Effects of Polychlorinated Biphenyls on Liver Ultrastructure, Hepatic Monooxygenases, and Reproductive Success in the Barbel

Ecotoxicology and Environmental Safety 42, 265—273 (1999) Environmental Research, Section B Article ID eesa.1998.1761, available online at http://www.idealibrary.com on

Effects of Polychlorinated Biphenyls on Liver Ultrastructure, Hepatic Monooxygenases, and Reproductive Success in the Barbel J. L. Hugla and J. P. Thome´ Institut de Zoologie, Laboratoire d+Ecologie animale et d+Ecotoxicologie, Universite´ de Lie` ge, 22 quai Van Beneden, B-4020 Lie` ge, Belgium Received March 5, 1998

Polychlorinated biphenyls (PCBs) are organochlorinated micropollutants ubiquitously distributed in the environment. They are known to be strong inducers of hepatic monooxygenases in fish. This can adversely affect reproduction by increasing steroid metabolism. In this work, adult barbels were contaminated with food containing Aroclor 1260, a commercial PCB mixture from Monsanto, at environmentally relevant concentrations. A significant increase in cytochrome P450 was observed, and two particularly sensitive enzymes, ethoxyresorufin o-deethylase (EROD) and ethoxycoumarin o-deethylase (ECOD), were strongly induced. Electron microscopy revealed alterations in liver ultrastructure in contaminated fish, principally an increase in the number of cisternae of the rough endoplasmic reticulum, drastic glycogen depletion, dissolution of mitochondrial contents, and appearance of myelin figures. Contamination was also studied in relation to reproductive success in a hatchery. Contaminated males displayed no alteration in milt quality, but PCBs did alter female reproductive parameters. Total mortality of eggs and larvae increased significantly with the level of PCBs in the eggs. The most highly contaminated fish did not even spawn. All the adverse effects recorded here tended to be reversible when the intoxication ended, sometimes after only a 1-year detoxication period.  1999 Academic Press Key Words: hepatocyte; ultrastructure; liver monooxygenases; reproduction; polychlorinated biphenyls.

INTRODUCTION

The use of physiological and biochemical parameters as indicators of water quality has recently been developed to detect sublethal impacts of pollutants. The usefulness of hepatic cytochrome P450-dependent monooxygenases (MOs) as biomarkers in fish has been clearly demonstrated, as these are well known to be induced by chemicals like

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polyaromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) (Masfaraud et al., 1990; Haasch et al., 1993; Palace et al., 1996). In the case of some xenobiotics, MO activities are related to the degree of contamination of the organism (Payne et al., 1987; Goks+yr et al., 1994; Stagg et al., 1995). As steroid hormones are substrates for these enzymes, their induction due to the presence of pollutants also produces antiestrogenic responses (Klotz et al., 1986; DeVito et al., 1992; Jansen et al., 1993) and thus affects the reproductive success of fish species (Freeman and Idler, 1975; Thomas, 1990; Singh and Singh, 1992). Reproduction, moreover, may be affected by some PCBs that are competitive ligands of the estrogen receptor (Lundholm, 1988; Jansen et al., 1993; Waller et al., 1995). Xenobiotics like PCBs are thus suspected to have played a major role in the rarefaction of wild fish species (Niimi, 1983; Gilbertson, 1992). This work is an ecotoxicological study of PCB contamination in a sensitive wild fish species, the common barbel, Barbus barbus, the reproduction and development of which have been extensively studied and are particularly well monitored (Philippart et al., 1989). This species has undergone major regression in Europe, although numerous local populations subsist. It is considered a threatened species in the European Union (EU) (Philippart, 1987). It also ranks high in PCB contamination among cyprinids and salmonids (Keck and Raffenot, 1979; Vindimian et al., 1991; Hugla et al., 1995). The impact of PCBs on the hepatic cytochrome P450 content of barbels and on the activities of two monooxygenases, ethoxycoumarin o-deethylase (ECOD) and ethoxyresorufin o-deethylase (EROD) was investigated. As PCBs generate alterations in hepatic tissue and hepatocyte ultrastructure in fish (Hacking et al., 1977; Weibel and Paumgartner, 1978; Hugla et al., 1995), the liver ultrastructure of contaminated barbels was observed and compared with that of controls. Also examined was the reproductive success of the species following intoxication.

265 0147-6513/99 $30.00 Copyright  1999 by Academic Press All rights of reproduction in any form reserved.

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MATERIAL AND METHODS

In wild barbels from the Belgian part of the River Meuse, the PCB composition expressed in terms of commercial mixtures is about 20% Aroclor 1254 and 80% Aroclor 1260 (Hugla et al., 1995). The fish were thus experimentally contaminated with a commercial mixture of Aroclor 1260. Adult barbels (3 to 5 years old) from the scientific hatchery of the Laboratory of Fish Demography and Aquaculture (University of Lie`ge) in Tihange (Belgium) were contaminated for 30 days by adding PCBs to their food. To develop ecotoxicological laboratory tests corresponding to natural conditions, 16 individuals (10 males and 6 females) were contaminated for 50 days with food containing 2.5 lg PCBs g\, an environmentally relevant concentration (Joaquim-Justo et al., 1995). Another batch of 16 fish received food containing 12.5 lg PCBs g\ for 75 days. The control batch consisted of 20 individuals. Finally, contaminated fish were kept in PCB-free water for a 1-year detoxication period.

the fluorometric method of Burke and Mayer (1974). All experimental values are specific activities (activity/protein content). The protein content was determined by the method of Lowry et al. (1951). Electron Microscopy Just after dissection, small pieces of liver (about 1 mm) were fixed for 24 h by direct immersion in a 1.25% glutaraldehyde solution buffered with 0.1 M Na cacodylate at pH 7.4. After being rinsed in the same buffer, the samples were postfixed for 1 h at 4°C in a Na cacodylate-buffered 1% OsO solution, rinsed in distilled water, dehydrated with an  ethanol and propylene oxide series, and embedded in Glycidether 100 (Serva). Thin sections cut with a diamond knife, deposited on collodion-coated grids, and contrasted with uranyl acetate and lead citrate were examined with a JEOL TEM 100 SX electron microscope at 80-kV accelerating voltage. Glycogen patches were detected by the periodic acid—thiocarbohydrazide—silver proteinate (PATAg) method developed by Thiery and Rambourg (1974).

PCB Quantification PCBs were extracted according to a slight modification of EPA method 608 as previously described by Hugla et al. (1995). The acid and Florisil cleanup procedures and the chromatographic separation of the PCB congeners in fish liver, sperm, and eggs, by means of high-resolution capillary gas chromatography (HRGC), were also carried out according to Thome´ et al. (1987) and Hugla et al. (1995). Twentyfive individual congeners (from di- to nonachlorinated) were identified and quantified, and PCB concentrations were expressed in terms of Aroclor 1260. Enzyme Assays Immediately after killing, parts of each fish liver were wrapped in aluminum foil and frozen in liquid nitrogen where they were stored for a few days prior to microsome extraction. The liver samples were homogenized in ice-cold buffer (250 mM sucrose, 1 mM EDTA, 10 mM Tris—HCl, pH 7.4) with a Potter—Elvehjem homogenizer. Homogenates were centrifuged at 10,000g for 20 min and the resulting supernatants centrifuged at 105,000g for 60 min. Pellets were resuspended in buffer containing 0.1 mM EDTA and 100 mM Tris (pH 7.5) and stored in small aliquots at !80°C. Protocols for assaying hepatic monooxygenase activities were adapted for fish samples. Optimal incubation conditions have been described elsewhere (Hugla et al., 1995). The method described by Omura and Sato (1964) was used to measure the total amount of cytochrome P450. ECOD activity was measured by the fluorometric method of Greenlee and Poland (1978). EROD activity was determined by

Fertility and Reproductive Success For two successive reproductive seasons, female fish were checked daily for sexual maturity. Ripe eggs were stripped and inseminated with sperm from three control or contaminated males to ensure effective fertilization. The fertilized eggs were then incubated and hatched in small Zug bottles in a closed thermoregulated (20°C) and UV-treated recirculating system (Philippart et al., 1989). At this temperature, hatching occurs within 4 days and the yolk absorption stage is completed by the age of approximately 13 days (Penaz, 1973; Krupka, 1988; Philippart et al., 1989). The following parameters were determined: The amount of spermatozoa in the sperm, using Bu¨rker’s chamber (sperm used at a 1:900 dilution in 0.7% NaCl); The duration of the mobility phase of the spermatozoa, which gives an idea of their fertilising ability, [Hochman et al. (1974) have classified the movements of spermatozoa according to a four-step scale, defining ‘‘very quick,’’ ‘‘medium quick,’’ ‘‘slow,’’ and ‘‘stationary’’ movements;] The number and mean weight of stripped eggs for each spawning; The numbers of dead eggs, aborted larvae, dead larvae, normal larvae, and abnormal larvae 30 days post fertilization. Statistical Analysis One-way analysis of variance, Tukey’s multiple comparison tests, and linear regressions were applied to the data, using the SigmaStat 2.0 for Windows software (Jandel Scientific GmbH, Erkrath, Germany).

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RESULTS

PCB Concentrations and Liver Monooxygenases Intoxicated fish accumulated large amounts of PCBs. Contamination levels measured after 50 and 75 days differed significantly from the control values (Table 1). In whole fish, the PCB concentration appeared proportional to the contaminant level in the food, but this was less obvious in the liver. The liver monooxygenase system is clearly induced following intoxication. The P450 concentration is significantly higher in contaminated fish than in controls (Table 1), and both EROD and ECOD activities are clearly higher. No sex-related difference in induction levels was observed. After a 1-year detoxication period, the MO values were comparable to those of control fish, even though contaminated fish displayed almost unchanged PCB concentrations in the liver (Table 1). Liver Ultrastructure In control fish, the nucleus was round or ovoid and the nucleolus was sometimes visible (Fig. 1a). The rough endoplasmic reticulum (RER) appeared arranged in parallel stacks of cisternae, usually located around the nucleus and along the plasma membrane. The mitochondria were usually found in close association with the RER, their shape varying from circular to elongated, with well-developed cristae. Some lipid droplets were present, but the cytoplasm was mainly filled with large glycogen-containing areas, as revealed by the PATAg method (Fig. 1b). The smooth endoplasmic reticulum (SER) and the Golgi apparatus were never observed. Liver ultrastructure appeared markedly altered in fish intoxicated with PCB-contaminated food (12.5 lg g\) (Fig. 1c). The most common changes were a major increase in RER, drastic glycogen depletion, and changes in

mitochondrial morphology: dramatic alteration of the cristae (Fig. 1d) and degeneration into laminated concentric membrane arrays (also called myelin-like figures) (Fig. 1e). After a 1-year detoxication period, the hepatocytes of contaminated fish no longer displayed any signs of intoxication (Fig. 1f ): the number of RER cisternae had decreased and glycogen-containing zones again filled the cytoplasm. The mitochondria appeared normal, with well-developed cristae, and laminated concentric membrane arrays were seldom present. Fertility and Reproductive Success Results concerning sperm quality in contaminated barbels are summarized in Table 2. The PCB concentration is surprisingly low compared with that of whole fish (see Table 1), but appears to correlate well with the contaminant level in the food. The number of spermatozoa in barbel milt is about 22;10 mm\, and PCBs did not significantly alter this concentration. Accordingly, the period during which the spermatozoa remained motile was the same whether the fish were contaminated or not (Table 2). PCB contamination thus does not appear to reduce the fertilizing capacity of male barbels. PCBs did, however, have a dramatic effect on barbel eggs. During the first reproductive season, female barbels highly contaminated with PCBs failed to spawn. After a 1-year period of detoxication, however, these females spawned two or three times each, as did the control specimens and those subjected to low-level contamination. PCB concentrations were about five times as high in the ovaries as in the eggs (Table 2). Control fish displayed a fecundity similar to that recorded in a scientific hatchery, i.e., 9465$1495 eggs per kilogram of fish (n"81) (Poncin, 1984, in Poncin, 1988), but in PCB-contaminated fish, fecundity was much lower.

TABLE 1 PCB Concentrations and Liver Monooxygenase Activities in Control Fish and in Barbels Fed Aroclor 1260-Contaminated Food, (A) after Intoxication and (B) after a 1-year Period of Detoxicationa 2.5 lg PCBs g\ food, 50 days Group PCBs in fish (lg g\ dry wt) PCBs in liver (lg g\ dry wt) Cytochrome P450 (pmol mg\) EROD (pmol mg\ min\) ECOD (pmol mg\ min\)

Control

A

1.0$0.2 0.5$0.2 246$26 40.0$9.9 48.0$9.9

2.6$0.3 2.4$1.0 292$50 68.4$7.5* 71.1$9.9

B —@ 1.2$0.2 215$51 37.6$4.0** 61.3$12.8

12.5 lg PCBs g\ food, 75 days A

B

13.2$1.7* 7.1$0.6* 464$62* 142.7$13.6* 121.2$21.3*

— 5.5$2.1* 222$31** 45.0$8.4** 57.4$5.5**

? Means$SD, n"6. @ —, no data. * Significantly different from control group (one-way ANOVA, P(0.01; Tukey test, P(0.05). ** Significantly different from contaminated group after intoxication (A) (one-way ANOVA, P(0.01; Tukey test, P(0.05).

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PCB EFFECTS ON LIVER AND REPRODUCTION IN BARBEL

TABLE 2 Sperm and Egg Characteristics of Controls and PCB-Contaminated Barbels Having Reached Maturity 1 Year after Contamination a PCB-laced food Group PCBs in sperm (ng g\ dry wt) Number of spermatozoa (10 mm\) Cumulative duration of the mobility phases (s) Very quick Medium quick Slow Oscillating PCBs in ovary (ng g\ dry wt) PCB in eggs (ng g\ dry wt) Mean weight of eggs (mg) Fecundity (eggs kg\) Hatching rate (% laid eggs)

Control (n"6)

2.5 lg g\, 50 days (n"6)

12.5 lg g\, 75 days (n"6)

93.7$17.2 22.8$4.1

175.2$71.2 23.1$3.1

1007.0$566.7R 21.1$3.7

16.2$2.1 26.7$2.5 37.2$3.8 69.7$12.9 699$137 124$23 9.8$1.1 9688$798 55.4$3.7

17.2$1.2 26.2$2.7 37.2$4.9 80.8$5.7 1561$770 252$116 10.3$0.7 4629$300* 50.3$0.2

18.2$2.4 28.4$3.8 40.9$3.7 71.1$17.7 5142$2047 1289$450* 8.8$0.7 4521$1770* 4.4$5.1**

? Means$SD. * Significantly different from control group (one-way ANOVA, P(0.05; Tukey’s test, P(0.05). ** Significantly different from control group (Kruskal—Wallis one-way ANOVA, P(0.01; Dunn’s test, P(0.05).

Surprisingly, the mean weight of the eggs was not significantly reduced in spawnings from treated fish (Table 2). On the contrary, eggs from the most highly contaminated barbels displayed a considerably reduced hatching rate (Table 2). Lastly, a good correlation was observed between the total mortality of eggs and larvae and egg PCB content (Fig. 2). The mortality observed for control eggs was quite high (52.4$9.2%) but in good agreement with previous results obtained for the same species (Absil, personal communication) and rainbow trout (Walker et al., 1992).

The MO induction levels recorded in intoxicated barbels are similar to those previously measured in wild specimens from the River Meuse, which were found to correlate significantly with the PCB concentration in the liver (Table 3); (Hugla et al., 1995). Such a correlation and MO induction in the same range are well documented in other fish species, both in vitro (Fo¨rlin and Lidman, 1978; Nasci et al., 1991) and in situ (Spies et al., 1988; Masfaraud et al., 1990; Haasch et al., 1993; Burgeot et al., 1994; Eggens et al., 1995). The

DISCUSSION

PCB Concentrations and Liver Monooxygenases The PCB concentrations measured in contaminated fish were in the range of those previously detected in 7- to 9-year-old barbels from the Belgian part of the River Meuse, but lower than the levels measured in older individuals (11—14 years), i.e., around 25 lg g\ dry wt in liver and muscle (Fig. 3). PCB accumulation in wild barbel is known to increase with age, even reaching levels similar to those detected in eels (Anguilla anguilla) in Northern Europe (Hendriks and Pieters, 1993; Haiber and Scholer, 1994), eels being considered among the most highly contaminated fish due to their high lipid content and carnivorous diet.

FIG. 2. Total mortality of eggs and larvae of control (open circle) and PCB-contaminated (filled circles) barbels, versus the PCB concentration in the eggs.

FIG. 1. Transmission electron micrographs of barbel hepatocytes. (a) Control fish. (b) Control fish, with glycogen displayed by means of the PATAg method. (c—e) Fish contaminated for 75 days with PCBs at a concentration of 12.5 lg g\ in food. (f ) Contaminated fish kept for 1 year under PCB-free conditions. G, glycogen; L, lipid droplet; m, mitochondria; my, myelin figure; N, nucleus; RER, rough endoplasmic reticulum. Bar"1 lm, except in Fig. 2e (0.1 lm).

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TABLE 3 Linear Regressions Between PCB Concentrations in the Liver and Monooxygenases (MO) Activities in Wild Barbels from the River Meuse MO EROD (n"17) ECOD (n"17)

FIG. 3. PCB concentrations in liver and muscle of wild barbels from the Belgian part of the river Meuse (after Hugla et al., 1995, modified).

results thus confirm the major impact of PCBs on liver oxidases in the barbel, as previously reported by Vindimian et al. (1991) in the River Rhoˆne. The absence of sex-related differences in MO induction has also been recorded previously in winter flounder (Pseudopleuronectes americanus) (Stegeman et al., 1987) and nase (Chondrostoma nasus) (Masfaraud et al., 1990). Other studies, however, have evidenced such differences (Spies et al., 1982; Lindstro¨m-Seppa, 1985). It is thus difficult to draw conclusions on this point, especially since steroids can modify liver enzyme activities (Stegeman and Chevion, 1980; Gray et al., 1991). The observations made after the detoxication period are consistent with the results of other studies in which a rapid decrease in liver enzyme induction was recorded when contamination ended, for example, in carp fed contaminated food (Kobayashi et al., 1987) or in rainbow trout (Oncorhynchus mykiss) contaminated by intraperitoneal injection (Andersson et al., 1985). Liver Ultrastructure The cell organization observed in control barbel hepatocytes is classic and quite comparable to that observed in other fish hepatocytes (e.g., Hacking et al., 1977; Sivarajah et al., 1978; Ko¨hler, 1990). The absence of SER and Golgi apparatus has also been noted previously (Chapman, 1981) in rainbow trout. The ultrastructural modifications observed in experimentally contaminated barbels are similar to but less marked than those detected in wild individuals from the River Meuse continuously exposed to various micropollutants (Hugla et al., 1995). Contrary to observations in mammals (Lipsky et al., 1978; Singh et al., 1996), birds (Williams et al., 1993; Stouvenakers et al., 1996), and catfish (Ictalurus punctatus) (Lipsky et al., 1978), no development of the SER was ob-

Regression equation

Correlation coefficient

EROD activity"4.90;[PCB] #78.8  ECOD activity"3.95;[PCB] #89.5 

r"0.80, P(0.01 r"0.76, P(0.01

served. This finding is in good agreement, however, with previous observations on PCB-contaminated carp (Cyprinus carpio) (Sivarajah et al., 1978) and DDT-contaminated zebrafish (Brachidanio rerio) (Weis, 1974). Hypertrophy of the RER as seen here is frequently observed in fish after PCB intoxication, for example, in rainbow trout (Hacking et al., 1977) and sea bass (Dicentrarchus labrax) (Lemaire et al., 1992). It is also classically associated with a disorganization into sinuous or circular profiles (Weis, 1974; Sivarajah et al., 1978; Lipsky et al., 1978; Holm et al., 1993) and breakage of the cisternae (Ko¨hler, 1990). Such modifications clearly reflect the initiation of hepatic detoxication mechanisms (Klaunig et al., 1979; Lemaire et al., 1992) and confirm the MO induction observed. Alterations of the mitochondria similar to those observed in the barbel have been described as reversible changes affecting liver ultrastructure in contaminated fish, including rainbow trout and flounder (Platichthys flesus) (Hacking et al., 1977; Ko¨hler, 1990). The drastic reduction in liver glycogen mentioned here is also frequently observed, not only after intoxication with PCBs, as for example in rainbow trout (Hacking et al., 1977) and sea bass (Lemaire et al., 1992), but also after contamination with other organochlorinated xenobiotics such as DDT and dioxin (2,3,7,8-TCDD) (Baruffaldi and Cucchi, 1989; Spitsbergen et al., 1991). This depletion may be due to a direct effect of PCBs on carbohydrate metabolism. It is considered an indicator of a hypoenergetic status (Mac and Edsall, 1991), probably linked to the energy cost of detoxication. Fertility and Reproductive Success The number of spermatozoa in barbel milt is large compared with the 7;10 mm\ recorded in Coregonus pollan (Hochman et al., 1974) and the 10;10 mm\ recorded in tench (¹inca tinca) (Zuromska, 1981). Anyway, the lack of impact of PCBs on fertilizing capacity of male fish was also mentioned by Von Westernhagen et al. (1987) in a study in which in four Baltic Sea species, the measured mean PCB concentration in the sperm was only 90 ng g\ wet wt. As noted for the most contaminated female barbels, Freeman et al. (1982) likewise observed no spawning in cod

PCB EFFECTS ON LIVER AND REPRODUCTION IN BARBEL

(Gadus morhua) receiving food contaminated with 1 to 50 lg PCBs g\. Moreover, a decrease in fecundity was also previously observed in DDT-intoxicated brook trout (Salvelinus fontinalis) (Macek, 1968) and on zebrafish intoxicated with 2,3,7,8-TCDD (Wannemacher et al., 1992). The only author to have considered the mean weight of treated fish eggs is Macek (1968), who found, as in the experiment presented here, the diameter of brook trout eggs to remain constant following DDT contamination. The reduced hatching rate observed in highly PCBtreated barbel eggs is in good agreement with observations on contaminated flounder (Platichthys flesus) eggs (120 ng PCBs g\ wet wt) (Von Westernhagen et al., 1987) and lake trout (Salvelinus namaycush) eggs (300 ng PCBs g\) (Mac and Edsall, 1991). Some authors, however, found that markedly higher PCB concentrations (on the order of 5 lg g\) were required to reduce the hatching rate of eggs from Atlantic salmon (Salmo salar) (Stalling and Mayer, 1972) and minnow (Phoxinus phoxinus) (Bengtsson, 1980). Finally, on the basis of the current results, survival of barbel larvae appears PCB dose dependent, as demonstrated in many other species like the starry flounder (Platichthys stellatus) (Spies and Rice, 1988), charr (Salvelinus alpinus) (Monod, 1985), and rainbow trout (Walker and Peterson, 1991). CONCLUSION

As demonstrated here, PCBs, even at environmental levels, clearly affect various physiological and biochemical parameters in the barbel. These micropollutants induce hepatic monooxygenases, alter liver ultrastructure, and reduce the fecundity and hatching rate of the species. These different phenomena appear closely related and are quite reversible on detoxication. The parameters studied here appear to be good biomarkers for assessing fish contamination by PCBs. From a general ecological standpoint, of course, weather conditions and local pollution clearly play a major role in barbel rarefaction. Factors such as the disappearance of spawning grounds, the dragging of river beds, and the sudden rise of a river may reduce the recruitment of some species into particular zones. PCBs are not the only xenobiotics present in rivers and thus cannot be considered solely responsible for the decline of wild fish populations. Yet undeniably, the chronic effects of PCBs have played and still play a role in the regression of some barbel populations in polluted areas, as DDT did for birds in the 1960s. Editor’s Note: In the 1960s and previously, PCBs and DDT were completely confused in biochemical analysis. The early gas chromatograph machines could not distinguish between the two.

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ACKNOWLEDGMENTS The authors appreciate the excellent assistance of Mrs. M. Louvet (technical help), N. Decloux (ultramicrotomy), Mrs. A. Todaro (biochemistry), Mrs. Ch. Breeur (iconography), and Mrs. C. Adam (undergraduate student). They also thank Professor G. Goffinet (Laboratory of General Biology and Ultrastructural Morphology, University of Lie`ge) and Dr. P. Kremers (Laboratory of Medical Chemistry, University of Lie`ge) for their helpful suggestions throughout this work, and Dr. J.C. Philippart (Laboratory of Fish Demography and Aquaculture, University of Lie`ge) and Dr. P. Poncin for sharing their knowledge on the barbel. This work was made possible by the financial support of the SSTC (HH/46 Health Hazards Impulse Programme). J.L.H. was the recipient of an IRSIA grant for this study.

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