Environ. Sci. Technol. 2010, 44, 7213–7219
Metabolites of the Aquatic Pollutant Diclofenac in Fish Bile JENNY-MARIA KALLIO,† MARJA LAHTI,‡ A I M O O I K A R I , ‡ A N D L E I F K R O N B E R G * ,† Åbo Akademi University. Laboratory of Organic chemistry, Biskopsgatan 8, FI-20500 Åbo, Finland, and University of Jyva¨skyla¨, Department of Biological and Environmental Sciences, FI-40014 University of Jyva¨skyla¨, Finland
Received November 10, 2009. Revised manuscript received March 1, 2010. Accepted March 10, 2010.
The uptake and metabolism of anti-inflammatory drug diclofenac (DCF) was studied by exposing rainbow trout (Oncorhynchus mykiss) to DCF intraperitoneally, and via water at concentration of 1.7 µg L-1. The bile was collected and the formed metabolites were identified. The identification was based on the exact mass determinations by a time-of-flight mass analyzer and on the studies of fragments and fragmentation patterns of precursor ions by an ion trap mass analyzer. The main metabolites found were acyl glucuronides of hydroxylated DCFs. In addition, one ether glucuronide of hydroxylated DCF was found. Also, unmetabolized DCF was detected in the bile. The total bioconcentration factors (BCFtotal-bile for DCF and its metabolites) in rainbow trout bile, varied between individuals and was roughly estimated to range from 320 to 950. These findings suggest that fish living downstream the wastewater treatment plants (WWTPs) and which are chronically exposed to the drug may accumulate the drug and its metabolites in the bile.
Introduction The anti-inflammatory drug diclofenac (DCF) is one of the most widely used pharmaceutical worldwide. The drug and the metabolites formed in the human body enter the wastewater distribution systems and are transported to wastewater treatment plants (WWTPs). In WWTPs, DCF and the metabolites are rather poorly eliminated and consequently the compounds will end up in the recipient waters (1-7). Although DCF is detected at low concentrations (normally from ng to µg L-1 levels) in recipient waters, the pharmaceutical may possess risks for the aquatic ecosystems. Since DCF (8, 9) has been shown to have a rather low acute lethality or toxicity, the possible risks possessed are mainly attributed to the steady input to the recipient waters, that is, DCF can be characterized as being a pseudo persistent compound. This results in a chronic and even life-long exposure of aquatic organisms to DCF. Long-term (28 days) aquaria exposures have shown that DCF induces cytological alterations in rainbow trout at concentrations of a few µg L-1 (10-12). At exposure concentration of 0.5 µg L-1 for 21 days, subtle effects have been found in the liver of brown trout (13). Rainbow trout have been shown to take up DCF in tissues when exposed to a concentration of 1 µg L-1 of DCF (10). * Corresponding author phone: +358 2 215 4138; fax: +358 2 215 4866; e-mail:
[email protected]. † Åbo Akademi University. ‡ University of Jyva¨skyla¨. 10.1021/es903402c
2010 American Chemical Society
Published on Web 04/09/2010
Brown et al. (14) noticed that rainbow trout accumulates DCF in the blood plasma when exposed to the compound through sewage treatment plant effluents. On the basis of reported human therapeutic concentrations and on the assumption that the DCF acts on the same targets in fish and humans, it was calculated that DCF may present a higher risk for fish than for example ibuprofen or naproxen (14). In mammals, the primary phase I metabolites of DCF are 4′-hydroxydiclofenac (4′-OH-DCF) and 5-hydroxydiclofenac (5-OH-DCF), whereas the main phase II metabolites are the acyl glucuronides of the parent compound and of the phase I metabolites (15-18). Also sulfate conjugates of OH-DCFs have been detected (19). To our knowledge, the hepatobiliary metabolism of DCF has not been studied in fish this far. Generally a lower capacity of biotransformation may be expected for fish species, and the biotransformation pathway may vary from species to species. The current work is concerned with the determination of the identity of the major biliary metabolites of DCF in rainbow trout and with a study of the uptake of DCF in fish exposed to the drug in water at concentrations reported to be found in the environment. Comparative knowledge on the uptake and metabolism of the key pharmaceuticals entering the environment through wastewater discharges is important for ecotoxicological risk assessments of the pharmaceuticals (20).
Material and Methods Chemicals. Diclofenac (purity >99%; CAS 15307-79-6), type B-1 β-glucuronidase from bovine liver (CAS 9001-45-0) and tricaine methane sulfonate (MS222, CAS 886-86-2) were purchased from Sigma-Aldrich (Steinheim, Germany). 4′hydroxydiclofenac, 4′-OH-DCF (purity 98%; CAS 64118-84-9), 5-hydroxydiclofenac, 5-OH-DCF (purity 98%; CAS 69002-84-2), and 1-β-O-acyl glucuronide of diclofenac (purity 98%; CAS 64118-81-6) were purchased from Toronto Research Chemicals Inc. (North York, Canada). The internal standards racibuprofen-d3 (purity g98.0%, 99 atom % D; CAS 121662-14-4) and fenoprop (purity 99.0%; CAS 93-72-1) were obtained from Fluka (Buchs, Switcherland) and Riedel-de Hae¨n (Seelze, Germany), respectively. The water used in the LC-MS analysis was purified using a Millipore Simplicity 185 system (Millipore S.A.S., Molsheim, France). Fish Experiments. Rainbow trout (Oncorhynchus mykiss, 1- and 1.5-year-old) were purchased from the hatchery of Savon-Taimen Oy (Rautalampi, Finland, 1-year-old) and from the Finnish Game and Fisheries Research Institute (Laukaa, Finland, 1.5-year-old). Before the experiments, fish were acclimatized to laboratory conditions in continuously changing nonchlorinated artesian well water (pH 7.5 ( 0.2, temperature 13.5 ( 0.5 °C, and 8.6 ( 0.6 °C, respectively, for 1- and 1.5-year-old-fish) for two weeks. Fish were fed every other day ad libitum (ca. 0.5% of fish biomass, Vital Plus 3.5 mm, Rehu Raisio, Raisio, Finland). Feeding was stopped three days before the experiment. Four 1.5-year-old rainbow trout (average weight 249 ( 46 g) were anesthetized with MS222 (100 mg L-1), and subsequently 0.25 mg diclofenac/100 g fish biomass (2.5 mg mL-1 dissolved in a 1:1 ethanol/0.7% NaCl-solution, injection volume 100 µL/100 g fish biomass) was intraperitoneally (i.p.) injected to abdominal cavity of the fish. Four control fish were injected with 100 µL of a 1:1 ethanol/0.7% NaCl-solution per 100 g fish biomass. After injection, the fish were allowed to recover and after 24 h the injections were repeated. Water temperature in the aquarium (490 L) was kept at 8.5 ( 0.5 °C and the oxygen concentration was over 8.5 ppm. The VOL. 44, NO. 19, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Retention Times and the Multiple Reaction Monitoring (MRM) Parameters Applied in the Analyses by the QqQ Mass Analyzera name standards
analytes
a
diclofenac (1) 4′-hydroxydiclofenac (2) 5-hydroxydiclofenac (3) acyl glucuronide of diclofenac (7) d3-ibuprofen sulfate conjugate of 5-hydroxydiclofenac (5) acyl glucuronides of hydroxydiclofenacs (8, 9, 10) ether glucuronide of 4′-hydroxydiclofenac (11)
cone voltage (V)
collision energy (eV)
294 310 310 470 208
250 266b, 230 266b, 230 175b 164
22 22 22 22 17
14 14 14 10 8
11.3
390
310b, 266
22
14
6.4, 7.9, 13.1-15.1
486
193, 175b
22
10
22
10
9.4
Numbers in brackets refer to structures in Figure 3.
9
product ion (m/z)
17.2 14.7 15.6 15.9, 16.9 17.5
486 b
b
442, 193, 175
Product ion used for the quantification.
pH of the water was measured daily and found to be 7.8 ( 0.1. After two days, the fish were immobilized and the bile samples were collected, frozen in liquid nitrogen and stored at -80 °C prior to analysis. For the study of the uptake of DCF at environmentally realistic levels, four 1-year-old rainbow trout were exposed to the drug at a concentration of 1.7 µg L-1 for 10 days. The weights of the trout were 169 ( 33 g, and lengths 24.3 ( 1.0 cm. A flow-through system was used, where half of the water was changed daily with the aid of a pumping system. Water temperature was 13.7 ( 0.2 °C and its oxygen saturation was over 90% (9.8 ( 0.3 mg L-1). The pH of the water was measured daily and found to be 7.7 ( 0.2 throughout the study. Four control fish were kept under similar water conditions as fish exposed to DCF. Following the exposure, the bile samples were collected from immobilized fish, frozen in liquid nitrogen, and stored at -80 °C prior to analysis. Work-Up Procedure for the Bile Samples and the Aquarium Water Samples. Bile samples (50 µL) from the i.p. experiments were diluted with 500 µL of 5% acetonitrile in water, and the solutions were analyzed by LC-MS (ion trap mass analyzer and time-of-flight mass analyzer). A volume of 20-100 µL of the bile of fish exposed to the DCF at environmentally relevant conditions in aquarium was diluted to 1 mL with water at pH 2.5 and 100 ng of rac-ibuprofen-d3 (d3-IBF) was added as an internal standard. Solid phase extraction (SPE) applying Oasis HLB 1 cc (30 mg) cartridges (Waters, Milford, MA) was used for the isolation and purification of the metabolites. The cartridges were preconditioned with 1 mL of methanol and 1 mL of water at pH 2.5. Following the sample loading, cartridges were washed with 1 mL of 2% formic acid, and the material of interest was eluted with 2 × 0.5 mL of 2% ammonium hydroxide in 60% methanol. The extracts were evaporated to dryness under a stream of nitrogen, redissolved in 200 µL of 5% ACN in 0.01 M ammonium acetate and analyzed by LC-MS (triplequadruple mass analyzer). Water samples, 30 mL, were taken once per day from the aquarium. Subsequently to the addition of the internal standard (53 ng of fenoprop) and adjustment of pH to 2, the water samples were passed through Oasis HLB 3 cm3 (60 mg) cartridges (Waters, Milford, MA). Cartridges were preconditioned with 2 mL of hexane, 2 mL of acetone, 3 mL of methanol, and 3 mL of water at pH 2. Samples were eluted from the cartridges with 4 × 0.5 mL of methanol. The extracts were evaporated to dryness under a stream of nitrogen, redissolved in 500 µL of 30% acetonitrile in 0.01 M ammonium acetate and analyzed by LC-MS (triple-quadruple mass analyzer). Enzymatic Hydrolysis of 1-β-O-Acyl Glucuronides. Enzymatic hydrolysis of 1-β-O-acyl glucuronides was adapted 7214
precursor ion (m/z)
tR (min)
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from Aresta et al. (21). Fifty µL of the hydrolysate was diluted with 250 µL of 5% acetonitrile in water and analyzed by LCMS (ion trap mass analyzer). Samples were analyzed before and after incubation. Chromatographic Methods. In the work concerned with the identification of metabolites (the i.p. bile samples), the chromatographic separations were performed on a Zorbax Eclipse C18 analytical column (5 µm, 2.1 × 50 mm column) to which a Zorbax Eclipse C18 narrow-bore guard column (5 µm, 2.1 × 12.5 mm) was connected (Agilent Technologies, Palo Alto, CA). The chromatographic separations of the SPE extracts (bile and water) were performed on an XBridge C18 column (3.5 µm, 2.1 × 50 mm) equipped with a guard column of the same brand (3.5 µm, 2.1 × 10 mm; Waters, Milford, MA). Both columns were eluted with a mobile phase consisting of 0.01 M ammonium acetate (A) and 0.01 M ammonium acetate in 90% acetonitrile (B) and at a flow rate of 300 µL min-1. The gradient used for the separation of the biliary metabolites started with 5% of B (0-1 min) and ended at 33% of B (1-15.5 min), while the gradient applied for the water samples started with 30% of B, and ended at 70% after 16.5 min. The injection volume varied from 10-30 µL. LC-MS Methods. The exact mass determinations were performed on a Bruker electrospray ionization quadrupoletime-of-flight mass analyzer (Q-ToF) (micrOTOF-Q, Bruker Daltonics, Bremen, Germany) operating at a resolution of 10 000. Nitrogen was used as a nebulizing gas (1.6 bar) and as a drying gas (9.0 L min-1, 200 °C). Capillary voltage was set to +4.5 kV. The analytes were transferred to the mass analyzer by an Agilent 1200 Series LC system consisting of a binary pump, a vacuum degasser, an autosampler, a thermostatted column (30 °C), and an UV detector (set at 275 nm) (Agilent Technologies, Palo Alto, CA). The data was collected and handled with the Bruker Compass DataAnalysis 4.0 software. For structural elucidation of analytes an Agilent 1100 Series LC/MSD Trap SL was used (an ion trap mass analyzer, IT). The system consisted of a binary pump, a vacuum degasser, an autosampler, a thermostatted column (30 °C), an UV detector (set at 275 nm), electrospray ionization source, and Agilent ChemStation data system (Agilent Technologies, Palo Alto, CA). Nitrogen was used both as a nebulizing gas (40 psi) and as a drying gas (12 L min-1, 325 °C). The capillary exit offset was set at -128.5 V, skim 1 voltage had the value of -40 V. The maximum ion accumulation time was 20 ms and the target value was 20000. Collision induced dissociation experiments coupled with multiple tandem mass spectrometry (MSn) employed helium as collision gas. The fragmentation amplitude was set at 1.0 V. A Quattro Micro triple-quadrupole mass analyzer (QqQ) equipped with electrospray ionization source (Waters, Mil-
FIGURE 2. LC-Q-ToF extracted ion chromatograms (EIC) of exact masses of bile samples from rainbow trout exposed to diclofenac by intraperitoneal injection. The chromatographic peaks represent diclofenac (1) and two geometric isomers of the acyl glucuronide of diclofenac (7a and 7b).
FIGURE 1. LC-UV and LC-IT extracted ion chromatograms (EIC) of bile samples from rainbow trout exposed to diclofenac by intraperitoneal injection. The numbers above the peaks corresponds to the structures of the metabolites depicted in Figure 3. ford, MA) was used for the quantification and the detection of trace amounts of the analytes in water and in the bile samples. The mass analyzer was operated in the multiple reaction monitoring mode (MRM) with a dwell time of 0.2 s and interchannel delay of 0.05 s. Nitrogen was used as a nebulizing gas (30 L h-1) and as desolvation gas (630 L h-1, 325 °C), and argon was used as the collision gas with a collision cell pressure 4.7 × 10-3 mbar. The source block temperature was 120 °C. The instrument was hyphenated with a Waters Alliance 2795 LC consisting of a tertiary pump, a vacuum degasser, an autosampler and a thermostatted column (30 °C). The MassLynx 4.0 sofware was used for data collection and handling. The cone voltages, the collision energies, the precursor and product ions (Table 1) were optimized by direct infusion of the pure standard compounds DCF, 4′-OH-DCF, 5-OH-DCF, acyl glucuronide of DCF, and the internal standard, d3-IBF. The cone voltages and collision energies applied for DCF were also used for the multiple reaction monitoring (MRM) mode transitions of the sulfate conjugates
FIGURE 3. Main identified metabolites of diclofenac (1): 4′-hydroxydiclofenac (2), 5-hydroxydiclofenac (3), sulfate conjugate of 4′hydroxydiclofenac (4), sulfate conjugate of 5-hydroxydiclofenac (5), sulfate conjugate of 4′,5-dihydroxydiclofenac (6), acyl glucuronide of diclofenac (7), acyl glucuronide of 4′-hydroxydiclofenac (8), acyl glucuronide of 5-hydroxydiclofenac (9), acyl glucuronide of 3′hydroxydiclofenac (10), and ether glucuronide of 4′-hydroxydiclofenac (11). Only the biosynthetic 1-β-O-acyl glucuronide isomers are depicted in the figure. However, due to acyl migration several chromatographic peaks or peak broadening were observed for the acyl glucuronides. and the glucuronides of OH-DCFs. Calibration curves were prepared in blank bile matrix by the use of the standard VOL. 44, NO. 19, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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compounds. The acyl glucuronide of DCF was used as standard for the quantification of all the DCF glucuronides and 5-OH-DCF for the sulfate conjugate of 5-OH-DCF.
Results and Discussion Fish were exposed intraperitoneally and via aquarium water to DCF. The purpose of the intraperitoneal exposure was to produce metabolites in high enough amounts to enable their structural elucidation with mass spectrometric methods, whereas the objective of the aquarium experiment with was to find out whether fish may accumulate the drugs at conditions that may prevail downstream wastewater treatment plants. Identification of DCF Metabolites. LC-UV, LC-IT, and LC-Q-ToF analyses of the bile of rainbow trout exposed to DCF through injection to the abdominal cavity showed the occurrence of compounds that represented DCF metabolites and unmetabolized DCF. Extracted negative ion chromatograms collected by the IT mass analyzer (Figure 1) showed the occurrence of two peaks with masses corresponding to OH-DCFs (peaks 2 and 3, m/z 310, retention times 15.4 and 16.1 min, respectively), two peaks with the masses corresponding to the sulfate conjugates of OH-DCFs (peaks 4 and 5, m/z 390, retention times 11.6 and 12.0 min, respectively), one peak with a mass corresponding to the sulfate conjugate of di-OH-DCF (peak 6, m/z 406, retention time 12.7 min) and several peaks of masses attributable to acyl and ether glucuronides of OH-DCFs (peaks 8, 9, 10, and 11, m/z 486, retention times 5.1, 8.3, 15.4-16.5, and 9.9 min). The peaks of unmetabolized DCF (peak 1, m/z 294, retention time 18.7 min) and of two geometric isomers of acyl glucuronides of DCF (peaks 7a and 7b, m/z 470, retention times 15.6 and 16.0 min) were observed only in extracted ion chromatograms of exact masses provided by the Q-ToF mass analyzer due to coelution with constituents of the bile (bile acids) (Figure 2). The structures of the metabolites and the parent compound are depicted in Figure 3. DCF or its metabolites were not detected from any control fish. Further proof for the structural identification of the metabolites were obtained by the determination of the exact masses of the compounds using the Q-ToF mass analyzer and from the fragmentation pattern observed in the spectra
recorded by the IT mass analyzer (Table 2). The errors of the exact mass measurements were less than ( 1.9 ppm for all of the compounds. Upon fragmentation, the acyl glucuronides (8, 9, and 10) lost initially 176 mass units [M-Hanhydroglucuronic acid]-, which was followed by a loss of 44 mass units [M-H-anhydroglucuronic acid-CO2](Scheme 1). On the other hand, the initial loss from the ether glucuronide (11) was 44 mass units and only subsequent to this loss the glucuronide unit was split off. The sulfate conjugates (4, 5, and 6) showed a characteristic loss of 80 mass units, that is [M-H-SO3]-. These fragmentation patterns are in accordance with those commonly reported for phase II metabolites (22). The phase I metabolites 4′-OH-DCF (2) and 5-OH-DCF (3) were identified by comparing the retention times and mass spectral data with authentic reference compounds. It was possible to distinguish between 4′-OH-DCF and 5-OHDCF by observing the fragments generated in the MS2 spectra. Both compounds readily produced a fragment ion at m/z 266 due to the loss of CO2 (m/z 310 f m/z 266), but 4′OH-DCF produced additionally intense ions at m/z 230 and 194 through loss of CO2 + HCl and CO2 + 2 × HCl, respectively (Figure 4, A1 and A2). These differences in fragmentation patterns were also observed in the MS3 spectra for the compounds representing peaks 8 and 9 (Figure 4, C1-D1, and C2-D2, respectively), and on the basis of these observations, it was suggested that 8 represented the acyl glucuronide of 4′-OH-DCF and 9 represented the acyl glucuronide of 5-OH-DCF. The ether glucuronide (11) showed a similar fragmentation pattern as 4′-OH-DCF (Figure 4, E1) and consequently was supposed to be the phase II metabolite of this compound. The major sulfate conjugate 5 showed only a weak fragment ion at m/z 230 (Figure 4, E2) and the metabolite was proposed to be the sulfate conjugate of 5-OH-DCF. Since 3′-OH-DCF is the phase I metabolite reported in the literature to be formed in lowest amounts of different isomers of OH-DCFs (e.g., ref 15), it was supposed that the peaks marked 10 in Figure 1 represent the acyl glucuronides of 3′-OH-DCF. In addition, peak 6 is probably the mono sulfate conjugate of 4′,5-dihydroxydiclofenac, but the position of the sulfate group could not be established.
TABLE 2. Identified Diclofenac Metabolites, Accurate Mass Data Recorded by the Q-ToF Mass Analyzer and Major Fragment Ions Observed in the Spectra Recorded by the IT Mass Analyzer cmpd nra 8
acyl glucuronide of 4′-hydroxydiclofenac acyl glucuronide of 5-hydroxydiclofenac ether glucuronide of 4′-hydroxydiclofenac sulfate conjugate of 4′-hydroxydiclofenac sulfate conjugate of 5-hydroxydiclofenac monosulfate conjugate of dihydroxydiclofenac 4′-hydroxydiclofenac acyl-migrated isomers of acyl glucuronide of 3′-hydroxydiclofenac acyl-migrated isomers of acyl glucuronide of diclofenac 5-hydroxydiclofenac diclofenac
9 11 4 5 6 2 10 7 3 1 a
7216
name
tR (min)
error (ppm)
mSigma value
Formula [M-H]-
486.0364
486.0361
0.8
3.1
8.3
486.0364
486.0362
0.4
2.6
9.9
486.0364
486.0356
1.7
4.2
11.6
389.9611
389.9609
0.6
11.4
310, 194, C20H18Cl2NO9 310, 193, C20H18Cl2NO9 442, 230, C14H10Cl2NO6S 310
12.0
389.9611
389.9611
0.1
11.8
C14H10Cl2NO6S 310, 266, 230
12.7
405.9561
405.9557
1.0
37.7
C14H10Cl2NO7S 370, 326, 246
15.4 310.0043 15.4-16.5 486.0364
310.0048 486.0355
-1.7 1.9
15.6, 16.0
470.0415
470.0418, 470.0424
-0.7, -1.9 15.0, 12.6 C20H18Cl2NO8
294, 250
16.1 18.7
310.0043 294.0094
310.0032 294.0095
C14H10Cl2NO3 C14H10Cl2NO2
266, 230 250
b
-0.4
C20H18Cl2NO9
major ESIfragment ions, IT
5.1
The numbers refer to Figure 3.
9
[M-H]calculated [M-H]- exper.
b
92.9 120.3b
2.9
C14H10Cl2NO3 C20H18Cl2NO9
Could not be exactly determined due to low peak intensity.
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266, 230, 175 266, 230, 175 266, 175
266, 230, 194 310, 266, 175
SCHEME 1. Fragmentation Pattern of Deprotonated Acyl Glucuronide of 4′-Hydroxydiclofenac (8), Ether Glucuronide of 4′-Hydroxydiclofenac (11) and Sulfate Conjugate of 4′-Hydroxydiclofenac (4) as Observed in the Ion Trap Mass Analyzer
A further support for the presence of acyl glucuronides was obtained by incubating the bile samples with the enzyme β-glucuronidase which selectively cuts of the glucuronide unit from the conjugates. After β-glucuronidase incubation, LC-UV analyses showed that the area of the chromatographic peaks of the acyl glucuronide of 4′-OH-DCF (8) and acyl glucuronide of 5-OH-DCF (9) decreased, while the area of the peaks of 4′-OH-DCF (2) and 5-OH-DCF (3) increased. Also a small increase in peak intensity was observed for the peak of DCF. The slight broadening of the chromatographic peaks representing the acyl glucuronides of 4′-OH-DCF at 5.1 min (8) and of 5-OH-DCF at 8.3 min (9) were most likely due to the presence of several geometric glucuronide isomers, which had been formed through acyl migration (e.g., ref 23). The acyl-migrated isomers of 10 were presented by the partly separated peaks at tR between 15.4 and 16.5 min. Biliary Metabolites in Fish Exposed to Environmentally Relevant Concentrations of DCF. Occasionally, DCF has been found in surface waters at concentrations slightly above 1 µg L-1 (24, 25). To examine whether DCF is taken up and metabolized in the liver of the fish at these concentration levels, the compound was added to the aquarium to receive a nominal concentration of 1.9 µg L-1. The exact concentration of the drug in the water was determined daily and was found to be 1.76 ( 0.2 µg L-1. Due to the low amount of the biliary metabolites, the compounds were isolated and purified with a solid phase extraction procedure and analyzed by an LC hyphenated with a triple-quadrupole mass analyzer (LCMS/MS) in the MRM mode. It was found that metabolites and unmetabolized DCF were present in the bile, although large variations in up-take and metabolism were obvious between the individuals (Table 3 and Figure 5). These findings prove that DCF present in water at concentration reported to be found in the environment can be taken up by fish. The exact mechanism of the
FIGURE 4. MS/MS and MS3 mass spectra recorded by the IT mass analyzer of 4′-OH-DCFs (left column) and 5-OH-DCFs (right column). The numbers in spectra corresponds to the structures of the metabolites depicted in Figure 3. uptake is not known but, in fish a variety of anionic, cationic, and neutral transporters exists, which allow for the active and passive uptake of polar xenobiotics. By the use of authentic reference compounds it was possible to determine the concentration of DCF, 4′-OH-DCF and 5-OH-DCF in the bile. The acyl glucuronide of DCF was used as a surrogate reference compound for the quantification of the acyl glucuronides of DCF and of the OH-DCFs. Since no suitable sulfate conjugates were available as reference compounds, 5-OH-DCF had to be used as a standard for the quantification of sulfate conjugate of 5-OHDCF. Therefore the quantification of the sulfate conjugates should be considered as semiquantitative. The acyl glucuronides of the phase I hydroxylated metabolites of DCF were the main metabolites of DCF. The total concentration of unmetabolized DCF and its metabolites was estimated to vary from 570 to 1670 µg L-1 and on the basis of these figures, the bioconcentration factors (BCFtotal-bile, that is, the ratio between the total concentration of DCF plus its metabolites in the bile and the concentration of DCF in aquarium water) ranged from 320 to 950 (Table 3). Previously, bioconcentration factor of 5 for DCF has been reported in blood of fish exposed to sewage effluent, where the DCF concentration was found to be 2.32 µg L-1 (14). Because the bioconcentration in the bile (the parent compound and the metabolites) is much higher than VOL. 44, NO. 19, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 3. Diclofenac and Its Metabolites in Rainbow Trout Bile Exposed to 1.76 µg L-1 of Diclofenac for 10 days at 14 °Ca name
fish 1 µg L-1
fish 2 µg L-1
fish 3 µg L-1
fish 4 µg L-1
diclofenac (1) 4′-hydroxydiclofenac (2) 5-hydroxydiclofenac (3) sulfate conjugate of 5-hydroxydiclofenac (5) acyl glucuronide of diclofenac (7) acyl glucuronide of 4′-hydroxydiclofenac (8) acyl glucuronide of 5-hydroxydiclofenac (9) acyl glucuronide of 3′-hydroxydiclofenac (10) ether glucuronide of 4′-hydroxydiclofenac (11) total concentration in fish bile BCFtotal-bile
110 70 40 ∼50 220 ∼620 ∼400 ∼120 ∼40 ∼1670 ∼950
nd 50 nd ∼40 nd ∼520 ∼190 nd nd ∼800 ∼450
nd 30 10 ∼20 60 ∼310 ∼120 ∼60 ∼10 ∼620 ∼350
40 20 20 ∼20 40 ∼290 ∼90 ∼40 ∼10 ∼570 ∼320
a Total bioconcentration factors (BDFtotal-bile) were calculated as a ratio between the measured total concentration (parent compound and metabolites) in the bile and measured exposure concentration in water. The standard used for the determination of compounds 8, 9, 10, and 11 was 7, and the standard for compound 5 was 3. (nd ) not detected). Numbers in brackets refer to structures in Figure 3.
to the aquatic life is likely not attributable to a single compound, but rather to the mixture of compounds.
Acknowledgments This study was funded by Maj and Tor Nessling Foundation, the Finnish Graduate School in Environmental Science and Technology (EnSTe) and the Academy of Finland (No. 7109823). Antti Jylha¨ is acknowledged for his contribution in conducting the flow-though exposures.
Literature Cited
FIGURE 5. LC-QqQ MRM chromatogram of compounds in bile sample from rainbow trout (fish 3) exposed to 1.76 µg L-1 of diclofenac in the aquarium for 10 days. The numbers above the peaks corresponds to the structures of the metabolites depicted in Figure 3. in the blood, the analyses of biliary metabolites could be a useful way of monitoring fish exposure to DCF. This study shows that the pharmaceutical DCF, when present in µg L-1 levels in water, is taken up and metabolized by fish. These findings are in accordance with the findings showing that the drug causes cytological alterations in rainbow trout and brown trout at the same level of concentration (10-13). However, it has to been recognized that fish and other aquatic organisms living downstream of wastewater discharges are exposed to a huge number of pharmaceuticals and other chemicals and the possessed risk 7218
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(1) Halling-Sørensen, B.; Nors Nielsen, S.; Lanzky, P. F.; Ingerslev, F.; Holten Lu ¨ tzhøft, H. C.; Jørgensen, S. E. Occurrence, fate and effects of pharmaceutical substances in the environmentsA review. Chemosphere 1998, 36, 357–393. (2) Daughton, C. G.; Ternes, T. A. Pharmaceuticals and personal care products in the environment: agents of subtle change. Environ. Health Perspect. Suppl. 1999, 107, 907–938. (3) Lindqvist, N.; Tuhkanen, T.; Kronberg, L. Occurrence of acidic pharmaceuticals in raw and treated sewages and in receiving waters. Water Res. 2005, 39, 2219–2228. (4) Vieno, N. M.; Tuhkanen, T.; Kronberg, L. Seasonal variation in the occurrence of pharmaceuticals in effluents from a sewage treatment plant and in the recipient water. Environ. Sci. Technol. 2005, 39, 8220–8226. (5) MacLeod, S. L.; McClure, E. L.; Wong, C. S. Laboratory calibration and field development of the polar organic chemical integrative sampler for pharmaceuticals and personal care products in wastewater and surface water. Environ. Toxicol. Chem. 2007, 26, 2517–2529. (6) Perez, S.; Barcelo, D. First evidence for occurrence of hydroxylated human metabolites of diclofenac and aceclofenac in wastewater using QqLIT-MS and QqTOF-MS. Anal. Chem. 2008, 80, 8135–8145. (7) Stuelten, D.; Zuehlke, S.; Lamshoeft, M.; Spiteller, M. Occurrence of diclofenac and selected metabolites in sewage effluents. Sci. Total Environ. 2008, 405, 310–316. (8) Ferrari, B.; Paxeus, N.; Lo Giudice, R.; Pollio, A.; Garric, J. Ecotoxicological impact of pharmaceuticals found in treated wastewaters: study of carbamazepine, clofibric acid, and diclofenac. Ecotoxicol. Environ. Saf. 2003, 55, 359–370. (9) Haap, T.; Triebskorn, R.; Ko¨hler, H. Acute effects of diclofenac and DMSO to Daphnia magna: Immobilisation and hsp70induction. Chemosphere 2008, 73, 353–359. (10) Schwaiger, J.; Ferling, H.; Mallow, U.; Wintermayr, H.; Negele, R. D. Toxic effects of the non-steroidal anti-inflammatory drug diclofenac: Part I: histopathological alterations and bioaccumulation in rainbow trout. Aquat. Toxicol. 2004, 68, 141–150. (11) Triebskorn, R.; Casper, H.; Heyd, A.; Eikemper, R.; Ko¨hler, H.; Schwaiger, J. Toxic effects of the non-steroidal anti-inflammatory drug diclofenac: Part II. Cytological effects in liver, kidney, gills and intestine of rainbow trout (Oncorhynchus mykiss). Aquat. Toxicol. 2004, 68, 151–166. (12) Triebskorn, R.; Casper, H.; Scheil, V.; Schwaiger, J. Ultrastructural effects of pharmaceuticals (carbamazepine, clofibric acid, metoprolol, diclofenac) in rainbow trout (Oncorhynchus mykiss)
(13)
(14)
(15)
(16)
(17)
(18)
(19)
and common carp (Cyprinus carpio). Anal. Bioanal. Chem. 2007, 387, 1405–1416. Hoeger, B.; Ko¨llner, B.; Dietrich, D. R.; Hitzfeld, B. Water-borne diclofenac affects kidney and gill integrity and selected immune parameters in brown trout (Salmo trutta f. fario). Aquat. Toxicol. 2005, 75, 53–64. Brown, J. N.; Paxe´us, N.; Fo¨rlin, L.; Larsson, D. G. J. Variations in bioconcentration of human pharmaceuticals from sewage effluents into fish blood plasma. Environ. Toxicol. Pharmacol. 2007, 24, 267–274. Sawchuk, R. J.; Maloney, J. A.; Cartier, L. L.; Rackley, R. J.; Chan, K. K. H.; Lau, H. S. L. Analysis of diclofenac and four of its metabolites in human urine by HPLC. Pharm. Res. 1995, 12, 756–762. Bort, R.; Mace´, K.; Boobis, A.; Go´mez-Lecho´n, M.; Pfeifer, A.; Castell, J. Hepatic metabolism of diclofenac: role of human CYP in the minor oxidative pathways. Biochem. Pharmacol. 1999, 58, 787–796. Kumar, S.; Samuel, K.; Subramanian, R.; Braun, M. P.; Stearns, R. A.; Chiu, S. L.; Evans, D. C.; Baillie, T. A. Extrapolation of diclofenac clearance from in vitro microsomal metabolism data: role of acyl glucuronidation and sequential oxidative metabolism of the acyl glucuronide. J. Pharmacol. Exp. Ther. 2002, 303, 969–978. Kenny, J. R.; Maggs, J. L.; Meng, X.; Sinnott, D.; Clarke, S. E.; Park, B. K.; Stachulski, A. V. Syntheses and characterization of the acyl glucuronide and hydroxy metabolites of diclofenac. J. Med. Chem. 2004, 47, 2816–2825. Corcoran, O.; Nicholson, J. K.; Lenz, E. M.; Abou-Shakra, F.; Castro-Perez, J.; Sage, A. B.; Wilson, I. D. Directly coupled liquid
(20)
(21)
(22)
(23)
(24) (25)
chromatography with inductively coupled plasma mass spectrometry and orthogonal acceleration time-of-flight mass spectrometry for the identification of drug metabolites in urine: Application to diclofenac using chlorine and sulfur detection. Rapid Commun. Mass Spectrom. 2000, 14, 2377–2384. EMEA. Guideline on Environmental Impact Assessment for Veterinary Medicinal Products in Support of the VICH Guidelines GL6 and GL38; EMEA/CVMP/ERA/418282/2005-Consultation: London, 2006; pp 1-59. Aresta, A.; Carbonara, T.; Palmisano, F.; Zambonin, C. G. Profiling urinary metabolites of naproxen by liquid chromatographyelectrospray mass spectrometry. J. Pharm. Biomed. Anal. 2006, 41, 1312–1316. Levsen, K.; Schiebel, H.; Behnke, B.; Do¨tzer, R.; Dreher, W.; Elend, M.; Thiele, H. Structure elucidation of phase II metabolites by tandem mass spectrometry: An overview. J. Chromatogr., A 2005, 1067, 55–72. Mortensen, R. W.; Corcoran, O.; Cornett, C.; Sidelmann, U. G.; Troke, J.; Lindon, J. C.; Nicholson, J. K.; Hansen, S. H. LC-1H NMR used for determination of the elution order of S-naproxen glucuronide isomers in two isocratic reversed-phase LC-systems. J. Pharm. Biomed. Anal. 2001, 24, 477–485. Ternes, T. A. Occurrence of drugs in German sewage treatment plants and rivers. Water Res. 1998, 32, 3245–3260. Scheurell, M.; Franke, S.; Shah, R. M.; Hu ¨hnerfuss, H. Occurrence of diclofenac and its metabolites in surface water and effluent samples from Karachi, Pakistan. Chemosphere 2009, 77, 870–876.
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