise from direct uptake from effluent, the possibility of ligand formation from metabolic modification of accumulated compounds cannot be discounted at this time. Evidence supporting direct accumulation of AhR ligands from effluent is provided by MFO induction measured on hepatic tissues of caged fish and accumulation of specific PCDD/DFs. It is not currently known if metabolic formation contributed to the ER, AR, and SSBP ligands that were detected in this study, but a further advantage in studying compounds present in tissues is that both pathways are considered. Future experiments will examine the role of metabolic activation as well as the influence of historical exposure on metabolic rates. Binding of Liver Fractions to Rainbow Trout Hepatic ER. Compounds of log Kow 3 to 5 in liver tissues from reference fish held in effluent bound to trout ER; the absence of any activity in the same fractions from preexposed fish held in effluent suggests preexposed fish metabolize these compounds at rates g accumulation rates. The implications of differential metabolism between naı¨ve fish and fish historically exposed to effluent are not known at this time. It is possible that the excretory mechanisms are inducible, with an associated lag phase, and were maintained in preexposed fish. Bioavailability and rapid accumulation of effluent constituents with the potential to interact with the ER is supported by earlier studies which detected weak estrogenic activity in MCF-7 cells incubated with muscle extracts of whitefish (Coregonus lavaretus) held in 7% secondary treated bleached kraft effluent (36) and estrogenic activity associated with dissolved phase of final effluent from a bleached kraft mill in Ontario (7). Wood extractives with established estrogenic activities may be accumulated directly from effluent and contribute to estrogenic activity measured as binding to rainbow trout hepatic ER. β-Sitosterol, known to induce vitellogenin production in rainbow trout (27) and bind to rainbow trout ER (12), would be expected in the log Kow > 5 fraction. Additional extractives such as abietic acid, pinosylvin, and betulin have estrogenic activity (13) and could contribute to the activity observed in this study. Despite the presence of chemicals in final effluents that have the potential to interact with the ER, induction of vitellogenin has not been observed in wild fish collected near effluent outfalls (2). Recent studies have detected estrogenic responses in caged fish and laboratory exposures. Mellanen et al. (37) observed expression of vitellogenin mRNA in juvenile whitefish (Coregonus lavaretus) caged in diluted effluent for 30 d from an elemental chlorine free (ECF) kraft mill in southern Finland. Vitellogenin has been recently observed in plasma of immature rainbow trout following laboratory exposures to effluent from a bleached kraft mill for 21 d (27). It is possible that antiestrogenic effects of effluent constituents, some also functioning as AhR ligands, may affect the cumulative estrogenic response at some mills. For example, Zacharewski et al. (7) found that the methanol extract of final effluent particles and colloids > 0.1 µm from a bleached kraft mill contained AhR ligands which displayed antiestrogenic effects in vitro. Assessment of antagonistic activities of ligands for the AR, ER, and SSBP will be examined in future studies.
Binding of Liver Fractions to Goldfish Testicular AR. This study represents the first evidence that fish exposed to pulp mill effluent contain compounds that interact with the AR. Compounds spanning a broad range of Kow functioning as AR ligands were present in all fish held in effluent. Fractions of preexposed fish held in clean water showed no affinity for the AR, demonstrating a metabolism of AR ligands after shortterm removal from effluent. Androgenic responses in preexposed wild fish at the time of the study were indicated by an apparent recovery in secondary sex characteristics in male sucker and tubercle development in 50% of females (3). It is not known whether continuous exposures to AR ligands would result in the observed wild fish responses, but our findings have determined a plausible mechanism. Androgenic responses represent a pathway by which reproductive impairment could occur and has received little attention, primarily due to the inability to detect responses. One exception involves masculinization of mosquitofish (Gambusia affinis), which has been associated with exposure to effluent from a kraft mill in northwest Florida and has persisted after mill process changes that included implementation of oxygen delignification, ECF bleaching, and tall oil recovery (38). Masculinization, measured as elongated female anal fins, has more recently been associated with kraft mill effluent from a New Zealand mill (39). Studies now also indicate the potential of effluents from different mill processes to interact with the AR from trout and goldfish (40). Although it is possible that species-specific thresholds for androgenic responses may exist, the relationship between the androgenic activities observed in this study and responses in wild fish requires further investigation. Binding of Liver Fractions to Goldfish SSBP. Our findings represent the first evidence that components accumulated from pulp mill effluent could affect hormone transport on carrier proteins and represents an additional pathway for reproductive dysfunction in wild fish. At this time the identities of the chemicals interacting with SSBP are not known, but there is evidence that natural products and anthropogenic chemicals can affect hormone binding to SSBP. The isoflavonoid phytoestrogen equol inhibits binding of 17β-estradiol and testosterone with human SSBP (41). It is possible that related isoflavonoids may be involved with the binding of fractions with log Kow 2 to 3 and 3 to 4 since these compounds have log Kow < 3. Polyunsaturated nonesterified fatty acids compete for goldfish SSBP (17), but their hydrophobicity would place them in the log Kow > 5 fraction where there was no significant binding observed. Organochlorine pesticides and nonylphenol have also shown weak competitive affinities for SSBP (42, 43). Displacement curves of fractions demonstrating interactions with SSBP indicate that compounds present in tissues of exposed fish are interacting at a common binding site with differential affinities.
Acknowledgments Support for this project was provided by Environment Canada, The Canadian Network of Toxicology Centres, and the Toxic Substances Research Initiative (TSRI), a research program managed jointly by Health Canada and Environment Canada (project #199). Thanks to M. Baker, S. Backus, N. Jones, and L. Luxon for technical support and A. Lister for constructive revisions. Thanks to Roger Ferguson for mill production conditions and to Al Haydon, Jim Murphy, and Ian Smith for assistance providing effluent contaminant data. Thanks to Klaus Kaiser for helpful discussions on partition coefficients.
Literature Cited (1) McMaster, M. E.; Portt, C. B.; Munkittrick, K. R.; Dixon, D. G. Ecotoxicol. Environ. Safety 1992, 23, 103-117. VOL. 34, NO. 20, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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Received for review March 22, 2000. Revised manuscript received June 26, 2000. Accepted July 13, 2000. ES0011212
