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The lipid-lowering agents bezafibrate and clofibric acid, which occur at concentrations up to 3.1 and 1.6 μL, respectively, are among the most frequently found human pharmaceuticals in the aquatic environment. In contrast to knowledge about their environmental occurrence, little is known about their effects in the environment. The aim of the present study was to analyze effects of these lipid-lowering agents in fish by focusing on their modes of action, lipid metabolism. Fathead minnows were exposed in aquaria to measured concentrations of 0.1, 1.27, 10.18, 101.56, and 106.7 mg/L bezafibrate and to 1.07, 10.75, and 108.91 mg/L clofibric acid for 14 and 21 d, respectively. After exposure, fish liver was analyzed for expression of peroxisome proliferator-activated receptor α (PPARα) by quantitative polymerase chain reaction (PCR), and the PPAR-regulated enzyme fatty acyl-coenzyme-A oxidase (FAO) involved in fatty acid oxidation. Bezafibrate had no effect, either on PPARα expression or on FAO activity, at all concentrations. In contrast, clofibric acid induced FAO activity in male fathead minnows at 108.91 mg/L. No increase in expression of PPARα messenger ribonucleic acid was observed. Egg production was apparently decreased after 21 d of exposure to 108.91 mg/L clofibric acid. The present study demonstrates that bezafibrate has very little or no effect on PPARα expression and FAO activity, but clofibric acid affects FAO activity.
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A considerable number of diverse pharmaceuticals have been detected in the aquatic environment at concentrations up to several micrograms per liter . Pharmaceuticals are designed to evoke biological response in humans, but little is known about their effects to aquatic organisms. Pharmaceuticals are tested mainly for their acute toxicities in classical ecotoxicological tests, but rarely do tests focus on their mechanisms of action and target organs . Due to the evolutionary conservation of many receptors between humans and fish, mechanisms of action of a given pharmaceutical may also be similar [1–3]. Such a mode-of-action-based concept often reveals more specific effects than classical tests. This has been demonstrated by the nonsteroidal anti-inflammatory compound diclofenac affecting liver and kidney in rainbow trout . Moreover, unexpected effects may be discovered, as shown with the lipid-lowering agent clofibric acid (CA) showing adverse effects on reproductive parameters such as spermatogenesis in fathead minnows . Therefore, studies focusing on the drug's modes of action and its biological targets may be more informative in discovering potential adverse effects than classical ecotoxicity tests alone. This hypothesis led us to study potential adverse effects of the human lipid-lowering agents bezafibrate (BF) and CA in fish by focusing on lipid metabolism.
Blood-lipid-lowering agents such as BF and the metabolite of different fibrates, CA, are among the most frequently prescribed drugs and also the most frequently found human pharmaceuticals in the aquatic environment [1,2]. Fibrates are widely used in human medicine to treat lipidemic diseases such as hypercholesterolemia and to prevent heart attack. Bezafibrate was found in sewage treatment plant effluents and surface waters up 4.6 μL  and 3.1 μL , respectively, in Germany. Also in Germany up to 1.6 μL  CA has been detected in sewage treatment plant effluents, and up to 0.55 μL CA has been detected in surface water . Clofibric acid is also the main metabolite of lipid-lowering agents including clofibrate, etofylline clofibrate, and etofibrate and is very persistent in the aquatic environment .
Bezafibrate and CA have been analyzed in classical ecotoxicity tests for acute effects in Daphnia magna (median effective concentration, EC50 > 200 mg/L)  and Anemia  and on several enzymes in mosquito fish [9,10]. Subchronic toxicity of CA was tested in algae (lowest-observed-effect concentration [LOEC] = 150 mg/L), Ceriodaphnia dubia (LOEC = 2.56 mg/L) and zebrafish early life stages (LOEC = 140 mg/L) . Bezafibrate and gemfibrozil affected the immune function, glycolysis, redox balance, and peroxisomal function in the bivalve mollusc Mytilus galloprovincialis . Lipid-lowering agents were found to have low cytotoxicity in fish cells .
Effects of fibrates are mediated, at least in part, through alterations in transcription of genes encoding for proteins controlling lipoprotein metabolism. Fibrates probably act by activating the lipoprotein lipase enzyme, which is mainly responsible for the conversion of very low density lipoprotein to high density lipoproteins, decreasing therefore plasma triglycerides concentration . Binding of fibrates to peroxisome proliferator-activated receptors (PPARs), nuclear receptors known to be activated in different cellular pathways, stimulates the expression of several lipid regulatory proteins. After ligand activation, PPARs enter the nucleus, and after heterodimerization with the retinoid X receptor, they promote both the expression of different genes involved in lipid metabolism and the proliferation of peroxisomes . Consequently, gene expression and activity of some of the specific peroxisomal proteins are increased . As other vertebrates, fish such as Atlantic salmon and plaice [16–19] have three PPARs: Peroxisome proliferator-activated receptor α, PPARβ (or 5), and PPARγ have different functions and show different tissue distributions in zebrafish. Peroxisome proliferator-activated receptor α is mainly expressed in tissues catabolizing fatty acids such as liver, proximal tubules of kidney, and enterocytes, whereas PPARβ is less specifically expressed and found in many tissues . Peroxisome proliferator-activated receptor α is involved in peroxisome proliferation and plays a pivotal role in controlling hepatic lipid metabolism , whereas PPARβ has diverse roles in basic lipid metabolism and PPARγ plays a key role in the differentiation of adipocytes . Peroxisome proliferator-activated receptor γ is induced in response to clofibrate and BF in salmon hepatocytes , but fish PPARγ seems to be less responsive than that in rodents . All three PPARs are expressed in larval zebrafish  and goldfish .
Activators of PPARα include a variety of endogenous compounds including fatty acids. Furthermore, fibrates are known to bind to PPARs, leading to induction of target genes involved in lipid metabolism. They interact with PPARs in salmon , zebrafish [23,25], and mussels . An increase in PPARγ messenger ribonucleic acid (mRNA) expression was demonstrated in salmon hepatocytes . Some organochlorine pesticides  and plasticizers  may activate PPARs in zebrafish, and perfluorooctanoic acid in fathead minnows . In addition, PPAR agonists may induce a specific peroxisome membrane protein (PMP70) and result in adverse effects on reproduction [2,28].
Fatty acyl-coenzyme-A oxidase (FAO) plays a key role in the β-oxidation of fatty acids and is found in peroxisomes only. Fibrates increased FAO activity in primary hepatocytes of salmon (Salmo salar) , rainbow trout, and Japanese medaka . In the present study with fathead minnows (Pimephales promelas), we focus on PPARα expression and associated FAO activity to determine potential effects on lipid metabolism and apparent effects on reproduction. The aim was to broaden the knowledge of previous studies in goldfish [24,30], mosquitofish , and fathead minnows .
MATERIALS AND METHODS
Bezafibrate (purity >99%) was supplied by F. Hoffmann-La Roche. Clofibric acid (purity >99%) and dibutyl phthalate (DBP; purity >98%) were purchased from Sigma-Aldrich. Ethanol and dimethylsulfoxide (DMSO) were purchased from T.J. Backer. Additional chemicals used in the different assays are described below.
Analysis of experimental water samples was performed for all experiments based on a modified procedure of Ternes et al.  and Hernando et al. . In experiment A, different volumes of water samples were extracted using a Strata Screen-A cartridge (500 mg/6 ml, 8B-S019-HCH; Phenomenex). Conditioning and equilibration were performed prior to sample loading with 5 ml of methanol and 5 ml of 50 mM phosphate buffer (pH 7). The rate of sample loading was maintained at 4 to 8 ml/min. Analytes were eluted by 1% concentrated HCl in 70% 1:1 methanol/acetonitrile (ACN). The extracts were dried under a stream of N2 at 40°C for 1 to 1.5 h and 55°C for 1 to 2 h, respectively, and subsequently dissolved in 1 to 2 ml of 40% ACN/Milli-Q water. In experiments B and C, water containing high concentrations of CA and BF (100 mg/L) were either 10-fold diluted or directly analyzed (CA 1 and 10 mg/L) without prior extraction.
Extracts (20 μl) or water samples were injected into a high-pressure liquid chromatograph (HPLC; Hewlett-Packard HP 1090 or 1050), both equipped with a Spherisorb RP-C18 250 × 4.6 mm column, with a flow rate of 1 ml/min. The mobile phase consisted of a binary mixture of Milli-Q water and ACN. The linear gradient program began at 5% ACN hold for 1 min to end at 40% ACN after 8 min. The peaks determined by ultraviolet detection at 225 nm were detected after 8 min (CA) and 11.5 min (BF), and quantification was performed using the external standard method. Recoveries were between 84 and 98%.
Acclimatization. Adult 2-year-old fathead minnows (Pimephales promelas) in experiment A were purchased in summer 2005 from Osage Catfisheries and were kept in a water tank (300 L) for 3 weeks for acclimatization prior to the beginning of the experiment. Fathead minnows in experiments B and C were 11 months old and cultured in-house at Springborn Smithers Laboratories from January to November 2006. Experiment A was conducted in October 2005, experiment B in January 2006, and experiment C in November 2006, and acclimatization was also 2 weeks prior to the experiment. Temperature (25 ± 2°C) and daylight photoperiod (16 h) were held constant during the acclimatization period. The water tanks were supplied with a carbon filtration system.
The experimental procedure was the same for all experiments and adapted from Kunz et al.  using a semistatic procedure. Table 1 summarizes the experiments performed and end points analyzed. Fish were exposed in aquaria containing reconstituted tap water (alkalinity 25–30 mg/L CaCO3, conductivity 500 μS/cm) at a constant temperature of 25 ± 1°C. Two replicates were included in experiments A and B, and three replicates were included in experiment C. Bezafibrate and CA were directly dissolved in tap water without solvent. Bezafibrate solutions were prepared 1 d prior to water renewal and kept under agitation overnight, whereas CA solutions were freshly prepared when water renewal took place. After 24 h, one-third of water was replaced with fresh medium to eliminate food residues and feces, and every 48 h, water in all aquaria was completely renewed. Each aquarium contained two steel tunnels for fish for spawning. Fish were fed twice daily with commercial flakes (Tetramin). Mortality and swimming behavior as well as egg production were recorded daily. Dissolved oxygen and pH were recorded twice during the renewal from fresh (0 h) and old solutions (48 h).
In experiments A and C, 12 fish (six males and six females), and in experiment B eight fish (four males and four females), were randomly distributed in stainless steel aquaria containing control water, DBP (dissolved in DMSO), or different concentrations of BF and CA. The aquaria were filled with 15 L of water in experiments A and C and with 10 L in experiment B. Plastic top covers were used, and air was delivered through air pumps connected to an appropriate plastic tube ending in a Pasteur pipette that was dipped into the water.
Table Table 1.. Experiments and endpoints analyzed designated with a check mark (✓)
Experiment A. Fish were exposed for 14 d in two replicates of four BF concentrations with a separation factor of 10, and FAO activity in liver was determined at the end of exposure. As a positive control for FAO activity, DBP (0.5 mg/L prepared in <0.01% DMSO) was used, because this concentration led to slight induction in zebrafish . For analysis of actual BF concentrations, 1-L water samples were collected from each aquarium at time points 0 and 48 h throughout the 14-d exposure period. Aliquots of the fresh stock solution (100 mg/L) were collected at each water renewal. Water samples were extracted and analyzed for BF or CA concentrations as described above; DBP was not determined by chemical analysis, however.
Experiment B. This experiment was conducted to determine PPARα expression, which was not determined in experiment A. The 14-d exposure experiment consisted of two replicates (water control and 100 mg/L BF). For determination of actual BF concentrations, 100 ml of water was collected from the control and the 100 mg/L BF dose group at 0, 24, and 48 h. Control water samples were directly injected into the HPLC for analysis, whereas BF samples were diluted 1:10 with fish medium prior to injection.
Experiment C. Three replicates each consisting of six male and six female fish of water control, and 1, 10, and 100 mg/L CA were used. At the end of the exposure, FAO activity and PPARα transcription were analyzed in the liver of fish. For the analysis of actual CA concentrations, water samples (50 ml) were collected from the control and different CA groups at 0, 24, and 48 h during the 21-d exposure. Controls and samples from the 1 and 10 mg/L CA dose groups were injected directly into the HPLC for analysis, whereas samples from the 100 mg/L CA dose were diluted 1:10 with tap water prior to injection.
After exposure, fish were anesthetized with 100 mg/L MS-222 (tricaine methanesulfonate; Fluka), and lengths and weights were measured. Blood was collected with 20 or 50 μl heparinized capillary tubes (KABE Labortechnik) from the caudal vein and kept on ice for a maximum of 3 h; 20 μl at 2 units/ml of protease inhibitor (aprotinin; Fluka AG) were added. Plasma was collected after centrifugation (10 min at 3,000 g and 4°C) and stored at −80°C until analysis. Liver of fish were either removed and directly fast-frozen in liquid nitrogen and stored at −80°C until determination of FAO activity or kept in RNAlater solution (Qiagen) according to the manufacturer's protocol for real-time polymerase chain reaction (RT-PCR).
PPARα expression, FAO activity, and vitellogenin
Quantitative PPARα expression analysis. The RNA was extracted from liver samples with the RNeasy extraction kit (Qiagen) according to the manufacturer's protocol. Extracted RNA was resuspended in 3 to 45 μl of nuclease-free water (Qiagen). The reverse transcription reaction was performed according to the manufacturer's protocol using reverse transcriptase (Roche Diagnostics). A quantity of 2 μg per reaction (0.1 μμl RNA), which was ensured by spectrophotometric detection at 260 nm (NanoDrop ND-1000 full-spectrum [220–750 nm]; Witec). For reverse transcription, poly-dT primers (Roche Diagnostics) were used for nonspecific transcription of total RNA into complementary deoxyribonucleic acid (cDNA).
A first set of degenerated primers was used to amplify and clone the PPARα and the β-actin cDNA of fathead minnows. The products were subsequently sequenced (Synergene Biotech), and sequences were submitted to Genbank (accession numbers EU195886 and EU195887, respectively). According to the sequences obtained, a second set of primers was designed to provide a shorter band for each gene for quantitative PCR. The sequences of the primers were 5′-GCGTCCTGCATGAATAAAGA-3′ and 5′-GTCCAGCTCGAGAGCGTT-3′ for PPARα and 5′-TCCGTAAGGATCTGTATGCC-3′ and 5′-GATCCAGACGGAGTATTTGC-3′ for β-actin. β-Actin is most suitable for normalization of gene expression in fish. These sets of primers were tested with a standard Corbett PCR machine (45 cycles of 30 s at 94°C, 45 s at 55°C, and 45 s at 72°C) and analyzed on a DNA chip 1000 Bioanalyzer (Agilent Technologies) prior to the start with quantitative PCR analysis. Bands were detected at 158 base pairs (β-actin) and 150 base pairs (PPARα). No nonspecific amplifications occurred. All bands were sequenced for confirmation.
After reverse transcription, the resulting cDNA samples were diluted for real-time analysis. A standard dilution sequence (1:20 to 1:5,120, with a factor of 4 of separation between each step) as well as a 1:320 dilution of each sample were prepared for both β-actin (housekeeping gene for normalization) and PPARα. The fluorescent dye FastStart SYBR Green (Roche Diagnostics) was chosen for the real-time procedure. The RT-PCR program was performed with 50 cycles and an annealing temperature of 58°C for 45 s. The total reaction volume was 20 μl, and the concentrations of the primers were 0.3 μM. At the end, a melting procedure for quality analysis was also performed between 50 and 95°C, with 1°C per 5 s. All real-time reactions were performed on a Corbett RotorGene TM 6000 (Corbett Life Sciences).
FAO activity. The FAO activity was measured according to the method of Small et al. , with some minor modifications [17,26]. Livers were thawed on ice, weighed on an analytical balance, and placed in 100 μl of ice-cold buffer (0.6 M Trizma base, 0.25 M sucrose, and 0.01% Triton X-100) and a protease inhibitor cocktail (F. Hoffmann-La Roche). Livers were homogenized with an ultrasonic homogenizer (Labsonic), and supernatants were collected after centrifugation at 600 g for 20 min. Before the start of the reaction, protein concentration was measured with a Bio-Rad protein assay. Briefly, liver samples were prepared at a protein concentration of 400 μml in a dilution buffer (phosphate-buffered saline at pH 7.4 with 0.02% Tween [Sigma-Aldrich] and 0.6 mg/ml bovine serum albumin) and added in triplicate on a microtiter plate. Forty microliters of reagent mixture (12 units/ml horseradish peroxidase, 0.05 mM 2′,7′-dichlorodifluorescein diacetate, 0.015 mM flavin adenine dinucleotide, and 40 mM aminotriazole; all from Sigma-Aldrich) were added to each sample and incubated in the dark for 5 min prior to measurement at 502 nm. Ten microliters of palmitoyl CoA (Sigma-Aldrich) at a final concentration of 30 μM were then added to start the reaction, and the kinetics of the enzyme (product formation) were monitored for 1 h (13 cycles of 5 min) at 502 nm using a spectrophotometric microtiter plate reader (Infinite M200; Tecan). A serial dilution of the dichlorofluorescein standard was used to quantify the amount of dichlorofluorescein produced from dichlofluorescein diacetate during the reaction. The FAO activity is expressed as nanomolar dichloro-fluorescein per minute per milligram of protein.
Table Table 2.. Measured exposure concentrations (mean ± standard deviation of 0 and 48 h samples) in experiments
Experiment A (bezafibrate)
Experiment B (bezafibrate)
Experiment C (clofibric acid)
Nominal concentration (mg/L)
Measured concentration (mg/L)
Nominal concentration (mg/L)
Measured concentration (mg/L)
Nominal concentration (mg/L)
Measured concentration (mg/L)
a Sample size is in brackets. NA = not applicable.
0.1 ± 0.01 (13)
1.27 ± 0.01 (16)
1.07 ± 0.02 (16)
10.18 ± 1.54 (12)
10.75 ± 0.09 (15)
101.56 ± 6.57 (14)
106.81 ± 2.85 (8)
108.91 ± 2.34 (18)
Vitellogenin. Vitellogenin analysis was performed with a commercial enzyme-linked immunosorbent assay kit (Biosense Laboratories) in experiment A following the manufacturer's instructions. The kits were provided with monoclonal antibodies against vitellogenin, and a sandwich enzyme-linked immunosorbent assay analysis was performed. Briefly, purified vitellogenin supplied in the kits was freshly prepared each day as a standard for vitellogenin quantification. Fish plasma samples were first diluted with sample buffer delivered with the commercial kit to achieve a 1:2 ratio of plasma to buffer. Further dilution steps were 1:5,000, 1:500,000, and 1:5,000,000 for female plasma and 1:50, 1:5,000, and 1:500,000 for male plasma. Measurements were performed in duplicate, and color was measured at 450 nm in a spectrophotometric microtiter plate reader (Tecan, Infinite M200). Fish from water control, positive control, and 100 mg/L BF dose were analyzed.
An analysis of variance test (ANOVA) with post-hoc Dunnett's t test was performed to determine the effects of the lipid-lowering agents BF and CA on the investigated parameter. The level of statistical significance was p < 0.05.
Chemical analysis confirmed that in all experiments concentrations of BF and CA were close to nominal values. The lipid-lowering agents were stable; the maximal decrease during exposure periods of 48 h was only 1.5%. We therefore give the mean of 0 and 48 h measurements in Table 2. Data for 0 and 48 h are given in Supporting Information Table S1 (http://dx.doi.org/10.1897/09–087.S1). To take the concentration variability into account, mean levels were taken as exposure concentrations in each dose group. Water of control was not contaminated by lipid-lowering agents.
Survival, weight, and length
Bezafibrate and CA did not interfere with survival, weight, or length of fathead minnows. In Table 3, the effects on condition factors (CF = 105 × W/L3, where W is the weight of the fish expressed in grams and L is the length expressed in millimeters) are summarized. No effects were observed, except in males exposed to 10.75 mg/L CA. Weight and length of the fish are given in Supporting Information Table S2 (http://dx.doi.org/10.1897/09–087.S1).
PPARα expression. In experiments B and C, PPARα expression was measured in the livers of fish and normalized for RNA concentration and β-actin, which is the most suitable gene for normalization. Bezafibrate exposure did not significantly alter PPARα expression at 106.7 mg/L BF in males and females (Fig. 1a). Similar to BF, exposure to CA did not result in a significant induction of PPARα expression (Fig. 1b).
Table Table 3.. Condition factors of fish in experiments A, B, and C (mean ± standard deviation)
a Significant difference to controls (p < 0.05). BF = bezafibrate; CA = clofibric acid; DBP = dibutyl phthalate (positive control).
1.073 ± 0.096
1.142 ± 0.226
DBP (0.5 mg/L)
1.035 ± 0.040
1.114 ± 0.155
BF (0.1 mg/L)
0.970 ± 0.027
1.077 ± 0.127
BF (1.27 mg/L)
1.017 ± 0.183
1.148 ± 0.138
BF (10.18 mg/L)
0.944 ± 0.043
1.054 ± 0.115
BF (101.56 mg/L)
0.953 ± 0.086
1.030 ± 0.150
0.984 ± 0.004
1.021 ± 0.024
BF (106.7 mg/L)
1.016 ± 0.080
0.995 ± 0.005
1.164 ± 0.062
1.172 ± 0.084
CA (1.07 mg/L)
1.128 ± 0.025
1.104 ± 0.007
CA (10.75 mg/L)
1.232 ± 0.029a
1.092 ± 0.072
CA (108.91 mg/L)
1.150 ± 0.027
1.091 ± 0.050
The FAO activity was measured in experiments A (male fish) and C (male and female fish). Fatty acyl-CoA oxidase activity was not significantly affected by BF after 14 d of exposure (Fig. 2a). In contrast, exposure to 108.91 mg/L CA for 21 d led to a significant increase in FAO activity in male (p < 0.05) but not female fish (Fig. 2b). The 1.5-fold increase was significant at 108.91 mg/L CA as compared with both control and 10.75 mg/L CA (p < 0.05).
Plasma vitellogenin. Vitellogenin (VTG) concentration in the blood of male and female fish was analyzed in experiments A and C. No alteration in VTG was observed for BF (Fig. 3a and b), and CA (Fig. 4a and b) in both males and females, suggesting that BF and CA do not have estrogenic activity in fathead minnows at the concentrations evaluated. Similar to zebrafish, no induction of VTG occurred by 0.5 mg/L DBP .
Egg production. Mating could take place in adult male and female fish, although egg production was very low and sporadic, probably due to the age of fish. In experiment A, fish exposed to 101.56 mg/L BF produced eggs only twice (data not shown). Fish of all groups in experiment B did not produce enough eggs to perform a statistical analysis. However, sporadic egg production was observed in all groups (data not shown). Only in experiment C was egg production higher, allowing a comparison between exposure groups. Fecundity apparently decreased in fish exposed to 1.07 and 10.75 mg/L CA, but reduction was statistically significant only at 108.91 mg/L CA. Data in Supporting Information Table S3 (http://dx.doi.org/10.1897/09–087.Sl) show the averages of eggs laid per day and last day of egg laying. Egg production took place in only two replicates at low and medium doses, whereas all replicates showed egg production during the 21 d in control fish. However, the very low egg production does not allow conclusions to be drawn about the effects of lipid-lowering agents on reproduction.
The main objective of the present study was to determine the effects of waterborne BF and CA exposures in fathead minnows by focusing on the modes of action of these compounds, the lipid metabolism in the liver. As peroxisome proliferators and PPARα ligands, these compounds were expected to affect PPARα mRNA levels and its target gene FAO and to cause peroxisome proliferation in the liver. Our study indicates that BF and CA had no effect on PPARα mRNA levels in the liver of fathead minnows after 14 and 21 d of exposure. Up to high concentrations, BF did not affect lipid metabolism, as determined by PPARα and FAO induction. In contrast, a 1.5-fold increase in FAO activity was found at 108.91 mg/L CA.
Hypolipidemic fibrate drugs bind preferentially to PPARα with weak binding to PPARβ and PPARγ . Because little is known about this binding in fish, we investigated the interaction of BF and CA with PPARα and its target gene FAO in fathead minnows. Exposure to CA increased FAO activity, with a significant induction at the highest concentration. Induction of FAO was apparently paralleled by an increase in PPARα mRNA level in the present study. In contrast to this PPARα-regulated enzyme, induction of PPARα was not significant. This is probably because of the transient nature of the induction of this receptor. Similar to the present study, an increase in FAO activity was observed in salmon (Salmo salar) hepatocytes exposed to BF and CA, and this was not paralleled by an increase in PPARγ mRNA level . In zebrafish primary hepatocytes, clofibrate led to induced PPARα and PPARγ determined immunohistochemically . Also similar to the present study, no induction of PPARα and PPARγ was observed in male goldfish exposed to 1.5 mg/L gemfibrozil for 14 and 28 d, but a significant reduction of hepatic PPARβ mRNA levels was observed . As with BF in the present study, FAO activity was not affected in goldfish exposed to gemfibrozil . An increase in FAO activity was observed in rainbow trout  but only when data were analyzed in nanomoles of dichlorofluorescein per milligram of liver (we found normalization to protein concentration to be more reliable). The activities found in the present study in fathead minnows were generally lower.
We selected DBP as a positive control for FAO activity based on results of a previous study in zebrafish, where an increase in FAO activity, but not VTG, was observed at 0.5 mg/L . However, we observed no significant changes at the same exposure concentration in any of the end points measured in fathead minnows. This may be explained by species differences, but potentially also by exposure concentrations that were too low to induce FAO or PPARα. The high lipophilicity of DBP may have resulted in considerable sorption to surfaces of aquaria and fish. Because we did not determine DBP in aquaria water, real exposure concentration may have been too low to elicit an effect. Moreover, fathead minnows may be less sensitive to this compound than zebrafish.
We also analyzed the fecundity in experiment C of the present study. However, the very low reproduction in all experiments did not allow us to reach any conclusions regarding reproductive effects. The lack of egg production in experiment A is likely due to the senescence of fish (they were 2 years old and previously used for breeding), but the reason for very low egg production in experiment B is unclear. In experiment C, egg production took place, although at a very low frequency. Exposure to CA seemed to decrease fecundity at the highest concentration (Supporting Information, Table S3), which may suggest an indirect effect on fecundity, although at very high concentrations.
Runnalls et al.  reported that CA affected the reproductive axis of fathead minnows. Spermatogenesis was impaired after exposure of 21 d at 1 mg/L CA, leading to a marked reduction in sperm counts. At lower concentrations of 0.001 and 0.01 mg/L, the occurrence of nonviable sperm counts increased. These effects were observed at much lower concentrations than effects on FAO activity (and potentially on fecundity), which occurred at 108.91 mg/L in the present study. Similar to the present study, where effects on lipid metabolism occurred only at very high concentrations, effects of CA on lipoprotein metabolism in fathead minnows were very minor in the study of Runnalls et al. . The surprising effects on spermatogenesis  suggest that CA may adversely affect androgen synthesis. This was reported by another fibrate, gemfibrozil, that led to reduced plasma testosterone concentrations . In our experiments, we did not analyze plasma testosterone concentrations but analyzed for potential estrogenic activity. However, we did not detect any estrogenicity of either BF and CA, because no induction of VTG (biomarker for estrogenicity) occurred.
Potential effects on fecundity as suggested by CA in the present study and demonstrated effects on reproduction  should further be investigated. Moreover, a decrease in plasma testosterone was reported after exposure of fish to gemfibrozil . Forthcoming studies should show whether fecundity and reproduction are affected by fibrates directly or by indirect pathways resulting from alteration of lipid metabolism.
The effects of CA on FAO activity, and potentially on fecundity, in fathead minnows occurred at concentrations several orders of magnitude higher than the maximal reported levels in treated wastewater. Up to 4.6 μL BF and 1.6 μL CA were reported in wastewater treatment plant effluents in Germany [5–7], whereas the effect level of CA in the present study was 108.91 mg/L. However, this does not rule out the possibility that other unexpected adverse effects, including reproductive effects may occur, because reduced sperm counts were found at 0.01 and 1 mg/L CA . In conclusion, the present study shows only minor effects of CA at high concentrations on lipid metabolism.
Table S1. Measured exposure concentrations (mean ± standard deviation) in experiments.
Table S2. Condition factors, weights, and lengths of fish in experiments A, B, and C (mean ± standard deviation).
Table S3. Egg production (daily mean ± standard deviation, n = 12) and last day of egg laying of fish exposed to clofibric acid in experiment C.
All found at DOI: 10.1897/09–087.S1 (16 KB PDF).
We thank Andreas Hartmann and Birgit Hoeger (Novartis Pharma AG, Basel, Switzerland) and Jürg O. Straub (F. Hoffmann-La Roche, Basel, Switzerland) for providing pharmaceuticals and reading the manuscript. The present study was funded by the Swiss Bundesamt für Berufsbildung und Technologie, Kommission für Technologie und Innovation (KTI-Project 7114.2 LSPP-LS, to K. Fent), Novartis International AG and Novartis Pharma AG, F. Hoffmann-La Roche Ltd., and Springborn Smithers Laboratories Europe.