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Removal of Antibiotics in
many surface water resources that receive discharges frommunicipal wastewater treatment plants (WWTPs) and ag- Wastewater: Effect of Hydraulic
ricultural runoff (3-6). A recent study showed that as highas 4 µg/L tetracycline and 1.2 µg/L chlortetracycline have and Solid Retention Times on the
been detected in municipal wastewater (7). Further, a Fate of Tetracycline in the Activated
reconnaissance study by the United States Geological Survey(USGS) reported detectable levels of tetracyclines in several Sludge Process
rivers and streams from many parts of the U.S. (4). Althoughtetracyclines are known to be highly sorbed to clay materials,soil, and sediments (8, 9), their occurrence in surface waters S U N G P Y O K I M , † P E T E R E I C H H O R N , ‡ suggests that their sorption to solids is not irreversible and that there are conditions that could favor their mobility in A . S C O T T W E B E R , † A N D D I A N A S . A G A * , ‡ Department of Civil, Structural, and Environmental The presence of low levels of antibiotics and their Engineering, State University of New York at Buffalo, 207 transformation products in the environment could provide Jarvis Hall, Buffalo, New York 14260, and Department of conditions for the transfer and spread of antibiotic resistant Chemistry, State University of New York at Buffalo, 611 determinants among microorganisms, an emerging issue in Natural Science Complex, Buffalo, New York 14260 public health (10). There is an increased interest in improvingthe removal efficiency of microcontaminants, such asantibiotics and other pharmaceuticals, in WWTPs (11, 12).
While existing treatment technologies produce water that A study was conducted to examine the influence of satisfies current regulatory standards, it has been demon- hydraulic retention time (HRT) and solid retention time strated that the removal of many emerging contaminants, (SRT) on the removal of tetracycline in the activated sludge including antibiotics, personal care products, and hormones, processes. Two lab-scale sequencing batch reactors is incomplete (13). Because of the need to provide sustainable (SBRs) were operated to simulate the activated sludge water supplies to meet the escalating water consumption process. One SBR was spiked with 250 µg/L tetracycline, associated with population growth and increased agriculture while the other SBR was evaluated at tetracycline and industrialization (14), the ability to recover water fromwastewater for reuse is critical. In this regard, it is crucial to concentrations found in the influent of the wastewater understand the fate of currently unregulated chemicals treatment plant (WWTP) where the activated sludge was obtained. The concentrations of tetracyclines in the influent The activated sludge process is the most common form of the WWTP ranged from 0.1 to 0.6 µg/L. Three different of secondary treatment employed in the U.S. (15). It is well- operating conditions were applied during the study known that activated sludge process operating conditions (phase 1sHRT: 24 h and SRT: 10 days; phase 2sHRT: such as solid retention time (SRT) can have a significant 7.4 h and SRT: 10 days; and phase 3sHRT: 7.4 h and SRT: effect on the biodegradation and adsorption of contaminants 3 days). The removal efficiency of tetracycline in phase during the treatment process (16). To date, only limited 3 (78.4 ( 7.1%) was significantly lower than that observed studies have investigated the influence of operating condi- in phase 1 (86.4 ( 8.7%) and phase 2 (85.1 ( 5.4%) at tions on the removal efficiency of emerging contaminants the 95% confidence level. The reduction of SRT in phase such as tetracycline. The study presented in this paper aimedto (i) estimate the tetracycline concentration in wastewater 3 while maintaining a constant HRT decreased tetracycline and the removal efficiencies in a laboratory-scale activated removal efficiency. Sorption kinetics reached equilibrium sludge process under various operating conditions and to within 24 h. Batch equilibrium experiments yielded an (ii) determine the extent of tetracycline removal resulting adsorption coefficient (Kads) of 8400 ( 500 mL/g and a from adsorption and biodegradation. Two sequencing batch desorption coefficient (Kdes) of 22 600 ( 2200 mL/g. No reactors (SBR) operated at different SRT and hydraulic evidence of biodegradation for tetracycline was observed retention times (HRT) were employed to simulate a typical during the biodegradability test, and sorption was found activated sludge process in the laboratory. On the basis of to be the principal removal mechanism of tetracycline in literature review, two major study hypotheses were tested: (i) the removal of tetracycline in biological WWTPs is afunction of operational conditions such as HRT or SRT and(ii) the fate of tetracycline in biological WWTPs is largelyinfluenced by adsorption processes rather than biodegrada- Introduction
The tetracycline group of antibiotics is the second most widely Although the persistence of tetracyclines has been dem- used antimicrobial in the world, with applications in human onstrated in agricultural soils that received antibiotic- therapy and the livestock industry (1). Only small portions containing manure (17, 18), there is little literature on the of tetracycline administered to the treated species are biodegradation of tetracyclines in secondary biological metabolized or absorbed in the body, with most of the wastewater treatment plants. Recently, the mechanisms of unchanged form of the drug being eliminated in feces and tetracycline adsorption on clays and the factors that affect urine (2). Residues of tetracyclines have been detected in their sorption in soil have been described (19, 20). However,the sorption behavior of tetracyclines in sludge may differsignificantly from that in clay or soil due to the high organic * Corresponding author phone: (716)645-6800 x2226; fax: (716)- matter content and complex nature of the mixed liquor 645-6963; e-mail: [email protected].
† Department of Civil, Structural, and Environmental Engineering.
present in biological wastewater treatment plants. To address the fate of tetracycline, additional experiments were per- day at the end of the aeration period. The biomass concen-tration, determined by the Standard Methods 2540D and2540E (21), and the pH of the wastewater in the SBR weremeasured frequently. The ranges and mean values arecompiled in Table 2.
The concentration of dissolved oxygen (DO) in each reactor was always >2 mg/L, as measured three times a weekduring the aeration time using a DO meter (Model 54A, YellowSprings Instrument, Yellow Springs, OH).
Determination of Tetracyclines in SBR Influent and
Effluent using ELISA. To monitor the tetracycline concen-
tration in SBR influent and effluent, a commercially available
96-well microtiter plate tetracycline enzyme-linked immu-
FIGURE 1. Schematic diagram of the sequencing batch reactor
nosorbent assay (ELISA) (R-Biopharm GmbH, Darmstadt, (SBR). [TC: tetracycline].
Germany) was employed. The ELISA procedure provided inthe instruction manual (RIDASCREEN Tetracycline, R- formed to evaluate the relative contribution of adsorption/ Biopharm GmbH, Darmstadt, Germany) was followed.
desorption and biodegradation on the observed removal oftetracycline.
Briefly, samples or standards (50 µL) were added to the BSA-tetracycline coated microwells, followed by a solution of anti- Materials and Methods
tetracycline antibodies (50 µL). The mixture was gently mixedin a plate shaker for 1 h at room temperature. After washing Design and Operation of Sequencing Batch Reactors. To
the wells with phosphate buffered saline and with Tween 20, evaluate the tetracycline fate in the activated sludge process, a solution of a peroxidase-conjugated secondary antibody two identical sequencing batch reactors (SBR-1 and SBR-2) (100 µL) against the anti-tetracycline antibodies was added were built. The operation of SBR-2 differed from SBR-1 in into each well and incubated for 15 min at room temperature.
that the wastewater was amended with tetracycline. The The wells were washed again, then, a 1:1-mixture (100 µL per schematic diagram of the experimental setup is presented well) of substrate (urea peroxide) and chromogen (tetra- methylbenzidine) was added and incubated for another 15 Each SBR consisted of an open 5 L Plexiglass cylinder min. Finally, the reaction was stopped by adding 100 µL of loaded with 4 L of wastewater. Liquid agitation of the SBR 1 M sulfuric acid into each well. The absorbance was contents was achieved through a Fisher direct-drive stirrer measured at 450 nm in a plate reader, and the amount of (Fisher Scientific Co, Pittsburgh, PA). Humidified compressed tetracycline present in the samples was calculated based on air was used for aeration, introduced into the water through the four-parameter fit calibration curve using the KC4 a Pyrex glass-fritted diffuser ensuring enhanced oxygen software (Bio-Tek instruments, Winooski, VT).
transfer efficiency. Influent wastewater of the reactor wasstored in a 20 L Nalgene carboy, which was continuously Sorption Kinetics of Tetracycline onto Activated Sludge.
stirred to maintain a mixed feed. Influent was pumped from Biomass from the first stage activated sludge aeration tanks the storage carboy through Tygon tubing (0.5 in i.d.) into the of the Amherst WWTP was collected for sorption experiments.
5 L SBR via a Masterflex pump (no. 17 head, 6-600 rpm, Amherst WWTP activated sludge was used in these studies Cole-parmer, Chicago, IL). The decanting procedure from because it has a low natural tetracycline loading. In addition, the SBR was controlled by a Masterflex pump (no. 14 head it was the source of the inocula for these studies and would 6-600 rpm) connected to Nalgene 890 Teflon FEP tubing be similar to the lab biomass because it has been developed (3/16 in. i.d). All pumping and mixing cycles in the SBR were with the same wastewater. One experiment was carried out controlled by a Chrontrol programmable timer (Model CD, with unwashed activated sludge (biomass concentration: Lindburg Enterprises, San Diego, CA). A Masterflex pump 3600 mg/L). This concentration is typical of Amherst WWTP (no. 13 head, 1-100 rpm) was used to deliver an aqueous operation and was used to represent adsorption phenomenon tetracycline solution (100 mg/L, freshly prepared each day) in a full-scale activated sludge process. In a second adsorption to achieve a SBR-2 influent concentration of 250 µg/L. The experiment, washed activated sludge at a biomass concen- aluminum foiled 125 mL Kimax Elenmeyer flask was used tration of 1000 mg/L was used to mimic the biomass for tetracycline stock solution storage. Activated sludge for concentrations in the studied SBRs (see Table 2). The washing initial inoculation of both reactors was collected from the procedure with 10 mM phosphate buffer (pH 7.2) was Amherst, NY first stage activated sludge aeration tanks, while performed three times as follows: biomass was separated the effluent from the primary clarifier at the same plant was from the water by centrifugation at 2000g for 3 min, the used as influent wastewater for both SBRs. Collection of the supernatant was discarded, and the biomass was resus- primary clarifier effluent for use as an SBR influent was pended in 10 mM phosphate buffer. In both experiments, conducted twice a week in 30 L carboys and stored at 4 °C 150 mL aliquots of the sludge were inoculated in duplicate until used. The two SBRs were subjected to three different in 250 mL Erlenmeyer flasks wrapped in aluminum foil to operating conditions during the course of the study according prevent possible photodegradation of tetracycline. As a control sample, a 10 mM phosphate buffer was used. To In phase 1, each SBR was operated with an SRT of 10 days minimize any tetracycline elimination due to biotic processes, and a hydraulic retention time (HRT) of 24 h. These conditions 0.15 g (0.1%, w/v) of sodium azide was added into each flask.
are similar to those of an oxidation ditch (HRT: 8-36 h and The flasks were shaken for 30 min at 150 revolutions per SRT: 10-30 days) (15). In phase 2, the reactors were operated minute (rpm) on an orbital shaking table and then a 100 µL under the same SRT as in phase 1 but with a shorter HRT aliquot of a tetracycline stock solution was added to each of 7.4 h. These operating conditions are consistent with a flask to achieve a concentration of 250 µg/L. Aliquots of 1500 conventional activated sludge process (HRT: 4-8 h and µL were withdrawn from each solution after 0.1, 1, 2, 4, 7, SRT: 5-15 days) (15). In phase 3, the SRT was set at 3 days and 24 h. After centrifugation at 2000g for 3 min, the while keeping the HRT at 7.4 h. This SRT is at the low end supernatants were transferred into 2 mL vials for analysis by of a conventional biological wastewater treatment. The target liquid chromatography-electrospray ionization-mass spec- SRT was maintained by manually wasting SBR biomass every trometry (LC-ESI-MS), which was done immediately. The TABLE 1. Operating Schedule of Sequencing Batch Reactorsa
operating times in each cycle
duration
aeration
settling
decanting
# of cycles
treated wastewater
(days-1)
a HRT: hydraulic retention time and SRT: solid retention time. b Actual sampling collecting periods. These periods exclude any transition period TABLE 2. Mean Biomass Concentration and pH in Sequencing Batch Reactors (SBR) during the Three Operational Phases
tetracycline concentrations were measured using external (<0.001%) and is therefore ignored in the calculations. The calibration and plotted against equilibration time.
final concentration in the liquid phase, Ce, was determined Activated Sludge Tetracycline Adsorption and Desorp-
using LC-ESI-MS. The initial concentration used in the tion Coefficients. On the basis of the results of the kinetic
calculations was the nominal amount (250 µg/L) of tetra- experiments, 24 h proved sufficient to reach the adsorption cycline added to the solution. The tetracycline Kads, defined equilibrium of tetracycline onto activated sludge. For the as the ratio of the equilibrium concentration of tetracycline equilibrium experiments, a total of ten 40-mL conical in the sludge relative to the concentration remaining in the polyethylene tubes were prepared. Of these tubes, eight were liquid phase (as expressed in eq 2) can be derived from the filled in duplicate at biomass concentrations of 500, 1000, slope of the plot of Cs (in mg/g) versus Ce (in mg/mL).
1500, and 2000 mg/L (biomass prewashed and resuspendedin 10 mM phosphate buffer pH 7.2 as described previously) and spiked with an aqueous tetracycline stock solution toachieve a final concentration of 250 µg/L. One of theremaining tubes, containing 10 mM phosphate buffer, was The desorption coefficient (Kdes) was determined by re- used as a control to monitor the tetracycline stability during equilibrating the biomass with a known solid-phase con- the experiment, while another tube was amended with centration of tetracycline (from the earlier adsorption tests) biomass at a concentration of 2000 mg/L but without adding in 40 mL of 10 mM phosphate buffer for 24 h. After tetracycline to account for any desorption of tetracycline reequilibrating the sample and determining the liquid-phase from the native sludge. As in the kinetic sorption test, 0.1% concentration (Ce), a mass balance was used to determine sodium azide was added into each tube to minimize any the new solid-phase concentration (Cs). Kdes was then tetracycline elimination due to biotic processes. All test calculated using the same equation used to obtain Kads.
mixtures were agitated using an orbital shaker for 24 h and Tetracycline Biodegradability. An additional batch ex-
were protected from light to prevent possible photodegra- periment was carried out to investigate the biodegradability dation of tetracycline. After a 24 h equilibration period, the of tetracycline and the possible formation of microbial supernatant was quantitatively separated from the biomass metabolites using biomass collected from SBR-2 in phase 3 by centrifugation at 2000g for 5 min. An aliquot was used for (SRT: 3 days). The procedure for the setup and operation of the determination of the tetracycline concentration in the the batch reactor was as follows (in duplicate): a 4 L amber dissolved phase employing LC-ESI-MS. For the desorption glass bottle was amended with a 200 mL aliquot of biomass experiment, the biomass in each tube from the adsorption from SBR-2 and diluted with 3800 mL of distilled water. Air work was resuspended with 40 mL of 10 mM phosphate buffer was introduced continuously into the test medium to and agitated for another 24 h. After centrifugation, an aliquot maintain aerobic conditions, and continuous mixing using of the supernatant was subjected to LC-ESI-MS analysis.
a 6 mm Teflon tubing with perforations at the bottom outlet For the determination of the adsorption coefficient (K was performed. An aliquot of a freshly prepared aqueous the adsorbed tetracycline concentration in the biomass, C tetracycline solution (1000 mg/L) was spiked into the reactor to achieve a test concentration of 200 µg/L. All tests wereconducted under dark conditions to prevent possible pho-todegradation of tetracycline. Two types of duplicate batch reactors were used for this additional experiment: biodeg- radation and control. The two control batch reactors wereamended with 0.1% sodium azide to minimize any tetra- where X is the total mass of tetracycline in the biomass, M cycline elimination by microbial activity. The first sample of is the total dried weight of the biomass, CB is the biomass 4500 µL was taken 5 min after spiking the tetracycline to concentration, V is the solution volume, C0 is the initial these reactors and was transferred into an amber vial tetracycline concentration, and Ce is the final tetracycline containing 500 µL of McIlvaine buffer (pH 4.0; added 0.1 M concentration in the liquid phase after 24 h of equilibration.
EDTA-Na2). Prior to analysis by LC-ESI-MS, a sample aliquot The change in volume of the test mixture in the bioreactors was centrifuged at 2000g for 4 min, and the supernatant was due to the added tetracycline stock solution is negligible transferred into an amber autosampler vial.
FIGURE 2. Time profile of tetracycline concentration in influent and effluent of SBR-1 and SBR-2.
TABLE 3. Tetracycline Concentrations (Mean Value ( Standard Deviation) and Removal Efficiencies during the Three Operational
a Spiked tetracycline concentration; b n.d.) not determined.
Liquid Chromatography-Electrospray Ionization-Mass
limit for tetracycline based on a signal-to-noise ratio of 3 Spectrometry. The liquid chromatograph used was an Agilent
was between 0.2 and 0.8 µg/L.
Series 1100 comprising the following modular components: Statistical Analysis. To test the significance of the
a quaternary pump, a microvacuum solvent degasser, and differences in the mean values of the results from the various an autosampler with thermostated 100-well tray, set to 4 °C.
experimental conditions, a t-test with one tail was performed Separations were achieved on a Thermo Hypersil-Keystone at the 95% confidence level. This test determines if the mean BetaBasic-18 100 mm × 2.1 mm (5 µm) column equipped value of detected tetracycline concentrations in the effluent with a 10 mm × 2.1 mm guard column of the same packing of the SBR in one phase is significantly higher or lower (one material. The mobile phases were (A) water acidified with tail) than with another phase tested. Before conducting the 0.3% formic acid and (B) acetonitrile. The gradient program t-test, data were tested for their normality by the Shapiro- started from 90% A to 10% B (1 min). The portion of A was Wilk method provided by the Origin pro 7.0 (Origin) software linearly decreased to 45% within 11.6 min and further to 5% program and showed to follow a normal distribution under within 0.1 min. These conditions were held for 3.5 min. The initial mobile phase composition was restored within 0.1min and maintained for column regeneration for another Results and Discussion
6.7 min resulting in a total run time of 23 min. The flow ratewas 250 µL/min, and the injection volume was 20 µL. During Behavior of Tetracycline at Different SBR Operating
the first 2 min and the last 6.7 min of each chromatographic Conditions. The tetracycline concentrations in the influent
run, the LC stream exiting the analytical column was directed and effluent of the SBRs were measured using ELISA. This to the waste via a programmable switching valve integrated technique detects all tetracycline derivatives including in the mass spectrometer. The mass spectrometric analysis tetracycline, chlortetracycline, doxycycline, oxytetracycline, was performed on an Agilent Series 1100 SL single-quadrupole and their transformation products (22). The results are instrument equipped with an electrospray ionization (ESI) therefore more appropriately reported as total tetracyclines.
source. A capillary voltage of +4000 V was applied to the The detection limit of the ELISA in wastewater is 0.1 µg/L nebulizer needle tip to generate protonated molecular ions total tetracyclines (Instruction Manual: RIDASCREEN Tet- [M + H]+ of the target analytes. Nitrogen was used as nebulizer racycline, R-Biopharm GmbH, Darmstadt, Germany). The gas (35 psi) as well as a drying gas at a temperature of 350 time profiles of total tetracycline concentrations in the °C and a flow rate of 10 L/min. Molecular ions of the target influent and effluent of SBR-1 and SBR-2 are presented in analytes were recorded at m/z 445 for tetracycline using Figure 2. Calculated average concentrations in the influent fragmentor values of 140. For confirmation purposes, the and effluents of the two reactors are given along with the m/z 410 for the tetracycline fragment ion was included.
removal efficiencies in Table 3. The background total Quantification was done by external calibration using tetracycline concentrations in the influent of the SBR (i.e., standard solutions in the range of 2-250 µg/L. The detection in the effluent from the primary clarifier of the Amherst FIGURE 3. Time profile of tetracycline residue percentages under two different biomass concentrations. (Error bars correspond to one
standard deviation.)
WWTP) were below 1 µg/L throughout the operation time of Adsorption and Desorption of Tetracycline. Removal of
tetracycline from the dissolved phase in SBR-2 as shown in Photodegradation is known as one of the main trans- Figure 2 may be achieved either through adsorption and/or formation reactions of tetracyclines in the environment.
biodegradation. Partitioning onto the suspended matter is However, the focus of this study was to determine the role expected to play a key role since tetracyclines, despite their of biomass for removing tetracycline in biological wastewater high water solubility and low n-octanol/water partition treatment plants; therefore, potential photodegradation was coefficients, are reported to sorb strongly onto soil (24). Ionic eliminated by protecting the test liquor from light. Other interactions and the metal-complexing properties of tetra- known abiotic transformations of tetracyclines are isomer- cyclines have been found to largely govern its adsorption ization and epimerization, which are highly pH dependent and reversible. The ELISA method measures total tetracy- To investigate the adsorption behavior of tetracycline, a clines, which include all the isomers and epimers of kinetic study was carried out at two different biomass tetracyclines. The tetracycline concentrations in Amherst concentrations. Figure 3 shows the time profile of tetracycline WWTP are similar to those previously reported in the at biomass concentrations of 1000 and 3600 mg/L. As can be literature. In monitoring studies conducted at six U.S.
seen, more than 75 and 95%, respectively, of the tetracycline treatment plants, which applied different treatment tech- initially present at 250 µg/L was removed from the dissolved nologies, the tetracycline concentrations were between 0.27 phase after an equilibration time of only 1 h, indicating a and 4 µg/L in the untreated sewage and between 0.23 and very fast sorption onto the sludge. Equilibrium concentrations 1.2 µg/L in the treated effluent samples (7, 23). In our study, were achieved quickly at 3600 mg/L biomass and stayed the total tetracycline concentrations in the SBR-1 effluent virtually unchanged over the 24 h study. On the basis of this appear to be generally lower as compared to the influent adsorption kinetic test, it was assumed that 24 h was sufficient wastewater, but it is difficult to assess if this difference was time to reach equilibrium for both adsorption and desorption due to elimination in the bioreactor or due to the intra-assay tests. Other researchers (9, 25) also used 24 h as an variability typical of ELISA analysis. Therefore, the removal equilibration time for tetracycline adsorption/desorption efficiency in SBR-1 was not determined. In the case of SBR- tests in soil. The sorption isotherm of tetracycline on activated 2, a substantial difference between the initial total tetracycline sludge is presented in Figure 4. The calculated Kads was 8400 concentration (spiking level 250 µg/L) and the final con- ( 500 mL/g (standard error of slope). This is about three centration in the effluent was obtained, as presented in Table times that reported for the more polar oxytetracycline on 3. Total tetracycline concentrations determined in the SBR-2 activated sludge (3020 mL/g) (26).
effluent ranged from 10 to 84 µg/L. On the basis of the average This value of Kads is substantially higher than has been concentrations given in Table 3 and an initial concentration reported for tetracycline in soils (400 and 1140 mL/g) (24).
of 250 µg/L (background concentration neglected), the The calculated desorption isotherm of tetracycline from removal efficiencies for SBR-2 amounted to 86% in phase 1, activated sludge is presented in Figure 4. The calculated 85% in phase 2, and 78% in phase 3. Statistical evaluation desorption coefficient (Kdes) was 22 600 ( 2200 mL/g and is of these data using t-tests showed that there was no significant more than three times higher than Kads. The difference differences at a 95% confidence level between phase 1 and between Kads and Kdes suggests that a portion of adsorbed phase 2 mean total effluent tetracycline concentrations (p ) tetracycline does not readily desorb from activated sludge, 0.366). These results suggest that lowering the hydraulic thereby displaying adsorption/desorption hysteresis. Ad- retention time from 24 to 7.4 h, which also resulted in an sortion/desorption hysteresis of trace chemicals such as increase in the mean SBR biomass concentration from 514 proteins and metals on activated sludge is well-documented to 1191 mg/L, did not influence tetracycline removal.
(27, 28). The sludge adsorption experiments indicated that However, decreasing the SRT of SBR-2 to 3 days in phase 3 elimination of tetracycline from the sewage in SBR is resulted in a significant reduction in tetracycline removal influenced strongly by the sorption onto the biomass.
when compared to the 10 days SRT used in phase 1 (p ) Biodegradability of Tetracycline. What is unclear from
0.029) and phase 2 (p ) 0.032).
the data presented thus far is the role of biodegradation in FIGURE 4. Adsorption and desorption isotherms for tetracycline on sludge. [Kads: sorption coefficient coefficient and Kdes: desorption
coefficient (error bars correspond to one standard deviation)].
difficult to rule out the potential competing effects of HRTand biomass concentration on tetracycline removal. In phase1, longer HRT resulted in low biomass concentrations, whilein phase 2, shorter HRT resulted in an increase in the biomassconcentration. The longer HRT could have promoted equi-librium or near equilibrium conditions (more complete) inphase 1, while the higher biomass concentrations in phase2 could have compensated for shorter reaction times.
The reduction of SRT from 10 to 3 days in phase 3, while maintaining a constant HRT of 7.4 h, did result in a significantreduction in tetracycline removal. There are a number ofplausible explanations for this reduction in tetracyclineremoval that are driven by changes in biomass physiologyand/or biomass quantity. It is well-documented that changesin SRT reduce biodegradation efficiency, and this loss ofefficiency is most notable for difficult to degrade compoundsthat support low biomass growth rates (15, 16, 30). In thisstudy, it was determined that under phase 3 SRT conditions,no biodegradation of tetracycline was observed. Tetracycline FIGURE 5. Time profile of tetracycline in a batch reactor spiked
biodegradation was not assessed directly in phases 1 and 2, with 200 µg/L.
which operated at a longer SRT. To date, there is little evidencein the literature to suggest biodegradation as a likely removal the observed removal of tetracycline. To examine whether mechanism. If biodegradation was not responsible for the the exposure of the sludge bacteria to elevated tetracycline reduced tetracycline removal efficiency in phase 3, then SRT concentrations over an extended period of time led to an influence on sorption is of interest. Reducing the SRT in acclimation to the substrate, a biodegradability assay was phase 3 did reduce the biomass concentration as shown in conducted using sludge from SBR-2 in phase 3. To this end, Table 2, and this reduction would favor less sorption and batch reactors containing 20-fold diluted sludge were spiked less removal assuming that the biomass sorption charac- with 200 µg/L tetracycline, and the concentration profile was teristics were unchanged between phases. Sorption char- determined by LC-ESI-MS analysis. Control reactors, acteristics of the biomass may have changed with SRT. Several amended with 0.1% sodium azide to inhibit microbial activity, researchers have observed increased biomass hydrophobicity were used to account for sorptive effects. The profiles shown at higher SRTs (31, 32). In fact, recent work by Harper and in Figure 5 reveal a decrease in concentration of tetracycline Yi (33) has shown that a bioreactor configuration can have in both reactor types. LC-ESI-MS analysis of the liquid phase a significant influence on biomass hydrohobicity and particle for known degradation products such as 4-epi-tetracycline size, which can affect the bioavailability and fate of phar- and anhydrotetracycline (29) as well as for novel metabolites maceuticals in WWTPs because of their impact on particle did not show the production of new compounds. From the floc characteristics. Even though tetracycline has a low tetracycline biodegradability test (Figure 5), the strong n-octanol/water partition coefficient, at certain pH values, similarity between inhibited and noninhibited biomass and hydrophobic interactions still play a role for the sorption of the lack of tetracycline metabolites strongly suggests that tetracycline on soil or clay (20). At the pH values observed sorption is the primary mechanism for tetracycline removal for the SBR in this study (Table 2), tetracycline is zwitterionic observed in phase 3 instead of biodegradation.
(no net charge); therefore, hydrophobic interactions with From the tetracycline removal data, it is tempting to sludge become a relatively important sorption mechanism.
conclude that SRT is a more important variable than HRT.
Finally, reductions in bacterially produced dissolved organic However, even though tetracycline removal efficiencies are matter (DOM) concentrations at lower SRTs (34) may have not statistically different between phase 1 and phase 2, it is reduced sorption at the lower SRT of phase 3. Which of the previous factors was most important in the reduction of (5) Kolpin, D. W.; Skopec, M.; Meyer, M. T.; Furlong, E. T.; Zaugg, tetracycline removal in this study is unclear and deserves S. D. Urban contribution of pharmaceuticals and other organic wastewater contaminants to streams during differing flow
conditions. Sci. Total Environ. 2004, 328, 119-130.
Implications in Wastewater Treatment. It is clear from
(6) Miao, X.-S.; Bishay, F.; Chen, M.; Metcalfe, C. D. Occurrence of the data obtained in this study that biomass is an important antimicrobials in the final effluents of wastewater treatment sink for tetracycline antibiotics in wastewater treatment plants in Canada. Environ. Sci. Technol. 2004, 38, 3533-3541.
systems. This study provides an important basis for optimiz- (7) Karthikeyan, K. G.; Bleam, W. F. DNR Project #163 Final Project ing operational parameters in WWTPs to improve removal Report; Wisconsin Department of Natural Resources; Wisconsin of other micropollutants in wastewater. For example, iden- Department Agriculture, Trade, and Consumer Protection:Wisconsin, October 2003. http://www.dnr.state.wi.us/org/ tification of the critical SRT values and temperature could water/dwg/gw/research/reports/169.pdf.
be crucial to effectively remove recalcitrant micropollutants ¨ mmerer, K. Pharmaceuticals in the Environment; Sources, from wastewater. It is highly likely that many of the Fate, Effects and Risks; Springer: New York, 2001.
pharmaceutical pollutants that are resistant to biodegrada- (9) Sithole, B. B.; Guy, R. D. Models for tetracycline in aquatic tion in WWTPs are also eliminated in the effluent by sorption environments: I. interaction with bentonite clay systems. Water, on sludge. For instance, the ciprofloxacin antibiotic, which Air, Soil Pollut. 1987, 32, 303-314.
has similar sorption characteristics to tetracycline (24), was (10) Boxall, A. B. A.; Kolpin, D. W.; Halling-Sørensen, B.; Tolls, J. Are veterinary medicines causing environmental risks? Environ. Sci. reported to be 95% adsorbed on the sludge or effluent solids Technol. 2003, 37, 286A-294A.
from a Swiss wastewater treatment plant (35). On the basis (11) Andreozzi, R.; Raffaele, M.; Nicklas, P. Pharmaceuticals in STP of these results, membrane bioreactor (MBR) technology effluents and their solar photodegradation in aquatic environ- could become more popular for treating municipal waste- ment. Chemosphere 2003, 50, 1319-1330.
water soon because the MBR process can be operated under (12) McArdell, C. S.; Molnar, E.; Suter, M. J.-F.; Giger, W. Occurrence conditions with very long SRT and high biomass concentra- and fate of macrolide antibiotics in wastewater treatment plants tions as compared to a conventional activated sludge process and in the Glatt Valley watershed, Switzerland. Environ. Sci.
Technol. 2003, 37, 5479-5486.
(31). Therefore, the influence of SRT and biosorption on the (13) Ternes, T. A.; Joss, A.; Siegrist, H. Scrutinizing pharmaceuticals removal of micropollutants in the WWTPs should be exam- and personal care products in wastewater treatment. Environ. ined for important micropollutants, particularly those that Sci. Technol. 2004, 38, 392A-399A.
have already been shown to be persistent in surface waters (14) Levine, A. D.; Asano, T. Recovering sustainable water from wastewater. Environ. Sci. Technol. 2004, 38, 201A-208A.
Finally, it is important to recognize that nonbiodegraded (15) Metcalf and Eddy, Inc. Wastewater Engineering: Treatment Disposal Reuse; MacGraw-Hill: Boston, MA, 2002.
tetracycline that sorb to biomass can become bioavailable (16) Grady, C. P. L.; Daigger, G. T.; Lim, H. C. Biological Wastewater when localized conditions are changed such that desorption Treatment; Marcel Dekker: New York, 1999.
becomes favorable. In such a case, the amount of tetracycline (17) Hamscher, G.; Sczesny, S.; Ho¨per, H.; Nau, H. Determination sorbed in the biomass will become an issue during the land of persistent tetracycline residues in soil fertilized with liquid application of biosolids because of the potential ecological manure by high-performance liquid chromatography with risks posed by antibiotics in the environment. Therefore, it electrospray ionization tandem mass spectrometry. Anal. Chem. is important to measure not only the overall percent removal 2002, 74, 1509-1518.
(18) Jacobsen, A. M.; Halling-Sørensen, B.; Ingerslev, F.; Hansen, S.
of pharmaceuticals in WWTPs but also the quantities of the H. Simultaneous extraction of tetracycline, macrolide, and sorbed portion in the biosolids. Finally, it is obvious from sulfonamide antibiotics from agricultural soils using pressurized this study that different activated sludge operational strategies liquid extraction, followed by solid-phase extraction and liquid play a key role in defining the removal efficiency of chromatography-tandem mass spectrometry. J. Chromatogr. tetracycline in wastewater. Engineering design and opera- A 2004, 1038, 157-170.
tional decisions can influence the ultimate fate of pharma- (19) Figueroa, R. A.; Leonard, A.; MacKay, A. A. Modeling tetracycline antibiotic sorption to clays. Environ. Sci. Technol. 2004, 38, 476-
ceuticals in WWTPs and deserve greater attention in phar- (20) Kulshrestha, P.; Giese, R. F., Jr.; Aga, D. S. Investigating the molecular interactions of oxytetracycline in clay and organic Acknowledgments
matter: insights on factors affecting its mobility in soil. Environ.
Sci. Technol. 2004, 38, 4097-4105.
The authors thank the Town of Amherst WWTP No. 16 facility (21) APHA. Standard Methods for the Examination of Water and for allowing us to access the plant and obtain samples from Wastewater; American Public Health Association, American the treatment tanks. Part of this work was funded by the Water Works Association, Water Environment Federation:Washington, DC, 1998.
National Science Foundation Grant 023700. Any opinions, (22) Aga, D. S.; Goldfish, R.; Kulshrestha, P. Application of ELISA in findings, and conclusions or recommendations expressed determining the fate of tetracyclines in land-applied livestock in this material are those of the authors and do not necessarily wastes. Analyst 2003, 128, 658-662.
(23) Antibiotics in New Mexico wastewater and groundwater: New Mexico Environment Department Groundwater Quality Bureau,New Mexico Department of Health Scientific Laboratory Divi- Literature Cited
sion, September 2003. http://www.nmenv.state.nm.us/gwb/Technical%20resourcesAntibiotics%20in%20NM%20Waters.PPT.
(1) Col, N. F.; O’Connor, R. W. Estimating worldwide current (24) Tolls, J. Sorption of veterinary pharmaceuticals in soils: a review.
antibiotic usage: report of task force 1. Rev. Infect. Dis. 1987,
Environ. Sci. Technol. 2001, 35, 3397-3406.
(25) Rabolle, M.; Spliid, N. H. Sorption and mobility of metronidazole, (2) Feinman, S. E.; Matheson, J. C. Environmental Impact State- olaquindox, oxytetracycline, and tylosin in soil. Chemosphere ment: Subtherapeutic Antibacterial Agents in Animal Feeds; 2000, 40, 715-722.
Department of Health, Education, and Welfare Report 372; Food (26) Stuer-Lauridsen, F.; Birkved, M.; Hansen, L. P.; Holten Lu¨tzhøft, and Drug Administration: Washington, DC, 1978.
H.-C.; Halling-Sørensen, B. Environmental risk assessment of (3) Hirsch, R.; Ternes, T.; Haberer, K.; Kratz, K. L. Occurrence of human pharmaceuticals in Denmark after normal therapeutic antibiotics in the aquatic environment. Sci. Total Environ. 1999,
use. Chemosphere 2000, 40, 783-793.
(27) Dueholm, T. E.; Andreasen, K. H.; Nielsen, P. H. Transformation (4) Kolpin, D. W.; Furlong, E. T.; Meyer, M. T.; Thurman, E. M.; of lipids in activated sludge. Water Sci. Technol. 2001, 43, 165-
Zaugg, S. D.; Barber, L. B.; Buxton, H. T. Pharmaceuticals, hormones, and other organic wastewater contaminants in U.S.
(28) Kodukula, P. S.; Patterson, J. W.; Surampalli, R. Y. Sorption of streams, 1999-2000: a national reconnaissance. Environ. Sci. cadmium and nickel in activated sludge. Water Qual. Res. J. Technol. 2002, 36, 1202-1211.
Canada 1995, 30, 277-297.
(29) Mitscher, L. A. The Chemistry of Tetracycline Antibiotics; Marcel (34) Pribyl, M.; Tucek, F.; Wilderer, P. A.; Wanner, J. Amount and nature of soluble refractory organics produced by activated (30) Lawrence, A. W.; McCarty, P. L. Unified basis for biological sludge microorganisms in sequencing batch and continuous treatment design and operation. J. Sanit. Eng. Div., Am. Soc. flow reactors. Water Sci. Technol. 1997, 35, 27-34.
Civ. Eng. 1970, 96 (SA3), 757-778.
(35) Golet, E. M.; Xifra, I.; Siegrist, H.; Alder, A. C.; Giger, W.
(31) Lee, W. T.; Kang, S.; Shin, H. Sludge characteristics and their Environmental exposure assessment of fluoroquinolone anti- contribution to microfiltration in submerged membrane biore- bacterial agents from sewage to soil. Environ. Sci. Technol. 2003,
actors. J. Membr. Sci. 2003, 216, 217-227.
(32) Liao, B. Q.; Allen, D. G.; Droppo, I. G.; Leppard, G. G.; Liss, S.
N. Surface properties of sludge and their role in bioflocculation Received for review January 2, 2005. Revised manuscript and settleability. Water Res. 2001, 35, 339-350.
(33) Harper, W. F., Jr.; Yi, T. Sorption of ethinyl estradiol in activated received May 7, 2005. Accepted May 10, 2005. sludge processes; Proceedings of the 25th Annual Alabama WaterEnvironment Association Conference: 2004.

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