Environ. Sci. Technol. 2002, 36, 2311-2321
Plasma Biomarkers in Fish Provide Evidence for Endocrine Modulation in the Elbe River, Germany M A R K U S H E C K E R , * ,† C H A R L E S R . T Y L E R , ‡ M E R V EÄ E H O F F M A N N , † SUE MADDIX,§ AND LUDWIG KARBE† Institute of Hydrobiology & Fisheries Science, University of Hamburg, Zeiseweg 9, 22765 Hamburg, Germany, School of Biological Sciences, The Hatherly Laboratory, University of Exeter, Exeter EX4 4PS, U.K., and Department of Biology & Biochemistry, Brunel University, Uxbridge, Middlesex UB8 3PH, U.K.
Blood plasma samples were collected from wild bream (Abramis brama L.) in the Elbe River, Germany, and analyzed for the yolk protein precursor vitellogenin (VTG), a biomarker for estrogen exposure, and the sex steroids 11ketotestosterone (11KT), testosterone (T), and 17βestradiol (E2) to investigate for evidence of endocrine modulation. In addition, the gonadal status and the prominence of spawning tubercles were investigated. Nine riverine sites were investigated on the Elbe that were influenced by different sources of endocrine-active substances. Bream were collected from a lake that received no domestic or industrial discharges as a control. Plasma VTG concentrations were significantly higher in male bream from the Czech border to the middle Elbe, with the highest concentrations in fish sampled at the locations near Magdeburg and downstream of Dresden (between 20 and 100 times higher than in the controls), regions that are characterized by high levels of effluent discharges into the river. Following the Elbe from this site to the sea, the concentrations of plasma VTG in males were lower than at Meissen but were still elevated above the controls. 11KT and E2 titers showed suppressions in their normal concentrations at some locations (those receiving the greatest industrial discharges). There were reciprocal relationships between inhibitory effects on gonadal growth, maturation, and plasma sex steroids and exposure to pollutants, such as organotins, pesticides, or metals. However, there was no single chemical that alone could explain the observed inhibitory effects on sexual development. The results indicate that the endocrine system in wild bream is disrupted in stretches of the Elbe River.
Introduction In recent years increasing international concern has been raised about pollutants that are able to disrupt reproductive function by interacting with the endocrine system. Numerous studies have been carried out to investigate the effects of * Corresponding author phone: +49-40-42838-6660; fax: +4940-42838-6696; e-mail:
[email protected]. Current address: Aquatic Toxicology Laboratory, Michigan State University, East Lansing, MI 48824. † University of Hamburg. ‡ University of Exeter. § Brunel University. 10.1021/es010186h CCC: $22.00 Published on Web 04/25/2002
2002 American Chemical Society
exposure to endocrine-modulating compounds in both humans and wildlife (1-3, 6). Most evidence for endocrine disruption (ED) in wildlife has come from animals living in or closely associated with the aquatic environment (4, 5, 8, 10). Studies conducted to date in fish have focused on exposures of wild or caged fish to effluents and in rivers or marine waters that receive high concentrations of effluents (11-16). Investigations on wild fish in British rivers and estuaries, however, have found widespread intersexuality in the ambient environment, albeit that at some of the study sites the river flow is comprised of a high proportion of effluent from sewage treatment works (STWs) (17, 18, 10). There is very little evidence for ED in aquatic wildlife populations in other parts of Europe. Many European rivers are unlike those in the U.K. because domestic and industrial discharges make up only a very small proportion of their flow. Nevertheless, these rivers often still receive a variety of potentially endocrine-active compounds from industrial activities and discharges that go back to the previous century, intensive agriculture and/or domestic effluents from densely populated regions. The Elbe was chosen as a European river to investigate ED because it is a large river receiving a series of complex effluents and it holds good fish populations. Furthermore, considerable information is available on both the pollution inputs (past and present) into the Elbe and on the river’s ecology. Indeed stretches of the Elbe River have routine chemical and biological monitoring programs that have been running for more than 10 years (19). To assess possible disturbances in the endocrine system in wildlife as a consequence of exposure to environmental chemicals, ideally a suite of biomarkers is needed. The use of plasma vitellogenin (VTG) to screen for estrogen exposure in oviparous vertebrates is well-described (20-22), and it has been employed to assess estrogen exposure in wild fish in U.K. and U.S. rivers (10, 13, 23, 24). Plasma steroid hormones have also been used in the study of ED in fish (11, 15), and recent data from laboratory studies have shown that exposure to a variety of ED chemicals suppresses the sex steroids estradiol (E2), testosterone (T), and 11-ketotestosterone (11KT) (25, 61-63). Steroids control sexual differentiation, maturation, spawning, sexual behavior, and expression of secondary sex characteristics, and they are therefore central in mediating successful reproduction. The balance between the sex steroids, E2 and 11KT, determines the phenotype in fish, and they mediate differentiation of the brain and influence behavior (26, 27). It is clear therefore that disturbances in sex steroid hormones may have considerable impacts on reproductive success. The aim of this study was to investigate whether exposure of wild fish to a complex mixture of chemicals of human origin, domestic sewage, and agricultural influences that are discharged into and/or impact on the Elbe River results in ED. Cyprinid fish have been shown to act as excellent sentinel species for endocrine influences in surface waters (10, 13, 28, 29). The fish species chosen for this study was another member of the cyprinid family, namely, the bream (Abramis brama L.). The bream is a species widely used for biomonitoring (30-32) because its ecological niche exposes it to sediment-bound pollutants as well as compounds in the water column. Another advantage of the bream to assess effects of aquatic pollutants in wild populations is that it is resident to specific locations and does not show a marked migration (33, 34). Furthermore, the required assays to measure VTG (22) and sex steroids (29) have been established for this species. VOL. 36, NO. 11, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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Blood and Tissue Sampling (Field). Blood samples were collected into heparinized tubes (Sarstedt, Lot No. 72411271) by cardiac puncture. A total of 1-2 TIU of aprotinin/mL of blood (Sigma, A6012) was added to prevent proteolysis. Samples were centrifuged at 9000g for 10 min, and from the resulting plasma, two aliquots were deep-frozen in liquid nitrogen for subsequent VTG and steroid analysis. Length and body weight were measured, and scales were collected to age the fish. The intention was to obtain a sample size of at least 10 male and 10 female bream from each location to fulfill the requirements for rigorous statistical evaluation. The gonads and livers were dissected from the fish, and the gonadosomatic index (GSI), and the hepatosomatic index (HSI) were calculated as described by Htun-Han (38):
GSI (%) ) gonad weight/body weight (without viscera) × 100 FIGURE 1. Sampling locations along the Elbe River, Germany.
Materials and Methods Sampling Locations. Nine sampling locations on the Elbe River were chosen based on their different influences from industries, agriculture, sediment, and soil-deposited pollutants as well as on their inputs from domestic sewages (35). The sampling sites following the river from the Czech border (Schmilka) downstream to the tidal area (Haseldorf) are illustrated in Figure 1. The site at Schmilka was located downstream of the Czech border and was influenced by a variety of inputs from several upstream petrol and chemical industries. Meissen had considerable inputs from the STWs of Dresden (which has a population of more than 500 000) that were located just upstream of the sampling site. Barby, Magdeburg, and Hohenwarte are located in the middle part of the Elbe and are areas characterized by heavy contamination from various industries, including (agro)chemical and petrochemical plants. Sediments and soils from this region have been reported to contain considerable amounts of residues of heavy metals and both organic and inorganic contaminants (36). The sampling site at Geesthacht was located in a dammed region that acts as a sink for suspended matter that flows down the river. Zollenspieker is located upstream of, Koehlbrand is located in, and Haseldorf is located downstream of the city of Hamburg. These sites were chosen in an attempt to assess the influence of urban and industrial effluents from Hamburg and its large harbor. Because the entire Elbe is polluted to a lesser or greater extent and no comparable pristine rivers exist in the same climate zone, it was not possible to obtain an unpolluted river site. Therefore, bream were sampled from a lake to act as the control. This control lake (Balksee) is a typical shallow lowland lake in a wildlife reserve, receiving no domestic or industrial waste. With the exception of the pollution influences, the Balksee and the Elbe River are comparable for to the ecological needs for bream reproduction (temperature, nutrition, etc.) (29). Sampling Fish. In the investigated latitudes, the bream reaches sexual maturity in the 3rd or 4th year of age and spawns in one or two batches in a period of 1-2 weeks during the spring (May/June) (64). Wild populations of bream of both sexes were sampled in 1999 in April/May just prior to the spawning season and in September/October at the locations along the Elbe River. To obtain fish at the same stage of maturation, all sites were sampled within a period of 2 weeks. The control fish from the control lake were caught in 1999 and 2000 in April/May and in early October, respectively. The fish were caught by electrofishing and were blood-sampled within 1 h of capture in order to maintain capture and handling stress at a minimum, as this can affect sex steroid titers (12, 37). 2312
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HSI (%) ) liver weight/body weight (without viscera) × 100 Histological Analysis. Both gonads were fixed in Bouins solution for 6-12 h and then removed to 70% ethanol in preparation for histological processing. From each fish, transverse sections were taken from different regions of the gonads. The sections were then processed histologically, embedded in paraffin wax, and sectioned at 2-4 µm. All sections were stained with Mayers Haematoxylin-eosin, mounted, and examined by light microscopy. The maturity stages (MS) were calculated as described by Kuzmin and Steptoe et al. (cited by Horvath in ref 69) for females and according to the method of Billard et al. (68) for males, respectively. Intersex Index. The assessment of intersexuality was conducted according to the methology described by Jobling et al. (10). Within this work, they devised an intersex index (ISI) that captures seven different levels of intersexuality ranging from 0 (normal male) to 7 (normal female). See Jobling et al. (10) for further details of the ISI. Analyses of Plasma for Selected Biomarkers (Laboratory). Vitellogenin. There is no antiserum available against bream VTG. However, antiserum against carp (Cyprinus carpio) VTG (c-VTG), a species that is a close relative of the bream, cross reacts well with bream VTG, and this antibody has been validated for use to measure VTG in bream plasma (22). Plasma from bream containing VTG dilutes parallel with the carp VTG standard and other plasma proteins did not appear to cross react with c-VTG antibodies. VTG titers in bream were measured using an ELISA protocol, described by Tyler et al. (39). The procedure is based on the competition of c-VTG coated on the walls of a 96-well Maxisorb microtitration plate (NUNC), with free bream vitellogenin (b-VTG) contained in the plasma for the c-VTG antibody. Throughout, plasma samples were diluted in phosphate buffer containing Tween 20 and BSA (bovine serum albumin) to dilute the plasma VTG to the working concentrations of the assay. Male plasma was diluted at least 1:10 (minimum dilution required for the working protocol of the c-VTG ELISA to prevent plasma effects). Female plasma was diluted 1:1000 or 1:10000 (depending on the maturation stage and amount of VTG present). Steroid ELISA. Frozen plasma samples were slowly defrosted on ice, and the free steroids were extracted twice with diethyl ether in conical glass tubes. The solvent extract was separated from the water phase by centrifugation at 2000g for 10 min, transferred in small glass vials, and left until the solvent had evaporated completely. Immediately before performing steroid ELISAs, the crystalline residue was dissolved in phosphate buffer containing BSA (0.1%). The
TABLE 1. Concentrations of Chlororganic Contaminants and Metals in Freshly Suspended Matters (SPM) at Six Different Sampling Sites along the Elbe River (45)a µg/g dry mass 6PCBb 8PAHc 6DDXd βHCH γHCH Zn
HA
KO
Z
B
ME
SCH
0.030/0.009 3.85/0.69 0.029/0.019 0.002/0.001 0.001/0.000 200/310
0.024/0.022 4.58/0.89 0.044/0.035 0.004/0.018 0.001/0.002 125/307
0.046/0.054 1.32/2.05 0.391/0.095 0.023/0.009 0.001/0.001 137/796
0.058/0.052 4.37/3.07 0.228/0.246 0.038/0.010 0.005/0.002 868/1204
0.137/0.166 4.39/5.27 0.240/0.398 nd/nd 0.001/nd 431/850
0.137/0.143 5.05/3.96 0.110/0.366 0.004/nd nd/nd 500/875
a The numbers represent the means of the monthly means for 2 months before the time of fish sampling (spring/autumn). Sampling sites (ARGE-Elbe site/fish site): SCH, Schmilka/Schmilka; ME, Zehren/Meissen; B, Magdeburg/Barby; Z, Bunthaus/Zollenspieker; KO, Seemannsho¨ ft/ Ko¨ hlbrand; HA, Grauerort/Haseldorf. nd, not determined. b 6PCBs: PCB 28; PCB 52; PCB 101; PCB 138; PCB 153; PCB 180. c 8PAHs: anthracene; benz[a]anthracene; chrysene; benzo[b]fluoranthene; benzo[k]fluoranthene; benzo[a]pyrene; dibenz[ah]anthracene; indeno[1,2,3-cd]pyrene. d 6DDX: p,p′-DDT; o,p′-DDT; p,p′-DDD; o,p′-DDD; p,p′-DDE; o,p′-DDE.
blood plasma concentrations of E2, T, and 11KT were measured by ELISA as described by Cuisset et al. (40) with modifications (Kime, personal communication). Antisera to T, 11KT, and E2 were kindly donated by Dr. D. E. Kime (Sheffield, U.K.) and the Institute for Hormone and Fertility Research, Hamburg. Cross reactivities of the androgene antisera are described in Nash et al. (9). The antiserum to E2 cross-reacted with estradiol-glucoronide (41%), 17β-estradiol-3-sulfate (13.3%), estriol (0.56%), estrone (0.4%), T (0.18%), and 5R-dihydrotestosterone (0.14%). For all other steroids, cross reactivities were below 0.02%. In these highly sensitive competitive enzyme immunoassays, the plasma steroid of the sample competes with a tracer (an acetylcholinesterase-labeled steroid) for the binding site on the polyclonal rabbit anti-steroid antibody. The steroid ELISAs were performed using COSTAR high binding plates (COSTAR). The working ranges of these assays for bream plasma were determined as follows: pg/mL plasma 17β-estradiol testosterone 11-ketotestosterone
40-20 000 40-20 000 40-40 000
After extraction, female plasma was diluted between 1:5 and 1:10 for the E2 and T ELISAs and 1:5 for the determination of 11KT. Male plasma was used at a dilution of 1:5-1:10 for the E2 and T ELISAs and at 1:10-1:100 for the determination of 11KT. Secondary Sex Characteristics (STI). The presence and prominence of spawning tubercles (only male fish) was assessed as a measure of sexual maturity. Development of spawning tubercles is dependent on sex steroids, and they have been used successfully to assess the effects of endocrinedisrupting compounds (EDCs) on sexual development in fish in the laboratory (41, 42). In this study, we devised a “spawning tubercle index” (STI) that captured five different states of the secondary sex characteristics: 0: no tubercles could be observed at all 1: 10 or less flat tubercles located above of the mouth region 2: more than 10 flat tubercles located above the mouth region and/or over the head 3: prominent tubercles (those raised above the skin surface) located all over the head and that start to extend down the dorsal surface of the fish 4: prominent tubercles distributed over the whole body The tubercles were counted, and depending on the number and/or prominence, the fish was assigned to one of the abovedescribed categories. Statistical Analyses. Data for plasma VTG, sex steroids, and GSI were not normal distributed (even after log transformation); therefore, the data were expressed as the median,
TABLE 2. Concentrations in Freshly SPM and Water at Six Different Sampling Sites along the Elbe River in June 1998 (44)a ng/g dry mass BPA NP NP1EO
HA
KO
Z
B
ME
SCH
96 483 760
68 367 886
187 852 712
194 448 464
278 387 323
343 485 568
ng/L of H2O
HA
KO
Z
B
ME
SCH
BPA NP NP1EO
57 12 10
22 9.5 12
27 7.2 18
57 12 35
776 8.7 205
76 52 46
a
See footnote a of Table 1 for definition of abbreviations.
( percentiles (0.25-0.75 ranges and 0.1-0.9 ranges). The results were subjected to a nonparametric test of variance (Mann-Whitney U-test) to analyze regional and seasonal differences in the parameters measured. Significance was accepted when p e 0.05. Rank correlations were calculated following the model described by Spearman (43).
Results Water Temperature. Water temperature in the Elbe and the control lake showed comparable seasonal variations. There were no marked differences in the absolute temperatures between the riverine sampling sites as well as between the Elbe and the control water body. During the spring sampling, the temperature (means of the 2 weeks before sampling ( standard deviation [SD]) ranged from 10.5 °C (no SD because single measurement) in the control lake to 11.3 ( 1.8 °C in middle and upper Elbe. The temperatures during the autumn samplings were between 14.5 ( 3.6 °C at Schmilka (Czech border) and 13.1 °C in the middle part of the river (45). Concentrations of Selected Chemicals Measured in the Elbe. Data on a selection of the chemicals that were measured in sediments and water of the Elbe River are given in Tables 1 and 2. As there was just a single special monitoring program for xenoestrogens such as alkylphenolics and bisphenol A that took place in 1998, the data for xenoestrogens presented in this manuscript are for that year. The results shown for the other toxicants (PAHs, PCBs, DDX, HCHs, Zn) represent the exposure situation at the different sampling sites during the 2 months before sampling the fish in 1999. The chemicals reported here are those routinely monitored substances that showed correlations with some of the physiological parameters investigated in this study. The eight PAHs presented here are believed to interact with the endocrine system (66, 67). A more complete picture of the pollution profiles for chemicals in this river are given in the reports from the ARGEElbe (44, 45). For the Balksee site, no data on pollutant concentrations in the water and/or sediments were available. VOL. 36, NO. 11, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 2. GSI (box plots) and maturation stages (data point lines; median values) in male (upper charts) and female (lower charts) bream collected during spring (left column) and autumn (right column) along the Elbe River and at a control site. Numbers above the upper triangles ) number of individuals sampled at this site. Triangles ) maximum and minimum values. Vertical lines ) percentiles. Bars ) quartiles. Horizontal lines ) median. Significances: ***(+++): p e 0.001; **(++): p e 0.01; *(+): p e 0.05; * ) GSI; + ) MS. Morphometric Data. All fish collected from the different sites and analyzed for plasma VTG and sex steroids were maturing. The median length of the fish caught was between 35 and 49 cm with a corresponding age of between 5 and 9 yr. The oldest fish were caught at Koehlbrand in the spring (9 ( 0.3 yr) and at Haseldorf in the autumn (9 ( 0.6 yr). In general, the median fish age was between 7 and 8 yr with the exception of bream at the site at Magdeburg in autumn, where male bream were 5 ( 0.00 yr and females were 6 ( 0.04 yr. Gonadosomatic Index. The GSI in all fish in the spring sampling period were significantly greater (p e 0.001) than those measured during autumn (Figure 2). The lowest GSIs occurred in both females and males at Magdeburg with median values 2-6 times lower than that of the controls. At this site there was also a significant delay in the maturation progress (as defined by lower MS). At Magdeburg most of the male fish (>90%) had GSI below that of more than 90% of the control bream. During the spring, significantly lower GSIs were also a feature of fish at Schmilka (females) and Geesthacht (males & females). Interestingly, in female fish at the sampling sites at Schmilka and Magdeburg there were no seasonal differences in the GSI, in contrast to that in the controls and in bream from the lower Elbe. There was a high degree of variation in the size of the gonads in males and females caught just before spawning (spring). The highest variation occurred at the locations of Zollenspieker, Hohenwarte (just females), Barby (just females), and Schmilka. There were also regional differences in the level of variability in gonad size within populations sampled, and this was highest in the fish sampled at Schmilka. The relative liver size (HSI) similarly showed marked regional differences (Figure 4) and was positively correlated with the GSI (p e 0.001). In both males and females, elevated HSIs (above controls) were observed in the upper and lower part of the Elbe. Interestingly, bream from the region 2314
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FIGURE 3. Incidence of intersexuality in male bream along the Elbe River and at a control site. (ISI 1, single oocytes in testes; ISI 2, cluster of oocytes; ISI 5 and 6, ovotestes, more than 45% of gonad was ovarian.) upstream, downstream, and at Magdeburg had similar indices to the controls (spring: males ) 1.51 ( 0.16, females ) 1.52 ( 0.09; autumn: males ) 1.48 ( 0.30, females ) 1.49 ( 0.02) (median ( standard error of median (SEM)). During the autumn, elevated HSIs occurred in bream at the sampling site at Meissen (males: 2.38 ( 0.23; females: 2.40 ( 0.34). No significant seasonal differences in the HSI were observed. Intersex. Due to the low incidence of intersex fish at all sampling sites, the whole data set, irrespective of season, are presented in one regional profile (Figure 3). Intersex fish were found at all sampling sites except from Geesthacht. The intersex score (degree of sexual disruption) found in bream from the Elbe were 1, 2, 5 and 6, and scores of 3 and 4 were not observed in any fish. Lower ISI scores occurred at sites along the whole river. Ovotestes (ISI 5 and 6), where considerable proportions (>45%) of the “testis” were ovarian
FIGURE 4. HSI in male (upper charts) and female (lower charts) bream collected during spring (left column) and autumn (right column) along the Elbe River and at a control site. tissues, were found downstream of the Czech border and at Zollenspieker. The incidence of intersexuality ranged from 0.5% at the control site to 6% at Meissen and Koehlbrand. Spawning Tubercles. Spawning tubercles were observed in male bream only. The STI showed clear seasonal differences, with tubercles significantly more advanced in the spring as compared with the autumn (as one would expect). Exceptions to this were in males collected from Magdeburg and Geesthacht. Highest STI values occurred at the control site and in fish from the tidal area (Figure 9). The STI was positively correlated with plasma 11KT (spring: R ) 0.777, p e 0.014; autumn: R ) 0.700, p e 0.052) and T (spring: R ) 0.703, p e 0.035; autumn: R ) 0.894, p e 0.003). Lowest STIs occurred at the sampling site Magdeburg where between 75 and 100% of the males showed no expression of any secondary sex characteristics. This correlated with high sediment concentrations of metals and pesticides. Plasma Biomarkers. Vitellogenin (VTG). The median concentration of plasma VTG in female bream from the control lake was 90 000 ( 16 888 ng/mL in the spring, and 55 000 ( 29 849 ng/mL in autumn. VTG concentrations in female bream in the control lake showed seasonal variations with lower concentrations in the autumn as compared with the spring, while at locations in the middle and upper Elbe no comparable differences were observed (Figure 5). At the sites at Haseldorf and Zollenspieker, seasonal changes occurred but concentrations were higher in the autumn than in the spring. Median concentrations of VTG at Magdeburg were suppressed throughout the seasons as compared with control females. In female bream at Magdeburg and also at Schmilka and Geesthacht, there was a very high degree of variability in the concentrations of plasma VTG between fish at the same reproductive stage (Figure 5). The highest plasma VTG concentrations occurred in female bream at Meissen (spring: 140 000 ( 1700 ng/mL, autumn: 240 000 ( 15 900 ng/mL; significance above controls p e 0.001).
In male bream from the control site, plasma VTG concentrations were just above the detection limit of the assay at both sampling time points (autumn: 15 ( 2.6 ng/ mL; spring: 12 ( 3.8 ng/mL). Plasma VTG in males from almost all riverine sites were significantly higher than that in males from the control lake. Male bream showed distinct regional differences in their plasma VTG concentrations, and these regional differences were consistent for both sampling periods (autumn and spring). The highest median concentrations (1200 ng/mL of plasma) of VTG were measured in males that were caught at the Meissen site just downstream Dresden. In the middle part of the Elbe there were also marked elevations in plasma VTG in male bream, with maximum median concentrations at Barby (260 ( 72 ng/mL) and Hohenwarte (265 ( 84 ng/mL) during spring and at Magdeburg (280 ( 61 ng/mL) during autumn. Downstream of these sites, VTG concentrations in male bream were progressively lower. The lowest concentrations of VTG in male riverine fish occurred in the tidal area downstream of Hamburg at Haseldorf. The induction of VTG in male fish was consistently accompanied by a high variability in plasma VTG concentrations within the population sampled. The relatively high VTG concentration measured in males at Meissen in the autumn fish were accompanied by an elevated HSI in these fish (Figures 4 and 5). Similar observations were not seen at the other specific locations. During this sampling period, however, there was a significant positive correlation between the induction of VTG and the HSIs across the sampling sites (R ) 0.788, p e 0.036). There was a weak but significant negative correlation between the induction of VTG and the GSI in male bream (spring: R ) -0.166, p e 0.019; autumn: R ) -0.299; p e 0.018), whereas in females there were either no or positive correlations (spring: R ) 0.363, p e 0.001; autumn: R ) 0.117, p e 0.205). Steroid Hormones. (a) 17β-Estradiol. In female bream from the control site, the highest concentrations of plasma E2 occurred in the autumn (median values ≈ 900 pg/mL, VOL. 36, NO. 11, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 5. VTG concentrations (ng/mL plasma) in male (upper charts) and female (lower charts) bream collected during spring (left column) and autumn (right column) along the Elbe River and at a control site.
FIGURE 6. E2 concentrations (pg/mL plasma) in male (upper charts) and female (lower charts) bream collected during spring (left column) and autumn (right column) along the Elbe River and at a control site. compared with 350 pg/mL plasma in April). Similar observations were made in female fish from the riverine sites except for those from Zollenspieker (Figure 6). At the Czech border, in the tidal part of the Elbe and in the control fish, the variability in plasma E2 concentrations were higher during 2316
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the spring than in the autumn. There was a significantly lower concentration of plasma E2 in female bream in the upper (except from Schmilka in the autumn) and middle part of the river, where there were elevated concentrations of metals and organochlorines (Table 1). The lowest con-
FIGURE 7. 11KT concentrations (pg/mL plasma) in male (upper charts) and female (lower charts) bream collected during spring (left column) and autumn (right column) along the Elbe River and at a control site (Balksee).
FIGURE 8. T concentrations (pg/mL plasma) in male (upper charts) and female (lower charts) bream collected during spring (left column) and autumn (right column) along the Elbe River and at a control site. centrations of plasma E2 in females occurred at the Magdeburg site (between 50% and 100% of the fish were below the assay detection limit of 40 pg/mL). Fish from the lower Elbe did not show any marked differences from the controls. In general, there were similar regional patterns of plasma E2 in
female bream for both seasons in the middle and the upper Elbe. In almost all male fish from the control lake, plasma E2 was below the detection limit of the assay for both seasonal samplings (spring: 90% e 40 pg/mL; autumn: 50% e 40 VOL. 36, NO. 11, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 9. Expression of spawning tubercles (STI) in male bream collected during spring (left column) and autumn (right column) along the Elbe River and at a control site. pg/mL). Most males from the riverine sampling sites had similarly low E2 titers (Figure 6). Exceptions to this were bream caught in the tidal area at Zollenspieker and Haseldorf in the spring, where plasma E2 concentrations (208 ( 260 pg/mL) were elevated above the baseline (p e 0.001). (b) 11-Ketotestosterone. The median concentrations of plasma 11KT in male bream from the control lake were higher than 20 000 pg/mL during the spawning season and 2154 ( 743 pg/mL in September/October. Male bream caught along the Elbe showed comparable seasonal differencessexcept in the middle part of the river where no differences between the two sampling periods were seen (Figure 7). Plasma 11KT concentrations in males from the control site and the lower river were distinctly higher than in male fish from the upper and the middle Elbe, areas that were characterized by elevated exposures to contaminants (Tables 1 and 2). The regional trends observed for plasma 11KT in males were similar to those seen for E2 in female bream (Figures 6 and 7). Lowest 11KT titers (spring: 55 ( 26 pg/mL, autumn: 91 ( 18 pg/ mL) occurred at the site Magdeburg (20-200 times below that in the control fish). At Schmilka in April/May there was an extreme variability in plasma 11KT. 11KT was present in females from both control and riverine sites (but the plasma concentrations were considerably lower than in males, and for the most part less than 500pg/msexcept from Haseldorf in April/May). (c) Testosterone. No marked sex specific differences could be observed in the plasma titers of T. In the control in April/ May (both sexes) variability in plasma T within a population was high. In the riverine fish, plasma concentrations of T in male and female bream in the spring were highly variable with no obvious regional patterns (as occurred for the other sex steroids). There was no apparent correlation between plasma T with GSI (Figure 8). In general, plasma T titers were the highest in fish from the tidal region of the Elbe. Furthermore, there were no clear seasonal variations (spring to autumn) in T concentrations in either sex in the controls or in the riverine bream.
Discussion
bream, the GSI in both sexes was significantly higher during the spawning season (April/May) as compared with in the autumn, and this is in accordance with the typical seasonal pattern for gonad growth and development in other temperate cyprinid fish (29, 46, 47, 48). The fact that VTG and E2 titers were lower in spring as compared with the autumn is probably because the sampling time in the spring was close to the spawning time when much of the VTG in the circulation would have been cleared to the ovary prior to final maturation (29). Plasma E2 concentrations in the female bream from the control site were similar to that found in an allied cyprinid, the gudgeon (Gobio gobio L.), which is similarly a batch spawning fish (47). Histological analyses of the ovaries from fish from the control site and riverine sampling locations in the spring found no signs of postovulatory follicles, showing that none of the females sampled had yet spawned that season. This showed that differences in GSIs within the populations of fish could not be attributed to spawning. Bream at Magdeburg had gonads with significant lower maturation stages as compared with the controls and almost all riverine sampling sites; therefore, the low GSIs in fish at this site are likely to be due to an inhibition of maturation. The phenomenon of endocrine modulation seen in bream along the Elbe is most likely to be as a consequence of estrogen influences characterized by an induction of VTG in male animals and/or other exposure to other endocrine-active or toxic chemicals resulting in a general suppression of reproductive functions. Weak estrogen influences seem to occur along the whole stretch of the Elbe studied. The greatest estrogenic influence (as determined by induction of VTG) occurred at Meissen, just downstream Dresden, and at the sampling sites of Barby and Magdeburg. The greatest suppression in reproductive function occurred in the middle part of the river and in fish at the Czech border. At the sampling locations at the lower tidal Elbe, no modulations of the endocrine system and/or alterations of the reproductive status could be observed. The very good gonad growth in bream from the tidal Elbe (even better than in the controls) was probably due to the good quality environment in this area (food, habitat structure, etc.).
The data presented show that bream living in the Elbe River have been subjected to endocrine modulation, probably as a consequence of exposure to a variety of domestic, agricultural, and chemical effluents. Differences in the biomarkers measured between the sampling sites as a consequence of water temperaturesa major environmental factor steering maturation in fishscan be excluded because there were no marked regional differences in this parameter. Assessing possible evidence for endocrine modulation in wildlife populations requires detailed baseline information on the parameters measured in the sentinel species. In the
Estrogenic and Endocrine Effects. The weak but significant induction of VTG in male bream at all riverine locationss with the exception of Haseldorfsindicates that there were estrogenic influences all along the Elbe River in Germany. Compared with results from Harries et al. (49) where plasma VTG concentrations in caged male trout in U.K. rivers were up to 82 000 ng/mL, even the highest median VTG concentrations in bream at Meissen were low and represent a relatively weak estrogenic effect. This is supported by the fact that just a weak suppression of gonad growth was seen in male bream from this site, which is known to occur when
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TABLE 3. Spearman Rank Correlations between the Biomarker Response in Bream and the Selected Contaminants in SPMa GSI compd 6PCB 8PAH 6DDX β-HCH γ-HCH TBT DBT MBT Zn a
VTG
11KT
T
E2
MS
STI
sex
spring
fall
spring
fall
spring
fall
spring
fall
spring
fall
spring
fall
M F M F M F M F M F M F M F M F M F
-0.126 -0.270 0.143 -0.314 -0.126 -0.270 -0.357 -0.571 -0.829 -1.000 0.500 0.500 -0.900 -0.900 -0.900 -0.900 -0.857 -0.829
0.435 -0.638 0.086 -0.143 0.486 -0.600 -0.600 -0.300 -0.700 -0.600 0.000 -0.600 0.400 -0.200 -0.600 0.000 -0.086 -0.429
0.829 0.833 0.649 0.257 0.450 0.500 -0.214 -0.179 0.086 0.714 -0.500 -1.000 -0.100 0.100 -0.100 0.100 0.036 0.714
1.000 0.900 0.900 0.700 0.900 0.700 -0.200 -0.200 0.200 0.700 0.500 0.500 -1.000 -1.000 0.500 0.500 0.300 0.100
-0.243 -0.062 -0.037 -0.410 -0.206 -0.030 -0.482 -0.759 -0.941 -0.827 0.564 0.775 -0.821 -0.775 -0.821 -0.775 -0.964 -0.677
-0.290 -0.551 -0.543 -0.486 -0.543 -0.257 0.500 0.100 -0.300 0.300 -1.000 0.400 0.200 0.800 -0.800 0.200 -0.943 0.257
0.072 0.319 0.143 -0.500 0.126 0.486 -0.536 -0.600 -0.714 -0.700 0.500 0.200 -0.900 -1.000 -0.900 -1.000 -0.786 -0.900
-0.290 -0.522 -0.086 0.600 -0.543 -0.771 0.500 -0.900 -0.100 0.200 -0.600 -0.800 -0.200 -0.400 0.000 -0.400 -0.543 -0.657
-0.135 -0.899 -0.045 -0.600 0.135 -0.429 -0.089 0.257 -0.541 0.100 0.354 0.600 -0.707 0.200 -0.707 0.200 -0.490 -0.600
nc -0.580 n.c. -0.371 n.c. -0.657 nc 0.100 nc -0.700 nc -1.000 nc 0.200 nc -0.800 nc -0.714
0.412 0.412 0.408 nc -0.103 0.000 -0.408 -0.408 -0.655 -0.655 0.000 0.000 -0.707 -0.707 -0.707 -0.707 -0.612 nc
n.c. -0.840 n.c. -0.828 nc -0.828 nc 0.707 n.c. 0.354 nc -0.258 nc 0.258 nc 0.258 nc -0.414
spring
fall
-0.057 -0.462 0.056 -0.152 -0.208 -0.638 -0.094
0.224
-0.765 -0.447 0.224 -0.600 -0.894 -0.200 -0.894
0.000
-0.617 -0.638
M, male; F, female. Significances: boldface numbers ) p e 0.05; italic numbers ) p e 0.01. nc, no correlations were made.
male fish are exposed to high concentrations of environmental estrogenics (3, 21). The highest induction of VTG in males occurred at sites that were influenced by high levels of communal effluents (Meissen: total population equivalents (PE) for discharges from the Dresden region into the Elbe ≈ 825 000; Barby and Magdeburg: total PE of discharges into the Saale downstream Leipzig/Halle ) 1 534 000). Other studies have clearly shown that effluents from STWs are estrogenic to fish (10, 16, 23). Interestingly, however, no marked induction of VTG was observed in bream at Koehlbrand, which is located just downstream of a large STW discharge (Hamburg) with a PE of more than 2 000 000. In the study on U.K. STWs most but not all of the river sites investigated just downstream of discharges were estrogenic to fish (16, 49). Factors such as level of treatment in the sewage works, retention times, and dilution of the effluent in the river all affect the estrogenic potency of the discharges (50). This might explain the lack of a marked estrogenic signal in fish from the Hamburg harbor, as the STW Koehlbrand/ Dradenau has a high standard of treatment, and the dilution factor in the river is between 2 and 4 times greater than for the Dresden and Saale effluents. Histopathological investigations found only low indices of intersex in bream from almost all parts of the Elbe River, and this is in accordance with levels of VTG induction in male bream. Interestingly, these parameters did not show significant regional correlations. This might be because the causal agents for VTG induction and intersex were different. Alternatively, it may simply relate to the fact that VTG is a biomarker for estrogen exposure and that the plasma titer will depend on how recently the fish was exposed to estrogenic compounds (and to what level), whereas intersex (altered germ cell development) may arise as a consequence of even a short exposure to estrogen at a particular life stage (e.g., early life). In addition to estrogenic biomarker responses in the middle part of the Elbe, there was a considerable suppression of other reproductive parameters. At Barby and Hohenwarte, bream had altered plasma titers of sex steroids (E2 in females and 11KT in males), and at Magdeburg all of the sex features (except from T) measured were affected. Bream at Magdeburg had smaller gonads (the GSI was between 50 and 100% less than that in more than in 90% of the control population). Although still at an age where they should reproduce (64), fish collected at Magdeburg in the autumn were younger than the control bream; therefore, the autumn results have to be interpreted with caution. Nevertheless, spring bream
(males and females) from Magdeburg were at the same age as those sampled in the control population, so that the suppression of gonad growth at Magdeburg does not appear to be an age-related difference. The low GSI (and corresponding immature germ cells) as compared to the control fish would indicate that in most of the bream from the Magdeburg site the reproductive capacity was compromised. A suppression of secondary sex characteristics in male bream at Magdeburg would also indicate a reduced sexual capacity of these fish. The variability in some of the physiological responses (e.g., VTG titer, 11KT and E2 concentrations, GSI, etc.) demonstrates that the mean response of wildlife populations exposed to environmental contaminants may not be adequate to identify a low level effect as appears to occur in fish in European rivers such as the Elbe. An increase in the variability of a biomarker within a population might give us a more informative indication of physiological disturbances that are not necessarily captured by the mean or median values. Possible Causation of Estrogenic and/or Reproductive Disruption Effects in Bream in the Elbe. A wide variety of environmental chemicals such as estrogenic compounds (natural and synthetic estrogens and xenoestrogens such as alkylphenolics and bisphenol A) and other pollutants (pesticides, PCBs, PAHs, organotins, heavy metals, etc.) that have been reported to be able to have effects on the endocrine system were measured along the Elbe (3, 7, 11, 18, 25, 5153). Most of the compounds measured in sediments and water column along the Elbe were present at concentrations below their biological effective concentrations detected in different in vivo and in vitro test systems (52, 54-56). It is becoming increasingly apparent, however, that when assessing biological effects, the mixture of pollutants has to be considered (57). In 1998 the Elbe downstream of Dresden (Meissen) was characterized by significant levels of xenoestrogens, such as BPA and alkylphenolic chemicals (Table 2). Heemken et al. (65) found that this is likely to originate from the effluent from the municipal STW in Dresden or a factory upstream. It is likely that the VTG induction seen here in male bream at Meissen can be attributed (or at least partly so) to these influences. Regions close to the Czech border and the Elbe at and upstream of Magdeburg are heavily contaminated with complex mixtures of chemicals. Some of these compounds, such as organotins, γ-HCH, Zn, etc., showed significant VOL. 36, NO. 11, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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negative correlations with the plasma androgen titers in males and E2 and VTG in females, whereas the exposure to PCBs and PAHs correlated positively with the VTG induction in males (Table 3). These relationships, however, were not always consistent over both sampling periods. The findings in this study clearly support the available evidence that endocrine modulation is occurring in wild fish from populations in the ambient riverine environment in Europe, but the study also highlights the complexity of identifying causation of a biological effect when wildlife is exposed to a complex mixture of chemicals as we find it in rivers such as the Elbe. The situation is even more complicated when it is appreciated that environmental chemicals can affect endocrine function through multiple mechanisms (51, 54, 56, 58-60). We can summarize this study by saying that there are a variety of chemicals present in stretches of the Elbe studied that are reported to be able to cause the physiological effects observed in the bream analyzed. The different modes of action of the specified chemicals as well as their possible agonistic or antagonistic interactions preclude any firm conclusions about cause-effect relationships. There do, however, seem to be some relationships between contamination in the river with γ-HCH, organotins, and zinc and suppressive effects on the endocrine axis. At least at Magdeburg the observed effects on fish are likely to result in a reduced fertility.
Acknowledgments We acknowledge David E. Kime for his scientific advice on steroid analyses and for kindly providing T and 11KT antibodies. Furthermore, we thank T. Gaumert (ARGE-Elbe) for his technical support providing bream from the upper Elbe River. The research was funded by the German Federal Environmental Agency (UBA) and the European Chemical Industry Council (CEFIC). This study is based on the Ph.D. theses of Hecker and Hoffmann, at the University of Hamburg.
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Received for review July 11, 2001. Revised manuscript received December 5, 2002. Accepted February 4, 2002. ES010186H
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