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Chapter 3

Downloaded by PENNSYLVANIA STATE UNIV on July 28, 2012 | http://pubs.acs.org Publication Date: July 30, 2001 | doi: 10.1021/bk-2001-0791.ch003

Occurrence and Fate of Fluoroquinolone, Macrolide, and Sulfonamide Antibiotics during Wastewater Treatment and in Ambient Waters in Switzerland Alfredo C. Alder, Christa S. McArdell, Eva M. Golet, Slavica Ibric, Eva Molnar, Norriel S. Nipales, and Walter Giger Swiss Federal Institute for Environmental Science and Technology (EAWAG) and Swiss Federal Institute of Technology (ΕΤΗ), Ueberlandstrasse 133, CH-8600 Dübendorf, Switzerland

The leading fluoroquinolone antibiotic in human medical use, ciprofloxacin, was measured in inflow and outflow of a mu nicipal wastewater treatment plant (WWTP). Samples composited over 24 hours were collected and analyzed by HPLC/fluorescence. Ciprofloxacin concentrations in the inflow and outflow ranged from 220-370 and 60-75 ng/L, respectively. These results show that only 70-80% of ciprofloxacin is eliminated in this W W T P and that residual amounts reach the receiving surface waters. In addition, selected macrolide and sulfonamide antibiotics were detected by L C / M S in similar concentrations in the outflows of different WWTPs. Occurrences of antibiotics in ambient waters indicate different exposure routes based on the various uses of these drugs in human and veterinary medicine.

56

© 2001 American Chemical Society

In Pharmaceuticals and Care Products in the Environment; Daughton, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2001.


57 The occurrence of pharmaceutical chemicals in the aquatic environment has become a topic of substantial scientific and public concern (1-4). Pharma ceuticals, which are incompletely eliminated in wastewater treatment, may enter the aquatic environment. Little is known on the risk of low levels of drugs in the environment because pharmaceuticals are not properly addressed by current environmental risk assessment methodologies. Antibiotics are applied in human medicine and for veterinary purposes with different exposure routes for entering the aquatic environment, i.e., through municipal wastewaters and soil run-off (Fig. 1). Studies on the fate of antibiotics in wastewaters and surface waters are motivated by the question of whether trace concentrations in the environment may pose a risk for aquatic organisms and microbial populations. For an environmental risk assessment, it also must be evaluated whether the occurrence of antibiotics in the environment contributes to the increasing resistance of microorganisms towards antibiotics.

Figure 1. Different exposure routes of human and veterinary pharmaceuticals to the aquatic environment (adapted from (I)).



58 In Switzerland, the annual consumption of antibiotics in 1997 was about 80 tons (Fig. 2, 5), of which about 35% is used in human medicine and up to 65% by veterinarians. In human medicine, 20% of the antibiotics are applied in hospitals; the rest is domestic consumption. The annual consumption of antibiotics in human medicine remained roughly constant in the past few years. Between 1992 and 1997, feed additives were reduced from 91 to 36 tons. This correlates with a reduction of the total consumption of antibiotics in these years from 125 to 80 tons. Within the same time frame the consumption of veterinary antibiotics for therapeutic purposes has doubled. Therefore, a shift from feed additives to therapeutic use has occurred. The application of antibiotics as growth promoters in feed additives has been forbidden in Switzerland since January 1999, which again creates a drastic change in the sales figures. Details on the use of antibiotics in veterinary medicine are difficult to obtain. In human medicine, the most highly consumed antibiotics belong to the compound classes of penicillins, ampicillins, and cephalosporins (i.e., /^-lactam antibiotics), macrolides and fluoroquinolones (Fig. 3). The tetracyclines are an additional important group of antibiotics. In veterinary medicine, the major antibiotic classes are again the ^-lactam antibiotics as well as chloramphenicol. Sulfonamides, which are also used in considerable amounts, were not included in these statistics (5).

Figure 2. Annual consumption of antibiotics in Switzerland from 1992 to 1997 (5).



59

Figure 3. Annual consumption of different types of antibiotics in human and veterinary medicine in Switzerland in 1997 (5).

Our study emphasizes antibiotics belonging to the fluoroquinolone, macrolide, and sulfonamide groups. The chemical structures of the main antibiotics investigated are given in Figure 4. These antibiotics are widely used in human or veterinary medicine. Although /^-lactam antibiotics are prescribed in the highest quantities, they are not very likely to occur in the aquatic environment because of their chemically unstable /^-lactam ring, which is readily susceptible to hydrolytic cleavage (6). Therefore, ^-lactam antibiotics were not included in our study. Fluoroquinolone (FQ) antibiotics inhibit the bacterial DNA-unwinding enzyme, gyrase. These totally synthetic chemicals are active against many gramnegative and gram-positive pathogenic bacterial species (7). They are licensed as important broad-spectrum antibiotics for the use in human medicine (e.g., ciprofloxacin, norfloxacin, ofloxacin, levofloxacin) and veterinary applications (e.g., enrofloxacin, danofloxacin).


60

COOH


ciprofloxacin

erythromycin Ri=0, R =H clarithromycin Ri= O, R = CH roxithromycin Ri= NOCH 0(CH )20CH3, R =H 2

2

3

2

2

2

ο·^^

Ο

Η

sulfamethoxazole R= HN H N-

I

2

sulfamethazine

R=

ν

S=0

trimethoprim

chloramphenicol

Figure 4: Chemical structures and names of the main fluoroquinolone, macrolide, and sulfonamide antibiotics investigated in this study.



61 Macrolides are mainly active against gram-positive pathogenic bacterial species, and act as inhibitors of protein synthesis. They are the major alternative for patients who are allergic to /^-lactam antibiotics. Sulfonamide antibiotics are active against many gram-negative and grampositive pathogenic bacterial species. They interfere in folic acid synthesis by competitive antagonism of p-aminobenzoic acid, resulting in a bacteriostatic effect on growing organisms. Nowadays, sulfonamides are mainly used in combination with a dihydrofolate reductase inhibitor such as trimethoprim. Sulfonamide antibiotics are applied in high quantities in human medicine (mainly sulfamethoxazole) and in veterinary medicine (mainly sulfamethazine). Chloramphenicol is a broad-spectrum antibiotic used in human medicine only in exceptional cases like meningitis. The veterinary use of this substance is forbidden in the European Union since 1995. It is also under discussion to be banned from the Swiss market, since it is believed to occasionally cause fatal aplastic anemia in humans due to its bone marrow damaging properties (8). In preceding investigations, concentrations of the predominant F Q in human medicinal use, ciprofloxacin, were determined in hospital wastewaters by H P L C with fluorescence detection (9) and qualitatively confirmed by electrospray mass spectrometry (10). Concentrations ranged from 5 to 90 μg/L. Here we report on field studies, which were performed in order to follow the input of several representative antibiotics of different groups into surface waters. Inflow and outflow concentrations of fluoroquinolone antibiotics were measured in municipal treatment plants in order to determine elimination rates. Daily variations of inflow and outflow were studied. For macrolide and sulfonamide antibiotics, first the results on measurements in W W T P outflows and in surface waters are presented. The focus of this investigation is to determine the occurrence of selected drug residues and to evaluate the different exposure routes of veterinary and human pharmaceuticals to the aquatic environment. Experimental

Fluoroquinolones Sampling Samples were taken from the inflow and outflow of the municipal W W T P of Zurich-Werdhôlzli. In this context, inflow means the effluent of the primary clarifier and outflow refers to the effluent of the W W T P after the secondary clarifier and flocculation filtration. In Switzerland, effluents of WWTPs are never disinfected. The W W T P of Zurich-Werdhôlzli serves approximately a population of 450,000 inhabitants (wastewater discharge = 170,000 m /d). The wastewater has a residence time of 16-18 h (activated sludge system incl. 3



62 secondary clarifier) and the total sludge age in the biological treatment is 10-11 d (the aerobic sludge age is 7-8 d, the anoxic zone is 28% of the total activated sludge tank volume). The facility is operated with chemical phosphate precipitation and flocculation filtration. Twenty-four hour flow proportional composite samples (1-L) of the inflow and outflow were collected over several days during a dry weather period. Samples were collected in plastic bottles containing 10 m L / L formaldehyde (37%) and were stored in the dark to avoid possible photodegradation. Samples were kept at 4°C until analysis, typically less than 24 h. In newer studies, samples are collected in amber glass bottles and stabilized by lowering the p H to 3. No differences were observed between plastic and glass bottles. Formaldehyde is no longer used for preserving the samples because it may react with the amino group of the piperazynil group of ciprofloxacin to give the Schiff s base.

Analyses The analytical methods used are summarized in Table I. Duplicate analyses were performed. Wastewater samples (200-400 mL) were adjusted to p H 4, filtered through a glass fiber filter with a nominal pore size of 0.7 μπι, and enriched by solid-phase extraction using strong cationic exchanger Empore disks. Elution of the disk was performed by 3 m L 5% aqueous N H (25%) in methanol. The extract was acidified with 200 μΙ, phosphoric acid (85%) and subsequently analyzed by reversed-phase liquid chromatography with fluorescence detection (Ex; 278 nm, Em: 445 nm). A n octadecylsilica column (Nucleosil 100, 5 μπι, 250 χ 3 mm) was operated at room temperature with a flow rate of 0.7 mL/min. The aqueous mobile phase (pH 2.4) was 20 m M K H P 0 , 30 m M ortho-phosphoric acid (85%) (eluent A ) , and 5 m M triethylamine. The organic modifier was acetonitrile (eluent B). Typically, elution started isocratically with 90% of eluent A for 1 min, followed by a linear gradient to 60% of eluent Β in 15 min. Final conditions of 90% eluent Β were reached in 2 min and held for 2 min before a 6-min linear gradient reestablished initial conditions, which were held for 4 min for column equilibration. 3

2

4

Quantification Quantification was carried out using ciprofloxacin-HCl (Bayer A G , Wuppertal, Germany) as an external standard. The limit of quantification for wastewater was 50 ng/L (S/N = 10). However, this was limited by interferences of the wastewater matrix, not by instrumental sensitivity. Recoveries of ciprofloxacin from inflow and outflow were 40-50 and 50-70%, respectively.


63


Macrolides and Sulfonamides Detailed descriptions of the analytical methods using L C / M S for the deteimination of macrolide and sulfonamide antibiotics in W W T P outflows and in surface waters will be published elsewhere (//). The method is a modified version of the procedure published by Hirsch et al. (12% who used L C / M S / M S for analyzing environmental samples. The limit of detection in groundwater samples was between 0.5 and 5 ng/L, except for spiramycin with a detection limit of 11 ng/L. Recoveries were in the range of 60 to 103%. Matrix interferences were small for macrolide antibiotics, but were of concern for sulfonamide antibiotics. Standard addition was therefore used for quantification if matrix effects needed to be considered.

Results and Discussion Fluoroquinolones Concentrations and massflowsof ciprofloxacin in wastewater The concentrations of ciprofloxacin in the inflow and outflow are given in Figure 5. The concentrations are based on two replicate determinations of a single wastewater sample. Over a 14-day sampling period ciprofloxacin concentrations in the inflow of the W W T P Zurich-Werdhôlzli ranged between 220 and 370 ng/L (m = 284, s ± 60). Outflow concentrations of ciprofloxacin varied from 60 to 75 ng/L (m - 70, s ± 4). The mass flow calculations are based on an average wastewater discharge of 170,000 m /d. The mass flow of ciprofloxacin in the inflow varied from 37.4 to 63.6 g/d (m = 48.0, s ± 10.7). In samples of the outflow, the mass flows were between 10.4 and 12.6 kg/d (m = 11.9, s ± 0.8). These results show that in this treatment plant ciprofloxacin is eliminated during activated sludge treatment with 70-80% efficiency, and that the residual amounts are discharged into ambient waters. Ciprofloxacin degradation is likely to occur during composting procedures. It was demonstrated that ciprofloxacin is degraded by a brown rot fungus (13). At present, it is unknown whether ciprofloxacin is degraded during wastewater treatment. A recent study has shown that wastewater bacteria were not able to degrade ciprofloxacin in a closed bottle test (14). Elimination due to adsorption to sludge during activated sludge treatment is another plausible mechanism. Because the log K of ciprofloxacin is approximately zero at p H 7 (15), hydrophobic partitioning into sludge cannot contribute significantly to the sorption. In wastewaters (pH 7-8), ciprofloxacin is present in the zwitterionic form with a positively charged amino group. Therefore, cationic exchange processes may dominate the sorption of ciprofloxacin to the negatively charged sludge. 3

o

w


64


Table I. Analytical methods for the determination of the antibiotics in wastewaters and ambient waters Antibiotic

Analytical methods

Fluoroquinolones

Enrichment Solid phase extraction with strong cationic exchanger Empore cation exchange disks, p H 4 elution with 5% aqueous N H in methanol HPLC Reversed-phase column, gradient elution with K H P O ( 2 0 mM), H P 0 (30 mM), ( C H ) N ( 5 mM) and acetonitrile Detection Fluorescence detection (extinction: 278 nm, emission 445 nm) 3

2

Macrolides

4

3

4

2

5

3

Enrichment Solid phase enrichment with LiChrolute E N / LiChrolute C (2:5), p H 7 HPLC Reversed-phase column, gradient elution with N H A c (10 mM) and acetonitrile Detection L C / M S , electrospray ionization 1 8

4

Sulfonamides

Enrichment Solid phase enrichment with Oasis H L B , p H 6 HPLC Reversed-phase column, gradient elution with N H A c (1 mM) and acetonitrile Detection L C / M S , electrospray ionization 4



65

Figure 5. Daily variation of the concentration of ciprofloxacin in inflow and outflow of the wastewater treatment plant Zurich-Werdhohli.

Plausibility estimation of the measured concentrations of ciprofloxacin To make a plausibility estimation of the found concentrations, predicted concentrations (PEC) were compared with measured concentrations (MEC) of ciprofloxacin in wastewaters (Table II). In a university hospital with 1000 beds, ciprofloxacin consumption is approximately 8 kg per year. Assuming a 50% excretion through the urine, and dividing this number by the mean annual wastewater discharge, an annual mean concentration of 8.7 μg/L can be predicted. This P E C corresponds well with the measured concentrations in grab samples of the hospital outflow. It must also be taken into account that most grab samples were collected in the morning when concentrations are assumed to be high. Four hospitals with a total of 2000 beds are situated in the catchment area of the investigated WWTP. This corresponds to an estimated consumption of 16 kg ciprofloxacin per year. According to the Swiss market statistics, the domestic consumption (not including hospitals) in 1998 was approximately 1,400 kg corresponding to an annual consumption of 184 mg per person. With a population of 450,000 inhabitants in the catchment area of the W W T P , this yields a private consumption of 83 kg/y. Assuming an excretion of 50% as parent compound, a ciprofloxacin concentration of 0.8 μg/L in the raw sewage of the W W T P can be predicted. This value correlates well with the measured concentrations between 0.25-0.37 μg/L in the inflow, especially i f we take into account that ciprofloxacin adsorbs to particles in the sewer and in the primary clarifier.


66

Table II. Predicted (PEC) and measured (MEC) environmental concentrations of ciprofloxacin Compartment Ciprofloxacin Wastewater consumption discharge [kg/y] [10 m /y] 6


1 Hospital (1000 beds)

4 Hospitals in catchment area (2000 beds)

3

PEC

MEC

[ g/LJ

f g/LJ M

5-90 (grab samples, wastewater)

0.459

16 ^ (estimated)

62.05 Domestic consumption in catchment

M

83

6

0.8*

0.25-0.37 (24 h samples, inflow)

area

"Assumption: 50% excreted as parent compound in urine. ^Annual private consumption in Switzerland (1998): 1.41, corresponding to 184 mg per person; population in the WWTP catchment area: 450,000.

Macrolides and Sulfonamides Macrolide and sulfonamide antibiotics were determined in the outflow of four wastewater treatment plants (24-h composite and grab samples) and in several surface water grab samples from various lakes and rivers in Switzerland. W W T P inflow samples were also collected. However, quantification proved impossible with the currently used method because of matrix effects. The ranges of measured concentrations in W W T P outflows and in surface waters are summarized in Figure 6. Minimum concentrations correspond to the lowest concentrations measured or to the limit of detection i f nothing was detected. From the group of macrolide antibiotics, erythromycin, clarithromycin, roxithromycin, tylosin, and spiramycin were determined. O f these five antibiotics, only clarithromycin and roxithromycin were found in the samples, at concentrations of up to 131 and 35 ng/L, respectively. These findings are not surprising, considering that in Switzerland clarithromycin is consumed in the


67 highest quantities of all macrolide antibiotics used in human medicine. Erythromycin was not detected in its original form but as a degradation product with an apparent loss of water (erythromycin-H 0). This degradation product no longer exhibits antibiotic properties (16). Spiramycin and tylosin were not detected. Both antibiotics are used in veterinary medicine, whereas erythromycin, roxithromycin, and clarithromycin are only used for humans. The three latter macrolide antibiotics were also found in the surface water samples. Concentrations were between 2 and 18 ng/L. The measurements indicate that these human-use antibiotics reach the surface water by the outflow of the W W T P and are found in ambient waters in concentrations corresponding to a dilution factor of two to ten. Results from measurements of sulfonamide antibiotics (sulfamethoxazole, sulfamethazine, sulfaguanidine, and sulfadiazine), trimethoprim, and chlor amphenicol are also summarized in Figure 6. Sulfamethoxazole, which is mainly used in human medicine, was found in relatively high concentrations of up to 473 ng/L in W W T P outflows. A dilution factor of approximately 15 fold was found for the concentrations in surface waters. The same behavior was observed for trimethoprim, which is normally used in combination with sulfamethoxazole in a ratio of 1:5.


2

A significantly different picture arises from the measurements of chloramphenicol and sulfamethazine, which are mainly used in veterinary medicine. Both antibiotics were found in higher concentrations in surface waters than in W W T P outflows. Chloramphenicol was detected in only one W W T P outflow sample at a level of 22 ng/L, and only two surface water samples showed concentrations of 10 and 30 ng/L, respectively. The relatively high concentration in one W W T P outflow sample may have been the result of a point input of chloramphenicol applied in human treatment. In general, chloramphenicol did not occur regularly in the aquatic environment. Sulfamethazine was also found in only one W W T P outflow sample in a concentration of 4 ng/L, which may have been caused by improper disposal of veterinary drugs into sewage. However, sulfamethazine was present in nearly all surface water samples in concentrations of up to 54 ng/L. Sulfamethazine is used in the highest quantities of all sulfonamides applied in veterinary medicine, but exact amounts are not available. Sulfaguanidine and sulfadiazine, both mainly used in veterinary medicine, were not found at detectable concentrations. These results indicate that the expected exposure route of veterinary antibiotics reaching surface water is mainly by run-off and field drains from manure application and animal excretion on soils.



68

Figure 6. Concentration ranges of antibiotics found in WWTP outflows and in ambient waters.

Conclusions and Outlook Antibiotics are ubiquitous trace contaminants in the aquatic environment. Due to their physico-chemical properties, their elimination in W W T P is not complete. Thus, residual amounts can reach the aquatic environment. This is the predominant exposure route for antibiotics used in human medicine. Antibiotics employed in veterinary medicine can enter surface waters by run-off and field drains from soils where they were applied by agricultural activity. Studies on several sulfonamide antibiotics used in human and veterinary medicine confirai these different exposure routes. The presented work shows the occurrence of selected macrolide and sulfonamide antibiotics in W W T P outflows and in ambient waters. Further research will focus on the elimination of these antibiotics in WWTPs depending on different treatment processes and will incorporate studies on degradation and sorption behavior. However, L C / M S / M S is needed to overcome problems with quantifying antibiotics in W W T P inflow samples due to matrix effects. Our findings indicate that ciprofloxacin occurs in treated wastewaters in concentrations that, in combination with other fluoroquinolone antibiotics, might have effects on some of the most susceptible aquatic organisms. Ciprofloxacin was used as a compound to investigate the daily input variations of human antibiotics in surface waters. However, it has to be considered that fluoroquinolone antibiotics comprise a group of individual chemicals. Therefore, for an exposure assessment for these antibiotics, the environmental fate of other fluoroquinolones also needs to be studied (17).


69

Acknowledgments We thank Bayer A G (Wuppertal, Germany) for supplying the reference compound ciprofloxacin and financial support. The ICSC-World Laboratory is gratefully acknowledged for the Wilhelm Simon fellowship provided to N . S . Nipales. We thank Abbott GmbH (Wiesbaden, Germany) for the supply of clarithromycin. We appreciate the comments on the manuscript by Erika Vye.


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