Environ. Sci. Technol. 2006, 40, 7200-7206
Occurrence and Fate of Barbiturates in the Aquatic Environment† MANUELA PESCHKA, JAN P. EUBELER, AND THOMAS P. KNEPPER* Europa University of Applied Sciences Fresenius, Limburger Strasse 2, D-65510 Idstein, Germany
Barbiturates have been widely used as sedative hypnotics in the mid-1960s and since then mainly as veterinary drugs. To monitor their presence and fate in the aquatic environment, a method based on gas chromatography-mass spectrometry (GC-MS) has been developed to quantify butalbital, secobarbital, hexobarbital, aprobarbital, phenobarbital, and pentobarbital, all with a limit of detection (LOD) down to 1 ng/L. From the various investigated waste and surface water samples, barbiturates were only, but regularly detected in the Mulde, a tributary of the river Elbe in Germany at relevant concentrations up to several µg/ L. Investigations of groundwater being affected with wastewater infiltration several decades ago also revealed a barbiturate pattern, indicating a strong recalcitrance of these drugs. To confirm this hypothesis, studies were carried out on biotic and abiotic degradation. Both, the biodegradability under aerobic conditions and hydrolysis did not show any degradation, implementing, that the investigated barbiturates, once released into the aquatic environment, show high stability over a long period of time.
or urine (15-19). Their metabolic pathway in human and animal organisms has been studied extensively, but very little is known about their biodegradability in the environment. Some studies dealt with abiotic degradation of barbiturates under various conditions such as hydrolysis at different temperatures and pH values (20, 21). Depending on the substituent at the C5 position, the properties, and therefore the behavior and stability varies, e.g. unsaturated side chains cause a higher resistance to hydrolysis. Barbiturates derive from barbituric acid, a product of malonic acid esters and urea (Table 1). The ease of substituting various groups at the C5 atom of the parent molecule yielded over 2500 barbituratesssome 60 of these were marketed (22). Barbiturates were used mostly as sedatives and hypnotics as well as narcotics and, which is still the case, as anaesthetics (thiopental) and antiepileptic (phenobarbital) drugs (23). Barbiturates were greatly sought and obtainable very easily with their peak of consumption during the mid1960s (22). Due to their narrow therapeutic range and their potential for abuse, benzodiazepines and other products were mainly used as replacements. Pollutants released into the aquatic environment need to be monitored over space and time. The aim of this work was thus to develop a sensitive and reliable method to monitor the fate and occurrence of barbiturates in the aquatic environment at the sub-µg/L level to follow up entry paths and to determine their resistance to biodegradation. Therefore investigations were carried out under abiotic and biotic laboratory conditions. A useful tool in investigating the biodegradability of a substance at environmental relevant concentrations is the fixed bed bioreactor (FBBR). This has already been shown for various degradation studies of a broad variety of pollutants, e.g. surfactants, pharmaceuticals, pesticides, or flame retardants in surface water and WWTP effluents (24).
Introduction
Materials and Methods
The permanent release of pharmaceuticals into the aquatic environment for many years has in the meantime initiated the development of a broad variety of analytical methods allowing their sensitive quantification. Going hand in hand with advancing analytical sensitivity as well as inclining monitoring programs, the amount of analytes detected increased formidable during the recent decades. Current investigations on pharmaceuticals in the environment, food, or drinking water concentrate on high consumption compounds such as anaesthetics (1, 2), analgesics (1), lipid regulators (3, 4), anticonvulsants (3, 5), hormones (6, 7), and antibiotics (8, 9). Main entry paths of pharmaceuticals into environmental compartments are generally through urban and industrial wastewater treatment plants (WWTPs) and manure (2, 10, 11). Up to now, there has been no thorough study upon the occurrence and fate of barbiturates, even if some derivates have been already detected in surface and groundwater. For example, pentobarbital and 5,5-dialylbarbituric acid were found in groundwater (12, 14) and phenobarbital, also the main metabolite of the antiepileptic primidone in the effluent of a WWTP at concentrations of 30 and 1000 ng/L, respectively (13). There are several forensic papers dealing with the determination of barbiturates in body fluids such as blood
Monitoring. For the monitoring campaign upon barbiturates, samples were taken from 8 WWTPs in Germany and Croatia, from 4 German rivers, and 2 groundwater sites. The WWTP were investigated with a total of 18 samples between 2004 and 2005 and differed all in the daily dry water flow, the population equivalents, and the ratio of municipal and industrial wastewater. The river Mulde was sampled 22 times from 2004 to 2005, the river Elbe three times in 2004. Samples from the river Rhine (Eltville, Germany) and Main (Bischofsheim, Germany) were taken every 2 weeks from February to May 2005 with a total of 10 in each case. The sampling points of the river Elbe (E1, Vockerode) and Mulde (M1 and M2) are given in Figure 1. Sampling point M1 (Pouch, Germany) is located upstream of point M2 (Dessau, Germany). Surface water samples were taken near the shores of rivers from the surface layer (0-0.3 m deep). All samples were filled in amber glass bottles, which were prewashed with water, demineralized water, and acetone, and dried at 70 °C before. One liter of river and groundwater and 0.5 L of WWTP effluent were taken and stored in the dark at 8 °C and enriched within 48 h after sampling. Groundwater was taken twice each from a pristine source at Niedernhausen, Hesse, Germany and a source where wastewater was infiltrated several decades ago (Berlin, Germany). Degradation Experiments. The fixed-bed bioreactor (FBBR) was developed in order to determine the biodegradability of single compounds under aerobic conditions (24).
†
This article is part of the Emerging Contaminants Special Issue. * Corresponding author phone: +49 (0)6126-935264; fax: +49 (0)6126-935210; e-mail:
[email protected]. 7200
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10.1021/es052567r CCC: $33.50
2006 American Chemical Society Published on Web 07/26/2006
TABLE 1. Names and Formulas of Some Selected Barbiturates
TABLE 2. Names, Retention Times, GC-MS Quantifier Ions, Recovery (n ) 5), and LODs (n ) 5) of Selected Barbituratesa
name
MW [g/mol]
RT [min]
ion m/z1 [amu]
ion m/z2 [amu]
ion m/z3 [amu]
recovery ground water [%]
recovery Rhine water [%]
recovery WWTP effluent [%]
LOD Rhine [ng/L]
LOD WWTP [ng/L]
butalbital secobarbital pentobarbital hexobarbital aprobarbital phenobarbital
224.3 238.3 226.3 236.3 210.2 232.2
22.4 27.9 25.6 31.2 20.4 36.4
168 168 156 221 167 204
167 167 141 155 168 117
181 195 157 81 124 232
81 ( 1 67 ( 1 79 ( 1 97 ( 2 101 ( 0 104 ( 1
105 ( 1 86 ( 1 103 ( 4 91 ( 1 64 ( 1 74 ( 2
82 ( 1 62 ( 3 88 ( 1 95 ( 1 52 ( 2 105 ( 2
5 5 1 5 1 1
20 20 10 10 10 10
a
Extraction volume for ground- and surface water: 1 L; extraction volume for water from the WWTP: 0.5 L.
Four FBBRs were run as pairs with river water (Rhine, 511 km) and effluent water from a WWTP (Wiesbaden, Germany), 5 L each. One pair was spiked with butalbital at a concentration of 1 mg/L each; the other pair was spiked with secobarbital-Na at the same concentration. The duration time of the degradation studies was 42 days at room temperature at neutral pH. To avoid photodegradation the experiment was set up in the dark. Another FBBR was set up for 21 days containing 6 barbiturates (aprobarbital, butalbital, hexobarbital, pentobarbital, phenobarbital, and secobarbital) spiked in water from the river Mulde that might contain microorganisms already adapted to the analytes. Hydrolysis studies were done at a pH range from 2 to 12. Pristine groundwater was adjusted to the appropriate pH with sulfuric acid and sodium hydroxide, respectively. Barbiturates (butalbital and secobarbital) were spiked at a concentration of 2 µg/L, and the solutions were left at room temperature for 15 h. Before solid-phase extraction (SPE) the solution was neutralized. FIGURE 1. Sampling points at the river Mulde and Elbe (Germany). A glass column filled with glass beads (18 cm filling level) forms the main part of an FBBR, enabling microorganisms to accumulate on the surface. The microorganisms derive from the water phase, circulating with a flow rate of 17 mL/ min in a closed loop. A membrane pump aerates the water in the storage bottle. Samples were taken via a three-way valve at the top of the fixed bed.
Sample Preparation. Native samples were filtered through glass fiber filters (0.45 µm; prewashed with methanol and Milli-Q water). SPE was performed at neutral pH on 1 L of surface water, 0.5 L of WWTP-effluent samples, and 0.5 L of water samples from hydrolysis experiments. Samples were spiked with 110 ng of atrazine D5 (internal standard, c ) 11 ng/µL) magnetically stirred for 10 min. The samples were filtered under vacuum (20 mL/min) through SPE-cartridges (Waters OASIS-HLB). Prior to extraction, the cartridges were VOL. 40, NO. 23, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 2. Mass spectra of butalbital and proposed fragmentation in EI (70 eV): A, r-cleavage of a side chain of butalbital; B, McLaffertyH-rearrangement and following r-cleavage; and C, thermal decomposition and ionization of the resulting molecule. conditioned with 6 mL of n-hexane, 6 mL of methanol, and 10 mL of pristine groundwater. After application and drying under a gentle stream of nitrogen gas for 60 min, the enriched compounds were eluted with 3 × 1.5 mL acetone:ethyl acetate (1:1; v:v). Ten minutes after applying the solvent the remaining extract was pressed out of the cartridge by compressed air. After eluting, the extracts were evaporated under nitrogen gas to approximately 100 µL, external standard was added (100 ng fluazifop-butyl, c ) 10 ng/µL), and the extract was made up to 200 µL final volume with acetone:ethyl acetate (1:1; v:v). Every second day 1 mL samples of each FBBR were taken and made up to 100 mL with pristine groundwater. This solution was treated the same way as native samples. All samples were stored at -18 °C until GC-MS analyses. Chemicals and Instrumentation. The standards used for the degradation experiments, butalbital, pentobarbital, hexobarbital, aprobarbital, thiopental, and secobarbital-Na, were 7202
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obtained from Sigma Aldrich, Germany. Phenobarbital was purchased from Th. Geyer GmbH, Germany. The carrier used in the FBBR, a SIRAN-carrier, was obtained from Schott Mainz, Germany. Used chemicals for SPE (acetone, methanol, n-hexane, ethyl acetate, sulfuric acid, sodium hydroxide) were bought from Merck/VWR-International, Darmstadt, Germany and SPE cartridges (OASIS-HLB 3 cm3) from WATERS, Eschborn, Germany. Samples were analyzed using GC equipped with a MS (6890-GC/5973inert-MSD from Agilent, U.S.A.). The instrument was fitted with an HP5ms capillary column (30 m × 0.25 mm i.d., 0.25 µm film thickness) from Agilent (Waldbronn, Germany). Helium was used as a carrier gas at a flow rate of 1.5 mL/min, and the following temperature program was applied: initial temperature of 50 °C for 0.75 min; increasing at 20 °C/min to 120 °C; then another ramp at 2 °C/min to 230 °C; and a final increase at 10 °C/min to 290 °C. The injector temperature was 230 °C, and all injections were made
FIGURE 3. Total ion chromatogram of a water sample from the river Mulde (April 02, 2004). in the splitless mode. The injected volume was 1 µL. The interface temperature was 280 °C. Measurements were carried out in the selected ion monitoring mode (SIM). Qualitative analysis of the individual substances was performed in the scan mode over a mass range from m/z 60 to 550 amu. Quantification was performed in SIM, based on retention times (RT) and three characteristic ions (see Table 2). To increase the sensitivity of the MS analysis six retention time windows were defined with a maximum of five ions in one window. Each measurement of a sample series commenced with an eight-point calibration performed for each of the ions selected for quantification. The LOD and recovery were determined for the barbiturates in groundwater (from Niedernhausen, Germany), surface water (from river Rhine), and WWTP effluent (Wiesbaden, Germany).
Results and Discussion Analytical Method. The results obtained demonstrate that GC-MS following SPE is a sensitive method for analysis of barbiturates. SPE proved to be a reliable enrichment procedure with 1% standard deviation of the mean (n ) 5). The recovery rates were calculated by using a comparison of spiked and enriched samples (100 ng) to pure standards (spiked but not enriched) at the same concentration. Good recoveries for selected barbiturates were obtained from spiked surface water samples, with values between 64 and 105% and in spiked groundwater and wastewater effluents with in general slightly lower values ranging from 67 to 104% and from 52 to 105%, respectively (Table 2). In general higher recoveries would be expected in groundwater rather than in surface water. For the calibration curves linear correlation coefficients between 0.995 and 0.999 were obtained. Possible fragmentation patterns were proposed for selected quantifier ions (Figure 2). Fragments are formed predominantly by the loss of side chains. A very characteristic pattern for barbiturates is the occurrence of two peaks with a difference of only 1 amu (Figure 2A,B). This is the result of two possible fragmentation mechanisms: (A) R-cleavage of the side chain
and (B) McLafferty rearrangement followed by R-cleavage. The third quantifier ion (Figure 2C) could be related either to thermal decomposition and following ionization or simply to alkyl cleavage. LODs were determined 5-fold in spiked surface water (from the river Rhine) and WWTP effluent. A signal-to-noise ratio of at least 3:1, retention time shift less than ( 0.05 min, and correct quantifier ion patterns were required for positive results. Monitoring Results. Barbiturates could be detected in all investigated samples taken from the river Mulde at Dessau (M2, Figure 1) with concentrations up to 5.4 µg/L for pentobarbital and 5.3 µg/L and 0.1 µg/L for butalbital and secobarbital, respectively (see Figure 4). An average concentration of 0.53 µg/L barbiturates was detected in all samples investigated between April 2004 and July 2005 in the river Mulde (M2) excluding the highest values found on April 02, 2004, September 30, 2004, and November 13, 2005. Next to pentobarbital and secobarbital also n-allylbarbital could be detected in samples taken on September 30, 2004. The presence of this substance was confirmed by comparing the mass spectra to NIST mass spectral library, but quantification was not possible since there was no analytical standard available for purchase. Because pentobarbital is not in use anymore and thiopental still is, it must be taken into consideration that the detected pentobarbital might be a metabolic product of thiopental (19), and there is a strong possibility that at least some proportion could originate from that source. Samples taken during 4 sampling campaigns (September 30, 2004, February 02, 2005, two times at July 02, 2005) from the sampling points in Dessau (M2) and Pouch (M1, Figure 1) show differences in the concentration of pentobarbital. No barbiturates could be found in the samples from Pouch, whereas e.g. pentobarbital was detected permanently in the water samples from Dessau with concentrations ranging between 0.09 and 5.4 µg/L. The results strongly indicate a point source situated between Dessau and Pouch (35 km apart). This area is influenced by industry with about 50 manufacturing companies as well as defective landfills. VOL. 40, NO. 23, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 4. Monitoring results of the barbiturates butalbital, secobarbital, pentobarbital, and phenobarbital in the river Mulde at sampling point M2 (sample 2005-02 mix ) weekly mixed sample).
FIGURE 5. Degradation study of barbiturates in the FBBR with water from the river Mulde. Barbiturates could be detected neither in samples from the rivers Main, Rhine and the Elbe upstream of the estuary of the river Mulde, nor in WWTP effluent samples, with a total sum of 41 investigated samples (data not shown). In samples taken at an irrigation field near Berlin, phenobarbital could be detected in concentrations between 0.2 and 1.3 µg/L. All other investigated barbiturates were in the range between 0.05 and 0.08 µg/L. A risk assessment of the amount of barbiturates detectable in the aquatic environment is quite difficult. Only few market data are obtainable for these products. Based on the data for the two barbiturates used in human medical applications a calculation provides some useful results. In 2004 the amount of sold barbiturates in Germany was 980 kg for thiopental and 70 kg for phenobarbital. The data were obtained in 7204
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communication with a local Hospital in Wiesbaden, Germany. To roughly estimate the order of magnitude in which barbiturates could be expected, a calculation for the river Nidda, a tributary of the river Main, and one for the river Rhine was performed. In both cases it was assumed that the compounds would enter the aquatic environment solely via wastewater treatment plants. With a population of 82 million people in Germany the applied amount of barbiturates would be 1.20 ‚ 10-5 kg/a for thiopental and 8.54 ‚ 10-7 kg/a for phenobarbital. The calculation for the river Nidda shows that we could expect 2.92 ng/L of thiopental and 2.08 ng/L of phenobarbital, with a population equivalent of a WWTP of 8.00 ‚ 104 and a velocity of the river of 10.4 m3/s. For the river Rhine, with a given velocity of 1500 m3/s at Wiesbaden a population equivalent
TABLE 3. Physical Chemical Properties of Investigated Barbiturates butalbital
secobarbital
pentobarbital
hexobarbital
aprobarbital
phenobarbital
1.87 237 -9.69 -6.71
2.36 235 -9.65 -6.64
2.00 478 -9.26 -6.58
2.02 488 -10.38 -7.69
1.38 454 -9.6 -6.93
1.33 105 -11.19 -7.84
log KOWa Swater [mg/L]b log Vp [atm]b log Henry constb (atmL/m) a
Calculated (www.syrres.com/esc).
b
Calculated (ibmlc2.chem.uga.edu/spa).
of 2.82 ‚ 105 for the WWTP Wiesbaden and an amount of sewage of 6.00 ‚ 104, the amount of thiopental and phenobarbital expected in the river Rhine would be 7.12 ‚ 10-2 ng/L and 5.09 ‚ 10-3 ng/L. This estimation also shows why no barbiturates would be detectable in WWTP effluent samples. It provides also an insight to the origin of the detected barbiturates in the river Mulde and in the samples obtained from the former irrigation field near Berlin. Either the amount of barbiturates used in veterinary medicine can be expected to be higher (unfortunately no data was available) or the contamination might originate from industry or old contaminated sites. After investigating barbiturates regarding their degradation behavior in simulated aquatic environments, the persistency of those compounds led to further examinations of native samples taken from a groundwater body from a former irrigation field near Berlin. Today, barbiturates have almost completely lost their potential applications in medicine. But in the mid-1960s the production of barbiturates reached 2000t per year in the United States. These pharmaceuticals were greatly sought, and it might explain why nowadays nothing in general can be found in surface water or WWTP effluent but rather in groundwater compartments entering through natural water flow from contaminated sites, via rivers or landfill sites, especially those containing old hospital wastes (14). Degradation Studies. Barbiturates are metabolized in mammal organisms by the hepatic microsomal enzyme system, mostly via enzymatic hydroxylation and carboxylation of aliphatic side chains following conjugation with glucuronides (25). It seems that the investigated barbiturates are not transformed by microorganisms present in water from river Mulde. This was used as a preparatory phase in the first FBBR degradation experiment (Figure 5), notwithstanding that one could reckon most of all in this case with degradation by already adapted microorganisms. Similar results were obtained from degradation studies carried out in different FBBRs run with WWTP effluent and river Rhine water. Spiked butalbital and secobarbital-Na could not be degraded by microorganisms present within 42 days (data not shown). Hydrolysis of butalbital and secobarbital did not occur within the pH range from 2 to 12. The spiked concentration of 1 µg/L could still be determined as expected in each sample after being left for 15 h at room temperature (data not shown). These results are consistent with the long-term studies of Thoma and Struwe (20) that focused on hydrolization of barbiturates under various conditions. The investigated barbiturates are soluble in water (105488 mg/L) and show low hydrophobicity. Distribution by volatilization can be excluded as a possible transportation pathway since barbiturates have low vapor pressures and Henry constants, respectively (Table 3). Due to the given recalcitrance of the investigated barbiturates and the low log Kow values, it is not surprising that these compounds can also be detected in groundwater possibly affected decades ago by infiltration of contaminated waters. It has to be postulated that the occasional presence of barbiturates in surface and groundwaters is related to past pollution and/or current production and not to discharges
of present municipal WWTPs as shown by the risk assessment for phenobarbital and thiobarbital as well as negative findings in the investigated effluents. Thus further investigations upon the occurrence of barbiturates in landfills leachates is recommended.
Acknowledgments The work was financially supported by Project EMCO (Emerging Contaminants) Contract No. INCO CT 2004509188. We thank Marijan Ahel from the Rudjer Boskovic Institute, Zagreb, Croatia for the sampling campaign of the Croatian wastewater treatment plants and Mr. Rauch, Landesbetrieb fu ¨ r Hochwasserschultz und Wasserwirtschaft Sachsen-Anhalt, for providing samples from the Mulde.
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Received for review December 22, 2005. Revised manuscript received May 22, 2006. Accepted June 9, 2006. ES052567R
