Upload
others
View
2
Download
0
Embed Size (px)
Citation preview
Research Collection
Doctoral Thesis
Occurrence and fate of sulfonamide and macrolideantimicrobials in wastewater treatment
Author(s): Göbel, Anke
Publication Date: 2004
Permanent Link: https://doi.org/10.3929/ethz-a-004930749
Rights / License: In Copyright - Non-Commercial Use Permitted
This page was generated automatically upon download from the ETH Zurich Research Collection. For moreinformation please consult the Terms of use.
ETH Library
Diss.ETHNo. 15703
Occurrence and Fate of
Sulfonamide and Macrolide Antimicrobials
in Wastewater Treatment
A dissertation submitted to the
SWISS FEDERAL INSTITUTE OF TECHNOLOGY ZURICH
for the degree of
Doctor of Science
presented by
ANKE GÖBEL
Lebensmittelchemikerin
University of Bonn
born on March 14, 1975
citizen of Germany
accepted on recommendation of
Prof. Dr. Walter Giger, examiner
Dr. Christa S. MeArdell, co-examiner
Prof. Dr. Hansruedi Siegrist, co-examiner
PD Dr. habil. Thomas A. Ternes, co-examiner
Zürich, 2004
I
Table of Contents
Summary V
Zusammenfassung VII
Abbreviations IX
1 Introduction 13
1.1 Pharmaceuticals in the Environment 15
1.2 Selection of Antimicrobials 18
1.3 Research Framework 21
1.4 Scope of this Study 23
1.5 Literature cited 25
2 Analytical Method for Wastewater 29
2.1 Introduction 31
2.2 Experimental Section 34
2.2.1 Chemicals and Reagents 34
2.2.2 Internal Standards 35
2.2.3 Sample Collection and Preparation 36
2.2.4 Liquid Chromatography 37
2.2.5 Tandem Mass Spectrometry 37
2.2.6 Method Validation 39
2.2.7 Identification and Quantification 40
2.3 Results and Discussion 41
2.3.1 Method Development 41
2.3.2 Method Validation 45
2.3.3 Wastewater Application 48
2.4 Conclusions 50
2.5 Literature cited 51
3 Analytical Method for Sewage Sludge 55
3.1 Introduction 57
3.2 Experimental Section 58
3.2.1 Chemicals and Reagents 58
3.2.2 Sample Collection 60
3.2.3 Sample Preparation 60
II
3.2.4 Chemical Analysis
3.2.5 Method Development
3.2.6 Method Validation
3.3 Results and Discussion
3.3.1 Method Development
3.3.2 Method Validation
3.3.3 Application to Sewage Sludge Sampl3.4 Conclusions
3.5 Acknowledgments
3.6 Literature cited
4 Occurrence in Wastewater Treatment
4.1 Introduction
4.2 Experimental Section
4.2.1 Wastewater Treatment Plants
4.2.2 Sample Collection
4.2.3 Analytical Methods
4.2.4 Estimation of Sorption Constants
4.2.5 Calculation of Loads
4.3 Results and Discussion
4.3.1 Occurrence in Wastewater Samples4.3.2 Daily Variations
4.3.3 Seasonal Differences
4.3.4 Occurrence in Sewage Sludge4.3.5 Sorption to Sewage Sludge
4.3.6 Mass Balances
4.4 Conclusions
4.5 Literature cited
5 Behavior in Wastewater Treatment
5.1 Introduction
5.2 Experimental Section
5.2.1 Wastewater Treatment Plants
5.2.2 Sample Collection
5.2.3 Chemical Analysis
5.2.4 Calculated Elimination
m
5.3 Results and Discussion
5.3.1 Primary Treatment
5.3.2 Secondary Treatment
5.3.3 Solid Retention Time
5.3.4 Substrate Dependencies
5.3.5 Anaerobic Compartment
5.3.6 Wastewater Temperature
5.3.7 Hydraulic Retention Time
5.3.8 Sand Filtration
5.4 Conclusions
5.5 Literature cited
6 Ozonation
6.1 Introduction
6.2 Experimental Section
6.2.1 Ozonation Pilot Plant
6.2.2 Wastewater Effluents
6.2.3 Spiking of Wastewater Effluents
6.2.4 Sample Collection and Chemical Analysis
6.2.5 Calculation of Relative Residual
6.3 Results and Discussion
6.3.1 Spiking of Wastewater Effluents
6.3.2 Oxidation Efficiencies
6.3.3 Influence of the Wastewater Matrix
6.3.4 Comparison of Spiked and Non-Spiked Sampl
6.3.5 Conclusions
6.4 References cited
7 Conclusions and Outlook
Seite Leer /Blank leaf
V
Summary
Antimicrobials are used worldwide in human medicine for the treatment of
infections. Their environmental occurrence and fate is of particular interest, due
to the potential spread and maintenance of antibacterial resistance. In
Switzerland, the annual human consumption of sulfonamides and macrolides,
two major groups of antimicrobials, amounts to approximately 6 and 4 t/a,
respectively. After consumption, human-use pharmaceuticals mainly enter
municipal wastewater treatment as the unchanged substances or as human
metabolites, and can eventually reach receiving surface waters. Their behavior
in wastewater treatment, as well as the efficiencies of distinct treatment steps
and technologies are largely unknown.
In this study, the occurrence, behavior and fate of sulfonamides, macrolides and
trimethoprim in wastewater treatment was investigated. Field studies were
performed in full-scale and pilot wastewater treatment plants, focusing on
activated sludge treatment, a fixed-bed reactor and a membrane bioreactor,
operated at three different solid retention times (SRT). In addition, the ozonation
of wastewater effluents was investigated as a procedure to reduce the loads of
antimicrobials entering the aquatic environment.
Analytical methods were developed and validated for the determination of
sulfonamides, macrolides and trimethoprim in aqueous and solid samples.
Pressurized liquid extraction was used for the extraction of activated and
digested sewage sludge. Solid-phase extraction and reversed-phase liquid
chromatography was applied to diluted sludge extracts and water samples. For
the detection and quantitative determination of the analytes, tandem mass
spectrometry with electrospray positive ionization in the multiple reaction mode
was used. Limits of quantification ranged between 3 and 214 ng/L in primary
effluent and between 1 and 23 ng/L in secondary and tertiary effluent. For
activated sludge samples they varied between 3 and 41 u,g/kg dry weight.
Relative recoveries were generally above 80% and the combined measurement
uncertainty ranged between 2.4 and 16% for aqueous samples.
Mass balances were performed, including sorption to sewage sludge, to assess
the elimination of sulfonamides, macrolides and trimethoprim in wastewater
treatment. In accordance to consumption data, clarithromycin and sulfa¬
methoxazole were the most predominant macrolide and sulfonamide,
respectively, found in Swiss wastewater. In the case of sulfamethoxazole it
VI
proved to be crucial to include the amount present as the main human
metabolite, A^-acetylsulfamethoxazole. No significant elimination was observed
for all investigated compounds in primary treatment. During secondary
treatment, the elimination observed depended on the treatment technology
investigated. Overall, sorption to sludge is of minor importance for all
compounds investigated. The predicted sorption constants for activated sludge
ranged below 500 L/kg, which results in an elimination of below 10%, assuming
a sludge production of 0.2 g/L.
Different treatment technologies were investigated for secondary wastewater
treatment concerning the elimination of the selected compounds. Similar results
were obtained in two conventional activated sludge systems and a fixed-bed
reactor. While no significant removal was observed for trimethoprim,
sulfamethoxazole, including the amount present as A^-acetylsulfamethoxazole,was eliminated by about 60%. For the macrolides investigated the results varied
irregularly between sampling campaigns in the two conventional activated
sludge systems and the fixed-bed reactor, with an elimination of up to 55% in
one single case. Approximately 80% of the total sulfamethoxazole load was
eliminated in the membrane bioreactor, independently of the SRT. Macrolides
and trimethoprim were generally eliminated by 25-50% at a SRT of 16 ± 2 and
33 ± 3 days in the membrane bioreactor. Significantly higher elimination of up
to 90% was observed at a SRT of 60 - 80 days for these compounds. High SRT
and resulting low substrate loading therefore seem to have an influence on the
diversity of the microbial population and consequently on the multitude of
degradation pathways being expressed. Elimination in tertiary treatment was
only observed for macrolides and trimethoprim in one of the sand filters
investigated and appeared to be oxygen limited.
Ozonation proved to be efficient for removing sulfonamides, macrolides and
trimethoprim from wastewater effluents. An ozone dose of 2 mg/L resulted in a
reduction of by over 90%, showing no dependence on the amount of suspended
solids present.
The presented study provides a representative example for the investigation of
human-use pharmaceuticals in wastewater treatment, necessary for a more
profound environmental risk assessment of these compounds. The results from
different treatment conditions and technologies show the significance of various
parameters for the elimination of sulfonamides, macrolides and trimethoprim in
wastewater treatment.
VII
Zusammenfassung
Antimikrobielle Wirkstoffe werden weltweit in der Humanmedizin zur
Behandlung von Infektionskrankheiten verwendet. Ihr Vorkommen und
Verhalten in der Umwelt ist von besonderem Interesse, da ein Zusammenhang
mit der Verbreitung und dem Erhalt von Antibiotikaresistenzen nicht aus¬
geschlossen werden kann. Der jährliche Verbrauch an Makroliden und Sulfon¬
amiden, zwei in der Humanmedizin wichtigen Antibiotikaklassen, beläuft sich
in der Schweiz auf ungefähr 6 bzw. 4 Tonnen pro Jahr. Arzneimittel gelangen
nach der Einnahme durch den Patienten als unveränderte Substanz oder in Form
von Metaboliten in die Abwasserreinigung und können auf diesem Weg
schließlich auch in die Oberflächengewässer gelangen. Ihr Verhalten in der
Abwasserreinigung sowie die Eliminationsleistung einzelner Behandlungsstufen
und verschiedener Reinigungstechnologien ist weitgehend unbekannt.
Vorkommen, Verhalten und Schicksal von Sulfonamiden, Makroliden und
Trimethoprim in der Abwasserreinigung war daher das Thema dieser Arbeit.
Dazu wurden Feldstudien in bestehenden Kläranlagen sowie in Pilotanlagen
durchgeführt mit dem Ziel, Belebtschlammverfahren, Biofilter sowie einen
Membranbioreaktor bei drei verschiedenen Schlammaltern zu vergleichen.
Zusätzlich untersucht wurde die Ozonung von Abwasser, als ein mögliches
Verfahren zur weiteren Verringerung der Umweltbelastung durch Antibiotika.
Analytische Methoden für die quantitative Bestimmung in Abwasser und
Klärschlamm wurden entwickelt und validiert. Belebt- und Faulschlammproben
wurden unter erhöhtem Druck und erhöhter Temperatur extrahiert. Die
verdünnten Extrakte und Abwasserproben wurden mittels Festphasenextraktion
und Flüssigchromatographie analysiert. Für die Detektion der Analyten wurde
Tandemmassenspektrometrie mit positiver Elektrospray-Ionisation verwendet.
Die Bestimmungsgrenzen bewegten sich zwischen 3 und 214 ng/L für Zuläufe
und zwischen 1 und 23 ng/L für Abläufe. Im Belebtschlamm lagen sie zwischen
3 und 41 u.g/kg Trockengewicht. Relative Wiederfindungsraten waren im
Normalfall größer als 80%, und die kombinierte Messunsicherheit betrug 2.4 bis
16% für Abwasserproben.
Mit Hilfe von Massenbilanzen wurde die Elimination der ausgewählten
Substanzen in der Abwasserreinigung untersucht. In Übereinstimmung mit den
vorliegenden Verbrauchszahlen, wurden Sulfamethoxazol und Clarithromycin
als Hauptvertreter der Sulfonamide, bzw. Makrolide in schweizerischem
VIII
Abwasser identifiziert. Für Sulfamethoxazol stellte sich das Einbeziehen des
mengenmäßig wichtigsten menschlichen Metaboliten A^-Acetylsulfa-methoxazol als unerlässlich heraus.
Keine signifikante Elimination der untersuchten Substanzen konnte in der
Vorklärung festgestellt werden. Unterschiedliche Eliminationsleistungen wurden
für die verschiedenen biologischen Abwasserreinigungsverfahren
beobachtet. Für alle untersuchten Substanzen stellte sich die Sorption an
Schlamm als unbedeutend heraus. Für die abgeschätzten Sorptionskonstanten
von unter 500 L/kg Überschussschlamm, liegt diese unter 10% bei einer
Schlammproduktion von 0.2 g/L. Vergleichbare Eliminationen wurde in zwei
Belebtschlammsystemen und dem Biofilter ermittelt. Im Fall von Trimethoprim
konnte kein signifikanter Abbau festgestellt werden, während Sulfamethoxazol
inklusive A^-Acetylsulfamethoxazol zu ca. 60% eliminiert wurden. Für die
Makrolide wurden stark schwankende Eliminationsleistungen in den
verschiedenen Probenahmen für diese drei Systeme ermittelt, mit einzelnen
Höchstwerten von 55%. Im Membranbioreaktor wurde unabhängig vom
Schlammalter eine Elimination von ca. 80% für die Gesamtmenge an
Sulfamethoxazol beobachtet. Makrolid- und Trimethoprim-Frachten wurden
zwischen 25 und 50% im Membranbioreaktor reduziert bei Schlammaltern von
16 ± 2 und 33 ± 3 Tagen, während signifikant höhere Eliminationsleistungen
von bis zu 90% bei 60 - 80 Tagen für diese Substanzen beobachtet wurden. Ein
erhöhtes Schlammalter und die daraus resultierende reduzierte
Schlammbelastung scheinen einen Einfluss auf die mikrobielle Vielfalt und
damit auf die Anzahl der möglichen Abbauwege zu haben. Eine Elimination der
untersuchten Substanzen während der abschließenden Filtration wurde nur für
Makrolide und Trimethoprim und nur auf einem der untersuchten Sandfilter
beobachtet. Die Elimination scheint durch Sauerstoff limitiert zu sein.
Die Ozonung von Abwasser hat sich als wirkungsvolles Verfahren für die
Oxidation von Sulfonamiden, Makroliden und Trimethoprim herausgestellt.
Bereits bei Ozongehalten von 2 mg/L wurde eine Elimination von über 90%
festgestellt, unabhängig von der Menge an Schwebstoffen im Abwasser.
In dieser Arbeit wird exemplarisch die Untersuchung von Humanarzneimitteln
in der Abwasserreinigung gezeigt, deren Ergebnisse zu einer umfassenderen
Risikobeurteilung dieser Substanzen beiträgt. Weiterhin konnte der Einfluss
verschiedener Faktoren auf die Elimination von Sulfonamiden, Makroliden und
Trimethoprim in der Abwasserreinigung aufgezeigt werden.
IX
Abbreviations
1°EFFL primary effluent
2°EFFL secondary effluent
3°EFFL tertiary effluent
13C6SMZ sulfamethazine-phenyl-"CtfASE accelerated solvent extraction
AZI azithromycin
BOD5 biological oxygen demand in 5 days
v^/\Ï3 conventional activated sludge
CASRN chemical abstract services registry number
CLA clarithromycin
CMU combined measurement uncertainty
COD chemical oxygen demand
d4SDZ sulfadiazine-«/
d4SMX sulfamethoxazole-«/
d4STZ sulfathiazole-«/
d5N4AcSMX A^-acetylsulfamethoxazole-d5DOC dissolved organic carbon
dw dry weight
EAWAG Swiss Federal Institute of Environmental Science and
Technology
ERY erythromycin
ERY-H20 dehydro-erythromycin
ETH Swiss Federal Institute of Technology
FBR fixed-bed reactor
H NMR proton nuclear magnetic resonance spectroscopy
HPLC high performance liquid chromatography
HUMABRA acronym for the Swiss national research project (NRP 49)
entitled "Occurrence of human-use antibiotics and antibiotic
resistance in the aquatic environment"
JOS josamycin
Kd sorption constant
Koc organic carbon normalized sorption constant
LC liquid chromatography
LOD limit of detection
X
LOQ
m/z
MBR
MEC
MS
MS/MS
V
N4
N4AcSMX
N4AcSMZ
NRP
Ntot
PE
PLE
POSEIDON
PPCP
Ptot
R
RAW
ROX
RT
S/N
SD
SDZ
SF
SMR
SMX
SMZ
SPE
SPY
STZ
t/a
limit of quantification
mass-to-charge ratio
membrane bioreactor
measured environmental concentration
mass spectrometry
tandem mass spectrometry
nitrogen in the sulfonamide group of sulfonamides
nitrogen in the para-amino group of sulfonamides
A^-acetylsulfamethoxazole//-acetylsulfamethazinenational research project
total nitrogen concentration
population equivalent
pressurized liquid extraction
acronym for the European project entiteld "Assessment of
Technologies for the Removal of Pharmaceuticals and
Personal Care Products in Sewage and Drinking Water
Facilities to Improve the Indirect Potable Water Reuse"
(EVK1-CT-2000-00047)
pharmaceutical and personal care product
total phosphorus concentration
substituent
raw influent
roxithromycin
retention time
signal-to-noise ratio
standard deviation
sulfadiazine
sand filter
sulfamerazin
sulfamethoxazole
sulfamethazine
solid-phase extraction
sulfapyridine
sulfathiazole
tons per annum
XI
TRI trimethoprimTSS total amount of suspended solids
TYL tylosin
USE ultrasonic solvent extraction
WWTP wastewater treatment plant
WWTP-A municipal WWTP at Altenrhein, Switzerland
WWTP-K municipal WWTP at Kloten-Opfikon, Switzerland
WWTP-W municipal WWTP at Wiesbaden, Germany
1 W W 1 "W Vf* I #
^ i ris i i\ i<< rî % : l
Chapter 1
Introduction
1 V» 1 ,~=
8 1% ^i „'
Introduction 15
1.1 Pharmaceuticals in the Environment
The protection of water resources for drinking water production, agricultural
crops, recreational activities and natural reserves from contamination is one of
the main issues in a sustainable water policy. In ecosystems and drinking water,
the absence of contaminants is particularly required based on the precautionary
principle. Within the large group of contaminants identified until today, the
occurrence of pharmaceuticals in the environment is of special interest. Due to
their high biological activity, possible adverse effects may occur in the
environment, which are so far not covered by conventional ecotoxicological
testing procedures. In contrast to the large number of high volume chemicals
applied in agriculture, industry and households, a complete ban or even a
significant use reduction of pharmaceuticals can not be envisaged because of the
immense benefits of these substances for society.
Pharmaceuticals comprise a very broad and diverse spectrum of hundreds of
chemical substances, including prescription and over-the-counter therapeutic
drugs, diagnostic agents, and many others. In the last decade, the attention of
researchers, authorities and the public has been drawn to the occurrence and fate
of pharmaceuticals in the environment. This development is strongly correlated
to the simultaneous improvements in chemical analysis enabling the detection
and quantitative trace determination of these mostly highly polar and
hydrophilic contaminants. Therefore, pharmaceuticals are considered as
emerging contaminants, even though it must be assumed that they have been
present in wastewater and in ambient waters as long as they have been used.
In 1976, Garrison and co-workers firstly reported the presence of salicylic acid
and clofibric acid, the metabolite of Clofibrate, a lipid regulator, in domestic
wastewater.[1] Approximately one decade later, in 1985, a wide variety of
pharmaceuticals was detected by Richardson and Bowron in wastewater
treatment plant effluents and surface waters with estimated concentrations
ranging up to 1 u.g/L.l2J From the beginning of the 1990's the occurrence of
pharmaceuticals in the environment has gained more and more interest in
Europe, and later worldwide, starting with Stan and Linkenäger,[3] who found
clofibric acid in groundwater in Berlin, Germany. This led to comprehensive
monitoring studies, first performed by Ternes et al.[4] Until today, over 80
different pharmaceutical compounds have been measured in various samples
and the results are summarized and discussed in many articles and
16 Chapter 1
reviews (e.g. [4-13]). The main interest has been the occurrence and
concentration range of pharmaceuticals in mainly aqueous environmental
compartments. Further research, however, is necessary to better understand the
behavior and ultimate fate of pharmaceuticals in the environment after their
application.
A large number of pharmaceutical compounds is used today in human and
veterinary medicine, reaching the environment through their manufacture, use
and disposal. Figure 1.1 shows the main exposure routes of pharmaceuticals into
the environment.
After their medicinal application and excretion via urine and feces, human used
pharmaceuticals mainly enter municipal sewage treatment plants. In Switzerland
over 95% of the population is connected to sewer systems. However, direct
input of human used pharmaceuticals into natural waters is also possible during
rain events and by leaks in the sewers. The behavior and fate of pharmaceutical
compounds in wastewater treatment is mostly unknown. Their frequent
Figure 1.1 Principal routes ofenvironmental exposure to pharmaceuticals
veterinaryuse
fish
farming human
use
manure
run¬
off
over
flow
sewage
soil <H- + -h-
leaks
treatment
plant
-^
surface water <^>
disposal
municipalwaste
landfill
groundwater
drinking water
Introduction 17
detection in wastewater effluents and receiving surface waters (e.g. [4, 14-22]),
however, suggests an incomplete removal of many pharmaceuticals. Next to
chemical or biological transformation, sorption to sewage sludge can play a role
in the elimination of pharmaceuticals in wastewater treatment. The use of
sewage sludge as fertilizer on arable land may therefore be an alternative route
to the environment. Up to date, only little data is available on the occurrence of
human used pharmaceuticals in sewage sludge and soil.[23"27]
In veterinary medicine, pharmaceuticals are applied for therapeutic use and as
growth promoters in livestock production. Due to the ban of growth promoters
in many European countries, including Switzerland, the overall consumption has
decreased in recent years. They mainly reach the environment via animal
manure - through the direct urination or defecation on the fields or after the
application of stored manure to arable land. They may therefore reach
groundwater after soil passage, or surface waters due to field runoff during rain
events or through drainage systems. In the field of veterinary pharmaceuticals,
research activities have also increased, mainly focusing on the occurrence and
fate of veterinary pharmaceuticals, especially antimicrobials, in manure, soil and
surface waters (e.g. [28-31]) Through the use as feed additives in fish farming,
veterinary pharmaceuticals are also directly introduced into surface waters
(e.g. [32,33]).
After consumption, pharmaceutical substances are metabolized in the body to
different extents and are excreted only partly unchanged. Metabolism usually
consists of two phases (Figure 1.2). In a first phase pharmaceutical compounds
may undergo an oxidation, reduction or hydrolysis reaction, resulting in the
introduction of reactive functional groups. In a second phase the pharmaceutical
or its first phase metabolite can then be covalently bound to polar molecules,
e.g. acids, sulfates or sugars. As a conclusion, metabolism usually results in
compounds with a higher polarity. Being more hydrophilic than the original
compound, metabolites are more easily excreted from the body. The excreted
metabolites, exhibiting diverse biological activity, increase the already complex
mixture of pharmaceuticals. In some cases the predominant form of a
pharmaceutical in the environment may be its metabolite (e.g. clofibric acid).
Additionally, metabolites may be transformed back to the parent compound
under environmental conditions or during wastewater treatment
(e.g. deglucuronidation). However, metabolites are so far generally not included
18 Chapter 1
Figure 1.2 Metabolism ofpharmaceuticals
metabolite
e.g. oxidation,
reduction, hydrolysis
conjugation
e.g. glucuronidation,
acetylation
phase 1
i
pharmaceutical —
r
metabolite
phase II
in studies on the occurrence, behavior and fate of pharmaceuticals in the
environment, with a few exceptions (e.g. dehydro-erythromycin, clofibric acid
and salicylic acid). This can mainly be attributed to the usually high polarity of
the metabolites and the lack of reference substances, both rendering the
determination of these compounds difficult.
Overall, it can be stated, that residual concentrations of pharmaceuticals,
unchanged or partly transformed, are continuously discharged to the
environment. Although pharmaceuticals are usually not included in legal
regulation, e.g. the priority list of the European Water Framework Directive, the
precautionary principle implies an efficient removal of these potentially harmful
substances. In some cases, pharmaceuticals have also been detected in
groundwater and were generally connected to specific input sources, e.g.
wastewater, agricultural use, or landfill sites.l34"37] Occasionally pharmaceutical
concentrations in the lower nanogram per liter range were found in drinking
water, caused by the widespread occurrence of these compounds in the aquatic
environment.f38,39] Even though these concentrations are unlikely to cause
adverse health effects in humans, drinking water should be devoid of
anthropogenic contaminations based on the application of the precautionary
principles.
1.2 Selection of Antimicrobials
The presence of pharmaceuticals in the environment is of concern, since they are
designed to cause specific effects in humans or animals. Within this large group,
antimicrobials are of special concern due to the possible spread and maintenance
Introduction 19
of bacterial resistance. While resistant pathogens are mainly found in clinics,
little is known on the contribution of the low but continuously discharged
antimicrobial concentrations from wastewater treatment to the widespread
occurrence of antimicrobial resistance observed in the environment.[40"44J
Antimicrobials are used in large quantities in human and veterinary medicine,
mainly for the treatment of bacterial infections. The overall consumption of
antimicrobials amounted to ~90 tons in Switzerland in 1997 and -40% thereof
were applied in human medicine.145"47^ Domestic consumption, i.e. during
ambulant treatment, accounts for 60 to 80% of the total human consumption
making urban wastewater the main source. With -18 tons per annum (t/a),
ß-lactam antimicrobials represent the largest fraction of human used
antimicrobials. They include penicillins and cephalosporins and seem to be
hydrolyzed shortly after excretion. Following the class of ß-lactams, macrolides
(4.3 t/a), sulfonamides (5.7 t/a) and fluoroquinolones (3.9 t/a) are mainly used in
human medicine. While the occurrence and fate of fluoroquinolone
antimicrobials in the environment has already been intensively studied,[23'48"50]
little is know regarding sulfonamide and macrolide antimicrobials.
The sulfonamide antimicrobials are a class of synthetic compounds derived from
sulfanilamide, whose antibacterial activity was discovered in the early 1930> s by
Domagk and Tréfouel.[51] Their core structure is shown in Figure 1.3. The acid
dissociation constants of the two amino groups in the molecule range between
1.7 and 2.4 for the protonated/>-amino group (TV4) and between 5.0 and 8.5 for
Figure 1.3 Core structures ofsulfonamide (A) and 14-membered-ring macrolide
antimicrobials (B)
A)
N4
X
M /
Nl
o
-s-
IIo
R1
/
\
V, TV4 = numbering of the nitrogens
RI, R2 = varying substituents
P—R1
20 Chapter 1
the sulfonamide nitrogen (TV1) in commonly used representatives. VaryingN1 substituents results in a large variety of compounds with a wide range of
pharmacological properties. Substitution on the /?-amino group, however, leads
to inactive compounds, since sulfonamides act as competitive antagonist of
/»-aminobenzoic acid in bacterial folic acid synthesis. Sulfonamides are
prescribed against a wide variety of bacterial infections, especially in the case of
a potential hypersensitivity to penicillins. However, the widespread bacterial
resistance to these compounds limits their application spectrum today. In
monotherapy, sulfonamides lead to a bacteristatic effect, while a combination
with trimethoprim, a diaminopyridin derivative, results in a bactericidal effect.
Trimethoprim, almost exclusively used in combination with sulfonamides, also
interferes with the bacterial folic acid synthesis through inhibition of the
bacterial dihydrofolate reductase.[52] Sulfonamides are metabolized to a varying
extent in the human body (e.g. by A^-acetylation and hydroxylation) and are
subsequently excreted mainly via the urine. Figure 1.4 shows the main human
metabolite of sulfamethoxazole, the most predominant sulfonamide in human
medicine. Approximately 50% of the administered dose is excreted as the
inactive metabolite A^-acetylsulfamethoxazole.[53] Sulfapyridine, the other
sulfonamide important in human medicine, is not administered as antimicrobial
agent directly, but in form of sulfasalazine, which is mainly used in the
treatment of ulcerative colitis and rheumatoid arthritis.[54] In sulfasalazine,
sulfapyridine is linked to 5-aminosalicylic acid via an azo bridge, which is
cleaved in the colon (Figure 1.5).
Figure 1.4 Main human metabolite ofsulfamethoxazole
c
/o
/o s phase I
H2N—<v />—S—NH—& I HN—(v /)—S—NH—<\
O
-NH—C
sulfamethoxazole A^-acetylsulfamethoxazole(50% of the administered dose)
Introduction 21
Figure 1.5 Chemical structure ofsulfasalazine
HOOC
~0-JhH~0Macrolide antimicrobials are derived from natural sources and are therefore also
classified as antibiotics.[55] They consist of a macrocyclic lactone ring attached
to one or more amino or neutral sugars. The ring structure is usually substituted
by various functional groups, e.g. hydroxyl, methyl or methoxy groups. In
Figure 1.3 the core structure of 14-membered-ring macrolides is given,
representative for the most commonly used macrolides in human medicine -
erythromycin and clarithromycin. The acid dissociation constant of the
protonated tertiary amine in the attached sugar ranges between 8.7 and 9.2,
making macrolides weakly basic. Their mode of action is based on the inhibition
of bacterial protein synthesis by a reversible interaction with the bacterial 50S
ribosomal subunit. Macrolides are active against a variety of gram-positive
bacteria and are mainly applied in the treatment of respiratory tract infections.
After consumption, macrolides are excreted mainly via the feces. Metabolism,
including 7V-demethylation of the amino groups and hydroxylation of the ring, is
generally of minor importance and macrolides are predominately excreted
unchanged.[55] As an example the main human metabolites of clarithromycin, the
most commonly used macrolide in human medicine are given in Figure 1.6.
1.3 Research Framework
The occurrence and fate of organic micropollutants in the environment has
already been a focus of the research at EAWAG for a long time. Field studies in
wastewater treatment plants, groundwater, rivers and lakes, and hospital
effluents as well as controlled laboratory studies were performed for a large
variety of water pollutants. In the area of human-use antimicrobials, the first
investigations dealt with the fluoroquinolone ciprofloxacin, for which genotoxic
studies in wastewaters of hospitals were performed.[56] Subsequently, several
fluoroquinolone antimicrobial agents were intensively studied in a dissertation
by Eva Golet.[57] After the development of analytical methods for aqueous
22 Chapter 1
Figure 1.6 Main human metabolites ofclarithromycin
clarithromycin
^\CH3
IH0
/
14-OH-(R)-clarithromycin
(20% of the administered dose)
*~ H0'**.../ Ö HO .
CH, "\OH
14-OH-(R)-N-demethyl-clarithromycin(13% of the administered dose)
and solid samples,[23] the occurrence and fate of flouroquinolones were
investigated in rivers, wastewater treatment and sludge-treated soil.[48,50] The
occurrence of sulfonamide and macrolide antimicrobials has been investigated
in several rivers and lakes as well as wastewater treatment plant effluents by
McArdell et al., followed by a detailed study on the environmental behavior of
macrolides in the Glatt valley watershed.[20] Within HUMABRA, a project in the
framework of the national research program NRP 49 on antibiotic resistance of
the Swiss National Science Foundation, the question of a potential
environmental risk of antimicrobials occurring as trace contaminants concerning
resistance is addressed.[43] In particular, residual levels of human-use antibiotics
are determined in wastewater, hospital effluents and in ambient waters.
Correlations between the measured concentrations and resistant bacteria strains
are investigated.
This dissertation is closely related to the European project POSEIDON, in which
eight research groups from seven countries are involved.[58'59] It is focused on the
Introduction 23
assessment and improvement of technologies for the removal of pharmaceuticals
and personal care products (PPCPs) in wastewater and drinking water treatment
facilities. Within POSEIDON eleven different compounds belonging to various
PPCP classes were chosen according to the amount consumed, chemical
properties, possible adverse effects and the availability of reference compounds.
Sulfamethoxazole and roxithromycin were included as antimicrobials. The
overall goal of the project is to reduce the contamination of receiving waters,
groundwater and drinking water with PPCPs from treated wastewater by
planned and unplanned indirect reuse. This clearly illustrates the need of
integrating different scientific disciplines, e.g. chemists and engineers, to
address the questions raised by environmental PPCP contamination. In
particular, in the field of water pollution, an integrated approach is required to
assure the sustainability of water use and re-cycling for various purposes.
Parallel to this study, Felix Wettstein has investigated in his dissertation
nonylphenoxy acetic acid and other nonylphenol compounds in wastewater
treatment field studies similar to those presented here.f60] Results obtained for
this very different class of organic substances allow interesting comparisons
with the selected antimicrobials.
1.4 Scope of this Study
This thesis project aimed at investigating the occurrence and behavior of
sulfonamides, macrolides and trimethoprim in municipal wastewater treatment.
The pathway of selected constituents was traced from the raw influents to the
final effluents of wastewater treatment plants. Different treatment stages and
technologies were evaluated with respect to their elimination efficiencies for
selected antimicrobials. Within this dissertation the following topics and
questions have been thoroughly addressed:
Analytical methods
Reliable and specific analytical methods for many different types of sample
matrices are essential to investigate the occurrence and fate of pharmaceuticals
in the environment. One of the main goals of this work was to develop the
methods necessary to investigate the selected substances in wastewater
treatment. Methods were validated for the simultaneous trace determination of
24 Chapter 1
sulfonamides, macrolide and trimethoprim in various wastewaters (Chapter 2),
and in sewage sludge (Chapter 3). Pressurized liquid extraction (PLE) was
chosen for efficient extraction from solid samples. Enrichment and clean-up of
sludge extracts or wastewater samples was achieved using solid-phase extraction
on polymeric sorbent cartridges. Analyses were performed by liquid
chromatography on a reversed phase column coupled to electrospray positive
mass spectrometry. Tandem mass spectrometry in the multiple reaction mode
provided the sensitivity and selectivity necessary for the analyses of complex
environmental samples, such as raw wastewater and sludge extracts.
Study of municipal wastewater treatment
In the field studies performed, the occurrence of sulfonamides, macrolides and
trimethoprim in municipal wastewaters from the raw influent to the final
effluent was one of the main questions addressed. Additionally, the studies were
designed to elucidate possible eliminations. Conventional activated sludge
treatment, being the most commonly applied wastewater treatment technology,
was investigated in detail (Chapter 4 and 5). In addition to aqueous matrices,
concentrations of the selected analytes were also measured in activated and
digested sewage sludge. Average daily loads were determined as well as daily
variations of antimicrobial loads entering wastewater treatment. By performing
complete mass balances the overall removal of the analytes was determined in
mechanical and biological municipal wastewater treatment. Furthermore, the
efficiency of individual treatment steps for the removal of sulfonamides,
macrolides and trimethoprim was investigated in depth. By attributing the
observed eliminations to different extents to transformation and sorption
processes, respectively, new information on the sorption characteristics of these
compounds could be gained.
Evaluation of different treatment technologies
The influence of various factors on the elimination of micropollutants was
addressed by studying the fate of sulfonamides, macrolides and trimethoprim
also in newly developed treatment technologies. Field studies were performed
on a fixed-bed reactor and on a membrane bioreactor (Chapter 5). In the case of
the membrane bioreactor, three different solid retention times were investigated
Introduction 25
in weekly sampling campaigns. The observed eliminations in the different
treatment technologies are discussed in relation to various parameters, e.g.
temperature and solid retention time. In two sand filters, used for tertiary
treatment, removal efficiencies concerning sulfonamides, macrolides and
trimethoprim are compared.
Ozonation of wastewater effluents
Since the studied antimicrobials are not completely eliminated in wastewater
treatment, other measures are necessary to further reduce their loads entering
ambient waters. Ozonation of wastewater effluents was investigated as a
possible additional treatment step (Chapter 6). The removal efficiencies were
determined for several sulfonamides, macrolides and trimethoprim at ozone
doses below 5 mg/L. The influence of suspended solids in the wastewater matrix
was particularly investigated. Using three different wastewater effluents, the
impact ofpH variations on the oxidation process could also be addressed.
1.5 Literature cited
[I] Garrison, A. W.; Pope, J. D.; Allen, F. R. In Identification & Analysis of
organic pollutants in water, Keith, L. H., Ed.; Ann Arbor Science: Ann
Arbor, 1976; pp 517-566.
[2] Richardson, M. L.; Bowron, J. M. J. Pharm. Pharmacol. 1985, 37, 1-12.
[3] Stan, H. J.; Linkerhäger, M. Vom Wasser 1992, 79, 75-88.
[4] Ternes, T. A. Water Res. 1998, 32, 3245-3260.
[5] Stan, H. J.; Heberer, T. Analusis 1997, 25, M20-M23.
[6] Halling-Sorensen, B.; Nors Nielsen, S.; Lanzky, P. F.; Ingerslev, F.; Holten
Lützenhoft, H. C; J0rgensen, S. E. Chemosphere 1998, 36, 357-393.
[7] Daughton, C. D.; Ternes, T. A. Environ. Health Perspect. 1999, 107, 907-
938.
[8] Kümmerer, K. Chemosphere 2001, 45, 957-969.
[9] Heberer, T. Toxicol. Lett. 2002,131, 5-17.
[10] Snyder, S. A.; Westerhoff, P.; Yoon, Y.; Sedlak, D. L. Environ. Eng. Sei.
2003, 20, 449-469.
[II] Debska, J.; Kot-Wasik, A.; Namiesnik, J. Crit. Rev. Anal. Chem. 2004, 34,
51-67.
26 Chapter 1
[12] Rooklidge, S. J. Sei. Total Environ. 2004, 325, 1-13.
[13] Giger, W.; Alder, A. C; Golet, E. M.; Kohler, H.-P. E.; McArdell, C. S.;
Molnar, E.; Siegrist, H. R.; Suter, M. J.-F. Chimia 2003, 57, 485-491.
[14] Stumpf, M.; Ternes, T. A.; Haberer, K.; Seel, P.; Baumann, W. Vom
Wasser 1996, 55,291-303.
[15] Sacher, F.; Lochow, E.; Bethmann, D.; Brauch, H.-J. Vom Wasser 1998, 90,
233-243.
[16] Zuccato, E.; Calamari, D.; Natangelo, M.; Fanelli, R. Lancet 2000, 355,
1789-1790.
[17] Alder, A. C; McArdell, C. S.; Golet, E. M.; Ibric, S.; Molnar, E.; Nipales,
N. S.; Giger, W. In Pharmaceuticals and Personal Care Products in the
Environment: Scientific and Regulatory Issues; Daughton, C. G., Jones-
Lepp, T., Eds.; Symposium Series 791; American Chemical Society:
Washington, D.C., 2001; pp 56-69.
[18] Kolpin, D. W.; Furlong, E. T.; Meyer, M. T.; Thurman, E. M.; Zaugg, S.
D.; Buxton, H. T. Environ. Sei. Technol. 2002, 36, 1202-1211.
[19] Yang, S.; Carlson, K. Water Res. 2003, 37, 4645-4656.
[20] McArdell, C. S.; Molnar, E.; Suter, M. J.-F.; Giger, W. Environ. Sei.
Technol. 2003, 37, 5479-5486.
[21] Metcalfe, C. D.; Miao, X.-S.; Koenig, B. G.; Struger, J. Environ. Toxicol.
Chem. 2003,22,2881-2889.
[22] Miao, X.-S.; Bishay, F.; Chen, M.; Metcalfe, C. D. Environ. Sei. Technol.
2004,55,3533-3541.
[23] Golet, E. M.; Strehler, A.; Aider, A. C; Giger, W. Anal. Chem. 2002, 74,
5455-5462.
[24] Küpper, T.; Berset, J. D.; Etter-Holzer, R.; Tarradellas, J. Chemosphere
2004,54,1111-1120.
[25] Ternes, T. A.; Herrmann, N.; Bonerz, M.; Knacker, T.; Siegrist, H.; Joss, A.
Water Res. 2004,35,4075-4084.
[26] Kreuzig, R.; Kullmer, C; Matthies, B.; Höltge, S.; Dieckmann, H. Fresen.
Environ. Bull. 2003,12, 550-558.
[27] Thiele-Bruhn, S. J. Plant Nutr. Soil Sei. 2003,166, 145-167.
[28] Haller, M. Y.; Müller, S. R.; McArdell, C. S.; Aider, A. C; Suter, M. J.-F.
J. Chromatogr., A 2002, 952, 111-120.
[29] Hamscher, G.; Sczesny, S.; Höper, H.; Nau, H. Anal. Chem. 2002, 74,
1509-1518.
Introduction 27
[30] Tolls, J. Environ. Sei. Technol. 2001, 35, 3397-3406.
[31] Stoob, K. "Fate and occurrence of veterinary sulfonamide antimicrobials in
the environment," PhD Thesis, ETH Zurich, in preparation.
[32] Björklund, H. V.; Rabergh, C. M. I.; Bylund, G. Aquaculture 1991, 97, 85-
97.
[33] Capone, D. G.; Weston, D. P.; Miller, J.; Shoemaker, C. Aquaculture 1996,
145, 55-75.
[34] Sacher, F.; Lange, F. T.; Brauch, H.-J.; Blankenhorn, I. J. Chromatogr., A
2001,935,199-210.
[35] Hirsch, R.; Ternes, T. A.; Haberer, K.; Kratz, K.-L. Sei. Total Environ.
1999,225,109-118.
[36] Holm, J. V.; Rügge, K.; Bjerg, P. L.; Christensen, T. H. Environ. Sei.
Technol. 1995, 29, 1415-1420.
[37] Ahel, M.; Jelicic, I. In Pharmaceuticals andpersonal care products ind the
environment - scientific and regulatory issues; Daughton, CD., Jones-
Lepp, T.L., Ed.; American Chemical Society, ACS Symposium Series 791,
2001; pp 100-115.
[38] Heberer, T.; Stan, H. J. Vom Wasser 1996, 86, 19-31.
[39] Bund/Länderausschuss für Chemikaliensicherheit (BLAC), "Arzneimittel in
der Umwelt - Auswertung der Untersuchungsergebnisse," Hamburg, 2003.
[40] Iwane, F.; Urase, T.; Yamamoto, K. Water Sei. & Technol. 2001, 43, 91-99.
[41]Bendt, T.; Pehl, B.; Gehrt, A.; Rolfs, C.-H. KA - Wasserwirtschaft,
Abwasser, Abfall 2002, 49, 49-56.
[42] Klare, T.; Konstabel, C; Badstübner, D.; Werner, G.; Witte, W. Int. J. Food
Microbiol. 2003, 55, 269-290.
[43] http://www.nrp49.ch/pages/.
[44] Husevag, B.; Lunestad, B. T.; Johannessen, B. J.; Enger, O.; Samuelsen, O.
B. J. Fish Dis. 1991,14, 631-640.
[45] Annual Report; Swiss Importers of Antibiotics (TSA): Berne, Switzerland,
1998.
[46] Pharmaceuticals Sold in Switzerland; Swiss Market Statistics, 1999.
[47] Antibiotics used in Veterinary Medicine; Swiss Federal Office for
Agriculture (BLW): Berne, Switzerland, 2001.
[48] Golet, E. M.; Alder, A. C; Giger, W. Environ. Sei. Technol. 2002, 36,
3645-3651.
25 Chapter 1
[49] Golet, E. M.; Alder, A. C; Hartmann, A.; Ternes, T. A.; Giger, W. Anal.
Chem. 2001,73, 3632-3638.
[50] Golet, E. M.; Xifra, I.; Siegrist, H. R.; Alder, A. C; Giger, W. Environ. Sei.
Technol. 2003, 37, 3243-3249.
[51] Vree, T. B.; Hekster, Y. A. Clinical pharmacokinetics ofsulfonamides and
their metabolites; Karger: Basel, 1987; Vol. 37.
[52] Poe, M. Science 1976,194, 533-535.
[53] Vree, T. B.; Hekster, Y. A. Pharmacokinetics of sulfonamides revisited;
Karger: Basel, New York, 1985; Vol. 34.
[54] Astbury, C; Dixon, J. S. J. Chromatogr. 1987, 414, 223-227.
[55] Bryskier, A. J.; Butzler, J.-P.; Neu, H. C; Tulkens, P. M. Macrolides;
Arnette Blackwell: Paris, 1993.
[56] Hartmann, A.; Alder, A. C; Koller, T.; Widmer, R. M. Environ. Toxicol.
Chem. 1998,77,377-382.
[57] Golet, E. M. "Environmental exposure assessment of fluoroquinolone
antibacterial agents in sewage, river water and soil.," PhD Thesis, No.
14690, ETH Zurich, 2002.
[58] http://www.eu-poseidon.com.
[59] Ternes, T. A. "Final Report ofPOSEIDON," 2004.
[60] Wettstein, F. "Auftreten und Verhalten von Nonylphenoxyessigsäure und
weiteren NonylphenolVerbindungen in der Abwasserreinigung," PhD
Thesis No. 15315, ETH Zurich, 2004.
Chapter 2
Analytical Method for Wastewater
An analytical method has been developed and validated for the simultaneous
trace determination of four macrolide antimicrobials, five sulfonamides, the
human metabolite A^-acetyl-sulfamethoxazole, and trimethoprim in wastewater.
The method was validated for tertiary, secondary, and - unlike in previously
published methods - also for primary effluents of municipal wastewater
treatment plants. This wide range of application is necessary to thoroughly
investigate the occurrence and fate of chemicals in wastewater treatment.
Wastewater samples were enriched by solid-phase extraction, followed by
reversed-phase liquid chromatography coupled to tandem mass spectrometry
using positive electrospray ionization. Recoveries from all sample matrices were
generally above 80%, and the combined measurement uncertainty varied
between 2.4 and 16%. Concentrations measured in tertiary effluents ranged
between 10 ng/L for roxithromycin and 423 ng/L for sulfamethoxazole.
Corresponding levels in primary effluents varied from 22 to 1 450 ng/L,
respectively. Trace amounts of these emerging contaminants reach ambient
waters, since all analytes were not fully eliminated during conventional
activated sludge treatment followed by sand flltration. In the case of
sulfamethoxazole, the amount present as human metabolite A^-acetyl-sulfamethoxazole had to be taken into account in order to correctly assess the
fate of sulfamethoxazole in wastewater treatment.
Göbel, A., McArdell, CS., Suter, M. J.-F., Giger, W.
Trace Determination of Macrolide and Sulfonamide Antimicrobials, a Human
Sulfonamide Metabolite, and Trimethoprim in Wastewater Using Liquid
Chromatography Coupled to Electrospray Tandem Mass Spectrometry
Analytical Chemistry, 2004, 76, 4756 - 4764.
Analytical Methodfor Wastewater 31
2.1 Introduction
Since 1997, interest in the occurrence and behavior of pharmaceuticals in the
aquatic environment has significantly increased.^1"71 One motivation for this
attention is the fact that these chemicals are designed to trigger specific
biological effects, and, hence, pose a potential threat for the aquatic
environment. In the case of antimicrobial agents (including both naturally and
synthetically derived compounds), the possible maintenance and spread of
bacterial resistance is a major point of concern. In 1997, the human consumption
of antimicrobials in Switzerland exceeded 30 tons per annum, (t/a).["
In
addition -55 t/a of antimicrobials were used in veterinary medicine. Macrolides
(4.3 t/a), sulfonamides (5.7 t/a), and fluoroquinolones (3.9 t/a) represent the
most important groups in human medicine, next to ß-lactams (17.5 t/a). The
latter include penicillins and cephalosporins and seem to be hydrolyzed shortly
after excretion.
In industrialized countries, most human used antimicrobials and other human
used pharmaceuticals reach the aquatic environment, unchanged or transformed,
mainly via discharge of effluents from municipal wastewater treatment plants
(WWTPs). The residual concentrations of these bioactive compounds in treated
effluents depend on their removal during wastewater treatment. They can
potentially pose a hazard for aquatic organisms if the removal is incomplete. In
addition, exposure via sewage sludge disposal on land could represent a hazard
for soil organisms.
Detailed knowledge of the behavior of antimicrobials in wastewater treatment
and the aquatic environment will help to achieve a reliable basis for
environmental risk assessment (e.g., by providing measured environmental
concentrations (MECs)). MECs can be used in environmental risk assessment
studies since they provide accurate indications of actual concentrations present
in environmental systems. Investigations on the occurrence and fate of
antimicrobial agents in various wastewater treatment steps can be exploited in
order to evaluate wastewater treatment technologies with respect to elimination
of specific contaminants. Reducing the release of residual pharmaceuticals into
the aquatic environment would presumably decrease any potential
environmental risks. By monitoring receiving surface waters as well as
wastewater treatment plants, locations of particular concern can be identified
and mitigated specifically.
32 Chapter 2
Table 2.1 Investigated compounds
compound acronym CASRN PKal/pKa2
sulfadiazine SDZ 68-35-9 64 [ii]/ 2.1 [12]
sulfathiazole STZ 72-14-0 7.2[11]/2.1[12]
sulfamethazine SMZ 57-68-1 7.4[11]/2.3[12]
sulfapyridine SPY 144-83-2 8.4[11J/2.6[13]
sulfamethoxazole SMX 723-46-6 5.7[1,]/1.8[12J
A^-acetylsulfamethoxazole N4AcSMX 5.0 [U1
trimethoprim TRI 738-70-5 7.2[14J
azithromycin AZI 83905-01-5 87[i5]/95[i5]
erythromycin ERY 114-07-8 8.8 [16]
clarithromycin CLA 81103-11-9 8.9[15]
roxithromycin ROX 80214-83-1 9.2[16]
tylosina TYL 1401-69-0 j i [15]
sulfamerazinea
SMR 127-79-7 7.0[,,]/2.2[12]
josamycina JOS 16846-24-5
Used as internal standard.
To reach the aims stated above, selective and sensitive analytical methods for
many different sample matrices are essential. Until now, published methods for
antimicrobial agents have focused on wastewater treatment plant effluents and
surface waters,tl7~231 with the exception of fluoroquinolones, which were studied
in detail by Golet et al.[2427] Analytical methods for wastewater matrices other
than final effluents including sludge extracts, however, are lacking. Another
important aspect that has not yet been sufficiently addressed is the presence of
human metabolites of antimicrobials in wastewaters. Sulfamethoxazole, for
example, is metabolized in the human body and -50% of the administered dose
is excreted as the inactive human metabolite A^-acetylsulfamethoxazole and
only 10% as the unchanged compound.[28] The retransformation of
A^-acetylsulfamethazine to the active sulfamethazine during the storage of
manure has already been shown by Berger et al., suggesting a similar cleavage
of A^-acetylated sulfonamides, for instance in wastewater treatment.[29]
Analytical Methodfor Wastewater 33
Observed elimination rates may be biased, if the possible retransformation to the
active pharmaceutical is not considered. To the best of our knowledge, only one
study included A^-acetylsulfamethoxazole in the analysis of surface water and
WWTP effluents - indicating concentrations of up to 2 200 ng/L in WWTP
effluents.[30J Unfortunately, the state of treatment has not been reported. This
clearly shows the importance of considering the main human metabolite of
sulfonamides when assessing the occurrence and fate of sulfamethoxazole in
wastewater treatment.
In this article, we present a reliable analytical method for the trace determination
of the most important macrolide and sulfonamide antimicrobials in the various
aqueous compartments of a WWTP, including primary effluent. In addition, the
human metabolite A^-acetylsulfamethoxazole and trimethoprim - frequently
used as a synergist to sulfamethoxazole - were measured. Table 2.1 lists the
selected macrolides and sulfonamides; their respective chemical structures are
given in Figure 2.1 and 2.2. Using solid-phase extraction combined with liquid
chromatography tandem mass spectrometry (positive electrospray ionization)
concentrations down to the low nanogram per liter range can be determined. The
Figure 2.1 Chemical structures ofmacrolide antimicrobials
K2
Ri
erythromycin (ERY) H
clarithromycin (CLA) CH3
roxithromycin (ROX) H
R2
O
O
HO,
H3CT
>ivrt azithromycin
(AZI)
'v/^oh y'""*CH3
H3C, *xCH3 dehydro-erythromycin
(ERY-H20)
/
\ CH3
°~7^CZ2o>
\
34 Chapter 2
Figure 2.2 Chemical structures ofsulfonamide antimicrobials and trimethoprim
pK.2 pKJ
R2
/N
\
N»
N MH2
trimethoprim
(TRI)
sulfadiazine
(SDZ)
sulfathiazole
(STZ)
sulfamethazine
(SMZ)
sulfapyridine
(SPY)
sulfamethoxazole
(SMX)
A^-acetyl-sulfamethoxazole
(N4AcSMX)
Ri
H
H
H
R2
N=v
-o
-0
H
H
-o
COCH3
-tf
CH3
CH3
presented method is feasible to study the occurrence and fate of the selected
compounds in all compartments of a wastewater treatment plant as well as for
environmental monitoring studies. Preliminary results on the occurrence of
macrolides and sulfonamides in Swiss wastewater treatment plants are
presented.
2.2 Experimental Section
2.2.1 Chemicals and Reagents
HPLC-grade methanol, acetonitrile, and water are purchased from Scharlau
(Barcelona, Spain). Analytical ethyl acetate, ammonia solution, 25% sulfuric
acid, sodium chloride, sodium hydroxide, ammonium acetate, and formic acid
were obtained from Merck (Darmstadt, Germany). Sulfamethazine,
sulfamethoxazole, sulfadiazine, and roxithromycin were purchased from Sigma-
Aldrich (Buchs, Switzerland). Sulfathiazole, sulfapyridine, trimethoprim,
Analytical Methodfor Wastewater 35
tylosin, josamycin, and erythromycin were obtained from Fluka Chemicals
(Buchs, Switzerland), and sulfamerazine was from Riedel-de Haën (Seelze,
Germany). Sulfamethazine-phenyl-13C6 was purchased from Cambridge Isotope
Laboratories (Andover, MA) and sulfamethoxazole-*/, sulfadiazine-*/,
sulfathiazole-d4, and A^-acetylsulfamethoxazole-d6 were purchased from
Toronto Research Chemicals (North York, ON, Canada). Clarithromycin was
kindly supplied by Abbott (Wiesbaden, Germany) and azithromycin by Pfizer
(Zurich, Switzerland). Azithromycin is also available from Sigma-Aldrich
(Buchs, Switzerland). Standard solutions for dehydro-erythromycin were
prepared from erythromycin as described by McArdell et al. The acidic
solution was readjusted to pH 6 after 4 h using IM NaOH to ensure stability
during storage. A^-acetylsulfamethoxazole was synthesized by acetylation with
acetic acid anhydrate according to Neumann with a yield of 70%.[31] Identity and
purity was confirmed by LC/UV, LC/MS/MS, and H NMR analysis.
2.2.2 Internal Standards
Deuterated sulfonamide standards were commercially available in most cases.
Erythromycin-13C2 was tested as an internal standard for the macrolides, but
proved to be unsuitable due to the significant natural contribution to M + 2 from
unlabeled erythromycin (11.6%). Similar observations were described by
Vanderford and co-workers for erythromycin-13Ci.[23] The absence in water
samples of all internal standards used was confirmed by enriching a
representative samples from each matrix, to which no surrogate or instrumental
standard was added. No peaks could be detected at the retention times of the
used internal standards. Surrogate standards were added prior to enrichment to
assess possible losses during the analytical procedure. Instrumental standards
were added to the final extracts prior to measurement. The following substances
were used as surrogate standards: sulfamethazine-phenyl- C6 ( C6SMZ) for
SMZ, TRI, and SPY, sulfamethoxazole-^/4 (d4SMX) for SMX, sulfadiazine-*/4
(d4SDZ) for SDZ, sulfathiazole-t/4 (d4STZ) for STZ, V-acetyl-sulfamethoxazole-^ (d5N4AcSMX) for N4AcSMX, and tylosin (TYL) for
ERY-H20, AZI, CLA, and ROX. As instrumental standards sulfamerazine
(SMR) was used for all sulfonamides and TRI, and josamycin (JOS) for all
macrolides. While the surrogate standards were used for quantification, the
instrumental standards were used to check the instrument performance during
36 Chapter 2
measurement. Its peak area was monitored over the whole measurement series in
order to detect problems with, for example, instrument sensitivity or injection
volume. If the area of the instrumental standard decreases significantly (signal
reduction of > 20% within same matrix), the series was stopped and the
instrument cleaned.
2.2.3 Sample Collection and Preparation
Flow-proportional composite samples of the primary effluent (1°EFFL) after
mechanical treatment, the secondary effluent (2°EFFL) after biological
treatment, and the tertiary effluent (3°EFFL) after sand filtration were collected.
The samples were transferred into amber glass bottles and filtered as soon as
possible but no later than 6 h after sampling through 0.45-um cellulose nitrate
filters (Schleicher & Schuell). The filtered samples were directly extracted or
kept at -20 °C in half-filled amber glass bottles in horizontal position until
extraction. The sample volumes were 250 mL for 2°EFFL and 3°EFFL samples
and 50 mL for 1°EFFL samples. The latter were diluted with 150 mL of water
prior to extraction. After addition of 1 g of sodium chloride, the pH was adjusted
to 4 with sulfuric acid, and the surrogate standard (50 - 100 ng) was added.
Solid-phase extraction was performed on 6-mL Oasis HLB sorbent cartridges
(200 mg; Waters, Bergen op Zoom, The Netherlands) using a 12-fold vacuum
extraction box (J.T. Baker, Phillipsburg, NY). The sorbent material is a
copolymer of two monomers, TV-vinylpyrrolidone and divinylbenzol. The
cartridges were preconditioned with 2 x 1.5 mL of methanol-ethyl acetate (1:1),
2 x 1.5 mL of methanol containing 1% (v/v) ammonia, and 2 x 1.5 mL of water
adjusted to pH 4 with H2S04. The wastewater samples were percolated through
the cartridges at a flow rate of less than 5 mL/min. After percolation, the
cartridges were washed with 1.5 mL of water-methanol (95:5) and the eluent
was discarded. Subsequently, the cartridges were dried completely in a nitrogen
flow for 1 h. The analytes were then eluted with 2 x 1.5 mL of methanol-ethyl
acetate (1:1) and 2 x 1.5 mL of methanol containing 1% ammonia into 10-mL
graduated glass vessels. Eluates were reduced to -50 pL by a gentle flow of
nitrogen at room temperature. After the addition of the instrumental standard
(100 ng), the sample volume was adjusted to 0.5 mL with water. Final extracts
were stored in amber glass vials at -15 °C until analysis.
Analytical Methodfor Wastewater 37
2.2.4 Liquid Chromatography
HPLC analyses were performed using a Rheos 2000 pump equipped with a
solvent degasser (Flux Instruments AG, Switzerland), a HTS Pal autosampler
(CTC Analytics, Zwingen, Switzerland), and a Jones Chromatography column
oven, Model 7956 (Omnilab AG, Mettmenstetten, Switzerland). Sample aliquots
of 20 pL were injected. Two analytical columns were tested for separation.
Initially, a 125 x 2 mm Nucleosil 100-5 C18HD end-capped column (Macherey-
Nagel, Dueren, Germany) equipped with a 8 x 2 mm precolumn containing the
same sorbent material was used (column 1). Gradient elution was performed
with water adjusted to pH 4.6 by acetic acid and acetonitrile, both containing
10 mM ammonium acetate. Later, a 150 x 2 mm YMC Pro Ci8, 120 Â, 3 urn
(Stagroma, Reinach, Switzerland) column equipped with a 10 x 2 mm
precolumn containing the same sorbent (column 2) was used. Optimal
separation was achieved using column 2 maintained at 30 °C and with a flow
rate of 0.15 mL/min. Solvent A was water acidified with 1% (v/v) formic acid,
resulting in a pH of 2.1, and solvent B was methanol acidified with 1% (v/v)
formic acid. The run (0.15 mL/min) started at 10% B for 5 min, followed by a 5-
min linear gradient to 15% B, a 5-min linear gradient to 40% B, and another 5-
min linear gradient to 45% B and was terminated by a 10~min linear gradient to
70% B. Afterward, the eluent was brought to 100% B in 2.5 min and the column
washed at a flow rate of 0.25 mL/min for 10 min. Initial conditions were
reestablished in 2.5 min, and the column was equilibrated for 10 min at a flow
rate of 0.25 mL/min prior to the next analysis. The total time per analysis was 55
min. Table 2.2 gives the retention times of the individual analytes. To prevent
sensitivity losses of the mass spectrometer, the eluate of the first 8 min and of
the last 20 min of the chromatographic run were bypassed and discarded.
2.2.5 Tandem Mass Spectrometry
A triple quadrupole mass spectrometer, TSQ Quantum Discovery (Thermo
Finnigan, San Jose, CA), equipped with electrospray ionization was used for
detection. Analyses were performed in the positive mode, with a spray voltage
of 3 500 V and an ion-transfer capillary temperature of 350°C. Nitrogen was
used as sheath gas (40 bar) and as auxiliary gas (10 bar), and argon as collision
gas (1.5 mTorr). Both mass analyzers were set to unit resolution.
38 Chapter 2
Table 2.2 Precursor ions, selected fragment ions, and retention times of the
measured compounds
analyte precursor ion product ions retention time
(m/z) ; (m/z) (min)
SDZ 251.06 156.01,
108.04 10.3
d4SDZa 255.08 160.01 112.04 10.0
STZ 256.02 156.01 108.04 12.7
d4STZa 260.05 160.01 112.04 12.4
SMZ 279.09 124.09 186.03 17.6
13C6SMZ a 285.09 124.09 186.03 17.6
SPY 250.07 156.01 184.09 12.6
SMX 254.06 156.01 108.04 20.4
d4SMXa 258.08 160.01 112.04 20.3
N4AcSMX 296.07 134.06 198.02 24.1
d5N4AcSMXa
301.10 139.06 203.02 24.0
TRI 291.15 123.07 275.14 17.1
AZI 375.26 591.40 158.12 21.1
ERY-H20 716.46 540.33 558.34 30.1
CLA 748.49 158.12 590.37 31.5
ROX 837.53 158.12 ; 679.41 31.6
TYLa 916.53 174.11.
772.45 29.1
SMRa 265.08 156.01 ; 172.02 14.9
JOSa 828.48 108.91
,174.11 31.1
Used as internal standard.
Usually, the protonated molecular ion ([M + H]+) of the compounds was
selected as precursor ion except for azithromycin, for which the doubly charged
molecular ion ([M + 2H] ) was chosen as precursor ion because of its greater
abundance under the given conditions. Detection was performed in multiple
reaction monitoring mode using the two most intense and specific fragment ions.
Table 2.2 lists the monitored transitions for the individual analytes. The
Analytical Methodfor Wastewater 39
detection of the compounds was divided in time windows during the course of
the chromatographic run with a dwell time of 100 ms. Figure 2.3 shows a
chromatogram of a 1°EFFL sample. In the case of SDZ, STZ, and SMZ, which
were not present in the sample, the peaks obtained from the measurement of a
1°EFFL sample spiked with 25 ng prior to sample preparation are included
(dashed lines).
Figure 2.3 Total ion chromatogram (sum oftwo transitions) ofan extractfrom a
primary wastewater effluent (1 °EFFL)
SPY
SDZ»
ill
TRI RQX
SMZ'
ERY-H20
100
c
at
^1 Vin*J*
II
i "WpftDwWfI
STZ«
50
Ï%
a.
SMX
AZI JfMüU: lé JIM kuAJuiiw
^'»^«,
N4AcSMX
! I
10
Jj.tf| --T-
15
.))ê*i
CLA
I" I f I
20 25
Retention "lime (min)
JWjJUj^jU 1^ A
T J "T>-r y --, |
30 35
aThe peaks shown for SDZ, STZ, and SMZ correspond to a 1°EFFL sample spiked
with 25 ng of these compounds since they were not present in the unspikcd sample.
2.2.6 Method Validation
For the method validation flow-proportional composite samples from the
respective effluents of a municipal wastewater treatment plant (WWTP Kloten-
Opfikon) were taken. Breakthroughs were determined by extracting spiked
40 Chapter 2
wastewater samples (duplicate analyses) using two stacked cartridges. A
breakthrough on the first cartridge triggered an enrichment on the consecutive
cartridge, which was then eluted separately. For the 1°EFFL a 250-mL sample
with a spiked analyte concentration of 2 000 ng/L was used, and for the 3°EFFL
a 500-mL sample with a spiked analyte concentration of 5 000 ng/L was
extracted. Complete elution of the cartridges was verified by eluting cartridges
of spiked samples for a second time with 1.5 mL of acetone as a stronger
solvent. The acetone extract was then treated as a separate sample. Instrumental
limits of detection (LODs) and limits of quantification (LOQs) were calculated
on the basis of standard deviation of the repeated measurement (n = 10) of a
standard mixture (100 pg on column). The LOD is defined as 3 times, and the
LOQ as 10 times the standard deviation. If the resulting value for the LOQ was
below the linear range, the lower limit of the linear range was set as LOQ.
Sample-based LOD and LOQ were defined as concentrations in a sample matrix
resulting in peak areas with signal-to-noise ratios (S/N) of 3 and 10,
respectively. Since samples typically contained analytes in higher amounts, the
concentration corresponding to the defined S/N was determined by scaling
down, using the measured concentration and the assigned S/N of the peak -
assuming a linear correlation through zero. Instrumental precision of the
measurement was assessed using an average of 10 independent injections of 100
pg on column of a standard mixture. The precision of the entire method was
determined using four replicates of each matrix investigated, spiked with 50 ng
of analyte prior to extraction. It is indicated by the relative standard deviation of
the measured concentrations of native plus spiked analyte. For recovery studies
over the entire procedure, wastewater samples (duplicate analyses) were spiked
prior to extraction with surrogate standard and with 25 and 50 ng of analytes,
respectively. The calculated amount of antimicrobials minus the amount already
present before spiking was then divided by the spiked concentration.
2.2.7 Identification and Quantification
For each substance, two transitions of the precursor ion were monitored.
Together with the retention times, they were used to ensure correct peak
assignment and to evaluate peak purity. For instrumental and surrogate
standards, peak purity was tested using the area ratio of the two product ions
monitored. Their individual ratio was calculated as well as the mean ratio of all
Analytical Methodfor Wastewater 41
samples and its relative standard deviation. The ratio in one sample was
compared to the mean ratio of all the samples measured in one series. The
variance had to be within the range of twice the standard deviation of the mean
sample ratio. The peak purity of the analytes was tested by calculating a
concentration (as described below) for both product ions measured. The
respective surrogate product ion was used. If no surrogate product ion resulting
from the same fragmentation reaction can be used, i.e., if no isotope labeled
surrogate standard is available, the sum of both product ions of the compound
assigned as surrogate standard was used for quantification to simplify the
procedure. The relative average deviation of the calculated concentrations from
the two product ions had to be less than 10%. Peaks not fulfilling the
requirements for peak purity were not used for quantification.
Quantification was performed using the ratio of the peak areas of the analytes
and of the surrogate standard. The sum of the two monitored product ions was
used. An external calibration curve, plotting ratio against concentration, was
obtained by diluting standards in HPLC water. A standard curve was acquired at
the beginning, at the end, and also in the middle of a measurement series. At
least five concentration points in the appropriate concentration range were used
for quantification.
Concentrations in the samples were calculated by comparing the peak area ratios
of the analytes and their assigned surrogate standards in the SPE extracts, to the
corresponding ratios in the standard solutions. These results were corrected with
the corresponding recovery rates obtained in the same matrix and sample batch
to provide accurate amounts. For routine determination, duplicate analyses of all
samples were performed. Procedural blanks, consisting of de-ionized water,
were analyzed with each set of 12 extractions as a control for laboratory
contamination. Additional instrumental blanks using de-ionized water were
checked with each calibration curve in order to uncover potential analytical
interferences.
2.3 Results and Discussion
2.3.1 Method Development
The crucial parameters for enrichment, separation, and detection of the analytes
were identified and optimized. The pH of the sample proved to be the most
42 Chapter 2
influential variable during sample extraction. A critical impact on the retention
of the analytes on the cartridge material was observed, especially for
sulfonamides caused by their amino groups. Our enrichment tests between pH 2
and 6 revealed, as expected, highest recoveries at pH 4 for the sulfonamides,
while the recovery of the macrolides and trimethoprim showed no strong pH
dependence. This behavior can be explained by the charge state of the
sulfonamides at the particular pH values (Table 2.1).[1M6] With a compound
specific pKa of 5 - 8 for the sulfonamino groups (pKa 1) and a pKa of 2 - 2.5 for
the arylamin (pKa 2), the sulfonamides are positively charged at pH 2 and
negatively at a pH above 5. The interaction with the cartridge material is
strongest for analytes in uncharged forms occurring at a pH of -4 in the case of
the sulfonamides. The dilution of the 1°EFFL samples prior to enrichment
additionally increased signal intensity provided by the mass spectrometer for the
sulfonamides - in most cases by a factor of 2. For the macrolides and
trimethoprim, no significant improvement was observed.
While A^-acetylsulfamethoxazole is stable during sample preparation,
erythromycin present in the samples is completely transformed to dehydro-
erythromycin at pH 4. This is in agreement with the reported instability of
erythromycin under acidic conditions resulting in the formation of the inactive
dehydro-erythromycin.[16] Erythromycin was therefore assessed as the main
environmental metabolite, dehydro-erythromycin.
Tandem mass spectrometric conditions were optimized for each analyte and
internal standard through automated tuning procedures implemented in the
instrument software. Figure 2.4 shows the breakdown curves for A^-acetyl-
sulfamethoxazole and its four most intense fragments as a function of the
collision energy. As expected, the collision energy, which gives the most intense
signal, increases for the formation of smaller fragments. Tentative product ion
structures are given also. These structures have not been reported previously but
are in agreement with transitions known to be typical for sulfonamides. '
During LC/MS/MS measurement, matrix compounds can be deposited on the
instruments sample interface, especially on the ion-transfer capillary, and can
thus significantly reduce instrument sensitivity. The higher the sample volume
the more matrix will be introduced into the mass spectrometer within one run.
On the other hand, a high enrichment factor is desirable to achieve the low limits
of detection, which are necessary for the environmental analysis of
antimicrobials. The sample volume was optimized by using a variable splitting
Analytical Methodfor Wastewater 43
Figure 2.4 Breakdown curves of N*-acetylsulfamethoxazole and proposed
product ion structures (absolute intensity 3.56e+0.5, collision pressure 1.5
mTorr)
IM
#
I
>
t8 4*-
«
1» 20
i 1 r
JO 40
Collision Energy (V)
o
-i r-=N
\ /©/
S -N.
[M+H]+m/z=296
H_
/H2N=/ N ©
O
v_/ \
m/z=65 m/z=108 m/z=134 m/z=198
if TT Chapter 2
device prior to the electrospray interface. For this experiment, higher sample
volumes were chosen. Therefore, 200 mL of 1°EFFL and 1 000 mL of 2°EFFL
and 3°EFFL, respectively, were enriched and measured in one series. The eluent
flow and split ratio were varied, so that the instrument remained sensitive
enough for the measurement of up to 30 samples of each matrix. The sample
volume used was then adjusted according to the split ratio. Subsequent samples
were measured without the additional splitting device that may pose as a source
for errors (for example, plugging of the capillary). The given sample volumes
therefore represent a compromise between method sensitivity and routine
analysis. Two columns were tested for the separation of the selected
antimicrobial agents. In both cases, a reversed-phase end-capped Ci8 column
was chosen - one belonging to the older (column 1) and one belonging to the
newer (column 2) generation of silica gels. On column 1, azithromycin produces
a peak with substantial tailing. To our knowledge, azithromycin has not been
included in analytical methods for environmental samples so far, likely also due
to analytical difficulties like this. The observed tailing on column 1 is probably
due to the interaction of the two basic functional groups with residual silanol
groups and metal impurities of the column material. In the case of the other
macrolides, which contain one amino group less than azithromycin in the
lactone moiety, this interaction was sufficiently suppressed by the addition of
ammonium acetate. On column 2, belonging to the new generation of silica gels
using a more efficient purification and end-capping procedure, however, good
separation was achieved with almost symmetrical peaks for all analytes. In
addition, the use of ammonium acetate in the eluent was no longer necessary.
This significantly increased the sensitivity of the method for sulfonamides,
which tend to form ammonium adducts during ionization.
In the case of some 1 °EFFL samples, the extracts needed to be diluted up to five
times in order to obtain good peak shapes for azithromycin, which seems to
form complexes with matrix compounds. For most analytes, the assumed loss of
sensitivity, due to dilution is partly compensated by the simultaneous reduction
of ion suppression, since signal intensities observed are reduced to a lesser
extent.
Analytical Methodfor Wastewater 45
2.3.2 Method Validation
The developed method was validated for primary effluents after mechanical
treatment, secondary effluents after biological treatment, and tertiary effluents
after sand filtration. For breakthrough studies, samples representing unnaturally
high concentrations and high loads of sample matrix were enriched on two
stacked cartridges. No quantifiable amounts of the analytes could be detected on
the second cartridge for both sample matrices (1°EFFL and 3°EFFL). When
testing for complete elution, no quantifiable amounts of analytes could be
measured in the acetone eluates of already eluted cartridges. Thus, the analytes
are quantitatively enriched by one cartridge and exhaustively eluted by the
procedure described above.
For the standard curves good linearity was observed with correlation factors
typically above 0.99. The linear range of the measurement varied with the
analyte due to differences of the ionization efficiencies (Table 2.3). The
instrumental LOQ ranges between 16 and 100 pg of analyte on column. In the
case of the sample-based LOQ and LOD the range and the average of the
resulting values for each matrix from different samples are given in Table 2.3.
Since the LOD and LOQ in an individual sample can be higher or lower than the
average LOD and LOQ, all peaks with an assigned S/N greater than or equal to
3 and 10, respectively, are considered valid.
The instrumental precision of the method was addressed for various aspects, and
the following relative standard deviations were obtained: the retention time
ranged between 0.06 and 0.35% and the peak area between 1.3 and 9.2%. The
peak area ratios of analyte versus surrogate standard varied to a lesser extent in
most cases (between 1.3 and 7%), since the surrogate standard compensates for
analytical variability. The precision of the entire method (reproducibility) is
indicated by the standard deviation of multiple analyses and ranged between 0.5
and 15%. Detailed results are shown in Table 2.4.
Accuracies of the method were determined by relative recovery studies over the
entire procedure (Table 2.4). The resulting recoveries obtained in all matrices
investigated were generally above 80%, with the exception of TRI where they
ranged between 30 and 47%. For TRI, this was caused by the use of a nonideal
surrogate standard (13C6SMZ), but none better suited could be found.
Recoveries, and thereby LODs and LOQs, of the analytes vary between samples,
mainly due to varying matrix effects, if no isotopically labeled surrogate
Table2.3Linearrangesand
limitsofquantification
(LOQ)
samplebasedLOQ
a
(ng/
L)
linearrange
primaryeffluent
seco
ndar
yeffluent
tertiary
effluent
anal
yte
(pgoncolumn)
average
range
average
range
average
range
SDZ
20-
30000
68
60-77
11
6-32
75-11
STZ
20-
30000
214
194-236
21
15-30
16
10-22
SMZ
10-30000
42
35-48
97-12
94-17
SPY
50-
30000
96
51-150
14
8-31
96-19
SMX
10-30000
62
35-
104
12
8-17
11
6-15
N4AcSMX
100-30000
212
155-288
23
17-29
22
16-28
TRI
5-
2000
21
15-27
64-9
43-7
AZI
10-4000
74.6-9.9
32.0-3.4
21.6-2.6
ERY-H20
5-
4000
19
4.4-13
53.5-8
63.4-9.5
CLA
10-6000
42.3-5.7
21.3-2.9
21.3-3.1
ROX
10-4000
31.2-6.3
10.3-2.0
10.4-1.4
Concentrationestimatedfrommeasuredsamplesforasi
gnal
-to-
nois
eratioof10.
Table2.4Methodpr
ecis
ions
,accuracies,andcombinedmeasurementuncertainties
precisio
naccuracy
combinedmeasurement
relativeSD
a
(%),
nb=4
relativerecovery±SD
a
(%),
nb=4
unce
rtai
nty(%)
prim
ary
secondar
ytertiary
prim
ary
seco
ndar
ytertiary
primary
secondary
tertiary
anal
yte
effluent
effluent
effluent
effluent
effluent
effluent
effluent
effluent
effluent
SDZ
3.4
0.5
1.0
102±2
95±2
98±5
3.1
2.7
4.0
STZ
0.7
5.2
2.0
101±2
102±2
103±3
2.6
3.3
5.7
SMZ
1.1
1.6
2.1
98±1
98±3
98±6
2.4
3.9
4.9
SPY
4.3
4.5
2.2
108±4
101±5
106±1
6.1
3.8
13
SMX
3.0
0.4
1.4
101±3
105±10
105±6
5.2
3.6
3.4
N4AcSMX
0.7
2.4
4.2
91±4
100±7
93±6
3.8
4.4
5.7
TRI
8.5
2.6
12
47±3
30±12
35±10
10
8.0
13
AZI
2.4
5.7
11
83±7
85±4
86±10
9.4
11
16
ERY-H20
5.6
4.1
8.9
91±1
82±1
86±8
4.8
3.8
15
CLA
5.9
3.0
15
89±8
81±8
78±6
6.5
7.4
12
ROX
6.6
5.9
15
100±5
108±2
124±3
8.6
8.7
14
aSD=
standarddeviation.
bn=numberofsamples.
48 Chapter 2
standard is available. Correct quantification can still be ensured if recovery
studies are performed in each matrix and sample batch as was the case within
the work presented here. The combined measurement uncertainty was quantified
using data from the method validation as described in Example A4 of the
EURACHEM/CITAG Guide Quantifying Uncertainty in Analytical
Measurement.1341 Therefore, all uncertainty sources were identified and
quantified. The main contributions result from the repeatability of the
measurement, calculated from duplicate sample analysis, and its accuracy,
represented by recovery studies. The relative values of all uncertainty sources
are finally combined using statistical methods. The values for the combined
measurement uncertainty vary between 2.4 and 16% with the analyte and the
matrix investigated (Table 2.4).
2.3.3 Wastewater Application
The developed method was successfully applied to the analyses of wastewater
samples from two urban wastewater treatment plants in Switzerland: WWTP
Kloten-Opfikon, located near the international airport of Zurich (WWTP-K),
and WWTP Altenrhein, which is located in the canton St. Gall close to the
border with Austria (WWTP-A). In both cases, mechanically treated wastewater
(primary effluent) passes through conventional activated sludge treatment,
followed by secondary settling (secondary effluent). After biological treatment,
both treatment plants use sand flltration as a tertiary treatment step (tertiary
effluent). Table 2.5 shows the results obtained from duplicate analyses of 72-h
flow-proportional composite samples of the primary, secondary, and tertiary
effluents. Samples were taken in February 2003. With 54 100 and 40 000
inhabitant equivalents, the two investigated treatment plants are of similar size
and have comparable volumes of wastewater inflow. This is also reflected in the
similar concentration ranges found at each plant, with the exception of
azithromycin. The latter appears to be more frequently used in the catchment
area of WWTP-A. Sulfamethoxazole and clarithromycin were found to be the
most commonly used sulfonamides and macrolides, respectively. Sulfadiazine -
which is very rarely applied in human medicine - could not be quantified in any
of the samples, nor could sulfathiazole - which is almost exclusively used in
veterinary medicine.
Table2.5Sulfonamideandmacrolideconcentrationsmeasured
intwomunicipalwastewatertreatmentpl
ants
inSwitzerland
sampleconcentrationa
(ng/L), n
=2
WWTP-Kc
WWTP-A
e
prim
ary
secondary
tertiary
prim
ary
secondary
tertiary
analyte
effluent
effluent
effluent
effluent
effluent
effluent
SDZ
ndd
nqd
nq
nd
nd
nd
STZ
nd
nd
nd
nd
nd
nd
SMZ
nd
nd
nd
39
18
19
SPY
72
82
88
135
63
85
SMX
343
344
352
641
352
352
N4AcSMX
518
86
82
943
nq
71
TRI
168
170
81
110
86
68
AZI
86
110
85
224
129
255
ERY-H20
67
96
75
44
54
55
CLA
234
374
329
160
188
220
ROX
22
11
10
30
21
23
aConcentrationmeasured
in
filtered72hflowproportional
composite
sample.Averageofdu
plic
atedetermination.
bn=number
of
measurements.cWWTP-K=
Kloten-Opfikon(cantonZu
rich
),WWTP-A=Altenrhein(c
anto
nSt
.Gall).
dnd=not
detected=
sign
al-t
o-no
isebelow
3;nq=not
quantifiable=
sign
al-t
o-no
isebelow
10.
50 Chapter 2
Sulfamethazine - another sulfonamide used mainly for veterinary applications -
was only present in one of the treatment plants (WWTP-A). A^-acetyl-
sulfamethoxazole is typically present in high amounts in the primary effluents,
but only small amounts can be found in the tertiary effluents. If the amount of
sulfamethoxazole present as acetyl metabolite is neglected, the elimination of
sulfamethoxazole will be underestimated. Concentrations of the analytes in both
tertiary effluent range between 19 and 352 ng/L. This clearly shows that the
compounds investigated are not eliminated completely and reach receiving
surface waters. Compared to results obtained in Germany/20'35 the
concentrations found are in the same range but generally lower.
2.4 Conclusions
Solid-phase extraction Oasis HLB cartridges coupled with reversed-phase liquid
chromatography and tandem mass spectrometry were successfully applied for
the determination of selected sulfonamides and macrolides, in addition to
trimethoprim and the human sulfonamide metabolite A^-acetylsulfamethoxazole,in municipal wastewater. As a result of this method's applicability to wastewater
samples spanning the whole treatment process (including primary effluent
samples), it can be used to investigate the fate of these compounds through the
various steps of wastewater treatment. The resulting information can be used to
evaluate the performance of wastewater treatment procedures and to highlight
options for the optimization ofWWTPs with the aim of minimizing the input of
pharmaceuticals into ambient receiving waters. Additionally, by including N4-
acetylsulfamethoxazole - the main human metabolite of sulfamethoxazole - the
fate of the most commonly used sulfonamide in human medicine can be
investigated more thoroughly.
The presented method provides the necessary basis for a comprehensive study
on antimicrobials in wastewater treatment including alternative wastewater
technologies such as biofilter technology and membrane flltration.[36] Our
ongoing studies are also aimed at achieving complete mass balances of
antimicrobials in wastewater treatment plants, including sewage sludge
treatment steps. Preliminary results show that the method can easily be adapted
for the analyses of sewage sludge extracts. Applications to drinking water,
ambient waters, and hospital wastewaters also seem to be possible judging from
first measurements without major changes in the procedure. With this method,
Analytical Methodfor Wastewater 51
we therefore present a powerful tool to fully assess the fate and occurrence of
macrolides and sulfonamides throughout their main pathways to and within the
aquatic environment.
Acknowledgments
Abbott GmbH (Wiesbaden, Germany) is acknowledged for supplying
clarithromycin and Pfizer AG (Zurich, Switzerland) for supplying azithromycin.
Partial financial support came from the EU project POSEIDON (EVK1-CT-
2000-00047)[37] and the EAWAG project on human-use antibiotics
(HUMABRA) within the framework of the National Research Program on
antibiotic resistance funded by the Swiss National Science Foundation.[38J We
thank Eva Molnar, Norriel Nipales, and René Schönenberger for their technical
assistance and advice. For helpful comments on the manuscript, we
acknowledge our colleagues Alfredo Alder, Michael Dodd, Stephan Müller, and
Krispin Stoob.
2.5 Literature cited
[I] Stan, H. J.; Heberer, T. Analusis 1997, 25, M20-M23.
[2] Ternes, T. A. Water Res. 1998, 32, 3245-3260.
[3] Halling-Serensen, B.; Nors Nielsen, S.; Lanzky, P. F.; Ingerslev, F.; Holten
Lützenhoft, H. C; torgensen, S. E. Chemosphere 1998, 36, 357-393.
[4] Daughton, C. D.; Ternes, T. A. Environ. Health Perspect. 1999, 107, 907-
938.
[5] Kümmerer, K. Chemosphere 2001, 45, 957-969.
[6] Kolpin, D. W.; Furlong, E. T.; Meyer, M. T.; Thurman, E. M.; Zaugg, S.
D.; Buxton, H. T. Environ. Sei. Technol. 2002, 36, 1202-1211.
[7] Heberer, T. Toxicol. Lett. 2002,131, 5-17.
[8] Annual Report; Swiss Importers of Antibiotics (TSA): Berne, Switzerland,
1998.
[9] Pharmaceuticals Sold in Switzerland; Swiss Market Statistics, 1999.
[10] Antibiotics used in Veterinary Medicine; Swiss Federal Office for
Agriculture (BLW): Berne, Switzerland, 2001.
[II] Vree, T. B.; Hekster, Y. A. Clinical pharmacokinetics ofsulfonamides and
their metabolites; Karger: Basel, 1987; Vol. 37.
i
52 Chapter 2
[12] Lin, C.-E.; Chang, C.-C; Lin, W.-C. J. Chromatogr., A 1997, 768, 105-
112.
[13] Petz, M., Habilitation Thesis, Westfälische Wilhelms-Universität, Münster,
1986.
[14] Neuman, M. Antibiotika-Kompendium; Verlag Hans Huber: Bern, 1981.
[15] McFarland, J. W.; Berger, C. M.; Froshauer, S. A.; Hayashi, S. F.; Hecker,
S. J.; Jaynes, B. H.; Jefson, M. R.; Kamicker, B. J.; Lipinski, C. A.; Lundy,
K. M.; Reese, C. P.; Vu, C. B. J. Med. Chem. 1997, 40, 1340-1346.
[16] Bryskier, A. J.; Butzler, J.-P.; Neu, H. C; Tulkens, P. M. Macrolides;
Arnette Blackwell: Paris, 1993.
[17] Hirsch, R.; Ternes, T. A.; Haberer, K.; Mehlich, A.; Ballwanz, F.; Kratz,
K.-L. J. Chromatogr., A 1998, 815, 213-223.
[18] Sacher, F.; Lange, F. T.; Brauch, H.-J.; Blankenhorn, I. J. Chromatogr., A
2001,938,199-210.
[19] Lindsey, M. E.; Meyer, M.; Thurman, E. M. Anal. Chem. 2001, 73, 4640-
4646.
[20] Hartig, C; Storm, T.; Jekel, M. J. Chromatogr., A 1999, 854, 163-173.
[21] McArdell, C. S.; Molnar, E.; Suter, M. J.-F.; Giger, W. Environ. Sei.
Technol. 2003, 37, 5479-5486.
[22] Giger, W.; Aider, A. C; Golet, E. M.; Kohler, H.-P. E.; McArdell, C. S.;
Molnar, E.; Siegrist, H. R.; Suter, M. J.-F. Chimia 2003, 57, 485-491.
[23] Vanderford, B. J.; Pearson, R. A.; Rexing, D. J.; Snyder, S. Anal. Chem.
2003, 75, 6265-6274.
[24] Golet, E. M.; Aider, A. C; Hartmann, A.; Ternes, T. A.; Giger, W. Anal.
Chem. 2001, 73,3632-3638.
[25] Golet, E. M.; Strehler, A.; Aider, A. C; Giger, W. Anal. Chem. 2002, 74,
5455-5462.
[26] Golet, E. M.; Aider, A. C; Giger, W. Environ. Sei. Technol. 2002, 36,
3645-3651.
[27] Golet, E. M.; Xifra, 1.; Siegrist, H. R.; Aider, A. C; Giger, W. Environ. Sei.
Technol. 2003, 37, 3243-3249.
[28] Vree, T. B.; Hekster, Y. A. Pharmacokinetics of sulfonamides revisited;
Karger: Basel, New York, 1985; Vol. 34.
[29] Berger, K.; Petersen, B.; Büning-Pfaue, H. Arch. Lebensmittelhyg. 1986,
37, 85-108.
[30] Hilton, M. J.; Thomas, K. V. J. Chromatogr., A 2003,1015, 129-141.
Analytical Methodfor Wastewater 53
[31] Neumann, J. "Untersuchungen zur BioVerfügbarkeit und Pharmakokinetik
von Sulfasalazin und seinen Metaboliten," PhD Thesis, Free University of
Berlin, 1989.
[32] Volmer, D. Rapid Commun. Mass Spectrom. 1996, 10, 1615-1620.
[33] Haller, M. Y.; Müller, S. R.; McArdell, C. S.; Aider, A. C; Suter, M. J.-F.
J. Chromatogr., A 2002, 952, 111-120.
[34] http://www.measurementuncertainty.org/mu/guide/index.html, Quantifying
uncertainty in analytical measurement / prep, by the EURACHEM
Working Group on Uncertainty in Chemical Measurement (ISBN 0 948926
15 5).
[35] Hirsch, R.; Ternes, T. A.; Haberer, K.; Kratz, K.-L. Sei. Total Environ.
1999,225,109-118.
[36] Göbel, A. PhD Thesis, ETHZurich, No. 15703 2004.
[37] http://www.eu-poseidon.com.
[38] http://www.nrp49.ch/pages/.
Seite Leer /Blank 1er?
Chapter 3
Analytical Method for Sewage Sludge
Pressurized liquid extraction (PSE) was optimized and validated for the
determination of sulfonamide and macrolide antimicrobials and trimethoprim in
sewage sludge samples. A mixture of water/methanol (50:50 v/v) was found as
the most efficient extraction solvent. A temperature of 100 °C and a pressure of
100 bar were chosen for extraction. Two cycles of 5 minutes each, efficiently
extracted at least 97% of all studied analytes from activated sludge. The limits of
quantification (S/N = 10) varied between 3 and 41 pg/kg dry weight (dw).
Additionally the influence of pH and analytical method on the absolute
recoveries was assessed. Of the investigated antimicrobials sulfapyridin,
Sulfamethoxazol, trimethoprim, azithromycin, clarithromycin, and
roxithromycin were detected in municipal sewage sludge samples.
Concentrations in activated sludge ranged up to 197 pg/kg dw. In comparison,
results obtained by ultrasonic solvent were generally lower in the case of
sulfonamides and in tendency lower for macrolides.
Göbel, A., Thomsen, A., McArdell, CS., Alder, A.C., Giger, W., Theiß, N.,
Löffler, D., Ternes, T.A.
Extraction ofSulfonamide and Macrolide Antimicrobialsfrom Sewage Sludge
submitted to Journal of Chromatography A
Analytical Methodfor Sewage Sludge 57
3.1 Introduction
Antimicrobial agents are widely used in human and veterinary medicine. The
overall human consumption of antimicrobials amounts to over 30 tons per
annum (t/a) in Switzerland and over 400 t/a in Germany - resulting in a similarnil
consumption of approximately 5 g per person and year in both countries."
Sulfonamides (16-21% of the total human consumption) and macrolides (9-
12%) belong to the most important groups of human used antimicrobials
following the ß lactams (50-60%).
Human used pharmaceuticals, including antimicrobial agents, are excreted,
unchanged or metabolized, from the patients' body. They therefore mainly reach
wastewater treatment plants (WWTPs) through household wastewater. The
occurrence and fate of pharmaceuticals in WWTPs and receiving surface waters
has hence been of increasing interest in recent years.[410] In the case of
antimicrobials this is also motivated by the possible maintenance and spread of
resistance caused by the constant input of low concentrations of antimicrobials.
They have been detected in WWTP effluent and receiving surface waters
illustrating the importance of WWTPs as point sources and the almost
ubiquitous presence of these emerging contaminants.[1M6] The occurrence of
macrolides and sulfonamides in WWTP effluents also indicates an incomplete
removal during conventional wastewater treatment. No distinction between
sorption and degradation can be made since the studies performed so far focus
on the fate and occurrence in the aqueous phase, except for fluorochinolones.
Golet et al. showed that sorption to sludge is the main removal route of the
highly polar fluorochinolones in wastewater treatment.ll7] This clearly illustrates
the need for analytical methods for sewage sludge when assessing the fate and
occurrence of contaminants in wastewater treatment. Methods published so far
for the determination of other antimicrobials in environmental biosolids focus on
the veterinary use and on the spread of contaminated manure onto soil.
Analytical methods and studies performed range from animal food products[18]
over manure[19"21]
to soil.[2228] Additionally river sediments [29] and meat from
production animals [30'31]were analyzed for sulfonamide and/or macrolides. A
review on part of the literature available can be found in [32]. In most cases the
compounds of interest were extracted from the samples by ultrasonic solvent
extraction (USE) or blending with a suitable solvent. USE thereby represents a
simple and relatively low priced approach. In a few cases, pressurized liquid
58 Chapter 3
extraction (PSE), also known as accelerated solvent extraction (ASE; Dionex),
was applied.[17,25] Using PSE the sample is extracted under high pressure and
high temperature to enhance solubility and mass transfer effects. ] Further
advantages of PSE are the minimal solvent usage and automation, which enables
the simultaneous extraction of a high number of samples.
In this study we aimed at developing a sensitive and reliable method for the
extraction of macrolides, sulfonamides and trimethoprim from activated and
digested sewage sludge (Figure 3.1). By comparing different extraction
approaches (PSE and USE) and the application of different analytical methods
in two different laboratories, an expanded validation of the method is achieved.
Results from the analysis of municipal activated and digested sludge samples
from Germany and Switzerland are given to show the applicability of the
methods presented.
3.2 Experimental Section
3.2.1 Chemicals and Reagents
HPLC-grade methanol, acetonitrile, and water were purchased from Scharlau
(Barcelona, Spain). Analytical grade ethyl acetate, acetone, ammonia solution,
25% sulfuric acid, sodium chloride, sodium hydroxide, ammonium acetate, and
formic acid were obtained from Merck (Darmstadt, Germany).
Sulfamethazine, sulfamethoxazole, sulfadiazine, oleandomycin and
roxithromycin were purchased from Sigma-Aldrich (Buchs, Switzerland).
Sulfathiazole, sulfapyridine, trimethoprim, tylosin, josamycin, and erythromycin
were obtained from Fluka Chemicals (Buchs, Switzerland) and sulfamerazine
from Riedel-de Haën (Seelze, Germany). Sulfamethazine-phenyl-13C6 was
purchased from Cambridge Isotope Laboratories (Andover, MA, USA) and
sulfamethoxazole-*/, sulfadiazine-«/, sulfathiazole-d4 as well as A^-
acetylsulfamethoxazole-öf5 were purchased from Toronto Research Chemicals
(North York, ON, Canada). Clarithromycin was kindly supplied by Abbott
(Wiesbaden, Germany) and azithromycin by Pfizer (Zurich, Switzerland).
Azithromycin is also available from Sigma-Aldrich (Buchs, Switzerland).
Standard solutions for dehydro-erythromycin were prepared from erythromycin
as described by McArdell et al.[16] The acidic solution was readjusted to pH 6
after 4 h using IM NaOH to ensure stability during storage.
Analytical Methodfor Sewage Sludge 59
Figure 3.1 Chemical structures ofthe investigated sulfonamides, macrolides
and trimethoprim
P—Rl
""*o v^v N\
erythromycin (ERY)CASRN 114-07-8
roxithromycin (ROX)CASRN 80214-83-1
Ri
H
clarithromycin (CLA) CH3
CASRN 81103-11-9
R2
O
O
H/^/V-N/
\t
, .„.111*
'V/^OH \>
H3C^"*/''CH3
'"///,
azithromycin (AZI)
CASRN 83905-01-5
o^v^X \I \ CH3
0B-
^zS°~\
\dehydro-erythromycin
(ERY-H20)»««
VV v*'*«h'CH3
'"*/(
? HO
/N.
\ GH3
-0„
o^~~~^ZSw
H7N-
jj R3
\ nr\.
R.
N .NH2
trimethoprim (TRI)CASRN 738-70-5
sulfadiazine (SDZ)CASRN 68-35-9
sulfathiazole (STZ)CASRN 72-14-0
sulfamethazine (SMZ)CASRN 57-68-1
sulfapyridine (SPY)CASRN 144-83-2
sulfamethoxazole (SMX)CASRN 723-46-6
( /N—,J
CH3
N=<
CH3
N=\
-o
Tu-O
60 Chapter 3
3.2.2 Sample Collection
Grab samples were taken from the end of the nitrification compartment at
different municipal WWTPs in Germany and Switzerland (activated sludge). All
plants consist of primary clarification and a denitrification - nitrification cascade
with an internal recirculation of sludge as secondary treatment. Phosphate
removal is performed by the addition of iron salts to different treatment steps. In
WWTP-W, located at Wiesbaden, Germany, serving 350 000 population
equivalents (PE), Fe(II)Cl2 is added to the final clarification. Simultaneous
precipitation with Fe3+ in secondary treatment is performed at WWTP-K,
located in Kloten-Opfikon, Switzerland, near the international airport of Zurich
(55 000 PE), and at WWTP-A, located in Altenrhein, Switzerland, close to the
border with Austria (40 000 PE). Additionally, a grab sample was collected from
outlet of the anaerobic, mesophilic digester at WWTP-K containing a mixture of
primary and secondary sludges (digested sludge).
Activated sludge samples were filtered through glass fiber filters (GF8,
Whatman) and the solid fraction was frozen. Digested sludge was directly frozen
without filtration. Samples were subsequently freeze-dried and finely ground in
a mortar. They were stored in amber glass bottles at -25 °C until analysis (up to
2 years). Consequently, the results obtained for activated sludge are given in
pg/kg dry weight (dw), while those for digested sludge, including the aqueous
phase, are given in pg/L. The concentration of solids in the freeze-dried digested
sludge was determined to be 17 ± 6 g/L.
3.2.3 Sample Preparation
For USE an aliquot (500 mg) of freeze-dried sludge was successively extracted
with 4 mL and 2 mL methanol and then two times with 2 mL acetone (Table
3.1). In each extraction step, the sample slurry was ultrasonicated for 5 min.
Surrogate standards (see 3.2.4) were spiked into the slurry of the first methanol
extraction before ultrasonication. The slurries were centrifuged at 3 000 rpm for
5 min after each extraction step and the supematants collected. The solvent of
the combined supematants was evaporated to a volume of -200 pL, which was
then diluted with 150 ml of local ground water for solid phase extraction as a
clean-up step.
Analytical Methodfor Sewage Sludge 61
Table 3.1 Extraction procedurefor sulfonamides, macrolides and trimethoprim
from activated sludge
pressurized liquid ultrasonic solvent
parameter extraction extraction
(PSE) (USE)
sample amount 200 mg 500 mg
solvent methanol : water methanol
(1:1, v:v) acetone
time 3 cycles of 5 min 4 times for 5 minutes
(preheating time 5 min) (4 mL methanol, 2 mL methanol,
2 mL acetone, 2 mL acetone)
temperature 100 °C -
pressure 100 bar -
flush 120% of cell volume
for all 3 cycles
-
nitrogen purge 60 sec -
For PSE samples of freeze-dried sludge were weighed (200 mg) and transferred
into 11-mL extraction cells (Dionex) partly filled with quartz sand. During
mixing, more sand was added until the cell was completely filled. For extraction
an automated Dionex ASE 200 accelerated solvent extractor (Sunnyvale, CA,
USA) equipped with a solvent controller was used. A methanol-water mixture
(50/50, v/v) proved to be optimal as extraction solvent. An extraction
temperature of 100 °C and an extraction pressure of 100 bar were chosen as
operating conditions (Table 3.1). Preheating time and static time were set to 5
minutes each. A total flush volume of 120% the cell volume and a purge time of
60 sec with nitrogen was used. The final extraction volume was ~ 22 mL with 3
extraction cycles for activated sludge and 2 for digested sludge. The PSE
extracts were completely transferred to 500 mL amber glass bottles by rinsing
the collection vial with -100 mL of de-ionized water in 3 steps. They were
further diluted with -350 mL de-ionized or local ground water to reduce the
methanol content of the sample for solid-phase extraction to below 5%.
Surrogate standard (see 3.2.4) was spiked directly on the sludge in the extraction
cell or in the PSE extract prior to dilution.
62 Chapter 3
The respective extracts of both extraction methods were adjusted to pH 4 with
sulfuric acid or directly enriched (pH -7). Solid-phase extraction was performed
on 6 mL Oasis HLB sorbent cartridges (200 mg) (Waters, Bergen op Zoom, The
Netherlands). Detailed information on sample preparation can be found in
Chapter 2.[34]
3.2.4 ChemicalAnalysis
Different methods were used at two different laboratories for the separation and
detection of sulfonamides, macrolides and trimethoprim in sludge extracts
(Figure 3.2), both based on the method published for aqueous wastewater
samples.[34'35J In method 1, separation was achieved using a 150 x 2 mm YMC
Pro CI8 column (120 Â, 3 pm, Stagroma, Reinach, Switzerland) and a mobile
phase of methanol-water containing 1% (v/v) formic acid. Gradient elution was
used at a flow rate of 150 pl/min. A triple quadrupole mass spectrometer, TSQ
Quantum Discovery (Thermo Finnigan, San Jose, CA, USA), equipped with
electrospray ionization was used for detection. A spray voltage of 3 500 V and a
capillary temperature of 350 °C were applied. Analyses were performed in the
positive multiple reaction mode using two transitions per analyte. An external
calibration curve in de-ionized water was used for quantification. Results were
corrected by relative recoveries over solid-phase extraction and measurement
determined in the same experimental series. Therefore the following substances
(100 ng) were added to the ASE extracts: d4SDZ, d4STZ, d4SMX, ,3C6SMZ as
surrogate standards for sulfonamides and trimethoprim and tylosin (TYL) as
surrogate standard for macrolides.
In method 2, separation was achieved on a 100 x 4.6 mm Chromolith
Performance RP-18e column at a flow rate of 400 pL/min and a total mn time of
50 min. Gradient elution was performed with solvent A (water containing 10%
acetonitrile and ammoniumacetat (lOmM)) and solvent B being a mixture of
80% acetonitrile and 20% solvent A. Initial conditions were set to 100% A.
After 10 min the percentage of B was increased to 26% within 5 min and to 38%
in the following 2 min. After 7 min of 38% B, the percentage of B amounts to
100% in a time span of 6 min, where it was kept for 4 min. Within 2 min initial
conditions were restored and mn for another 14 min. Detection was performed
using a triple quadmpole mass spectrometer, API 4000 (Applied Biosystems,
Foster City, CA, USA), equipped with electrospray ionization. An ion source
Analytical Methodfor Sewage Sludge 63
Figure 3.2 Scheme for the extraction and analysis ofsulfonamides, macrolides
and trimethoprim in sewage sludge
0.5 g activated or digested sludge
(freeze-dried)
0.2 g activated or digested sludge
(freeze-dried)
ultrasonic solvent extraction (USE)see Table 1
(addition of surrogate standards prior to extraction)
pressurized liquid extraction (PSE)see Table 1
(addition of surrogate standards after (method 1 ) or prior
(method 2) to extraction )
dilluted sludge extract
(pH 4 or pH 7)
solid phase extraction
Oasis HLB, 6 mL, 200 mg
elution: methanol/ethyl acetate/ammonia
evaporation to -50 uL by N2-stream(addition of instrumental standard)
Method 1
liquid chromatography
column: YMCproC18
gradient: water/methanol/
formic acid
tandem mass spectrometry
(TSQ Quantum Discovery)
Method 2
liquid chromatography
column: Chromolith Performance
RP-18e
gradient: water/acetonitrile/
ammonium acetate
tandem mass spectrometry
(API 4000)
voltage of 5 000 V and a temperature of 750 °C were applied, while the
declustering potential was compound dependent and ranged between 56 and
106 V. Analyses were performed in the positive multiple reaction mode using
two transitions per analyte. Quantification was performed using an internal
calibration curve in local ground water. Surrogate standard (100 ng) was added
prior to extraction. d4SMX was used as surrogate standard for all sulfonamides
investigated and oleandomycin (OLE) for all macrolides. No surrogate standard
was used for azithromycin and sulfapyridine, which were subsequently
quantified by comparing peak areas of the samples and the calibration. In the
case of sulfapyridine, results obtained were additionally corrected by the
respective absolute recoveries obtained from spiking experiments.
64 Chapter 3
In both methods, the SPE extracts were mostly diluted up to 10-fold with de-
ionized water prior to measurement. Filtration of the final extracts prior to
measurement, lead to significant losses of the analytes, especially in the case of
the macrolide antimicrobials.
3.2.5 MethodDevelopment
For PSE method development, activated sludge from WWTP-K was filtered and
the solid fraction was spiked with an aqueous solution raising the concentration
of analytes by approximately 400 pg/kg dw .The mixture was stirred manually
for Vi h and subsequently freeze-dried. This was considered to be the best
substitute for native sludge where the interaction between compounds and
sludge may be different due to aging effects. Spiking was necessary since not all
compounds investigated were present in the sludge sample taken. By varying
extracting conditions the following parameters were optimized by duplicate
analyses in the order given: extraction solvent (9 solvents and mixtures),
extraction temperature (60/80/100/150/200 °C), cycle time (1/3/5/10/20 min),
extraction pressure (60/80/100/120/150 bar) and sample amount (100/200/400
mg). Multiple sequential extraction (4x5 min, n = 2) of the same sludge sample
(activated and digested) was performed to ensure quantitative extraction.
Therefore the extracts of the individual cycles were collected separately. The
maximum extractable amount was defined as the sum of the amounts measured
in the four cycles. The amount recovered in each cycle was expressed as a
percentage of this sum (extraction yield). To assess the stability of the
compounds investigated during PSE extraction, quartz sand as inert matrix was
spiked with analytes (100 ng) and extracted (n = 2) as described.
In the case of the USE method, parameters generally suitable for the extraction
of sewage sludge were chosen (Table 3.1).[36] Exhaustive extraction under the
given conditions was tested by prolonged extraction of activated sludge with
acetone.
3.2.6 Method Validation
Accuracy was assessed by relative recovery studies using area ratios
(analyte/surrogate standard) for quantification. To evaluate the whole method,
freeze-dried activated sludge was spiked prior to extraction in the extraction cell
Analytical Methodfor Sewage Sludge 65
with analytes (50-100 ng) in methanol and surrogate standard and subsequently
analyzed (n = 2-3). For relative recoveries over solid-phase extraction and
measurement, activated sludge extracts were spiked with analytes (50-100 ng)
and surrogate standard prior to solid-phase extraction (n = 2). The calculated
amount of antimicrobials minus the amount already present before spiking (n =
2-3) was related to the spiked concentration. Absolute recoveries were obtained
using absolute areas instead of area ratios. The areas obtained in spiked
activated sludge (50-100 ng, prior to or after extraction) minus the areas
obtained in the respective non-spiked samples, were compared to the areas
obtained from an external standard with the same concentration as the spike.
Breakthrough of the analytes on the SPE cartridges was determined by the
enrichment of spiked activated sludge (400 pg/kg dw) in duplicate analyses
using two stacked cartridges. A breakthrough on the first cartridge triggered an
enrichment on the consecutive cartridge, which was then eluted separately.
Complete elution of the cartridges was verified by eluting cartridges for a
second time with 1.5 mL acetone as a stronger solvent (n = 2). The acetone
extract was then treated as a separate sample. The precision of the entire method
was determined by extracting replicates (n = 3-6) of spiked activated sludge (90-
500 pg/kg dw). It was defined as the relative standard deviation of the amount
measured. Limits of quantification (LOQ) were defined by two methods. In the
case of PSE, the LOQ was defined as concentrations in a sample matrix
resulting in signals with signal-to-noise (S/N) ratios of 10. The concentration
corresponding to the defined S/N was determined by scaling down, using the
measured concentration and the assigned S/N ratio of the peak - assuming a
linear correlation through zero. Results from several samples (n = 6) were used
to yield an average value. In the case of USE, the second lowest concentration in
the linear range of the internal calibration curve in local groundwater with a S/N
ratio exceeding 10 was used to estimate the LOQ.
3.3 Results and Discussion
3.3.1 MethodDevelopment
For PSE the effect of the different extraction parameters on the extraction
efficiency was evaluated to obtain optimal relative extraction conditions for
sulfonamides, macrolides and trimethoprim from activated sludge (Table 3.1).
66 Chapter 3
Various solvents and mixtures were tested first. Once the optimum solvent
mixture was determined other extraction parameters, such as extraction
temperature and pressure, cycle time, number of cycles and sample amount,
were investigated.
Extraction solvent
Table 3.2 shows the results obtained from using water, organic solvents and
various mixtures as extraction solvent. A total of 10 substances was
investigated, however, only the results of the compounds mainly found in
activated sludge samples are presented: sulfamethoxazole, sulfapyridine,
azithromycin, clarithromycin, roxithromycin and trimethoprim.
Lower extraction efficiencies were observed for all compounds investigated,
especially macrolides, when mixtures of methanol and other organic solvents
(aceton or acetonitrile, 1:1) were used. Water itself proved to be a good
extraction solvent for the sulfonamides but resulted in low extraction
efficiencies for macrolides. More trimethoprim seems to be extracted with
increasing amounts of methanol, whereas no significant influence on the
sulfonamides was observed. For macrolides the highest extraction efficiencies
were observed using a mixture of water and organic solvent at a ratio of 1:1.
This is in accordance with previous findings of Salvatore & Katz[37] that
reported increasing solubility of macrolides to a maximum with increasing
solvent polarity. Mixtures of water with organic solvents other than methanol
(1:1) showed similar results for most analytes but resulted in lower extraction
efficiencies for sulfapyridine and trimethoprim. Methanol-water at a ratio of 1:1
was finally chosen as extraction solvent representing the best compromise for all
compounds investigated. With a pKa of -9 macrolides are weak bases that are
positively charged at neutral pH. Since the surface of most particles in sewage
sludge are negatively charged[381 ionic interactions may play a role in the
sorption of macrolides to sewage sludge. Therefore the effect of the pH of the
chosen extraction solvent was investigated. No significant change in extraction
efficiency for any of the analytes was observed when the pH of the water used
was adjusted to 10 with sodium hydroxide. This may be caused by the buffer
capacity of the sludge or indicate that hydrophobic interactions are
predominantly responsible for the sorption of macrolides to activated sludge.
Analytical Methodfor Sewage Sludge 67
Table 3.2 Solvent influence on the extraction of sulfonamides, macrolides and
trimethoprimfrom activated sludgea
concentrationb
(pg/kg dw)
extraction solvent SPY SMX TRI AZI CLA ROX
methanol/acetone (1:1) 116 527 138 113 42 112
methanol/acetonitrile (1:1) 120 572 139 133 74 121
methanol 268 594 321 252 180 195
methanol/water (3:1) 282 635 295 260 219 251
methanol/water (1:1) 287 667 225 368 337 351
methanol/water (1:3) 289 663 217 103 339 369
water 291 667 228 33 211 231
water/acetone (1:1) 125 652 144 485 341 364
water/acctonitrile (1:1) 214 698 222 375 314 343
aSelected operating condition in bold letters.
Mean of duplicate analyses using pressurized liquid extraction. Extraction
parameters: 100 °C, 100 bar, 1 cycle of 10 minutes, 150% flush. Extracts adjusted to
pH 4 prior to solid phase extraction. Chemical analysis: Method 1.
Similar conclusions for the macrolide tylosin were made by Tolls,[28] when
investigating the sorption of veterinary pharmaceuticals in soil.
Extraction temperature and pressure
The effect of extraction temperature on the extraction efficiencies of the analytes
turned out to be less profound. An extraction temperature of 100 °C was selected
as operating condition. Slightly lower extraction efficiencies (10 - 20%) were
observed for all analytes at temperatures below 100 °C. However, if the
extraction temperature was increased above 100 °C, the extracted amounts
decreased drastically. Compared to the chosen extraction temperature, only 60 -
80% of most sulfonamides and trimethoprim were measured at an extraction
temperature of 200 °C. For sulfamethoxazole a reduction by 95% was observed
and by 60 - 90% for the macrolides investigated. These findings may be ascribed
to a thermal degradation of the analytes at temperatures above 100 °C.
Additionally, it was observed that increasingly darker extracts were obtained at
higher extraction temperatures, indicating a larger extraction of soluble organic
i
68 Chapter 3
matter. This resulted in problems during solid-phase extraction due to the
clogging of the cartridges. An identical effect was observed when increasing the
extraction pressure from 60 to 150 bar. However, no significant impact of
increasing extraction pressure was observed on the extraction efficiencies of the
compounds investigated (data not shown).
Cycle time and sample amount
A cycle time of 5 min resulted in maximum extraction efficiencies for almost all
compounds. However, the effect of the extraction time observed was low
(variations below 20%) for the investigated sulfonamides, macrolides and
trimethoprim (data not shown). An influence of the cycle time on the extraction
efficiencies may be expected due to the higher extraction temperature used in
PSE resulting in a reduction of the viscosity of the solvent. It may therefore
penetrate further into the sample matrix, a process also facilitated by the
increased pressure. The extraction efficiencies may furthermore be enhanced by
the swelling of the matrix while in contact with the solvent. These processes can
also be influenced by the ratio of sample matrix to extraction solvent. However,
no significant influence on the extraction efficiency of the analytes from varying
sample amounts was observed (data not shown).
Number of cycles
Multiple sequential extractions of the same sample (activated and digested
sludge) were performed to evaluate the ability of the method to quantitatively
extract sulfonamides, macrolides and trimethoprim from the matrices
investigated. For all analytes, except azithromycin, no significant amounts
(>2%) were recovered from activated or digested sludge after the first cycle. As
shown in Figure 3.3 approximately 90% of azithromycin was recovered from
activated sludge in the first cycle. Another 7% were recovered in the second
cycle, whereas the amounts present in the last two cycles were not quantifiable.
Therefore 3 cycles were performed in the analyses of activated sludge to assure
quantitative extraction. In the case of digested sludge 82% of azithromycin was
recovered in the first cycle and another 12% in the second cycle. Even though
small amounts could still be detected in the third (4%) and forth (2%) cycle, two
extraction cycles were chosen for the extraction of digested sludge. The slightly
Analytical Methodfor Sewage Sludge 69
Figure 3.3 Results for azithromycin from the multiple sequential extraction of
activated and digested sludgea
100
80
S 6092">,
c
o
"S 40
CD
CD
20
h-H
i
I
1
,
activated sludge
Ddigested sludge
*r12 3 4
number of extraction cycle
aError bars represent the range of duplicate analyses. Pressurized liquid extraction:
parameters see Table 3.1. Extracts adjusted to pH 4 prior to solid phase extraction.
Chemical analysis: Method 1.
bPercentage ofthe total amount extracted in the four cycles.
incomplete extraction of azithromycin was neglected since severe problems
were encountered in solid-phase extraction (clogging of the cartridges) and
measurement (bad peak shape) when more than 2 cycles were performed. These
findings indicate that the extraction efficiency of azithromycin varies with the
sample matrix. It has to be noted however, that complete method development
for PSE was performed only for activated sludge.
Also in the case of ultrasonic solvent extraction, exhaustive extraction of
activated sludge was achieved with the chosen parameters (Table 3.1), since no
significant amounts of analyte could be detected in the acetone extract of an
already extracted sample.
Thermal degradation
Since thermal degradation seems to occur at elevated temperatures and longer
extraction times, the stability of the analytes under the chosen extraction
70 Chapter 3
conditions for PSE was of potential concern. However, recoveries from spiked
quartz sand (n = 2) varied around 100% for all substances giving no evidence of
thermal instability. Deviating results were obtained for trimethoprim (150%) and
azithromycin (81 %) and are probably due to a different behavior of the analytes
and the respective surrogate standards (13C6SMZ and TYL) during solid phase
extraction.
3.3.2 Method Validation
Accuracy
The accuracy of the method, expressed by relative recoveries, is influenced by
different parameters, e.g. the suitability of the surrogate standard used or the
method applied for chemical analysis. For pressurized liquid extraction, solid
phase extraction at pH 4 and method 1 for separation and detection for example
the relative recovery ranged between 78 and 106% for the sulfonamides and
trimethoprim and between 91 and 142% for the macrolides (Table 3.3). In that
case no major differences were observed between relative recoveries over the
entire method (including extraction) and over solid-phase extraction and
measurement (excluding extraction). The results from both studies were
therefore combined. The small variations obtained when combining both,
illustrate the thermal stability of the compounds during extraction. Additionally,
they indicate that the analytes spiked on the freeze-dried activated sludge are
extracted quantitatively with the selected extraction conditions. Since spiked
analytes are not exposed to the same active sites as native pollutants this result
cannot be extrapolated to native activated sludge samples. However, quantitative
extraction of native sulfonamides, macrolides and trimethoprim was shown for
activated sludge with the developed method by performing multiple sequential
extraction experiments.
In the case of absolute recoveries no correction by using surrogate standards is
performed. They therefore mirror possible losses during extraction, sample
preparation and variations in measurement due to matrix effects. From the
results obtained during method development and validation it seems that matrix
effects, e.g. ion suppression, are the most important factor. Absolute recoveries
were determined using two different methods for chemical analysis (see 3.2.4),
but the same method for sample preparation. In both cases PSE with identical
Analytical Methodfor Sewage Sludge 71
Table 3.3 Relative and absolute recoveries for sulfonamides, macrolides and
trimethoprim in activated sludge using method 1 for chemical analysisa
retention relative recoveryb absolute recoveryc
time (%) (%)
compound (min) average
106
t %SD
7
average
63
%SD
SDZ 10.3 6
STZ 12.7 99 5 55 7
SMZ 17.6 97 5 64 17
SPY 12.6 79 5 64 8
SMX 20.4 100 3 64 3
TRI 17.1 78 3 51 4
AZI 21.1 91 10 29 7
ERY-H20 30.1 112 9 37 14
CLA 31.5 110 13 33 24
ROX 31.6 142 16 45 27
a
Pressurized liquid extraction: parameters see Table 3.1. Extracts adjusted to pH 4
prior to solid phase extraction. Chemical analysis: Method 1.
Relative recoveries were determined using area ratios of analyte to surrogate
standard. Average and relative standard deviation (%SD) combing results from
experiments with surrogate standard added prior to or after extraction (n = 4).cAbsolute recoveries were determined using areas. Average and relative standard
deviation (%SD) combing results from experiments with surrogate standard added
prior to or after extraction (n = 4).
parameters was used and the extracts were adjusted to pH 4 prior to SPE.
Results obtained for method 1 are given in Table 3.3, while those for method 2
are included in Table 3.4 (PSE, pH = 4). Similar absolute recoveries were
obtained with both methods for sulfonamides and trimethoprim. In the case of
the macrolides, significantly lower values, and therefore higher ion suppression,
were obtained for method 1 compared to method 2. This could be caused by a
different separation of matrix and analytes during liquid chromatography, i.e. by
the choice of column and gradient. Differences in separation are also mirrored
by the varying retention times of the compounds in the two methods.
Additionally, two different mass spectrometers were used, which may also
Table3.4Absoluterecoveriesforsu
lfon
amid
es,macrolidesandtrimethoprim
inactivatedsludge
usingmethod2forchemical
analyis
absoluterecovery
a
(%)
ASE
bUSE
c
PH==4
pH=--1
pH=4
pH=--
1
compound
RT
average
83
%SD
12
average
54
%SD
6
average
41
%SD
13
average
53
%SD
SDZ
8.6
11
SMX
20.4
37
19
41
416
16
62
7
TRI
20.0
47
744
325
10
31
8
SMX-J4
d20.4
37
15
44
416
11
62
8
CLA
33.4
74
21
90
555
859
15
ROX
33.9
91
33
88
373
10
76
8
OLEd
25.3
93
995
367
557
14
a
Absoluterecoveriesweredeterminedusing
areas.Averageand
relativestandarddeviation(%SD)
isgiven
(n=
3)Re
spec
tive
relative
recoveriescanbecalculatedfromtheabsoluterecoveryratiooftheanalyteand
itssurrogatestandard.
bPressurizedliquid
extraction(P
SE):
parameters
seeTable
3.1.
Extractsadjusted
topH
4priortoso
lid-
phas
eextraction(pH=
4)or
directly
enriched(pH=
7).Chemical
anal
ysis
:Method
2.Surrogatestandardaddedpr
iortoextraction.
0
Ultrasonicsolventextraction(USE):parameters
seeTable
3.1.Extractsadjusted
topH4
priortoso
lid-
phas
eextraction(pH=
4)or
directly
enriched
(pH=
7).Chemical
anal
ysis
:Method
2.Surrogatestandardaddedpr
iortoextraction.
dUsed
assurrogatestandard.
Analytical Methodfor Sewage Sludge 73
influence the ionization efficiency of macrolides in the samples. Especially, the
differences in temperature applied and the amount of in-source fragmentation
may lead to different ionization efficiencies for the two methods. Further on, the
absolute recoveries were obtained from the analysis of different activated sludge
samples, which also has an effect on the matrix present.
Additionally, the influence of the sample pH during solid phase extraction (SPE)
on the absolute recoveries was investigated. No distinct influence was observed
on the absolute recoveries for the investigated antimicrobials (Table 3.4). The
strong pH dependence of the sulfonamide interaction with the SPE cartridge, as
described for aqueous wastewater samples in Chapter 2, seems not to occur in
sewage sludge extracts. More or less comparable absolute recoveries were also
observed for the investigated compounds at both pH values independently of the
extraction method used. However, a significantly higher relative standard
deviation, of up to 33%, was observed if the pH of the sample was adjusted to 4
prior to SPE. This is caused by an increased clogging of the SPE cartridges at
the lower pH, which made the enrichment of the total sample volume in some
cases impossible.
A dilution of the samples prior to analysis lead to a decrease of matrix effects,
since the areas obtained in diluted samples were reduced to a lesser extent than
expected by the respective dilution factor. In method 1, for example, absolute
recoveries in undiluted samples were 26-50% lower than in 6-fold diluted
samples for sulfonamides. For macrolides and trimethoprim the reduction
ranged between 40 and 80% compared to diluted samples.
Breakthrough and complete elution
Due to the simultaneous extraction of significant amounts of soluble organic
matter during extraction of sewage sludge, breakthrough of the analytes from the
cartridges and complete elution from the cartridges were investigated. No
quantifiable amounts of the analytes could be detected on the second cartridge,
which was eluted separately. When testing for complete elution, also no
quantifiable amounts of analytes could be measured in the acetone eluates of
already eluted cartridges. Thus, the analytes are quantitatively enriched by one
cartridge and exhaustively eluted in the case of activated sludge extracts by the
procedure applied.
74 Chapter 3
Table 3.5 Limits of quantification for sulfonamides, macrolides and
trimethoprim in activated sludge
limit of quantification (pg/kg dw)
pressurized liquid extractiona ultrasonic solvent extraction
compound average range
SDZ 4 3-7 4
STZ 41 31-51 -
SMZ 16 12-20 4
SPY 29 21-36 4
SMX 15 10-23 4
TRI 14 9-17 10
AZI 3 2-4 40
ERY-H20 6 5-8 -
CLA 4 3-6 10
ROX 3 2-4 10
aConcentration estimated from measured samples (Method 1) for a signal-to-noise
ofl0(n=6).bDefined as the second lowest linear concentration of the internal calibration curve in
local groundwater (Method 2).
Precision
Precision was characterized as the relative standard deviation resulting from the
multiple determination of the analytes in activated sludge. It ranged between 2
and 8% for pressurized liquid extraction and between 7 and 20% for ultrasonic
solvent extraction. The higher values for USE are probably caused by a higher
amount of matrix extracted with the solvents used for ultrasonic solvent
extraction. Another reason may lay in the series of manual extraction steps
necessary compared to the fully automated extraction during PSE.
Limit of quantification
The limits of quantification for the analytes in activated sludge were defined
using two different approaches for pressurized liquid and ultrasonic solvent
extraction, respectively (Table 3.5). Overall, it ranges between 3 and 41 pg/kg
Analytical Methodfor Sewage Sludge 75
dw for the investigated antimicrobials. The differences observed result from a
combination of various factors. Next to the different approaches applied for the
estimation of the LOQ, the higher sample amount used in USE compared to PSE
plays a role. Additionally, differences in the methods used for separation and
detection have an influence, e.g. via peak shape and matrix effects. The results
clearly indicate that the limits of quantification given can only be considered
rough estimates. In routine analysis all peaks with a S/N above 10 were
therefore considered valid results.
3.3.3 Application to Sewage Sludge Samples
The developed methods were applied to selected activated and digested sludge
samples from different wastewater treatment plants in Germany and Switzerland
(Table 3.6). The results for the most commonly detected sulfonamides,
sulfapyridine and sulfamethoxazole, and macrolides, azithromycin,
clarithromycin and roxithromycin, are given. Additionally results for
trimethoprim, used almost exclusively in combination with sulfonamides, are
included. The occurrence of antimicrobials in activated sludge generally
correlates well with the respective aqueous phase.[n'34] Higher concentrations
were generally determined in German activated sludge samples (WWTP-W),
ranging up to 197 pg/kg dw for sulfapyridine, indicating a lower wastewater
dilution compared to Switzerland. A maximum concentration of 73 pg/kg dw
was found for sulfamethoxazole in Swiss samples (WWTP-K and WWTP-A). A
more detailed discussion on the occurrence of sulfonamides, macrolides and
trimethoprim in Swiss municipal wastewater treatment is given in Chapter 4.
Overall, similar results were obtained in activated and digested sludge using
pressurized liquid extraction, independently of the sample pH and the method
used for chemical analysis (Table 3.6). However, using ultrasonic solvent
extraction, the concentrations determined are generally lower for the
investigated sulfonamides and in tendency lower for the investigated macrolides.
This may be caused by the less radical extraction conditions, e.g. temperature
and pressure, compared to pressurized liquid extraction. Additionally, the
extraction conditions used for USE, especially the choice of solvent, were not
optimized particularly for the extraction of sulfonamide and macrolide
antimicrobials.
76 Chapter 3
Table 3.6 Concentrations of sulfonamides, macrolides and trimethoprim in
activated and digested sewage sludge from different wastewater treatment
plants in Germany (WWTP Wiesbaden) and Switzerland (WWTP Kloten-
Opfikon and WWTP Altenrhein)
concentrationa
SPY SMX TRI AZI CLA ROX
activated sludges ug/kg dwb
WWTP-W ASE + pH 4c 57 113 91 127 34 46
sample 1 ASE + pH 7d
51 100 87 158 41 61
USE + pH 7c
26 41 79 127 34 45
WWTP-W ASE + pH 4 197 41 107 151 27 131
sample 2 ASE + pH7 160 37 133 115 16 83
USE + pH 7 85 18 96 47 (9)e 50
WWTP-K ASE + pH 4 29 73 30 52 30 ndg
ASE + pH 7 24 51 (18)f (7)f 25 nd
USE + pH 7 nag 20 14 (21)f 12 nd
WWTP-A ASE + pH 4 (ll)f 60 21 56 63 nd
ASE + pH 7 nd 34 13 (5)' 32 nd
USE + pH 7 nd 27 nd 48 41 nd
digested sludges
ASE + pH 4
ug/Lh
WWTP-K 1.0 nd (0.1)f 2.3 0.8 nd
ASE + pH 7 0.8 nd nd 1.6 0.3 nd
USE + pH 7 1.2 nd nd 1.3 0.3 nd
a
Mean of duplicate analyses for pressurized liquid extraction (PSE) and single
analysis for ultrasonic solvent extraction (USE).b
Separation of solid and aqueous phase through flltration before freeze-drying.cPressurized liquid extraction: parameters see Table 3.1. Extracts adjusted to pH 4
prior to solid-phase extraction. Chemical analysis: Method 1.
dPressurized liquid extraction: parameters Table 3.1. Extracts not pH-adjusted prior to
solid-phase extraction. Chemical analysis: Method 2.
e
Ultrasonic solvent extraction: parameters Table 3.1. Extracts not pH-adjusted prior to
solid-phase extraction. Chemical analysis: Method 2.
fEstimated concentrations below the limit of quantification (S/N < 10).
ê nd: not detected (S/N < 3), na: not analyzedhNo separation of solid (15-18 g/L) and aqueous phase through filtration before
freeze-drying.
Analytical Methodfor Sewage Sludge 77
3.4 Conclusions
A robust and selective method for the pressurized liquid extraction of
sulfonamides, macrolides and trimethoprim, from sewage sludge was developed
and validated. Several extraction parameters were investigated and the
optimized procedure is summarized in Table 3.1. The method was successfully
applied to activated and digested sewage sludge. Even though comparable
results were obtained for different sample pHs, it is suggested to not adjust the
pH of the extracts prior to solid-phase extraction, to minimize the clogging of
the cartridges. The method presented can be used to investigate the occurrence
and fate of sulfonamides, macrolides and trimethoprim in wastewater treatment,
including the sorption to sewage sludge. Additionally, it may serve as the basis
for the determination of pharmaceuticals in general in sewage sludge and other
biosolids. Ultrasonic solvent extraction seems to be equally or slightly less
efficient for the extraction of macrolides and trimethoprim, while significantly
lower extraction efficiencies seem to result for sulfonamides compared to
pressurized liquid extraction.
3.5 Acknowledgments
Abbott GmbH (Wiesbaden, Germany) is acknowledged for supplying
clarithromycin and Pfizer AG (Zurich, Switzerland) for supplying azithromycin.
Financial support came from the EU project POSEIDON (EVK1-2000-00047,
www.eu-poseidon.com) and the EAWAG project on human-use antibiotics
(HUMABRA, www.nrp49.ch/pages/) within the framework of the National
Research Program on antibiotic resistance funded by the Swiss National Science
Foundation.[40] We thank Elvira Keller, Niccolo Hartmann and Matthias Ruff for
their technical assistance and advice. For helpful comments on the manuscript
we acknowledge M. Suter and M. Ruff.
3.6 Literature cited
[1] Annual Report; Swiss Importers of Antibiotics (TSA): Berne, Switzerland,
1998.
[2] Pharmaceuticals Sold in Switzerland; Swiss Market Statistics, 1999.
78 Chapter 3
[3] Bund/Länderausschuss für Chemikaliensicherheit (BLAC), "Arzneimittel
in der Umwelt - Auswertung der Untersuchungsergebnisse," Hamburg,
2003.
[4] Stan, H. L; Heberer, T. Analusis 1997, 25, M20-M23.
[5] Ternes, T. A. Water Res. 1998, 32, 3245-3260.
[6] Halling-Sorensen, B.; Nors Nielsen, S.; Lanzky, P. F.; Ingerslev, F.; Holten
Lützenhoft, H. C; Jorgensen, S. E. Chemosphere 1998, 36, 357-393.
[7] Daughton, C. D.; Ternes, T. A. Environ. Health Perspect. 1999, 107, 907-
938.
[8] Kümmerer, K. Pharmaceuticals in the environment: Source, fate, effects
and risks; Springer: Berlin, Heidelberg, New York, 2001.
[9] Heberer, T. Toxicol. Lett. 2002,131, 5-17.
[10] Giger, W.; Alder, A. C; Golet, E. M.; Kohler, H.-P. E.; McArdell, C. S.;
Molnar, E.; Siegrist, H. R.; Suter, M. J.-F. Chimia 2003, 57, 485-491.
[11] Hirsch, R.; Ternes, T. A.; Haberer, K.; Kratz, K.-L. Sei. Total Environ.
1999,225,109-118.
[12] Kolpin, D. W.; Furlong, E. T.; Meyer, M. T.; Thurman, E. M.; Zaugg, S.
D.; Buxton, H. T. Environ. Sei. Technol. 2002, 36, 1202-1211.
[13] Sacher, F.; Lochow, E.; Bethmann, D.; Brauch, H.-J. Vom Wasser 1998,
90, 233-243.
[14] Alder, A. C; McArdell, C. S.; Golet, E. M.; Ibric, S.; Molnar, E.; Nipales,
N. S.; Giger, W. In Pharmaceuticals and Personal Care Products in the
Environment: Scientific and Regulatory Issues; Daughton, C. G., Jones-
Lepp, T., Eds.; Symposium Series 791; American Chemical Society:
Washington, D.C., 2001; pp 56-69.
[15] Golet, E. M.; Aider, A. C; Giger, W. Environ. Sei. Technol. 2002, 36,
3645-3651.
[16] McArdell, C. S.; Molnar, E.; Suter, M. J.-F.; Giger, W. Environ. Sei.
Technol. 2003, 37, 5479-5486.
[17] Golet, E. M.; Strehler, A.; Aider, A. C; Giger, W. Anal. Chem. 2002, 74,
5455-5462.
[18] Di Corcia, A.; Nazzari, M. J. Chromatogr., A 2002, 974, 53-89.
[19] Haller, M. Y.; Müller, S. R.; McArdell, C. S.; Aider, A. C; Suter, M. J.-F.
J. Chromatogr., A 2002, 952, 111-120.
[20] Pfeiffer, T.; Tuerk, J.; Bester, K.; Spiteller, M. Rapid Commun. Mass
Spectrom. 2002,16, 663-669.
Analytical Methodfor Sewage Sludge 79
[21] Loke, M.-L.; Tjornelund, J.; Halling-Sorensen, B. Chemosphere 2002, 48,
351-361.
[22
[23
[24
[25
[26
[27
[28
[29
[30
[31
[32
[33
[34
[35
[36
[37
[38
Fedeniuk, R. W.; Shand, P. J. J. Chromatogr. 1998, 812, 3-15.
Hamscher, G.; Sczesny, S.; Höper, H.; Nau, H. Anal. Chem. 2002, 74,
1509-1518.
Boxall, A. B. A.; Blackwell, P.; Cavallo, R.; Kay, P.; Tolls, J. Toxicol. Lett.
2002,131, 19-28.
Schlüsener, M. P.; Spiteller, M.; Bester, K. J. Chromatogr., A 2003, 1003,
21-28.
Hartmann, N.; Diploma Thesis, ETH Zurich, Switzerland, 2003.
Thiele-Bruhn, S. J. Plant Nutr. Soil Sei. 2003,166, 145-167.
Tolls, J. Environ. Sei. Technol. 2001, 35, 3397-3406.
Löffler, D.; Ternes, T. A. J. Chromatogr., A 2003,1021, 133-144.
Horie, M.; Takegami, H.; Toya, K.; Nakazawa, H. Anal. Chim. Acta 2003,
492, 187-197.
Van Eeckhout, N.; Castro Perez, J.; Van Peteghem, C. Rapid Commun.
Mass Spectrom. 2000,14, 2331-2338.
Diaz-Cruz, M. S.; Lopez de Aida, M. J.; Barcelo, D. Trends Anal. Chem.
2003,22,340-351.
Dean, J. R. Extraction methodsfor environmental analysis, 1998.
Göbel, A.; McArdell, C. S.; Suter, M. J.-F.; Giger, W. Anal. Chem. 2004,
76, 4756 - 4764.
Hirsch, R.; Ternes, T. A.; Haberer, K.; Mehlich, A.; Ballwanz, F.; Kratz,
K.-L. J. Chromatogr., A 1998, 815, 213-223.
Ternes, T. A.; Bonerz, M.; Hermann, N.; Keller, E.; Bago Lacida, B.;
Aider, A. C. submitted to Journal ofChromatography A 2004.
Salvatore, M. J.; Katz, S. E. J. AOACInt. 1993, 76, 952-956.
Carberry, J. B.; Englande, A. J. Sludge Characteristics and Behavior;
Martinus Nijhoff Publishers: Boston, The Hague, Dordrecht, Lancaster,
1983.
P*" /
Ö
Chapter 4
Occurrence in Wastewater Treatment
The occurrence of sulfonamide and macrolide antimicrobials, as well as
trimethoprim, was investigated in conventional activated sludge treatment.
Average daily loads in untreated wastewater correlated well with those estimated
from annual consumption data and pharmacokinetic behavior. Considerable
variations were found during a day and seasonal differences seem to occur for
the macrolides, probably caused by a higher consumption of these substances in
winter. The most predominant macrolide and sulfonamide antimicrobials were
clarithromycin and sulfamethoxazole, respectively. In the case of
sulfamethoxazole, the main human metabolite, A^-acetylsulfamethoxazole, was
included as an analyte, accounting for up to 86% of the total load in untreated
wastewater. The results obtained illustrate the importance of considering
retransformable substances, for example human metabolites, when investigating
the behavior and fate of pharmaceuticals. Average concentrations of
sulfapyridine, sulfamethoxazole, trimethoprim, azithromycin, and clarithromycin
in activated sludge ranged between 28 and 68 pg/kg dry weight. Overall the
sorption to activated sludge was shown to be low for the investigated
antimicrobials, with estimated sorption constants below 500 L/kg. Elimination in
activated sludge treatment was found to be incomplete for all investigated
compounds. In final effluents median concentrations for sulfamethoxazole were
290 ng/L and 240 ng/L for clarithromycin.
i
Göbel, A., Thomsen, A., McArdell, CS., Joss, A., Giger, W.
Occurrence ofSulfonamides, Macrolides and Trimethoprim in Activated Sludge
Treatment including Sorption to Sewage Sludge
Environmental Science and Technology, 2005, in press.
Occurrence 83
4.1 Introduction
The occurrence and potential adverse effects of pharmaceuticals in the aquatic
environment have been the object of increasing interest in recent years, indicated
by the growing number of scientific publications published, for example review
articles.[111] For human-use pharmaceuticals the principle entry route to ambient
surface waters, as unchanged compound or after transformation through human
metabolism, is via municipal wastewater treatment plants (WWTP).
Antimicrobial agents, which are used in human and veterinary medicine to
similar extents, are a particularly important pharmaceutical group due to the
possible spread and maintenance of bacterial resistance. The overall human
consumption of antimicrobials in Switzerland exceeded 30 tons per annum (t/a)
in 1997, with ß-lactam antibiotics (18 t/a) as the largest group, including
penicillins, cephalosporins, penems and other smaller sub-classes.[12"4]
Additional important groups in human medicine are the sulfonamides (6 t/a),
macrolides (4 t/a) and the fluoroquinolones (4 t/a). Domestic consumption, that
is through prescription, accounts for 60 to 80% of the total consumption of
human used pharmaceuticals, making urban wastewater the main input source
into the aquatic environment.
Macrolides, which are mainly active against gram-positive bacteria, inhibit
ribosomal protein synthesis.^151 Their main application is in the treatment of
upper and lower respiratory tract infections, especially as an alternative to
penicillins. They are generally not metabolized to a large extent but are mainly
excreted with bile and feces as the unchanged parent substance (Table 1).
Sulfonamides, which are active against a wide range of gram-positive and gram-
negative bacteria, function as competitive antagonists to ^-aminobenzoate in
bacterial folate synthesis.[1617] They are excreted via urine mainly in the form of
metabolites and partly as the unchanged active compound. One class of
important human metabolites is the acetylated forms of sulfonamides, for
example A^-acetylsulfamethoxazole, which accounts for approximately 50% of
the administered dose. The retransformation of A^-acetylsulfamethazine to the
active parent compound sulfamethazine during the storage of manure has been
reported by Berger et al. indicating a possible analogous behavior for other /V4-
acetylated sulfonamides.1183 Some of the excreted metabolites may therefore be
retransformed to the parent compound during wastewater treatment.
Trimethoprim is almost exclusively used in combination with sulfonamides, in a
Table
4.1Characteristicsofsu
lfon
amid
es,trimethoprim
andmacrolides
compound
acronym
CASRN
main
applicationa
humanconsumption
[-
(Switz
erla
nd,1999,
-kg/a]
[13K
%excretedunchanged
inhumanurine
/feces
c
sulfadiazine
SDZ
68-35-9
V+H
68
25%
/-[
17]
sulfathiazole
STZ
72-14-0
V-
50%/-[17]
sulfamethazine
SMZ
57-68-1
V-
10%/
-[17
]
sulf
apyr
idine
SPY
144-83-2
H836
15%/
-tI7
]
sulfamethoxazole
SMX
723-46-6
H2572
10%/
-[17
]
A^-acetyl
sulfamethoxazole
N4AcSMX
humanmetabolite
-
(50%
/-)
d[17]
trimethoprim
TRI
738-70-5
V+H
522
50%
/-[
47]
azithromycin
AZI
83905-01-5
H318
<10%/>65%[15]
erythromyc
inERY-H20
114-07-8
H218
4-20%
/40-50%
[15]
clarithromyc
inCLA
81103-11-9
H1743
20-30%/4-
11%[
15]
roxithromyci
nROX
80214-83-1
H149
8%
/55%
aV=
veterinary
medicine,H=humanmedicine.
bConsumptioncalculatedfromtheusageofsu
lfas
alaz
ine,
assuming100%transformationtosulfapyridineinthecolon.
c
Percentageoftheadministereddose.
dPercentageoftheadministeredsulfamethoxazoledose.
Occurrence 85
fixed ratio of 1:5 with sulfamethoxazole for example, since it also interferes
with bacterial folate synthesis via direct inhibition of dihydrofolate reductase
enzyme. When used together, sulfonamides and trimethoprim produce a
bactericidal effect, as opposed to the bacteriostatic effect yielded by
monotherapy.[19]
Monitoring studies for antimicrobials have been performed, mainly in
wastewaters and surface waters. While ß-lactams, for example penicillins, seem
to be rapidly hydrolyzed after excretion, members of the other three important
antibacterial groups applied in human medicine (fluoroquinolones, macrolides,
sulfonamides) have been detected in the aquatic environment.[6'2023] Generally,
concentrations of up to low pg/L levels occur in WWTP effluents indicating the
importance of WWTPs as point source.[20'23"29] Most concentrations published
are results of the analyses of grab samples, which limits their significance for
assessing occurrence and fate of antimicrobials in wastewater treatment
facilities. Additionally, no data are published yet for the occurrence of
antimicrobials in sewage sludge, with the exception of fluoroquinolones^ 0]
Such data, however, are crucial to enable evaluation and modeling of their
behavior in wastewater treatment.
The aim of the present study was to investigate the occurrence of sulfonamides,
macrolides, and trimethoprim in municipal wastewater treatment plants in
Switzerland (Table 4.1). Wastewater samples from the raw influents to the final
effluents as well as activated and digested sewage sludge samples were
investigated. The results provided the basis for evaluating the behavior within
individual treatment steps of conventional wastewater treatment. Daily profiles
were investigated to assess the short-term variations of antimicrobial loads
entering wastewater treatment. Furthermore, sorption to sewage sludge is
discussed for the various analytes.
4.2 Experimental Section
4.2.1 Wastewater Treatment Plants
Samples were collected from two municipal wastewater treatment facilities in
Switzerland. The wastewater treatment plant Kloten-Opfikon (WWTP-K) is
near the international airport of Zurich, Switzerland. The plant handles 55 000
population equivalents (PE) combining the domestic wastewater of 25 900
86 Chapter 4
inhabitants in the catchment area and the wastewater from the international
airport, including an unknown number of commuters and passengers. During
sampling the average raw inflow was 16 500 ± 5 500 m là. The second
wastewater treatment plant is located at Altenrhein (WWTP-A) in the canton St.
Gall. It receives the combined sewage of 80 000 PE comprising that of 52 000
inhabitants in the catchment area. The average raw inflow amounted to 21 000
m3/d during dry weather. In both WWTPs primary treatment consists of a
screen, an aerated grit-removal tank, and a primary clarifier. In the case of
WWTP-K secondary treatment is performed in two consecutive activated sludge
units and the final effluent is discharged to the receiving surface water after sand
filtration as tertiary treatment. At WWTP-A, secondary treatment is performed
in two parallel operated treatment units: a conventional activated sludge system
and a fixed-bed reactor, receiving -50% of the primary effluent each. Both
systems are designed to provide wastewater treatment by nitrification and
denitrification. The secondary effluents of both units are combined and treated
by a sand filter before being discharged to the receiving river.
In both wastewater treatment plants (WWTP-K and WWTP-A), primary and
secondary sludges are sedimented in the primary clarifier, partially dewatered
and treated in anaerobic, mesophilic digesters with a residence times of 15 - 25
days. Further details on the treatment technologies applied in the two wastewater
treatment plants are given in Chapter 5.
4.2.2 Sample Collection
Sampling campaigns were performed on days without substantial rainfall in
March 2002, February 2003 and November 2003 at WWTP-K and in September
2002 and March 2003 at WWTP-A. Flow-proportional composite samples were
collected by means of automated samplers of raw influents after the screen, of
primary effluents after primary clarification, of secondary effluents after
conventional activated sludge treatment and of tertiary effluents after sand
filtration. Weekly campaigns comprised 3 composite samples, one combining
the 24 h integrated samples from Saturday and Sunday and two combining two
or three weekdays. For daily profiles at WWTP-K, flow proportional 8 h
composite samples of three consecutive intervals were collected in February
2003. All samples were transferred into amber glass bottles and filtered (0.45-
pm cellulose nitrate filters, Schleicher & Schuell) as soon as possible (no later
Occurrence 87
than 6 h after collection). For detailed information on sample treatment see
Chapter 2.
Grab samples of sewage sludge were taken at the end of the aeration tanks
(activated sludge) and at the outlets of the anaerobic, mesophilic digesters fed by
both primary and secondary sludges (digested sludge). In the case of the
activated sludge, grab samples were taken once per week (November 2003),
three and four times per week (September 2002 and March 2003, respectively)
or daily (March 2002 and February 2003), filtered (glass fiber filters, GF8,
Whatman) and freeze-dried. Subsequently, grab samples were mixed in equal
amounts to obtain weekly samples, which were further analyzed (refer to
Chapter 3). Digested sludge samples were taken in February, March, and
November between 20 and 25 days after the respective sampling campaigns
(taking the residence time in the digester into consideration) and directly freeze-
dried without flltration. Consequently, the results obtained for activated sludges
are given in pg/kg dry weight, while those for digested sludges, including the
aqueous phase, are given in pg/L. The concentration of solids in the freeze-dried
digested sludge was determined to range between 15 and 18 g/L.
4.2.3 Analytical Methods
Analytical details, including information on all materials and reagents used, are
described for wastewater samples in Chapter 2 and for sludges in Chapter 3.
Briefly, wastewater samples were enriched by solid-phase extraction (Oasis
HLB, Waters, Bergen op Zoom, The Netherlands), followed by reversed-phase
liquid chromatography - tandem mass spectrometry using positive electrospray
ionization. Recoveries from all sample matrices were generally above 80%, and
the combined measurement uncertainty varied between 2 and 18%. Sample-
based quantification limits depended on the analyte and the sample matrix and
ranged between 1 and 220 ng/L.
Freeze-dried sludge samples were extracted using pressurized liquid extraction
with methanol:water (1:1) as extraction solvent. Sludge extracts were diluted
with water to reduce the methanol content below 5%, and subsequently enriched
on Oasis HLB cartridges and analyzed as described for aqueous samples (see
above). Recoveries from activated sludge were above 80%> in all cases and the
overall precision of the method ranged between 3% and 8%. The limits of
quantification varied between 3 and 30 pg/kg.
88 Chapter 4
4.2.4 Estimation ofSorption Constants
Sorption constants were estimated for antimicrobials present in activated sludge
to characterize their respective distribution between sludge and wastewater
phases. Therefore, the amount present as sorbed fraction, that is associated with
the sludge, is related to the dissolved amount by two different approaches. In the
first approach, concentrations measured in the composite samples of the
secondary effluents from the conventional activated sludge systems at WWTP-K
and WWTP-A are used. These aqueous concentrations (n = 3) were related to
the amount present in the activated sludge sample (n =1) of the identical
sampling week (field experiments). In the second approach, grab samples from
the end of the nitrification compartment were taken in October 2003 and
February 2004 of WWTP-K. In the latter case, the amounts present in the filter
cake (sorbed fraction) and in the filtrate (dissolved fraction) were determined by
duplicate analyses. The pH of the filtrate ranged between 7.5 in October 2003
and 7.0 in February 2004. Since the filter cake is not completely dry after
filtration, it also contains some analytes in the water phase. Calculations showed
that the amount of analytes dried onto the sludge during freeze-drying ranged
between 2 - 5%o of the amount sorbed to the sludge. It was therefore neglected in
all cases.
4.2.5 Calculation ofLoads
Loads were calculated to take into account the influence of flow variations on
the measured antimicrobial concentrations (e.g. due to rain). The loads of the
three sampling intervals were summed to yield a weekly load, which was then
used to calculate an average daily load (g/d). For comparison reasons the results
were normalized to 1000 PE in Table 4.3. The dissolved loads were calculated
by multiplying the measured concentration at a specific sampling point and the
respective water flow during the sampled time period. The amount of
antimicrobials sorbed to suspended solids in the raw influent (130-200 mg/L)
and the primary effluent (80-100 mg/L) was estimated using the highest Kd
value observed for activated sludge for the respective compound. The sorbed
portion of antimicrobials in secondary and tertiary effluents was neglected,
based on the small concentrations of suspended solids (5-20 mg/L).
Occurrence 89
The uncertainty of the average daily loads was calculated using error
propagation. Therefore the uncertainty of the analytical method (see Chapter 2)
and the uncertainty of the water flow measurement (5-10%) was used. For
samples below the limit of quantification, a relative uncertainty of 100% was
applied to the concentration measured. A relative uncertainty of 50% was
assigned to the Kd value used for suspended solids in the raw influent and the
primary effluent, to account for the uncertainty connected with the extrapolation
from activated sludge. The resulting uncertainties for the average daily loads
therefore only represent measurement uncertainties and do not show expected
variations between days or sampling campaigns.
Additionally, theoretical loads of antimicrobials were calculated in the raw
influent using available sales data for Switzerland, the maximal amount of the
compound excreted unchanged (Table 4.1) and the number of inhabitants
(n= 25 900) in the catchment area of the investigated treatment facility.
4.3 Results and Discussion
4.3.1 Occurrence in Wastewater Samples
In part of this study the occurrence of sulfonamides (including one human
metabolite), trimethoprim, and macrolides in various compartments of two
municipal wastewater treatment plants was investigated. The median, minimum
and maximum concentrations found in wastewater samples are summarized in
Table 4.2.
While sulfathiazole and sulfamethazine were not detected in the wastewater
samples, all human used antimicrobials investigated were detected, except for
sulfadiazine, which is only prescribed in very low quantities (Table 4.1).
Sulfamethoxazole was the most commonly detected sulfonamide in our samples.
The fraction present as human metabolite, A^-acetylsulfamethoxazole was taken
into account to better assess the occurrence of sulfamethoxazole in wastewater.
Median concentrations of sulfamethoxazole including the measured amount
present as A^-acetylsulfamethoxazole were up to 1 900 ng/L in the raw influent
and 880 ng/L in the tertiary effluent. The frequent abundance of
sulfamethoxazole in our study, as well as described in the literature, is due to the
high consumption of this compound in human medicine. Maximal influent
concentrations of 520 ng/L in the United States [23], 232 ng/L in Austria [25],
Table
4.2
Concentrations
ofsu
lfon
amid
es,
trimethoprim
and
macrolides
in
wastewater
oftwo
municipal
wastewater
treatmentpl
ants
inSwitzerland
concentrationsmeasured(n
g/L)
raw
infl
uent
bpr
imar
yeffluentc
seco
ndar
yeffluentc
tertiary
effluentc
compounda
median
min
max
median
min
max
median
min
max
median
min
max
SPY
90
60
150
130
90
230
70
20
230
90
40
350
SMX
430
230
570
430
90
640
280
130
840
290
211
860
N4AcSMX
1400
850
1600
890
570
1200
40
<LOQ
150
10
<LOQ
180
SMX+N4AcSMX
1700
940
1900
1200
720
1600
380
190
880
400
210
880
TMP
290
210
440
230
80
340
200
80
400
70
20
310
AZI
170
90
380
150
80
320
140
40
380
160
80
400
ERY-H20
70
60
190
80
40
190
80
50
140
70
60
110
CLA
380
330
600
330
160
440
260
150
460
240
110
350
ROX
20
10
40
20
10
50
20
10
30
10
10
30
a
Sulfadiazine,sulfathiazole,
andsulfamethazinewerenotorveryra
rely
detected(d
atanotshown).
bMedian,minimum
andmaximum
valueofninemeasurements,
sinceraw
influentwas
notsampled
inMarch2002
andSeptember
2002.
c
Median,minimumandmaximum
valueof15measurements.
In
the
caseofAZI
(n=
12)no
resultsare
available
forMarch2002
becauseofan
alytical
interferences.ForN4AcSMX
8outof15
tertiary
effluentsamplesand6outof15secondaryeffluentsa
mple
swere
belowthelimitofqu
anti
fica
tion
(—20ng
/L).
Thesewereincludedas0.5xLOQ
inthecalculationofmedianvalues.
Occurrence 91
580 ng/L in Spain L31J, and 9 000 ng/L in Germany[ZI'^
are reported.
Concentrations for sulfamethoxazole available in literature for treated sewage
range from 50 to 4 700 ng/L.[20,23'25'27'29,311 Since none of the previous studies
included the main human metabolite A^-acetylsulfamethoxazole, these earlier
results must be cautiously interpreted. To the best of our knowledge, the
presence of this possibly re-transformable metabolite has been reported only
once. Hilton and Thomas detected up to 2 235 ng/L of A^-acetyl-sulfamethoxazole in a treated wastewater grab sample and up to 239 ng/L in
receiving surface waters.[32] Contrary to our data, the parent compound
sulfamethoxazole was not detected.
We also detected the synergist trimethoprim in all samples at median
concentration of 290 ng/L and 70 ng/L in raw influents and tertiary effluents,
respectively. Trimethoprim concentrations previously reported range from 9
ng/L to 1 500 ng/L for WWTP effluents. [20>24>25'28>32'33] ln human medicine the
prevailing form administered combines sulfamethoxazole and trimethoprim at a
fixed ratio of 5:1, which is reflected in the consumption data. This ratio was still
visible in the raw influent samples we analyzed (5.4:1). However, in the final
effluent (8.6:1) this ratio is altered presumably by the different behavior of
sulfamethoxazole and trimethoprim in wastewater treatment as discussed in
Chapter 5.
Sulfapyridine was also detected in all wastewater samples with concentrations
up to 150 ng/L in the raw influents and up to 350 ng/L in the tertiary effluents.
The higher concentration in the effluents is probably due to the presence of re-
conjugable substances as described below. A similar median concentration of
sulfapyridine (81 ng/L) was detected in grab samples from eight Canadian
wastewater effluents.[29] Sulfapyridine has otherwise not been investigated in
environmental samples, probably due to the fact that it is rarely used as an
antimicrobial agent itself. Its occurrence in wastewater samples results from the
application of sulfasalazine, which is mainly used for the treatment of ulcerative
colitis and rheumatoid arthritis.[34] In sulfasalazine, sulfapyridine is linked to 5-
aminosalicylic acid via an azo bridge, which is cleaved in the colon - thereby
yielding the two components. Of the administered dose only -10% are excreted
via the urine as sulfasalazine itself, while another 10 to 35% are excreted as
sulfapyridine and between 20 and 40% as V-acetylsulfapyridineJ351All four investigated macrolides were detected in wastewater with
clarithromycin being most abundant. Median concentrations measured for
Table
4.3
Daily
loads
ofsu
lfon
amid
es,
trimethoprim
and
macrolides
inprimary
effluentsoftwo
municipalwastewater
treatmentplants
inSwitzerland
averageda
ilyloada(mg/d/1000PE)
WWTP-K
WWTP-A
compoundb
March
February
November
September
March
2002
2003
2003
2002
2003
SPY
16±7C
38±
15
53±4
33±2
34±5
SMX
42±2
158±4
178±5
39±1
188±8
N4AcSMX
307±7
302±7
264±7
255±13
381±19
SMX+N4AcSMXd
305±7
416±9
404±7
258±13
516±20
TRI
89±7
82±7
84±7
33±4
90±9
AZI
e45±2
47±2
59±4
101±8
ERY-H20
49±2
44±2
24±2
15±1
26±1
CLA
149±7
160±7
96±4
59±4
125±8
ROX
16±2
13±2
5±2
9±1
5±1
a
Average
dail
yloadscalculatedfromweeklyloadsasdescribedintheEx
peri
ment
alSection.Thevalueswerenormalizedtopo
pula
tion
equivalents(PE),whichare55000PE
forWWTP-Kand80000PE
forWWTP-A.
bSulfadiazine,sulfathiazole,
andsulfamethazinewerenotorveryra
rely
detected(d
atanotshown).
cDeviationcalculatedfrommeasurementuncertaintiesasdescribedintheEx
peri
ment
alSection.
dSulfamethoxazoleload,in
clud
ingtheamountpresentasA^-acetylsulfamethoxazole.
eNo
resultsavailablebecauseofanalytic
alinterferences.
Occurrence 93
Clarithromycin were 380 ng/L in raw influents and 240 ng/L in final effluents.
For roxithromycin and dehydro-erythromycin median concentrations ranged
between 20 and 70 ng/L in the raw influents and between 10 and 70 ng/L in the
final effluents, respectively. These concentrations correspond well with previous
findings in Switzerland by McArdell et al., who reported between 57 ng/L and
328 ng/L for clarithromycin, up to 31 ng/L of roxithromycin, and up to 199 ng/L
of dehydro-erythromycin in final effluents of three conventional treatment
plants/261 Similar concentrations have also been observed in wastewater
effluents in other countries.[25'28,29'32] Median concentrations of dehydro-
erythromycin - the most predominant macrolide in the investigated wastewater
effluents - were found to be 137 ng/L in Germany, 109 ng/L in the UK, 80 ng/L
in Canada, and 394 ng/L in Austria.
Median concentrations of azithromycin were 170 ng/L in raw influents and 150
ng/L in tertiary effluents of conventional wastewater treatment. Thus,
azithromycin is the second most abundant macrolide after clarithromycin in
Swiss wastewaters.
Table 4.3 shows the average daily loads from the individual sampling campaigns
determined in the primary effluent of both municipal wastewater treatment
plants, WWTP-K and WWTP-A. The individual loads of the investigated
antimicrobials are similar in both wastewater treatment plants due to their
similarity in size, and range between 0.3 and 41 g/d. As a consequence of human
metabolism, the amount of sulfamethoxazole itself in the primary effluent is low
and varied between 14 and 44% of the total amount. Therefore, it is crucial to
include the fraction present as the human metabolite A^-acetylsulfamethoxazole.
Figure 4.1 illustrates the loads measured in the raw influents and tertiary
effluents of WWTP-K for all antimicrobials investigated. Additionally, it shows
that loads in the raw influent estimated from the available consumption data
available follow the same pattern as the measured data - both mirroring the
annual amounts (1999) used in human medicine in Switzerland (Table 4.1). A
very good agreement was obtained between the measured and calculated loads,
within the limits of uncertainty, with disagreements not greater than a factor of
two. The differences can be inferred by i) local variations in consumption as
compared to Swiss average, Ü) unknown loads of antimicrobials from daily
commuters in the catchment area, iii) unaccounted input loads from air traffic
passengers at the international airport of Zurich and iv) the uncertainty of the
available metabolism data (e.g. percentages excreted unchanged).
94 Chapter 4
Figure 4.1 Daily loads ofsulfonamides, trimethoprim and macrolides in raw
influents andfinal effluents ofthe municipal wastewater treatmentplant, Kloten-
Opfikon, Switzerland
30
26
20 -
115«8
O
10
Ml
X
I
X
I
jlT.
X
T
maximum
75% percentile
median
25% percentileminimum
X estimated bad
If raw influent (n=6)
final efluent (n=9)
intfÏMM 1—^~1
fJ__L _L^^
SPY SMX N4ACSMX SMX-t- TMP
N4ACSMX
AZI ERY-H20 CLA ROX
a
Theoretical antimicrobial loads calculated from consumption data as described in the
text.
4.3.2 Daily Variations
The loads determined in the raw influent of three consecutive 8 h time intervals
vary to different extents for the investigated antimicrobials (Table 4.4). These
variations were smoothed out during wastewater treatment, resulting in
approximately equal amounts in the tertiary effluents at all three time intervals
(data not shown). To illustrate the impact of human urine on the wastewater
composition, an average daily profile of ammonium in the WWTP inflow is also
included in Table 4.4. The values given for the water flow in the respective
sampling period represent a typical flow profile for a dry weather influent.
In the case of the sulfonamides and trimethoprim the distribution of the daily
load correlates well with the respective water flows and typical ammonium
Occurrence 95
Table 4.4 Daily variations of antimicrobial loads, water flow, and ammonium
load in untreated wastewater (WWTP Kloten-Opfikon, Switzerland)
daily load fraction of daily load (%)
parameter /
compound(g/d) 0-8 am 8-4 pm 4-12
SPY 1.7 26 39 35
SMX 3.5 28 42 30
N4AcSMX 20 23 44 33
SMX+N4AcSMX 21 24 43 33
TRI 4.7 34 36 30
AZI 3.9 9 61 30
ERY-H20 1.5 15 49 36
CLA 8.1 19 46 35
ROX 0.4 9 27 64
water flow 21939m7d 24 40 36
ammonium ~320kg/d 18 46 36
loads. With a half-life of -10 h in the human body and a typically prescribed
oral administration of twice a day, these findings correspond well with the
theoretically expected distribution pattern.[36]The macrolides showed a more variable daily profile with the lowest loads
occurring between midnight and 8 am (9 - 19% of the daily load). This is most
likely caused by the respective consumption patterns and excretion rates of these
compounds. In the case of azithromycin the observed pattern can be explained
by the usually prescribed oral consumption of once a day - presumably in the
morning, and a half-life in the human body of 10-14 h.[15] Good agreement is
also observed for clarithromycin, which is normally taken twice a day, assumed
to be in the morning and the evening, and which has a half-life in the human
body of only ~5 h. Erythromycin has a half-life in the human body of 1 -2 h and
is administered between 2-4 times a day, which also correlates well with the
observed daily variations. In addition to oral application, ~20%> of the annual
consumption of erythromycin is used in the form of facial lotions against acne.
Therefore, additional amounts of erythromycin are probably washed of directly
during the day. From the usual prescription pattern (twice a day) and the
96 Chapter 4
reported half-life (-10 h) for roxithromycin a correlation of the respective loads
with the water flow and typical ammonium loads would be expected. The poor
agreement observed in this case, may be due to the low consumption of
roxithromycin in Switzerland resulting in very few patients in the catchment
area.
One has to keep in mind that the daily variations observed only result from one
sampling day divided into three sampling periods. Further investigations using
smaller time intervals and covering multiple days would be necessary to confirm
these findings. However, the results clearly illustrate a possible daily variance
for antimicrobials in wastewater samples. It is therefore crucial to use flow
proportional composite samples over at least 24 h, as done in this study, when
assessing their fate and occurrence in wastewater treatment.
4.3.3 Seasonal Differences
Concerning possible seasonal difference in consumption no conclusive results
were obtained for sulfamethoxazole, including the amount present as i\T-
acetylsulfamethoxazole, and trimethoprim (Table 4.3). In the latter two cases,
higher loads were observed in the primary effluent of WWTP-A in March 2003
compared to September 2002. In WWTP-K, however, similar loads were
measured in all three sampling campaigns for sulfamethoxazole, including the
amount present as A^-acetylsulfamethoxazole, and trimethoprim. Also, no
conclusion can be drawn for sulfapyridine, where the presence of chemically
bound sulfapyridine, for example A^-acetylsulfapyridine, is very likely, but was
not assessed in this study.
For the macrolides the determined loads were generally higher in March and
February than in November and September by approximately a factor of two.
Relative monthly sales data available for the group of macrolides in total, show
a clear periodicity with two times lower consumption in summer compared to in
winter.[37] Highest amounts of macrolides are sold between February and April
each year. Even though no sampling campaign was performed directly in
summer, the different loads measured for macrolides in this study correlate well
with the consumption data. This variability is probably caused by the use of
macrolides primarily against respiratory tract infections. McArdell et al. already
related higher loads of macrolides in winter in the effluent of the same WWTP
to the distinct seasonal consumption.[26] Loads determined in influent samples,
Occurrence 97
as presented in this study, however are needed to check this correlation. A
similar annual fluctuation was reported by Strenn et al. for the concentration of
roxithromycin in the inflow of a wastewater treatment plant, however, the
influence ofpossibly varying water inflow was not taken into account.[38]
4.3.4 Occurrence in Sewage Sludge
The antimicrobials investigated in wastewater samples were also measured in
sewage sludge (Table 4.5) Most of the antimicrobials present in aqueous phase
were also found in the solid samples, with a few exceptions. Even though
A^-acetylsulfamethoxazole is present in high concentrations in the raw influent
of the wastewater treatment plant it could not be detected in any of the sewage
sludge samples. Spiking A^-acetylsulfamethoxazole on activated sludge (-100
pg/kg) results in an increase of the sulfamethoxazole concentration while no
A^-acetylsulfamethoxazole could be detected in the sludge (data not shown).
Since no degradation of A^-acetylsulfamethoxazole occurred, when extracted
under the same extraction conditions from silica sand only, a fast transformation
of this compound in the presence of sewage sludge must be assumed.
Sulfamethoxazole and trimethoprim, each of which were present in aqueous
samples, were also detected in activated sludge with average concentrations of
68 ± 20 pg/kg dry weight and 41 ± 15 pg/kg dry weight, respectively. In
digested sludge, however, no significant amounts of these two compounds could
be detected. Therefore, sulfamethoxazole and trimethoprim seem to be unstable
in anaerobic, mesophilic sludge digestion. Sulfapyridine, however, was detected
in activated (28 ± 3 pg/kg dw) and in digested (1.0 ± 0.1 pg/L) sludge. Two
possible explanations could be a significantly higher stability of sulfapyridine
compared to sulfamethoxazole during sludge digestion or the possible presence
of substances re-transformable to sulfapyridine (e.g. sulfasalazine) in sewage
sludge.
Among the macrolides only azithromycin and clarithromycin were detected in
sewage sludge samples. In activated sludge average concentrations of 64 ± 30
pg/kg dry weight azithromycin and 67 ± 28 pg/kg dry weight clarithromycin
were determined. Concentrations in digested sludge were 2.5 ± 1.0 pg/L for
azithromycin and 0.7 ± 0.4 pg/L for clarithromycin. The low concentrations of
roxithromycin present in the water phase most probably led to concentrations in
sewage sludge below the limit of quantification (LOQ = 3 pg/kg dry weight).
98 Chapter 4
Table 4.5 Concentrations of sulfonamides, trimethoprim and macrolides in
sewage sludges
concentration
compoundactivated sludge
(pg/kg dw)a
digested sludge
(ltg/L)b
average ± SD (n = 5) average ± SD (n = 3)
SPY 28 ±3 1.0±0.1
SMX 68 ±20 ndc
TRI 41 ±15 nq(0.1)c
AZI 64 ±30 2.5 ±1.0
CLA 67 ±28 0.7 ± 0.4
a
Separation of solid (~3g/L) and aqueous phase through flltration before freeze-
drying.bNo separation of solid (15-18 g/L) and aqueous phase through filtration before
freeze-drying.c
nd: not detected, nq: below limit of quantification (estimated value)
In the case of dehydro-erythromycin, high dissolved concentrations were
measured while no significant amounts were detected in sewage sludge samples
(LOQ = 6 pg/kg dry weight). Therefore no significant sorption of dehydro-
erythromycin to sewage sludge seems to take place.
4.3.5 Sorption to Sewage Sludge
The measured concentrations ofantimicrobials in activated sludge, during the
performed sampling campaigns and in grab samples (refer to 4.2.4), were also
used to estimate sorption coefficients (K^) for the particular analytes. The values
obtained, ranged between 114 and 460 L/kg for all compounds investigated
(Table 4.6). For the macrolides the results obtained in the field experiments or
grab samples are generally very similar. For sulfonamides and trimethoprim the
resulting K<j values range between 114 and 418 L/kg in activated sludge. From
literature, data on the sorption of antimicrobials to biosolids is available for
Occurrence 99
Table 4.6 Estimated sorption constantsfor sulfonamides, trimethoprim and
macrolides
Kdfield experiments
'
in activated sludge (L/kg)a
'
grab samples
compound average ± SD (n= 9 - 15) October 2003c February 2004c
SPY 295 ± 145 202 418
SMX 256 ±169 114 400
TRI 208 ± 49 157 375
AZI 376 ± 86 460 352
CLA 262 ± 93 300 400
a
Calculated as the ratio of the sorbed and dissolved fraction measured.
Calculated using concentrations from weekly sampling campaigns (dissolved
fraction, n=3; sorbed fraction, n=l).cCalculated using concentrations measured in grab samples from the end of the
nitrification compartment.
tylosin, a macrolide used in veterinary medicine.[39] Organic carbon normalized
sorption coefficients (Koc) were reported for tylosin in soil and manure ranging
between 110 and 7 990 L/kg. The same review gives K0c values in soil between
48 and 323 L/kg for sulfonamides in general and between 101 and 308 for
sulfapyridine in particular. Since activated sludge consists of about 40% organic
matter, reported KoC values might be expected to be larger than IQ values for
activated sludge by at least a factor of two. In general, however, the significance
of such a comparison is limited due to the structural and compositional
differences in the matrices investigated relative to soil samples used in the
previous study.
For the macrolides, the Kd values obtained in this study vary to a lesser extent
than for the sulfonamides. The results for sulfonamides vary up to 70% in the
field experiments and are between two to four times higher in the grab samples
from February 2004 than those from October 2003. This indicates a possible
non-equilibrium state in the samples. The change in solid to solution ratio may
be caused for example by a different degradation of the analytes in the two
phase or by the wastewater treatment processes combined with slow sorption
kinetics. Different sludge characteristics in the samples taken can also influence
the sorption of the investigated antimicrobials. For soil, there is a strong
100 Chapter 4
dependence of the adsorption of sulfonamides on the pH and on the quantity and
composition of the organic matter, namely the concentration of lipids and lignin
dimers, was described.[39] In the case of macrolides, hydrophobic interactions
were described to be mainly responsible for the sorption to soil. Due to the
predominantly negatively charged surface of activated sludge,[40] ionic
interactions are expected to be significant for the dissolved macrolides, being
positively charged through the protonation of the tertiary amino group (pKa >
8.7 [41]). The sorption of macrolides to activated sludge should therefore be
strong and higher than that of the negatively charged or neutral sulfonamides
(pKa = 5.7-8.4 [17]) However, this was not supported by our data (Table 4.6) and
ionic interactions therefore seem to be of minor importance for the sorption of
macrolides to activated sludge. Further experiments would be necessary to
investigate the parameters affecting the observed sorption of antimicrobials to
activated sludge. Additionally it has to be mentioned that sewage is a very
inhomogeneous medium that varies globally on both a spatial and temporal
scale. Therefore the results obtained primarily apply to the investigated systems.
4.3.6 Mass Balances
The loads determined now provide the basis to investigate the behavior of
Figure 4.2 shows the loads obtained in the second sampling campaign (February
2003) for three exemplary compounds. Average daily loads were calculated at
the different sampling points, with differentiation between the dissolved and
sorbed fraction.
A total load (sorbed and dissolved) of 24.4 g/d sulfamethoxazole was
determined in the raw influent including the fraction present as the human
metabolite /V-acetylsulfamethoxazole, which accounted for -75% of the load.
After primary treatment only -62% of the total load could be assigned to
/V-acetylsulfamethoxazole and after secondary treatment this metabolite was
almost completely absent. Thus, there is a strong indication of re-transformation
of A^-acetylsulfamethoxazole to sulfamethoxazole. Further experiments,
however, are needed to fully confirm this assumption. An elimination of
sulfamethoxazole, including the amount present as A^-acetylsulfamethoxazole,occurred mainly in secondary treatment (-55%), while no significant
elimination was observed in primary and tertiary treatment in February 2003. In
the case of clarithromycin and trimethoprim no significant elimination was
Occurrence 101
Figure 4.2 Mass balances for clarithromycin, sulfamethoxazole including the
fraction present as N4-acetylsulfamethoxazole, and trimethoprim in the
conventional wastewater treatment plant Kloten-Opfikon, Switzerland
(February 2003)
A. Sulfamethoxazole including the amount present as 7V4-acetylsulfamethoxazole
daily load
24,5 g/d
B. Trimethoprim
daily load
5.2 g/d
C. Clarithromycin
daily load
9.9 g/d
l%d
]%s
< 0.2%
l%d
3% s
<1%
d: dissolved, s: sorbed/particulate
102 Chapter 4
observed in primary and secondary treatment, but they were partly eliminated
during sand filtration. For clarithromycin a reduction by 15% was found on the
sand filter ofWWTP-K and by 60% for trimethoprim.
4.4 Conclusions
Overall, an incomplete removal of the selected antimicrobials is observed in
secondary wastewater treatment. The maximum concentrations determined in
the final effluents of two municipal wastewater treatment plants amounted to
over 800 ng/L for sulfamethoxazole (Table 4.2). Concentrations of
antimicrobials in the final effluent of wastewater treatment plants have to be
critically assessed concerning their impact on the aquatic environment. Most of
the microbial toxicity data available originates from acute toxicity studies and
falls in the mg/L range, see e.g. references [9,42-45]. In some cases sublethal
effects in algal species have been reported in the high pg/L range.[46] Therefore
antimicrobials are unlikely to cause acute adverse effects in aquatic
microorganisms even if the dilution factor is low, for example in summer.
However, effects due to the chronic low dose exposure or non-target effects and
mixture effects cannot be ruled out. Another aspect concerns the spread and
maintenance of antibacterial resistance. These latter issues require further
investigation.
A detailed investigation of the elimination observed for sulfonamides,
macrolides and trimethoprim in the individual treatment stages of conventional
activated sludge treatment is the focus of our further studies (Chapter 5).
Additionally, conventional activated sludge treatment will be compared to other
secondary treatment technologies, i.e. a fixed-bed reactor and a membrane
bioreactor operated at three different solid retentions times, with respect to the
elimination of the selected antimicrobials.
Acknowledgments
Abbott GmbH (Wiesbaden, Germany) is acknowledged for supplying
clarithromycin and Pfizer AG (Zurich, Switzerland) for supplying azithromycin.
Partial financial support came from the EU project POSEIDON (EVK1-CT-
2000-00047, www.eu-poseidon.com) and the EAWAG project on human-use
antibiotics (HUMABRA) within the framework of the National Research
Occurrence 103
Program on antibiotic resistance funded by the Swiss National Science
Foundation (www.nrp49.ch/pages/). We would also like to thank the Swiss
Agency for the Environment, Forestry and Landscape, the Swiss cantons of
Aargau, Basel Land, Bern, Luzern, Schaffhausen, Schwyz, St. Gallen, Thurgau,
Ticino, Zurich and the WWTPs of Kloten-Opfikon and Altenrhein for additional
financial support. We thank the technical staff of the WWTP Kloten-Opfikon
and of the WWTP Altenrhein for their assistance during sampling. For helpful
comments on the manuscript we acknowledge A. Alder, H. Siegrist, M. Suter, T.
Ternes and M. Dodd.
4.5 Literature cited
[1 ] Stan, H. J.; Heberer, T. Analusis 1997, 25, M20-M23.
[2] Ternes, T. A. Water Res. 1998, 32, 3245-3260.
[3] Halling-S0rensen, B.; Nors Nielsen, S.; Lanzky, P. F.; Ingerslev, F.; Holten
Lützenheft, H. C; J0rgensen, S. E. Chemosphere 1998, 36, 357-393.
[4] Daughton, C. D.; Ternes, T. A. Environ. Health Perspect. 1999, 107, 907-
938.
[5] Kümmerer, K. Chemosphere 2001, 45, 957-969.
[6] Kolpin, D. W.; Furlong, E. T.; Meyer, M. T.; Thurman, E. M.; Zaugg, S.
D.; Buxton, H. T. Environ. Sei. Technol. 2002, 36, 1202-1211.
[7] Heberer, T. Toxicol. Lett. 2002,131, 5-17.
[8] Tolls, J. Environ. Sei. Technol. 2001, 35, 3397-3406.
[9] Boxall, A. B. A.; Fogg, L. A.; Blackwell, P. A.; Kay, P.; Pemberton, E. J.;
Croxford, A. Reviews in Environmental Contamination and Toxicology
2004,180,1-91.
[10] Jones, O. A. H.; Voulvoulis, N.; Lester, J. N. Environ. Technol. 2001, 22,
1383-1394.
[11] Ayscough, N. J.; Fawell, J.; Franklin, G.; Young, W. "Review of human
pharmaceuticals in the environment," Environment Agency, 2000.
[12] Annual Report; Swiss Importers of Antibiotics (TSA): Berne, Switzerland,
1998.
[13] Pharmaceuticals Sold in Switzerland; Swiss Market Statistics, 1999.
[14] Antibiotics used in Veterinary Medicine; Swiss Federal Office for
Agriculture (BLW): Berne, Switzerland, 2001.
104 Chapter 4
[15] Bryskier, A. J.; Butzler, J.-P.; Neu, H. C; Tulkens, P. M. Macrolides;
Arnette Blackwell: Paris, 1993.
[16] Vree, T. B.; Hekster, Y. A. Pharmacokinetics ofsulfonamides revisited;
Karger: Basel, New York, 1985; Vol. 34.
[17] Vree, T. B.; Hekster, Y. A. Clinicalpharmacokinetics ofsulfonamides and
their metabolites; Karger: Basel, 1987; Vol. 37.
[18] Berger, K.; Petersen, B.; Büning-Pfaue, H. Arch. Lebensmittelhyg. 1986,
37,85-108.
[19] Poe, M. Science 1976,194, 533-535.
[20] Hirsch, R.; Ternes, T. A.; Haberer, K.; Kratz, K.-L. Sei. Total Environ.
1999,225,109-118.
[21] Sacher, F.; Lange, F. T.; Brauch, H.-J.; Blankenhorn, I. J. Chromatogr., A
2001,955,199-210.
[22] Aider, A. C; McArdell, C. S.; Golet, E. M.; Ibric, S.; Molnar, E.; Nipales,
N. S.; Giger, W. In Pharmaceuticals andPersonal Care Products in the
Environment: Scientific and Regulatory Issues; Daughton, C. G., Jones-
Lepp, T., Eds.; Symposium Series 791; American Chemical Society:
Washington, D.C., 2001; pp 56-69.
[23] Yang, S.; Carlson, K. Water Res. 2003, 37, 4645-4656.
[24] Andreozzi, R.; Marotta, R.; Paxeus, N. Chemosphere 2003, 50, 1319-1330.
[25] Scharf, S.; Gans, O.; Sattelberger, R. "Arzneimittelwirkstoffe im Zu- und
Ablauf von Kläranlagen," Report of the Umweltbundesamt, Wien, ISBN 3-
85457-624-2, 2002.
[26] McArdell, C. S.; Molnar, E.; Suter, M. J.-F.; Giger, W. Environ. Sei.
Technol. 2003, 37, 5479-5486.
[27] Hartig, C; Storm, T.; Jekel, M. J. Chromatogr., A 1999, 854, 163-173.
[28] Bund/Länderausschuss für Chemikaliensicherheit (BLAC), "Arzneimittel in
der Umwelt - Auswertung der Untersuchungsergebnisse," Hamburg, 2003.
[29] Miao, X.-S.; Bishay, F.; Chen, M.; Metcalfe, C. D. Environ. Sei. Technol.
2004,55,3533-3541.
[30] Golet, E. M.; Strehler, A.; Aider, A. C; Giger, W. Anal. Chem. 2002, 74,
5455-5462.
[31] Carballa, M.; Omil, F.; Lema, J. M.; Llompart, M.; Garcia-Jares, C;
Rodriguez, I.; Gomez, M.; Ternes, T. A. Water Res. 2004, 38, 2918-2926.
[32] Hilton, M. J.; Thomas, K. V. J. Chromatogr., A 2003, 1015, 129-141.
Occurrence 105
[33] Metcalfe, C. D.; Koenig, B. G.; Bennie, D. T.; Servos, M.; Ternes, T. A.;
Hirsch, R. Environ. Toxicol. Chem. 2003, 22, 2872-2880.
[34] Astbury, C; Dixon, J. S. J. Chromatogr. 1987, 414, 223-227.
[35] Neumann, J. "Untersuchungen zur Bioverfügbarkeit und Pharmakokinetik
von Sulfasalazin und seinen Metaboliten," PhD Thesis, Free University of
Berlin, 1989.
[36] Neuman, M. Antibiotika-Kompendium; Verlag Hans Huber: Bern, 1981.
[37] Truempi, B., Abbot AG, Baar, Switzerland, personal communication.
[38] Strenn, B.; Clara, M.; Gans, O.; Kreuzinger, N. In Water Pollution VII;
Brebbia, C. A., Ed.; WIT Press: Southampton, UK, 2003; Vol. ISBN 1-
85312-976-3, pp 273-282.
[39] Thiele-Bruhn, S. J. Plant Nutr. Soil Sei. 2003,166, 145-167.
[40] Carberry, J. B.; Englande, A. J. Sludge Characteristics andBehavior;
Martinus Nijhoff Publishers: Boston, The Hague, Dordrecht, Lancaster,
1983.
[41] McFarland, J. W.; Berger, C. M.; Froshauer, S. A.; Hayashi, S. F.; Hecker,
S. J.; Jaynes, B. H.; Jefson, M. R.; Kamicker, B. J.; Lipinski, C. A.; Lundy,
K. M.; Reese, C. P.; Vu, C. B. J. Med. Chem. 1997, 40, 1340-1346.
[42] Holten Lützh0ft, H. C; Halling-S0rensen, B.; Jorgensen, S. E. Arch.
Environ. Contam. Toxicol. 1999, 36, 1-6.
[43] Halling-S0rensen, B. Arch. Environ. Contam. Toxicol. 2001, 40,451-460.
[44] Jones, O. A. H.; Voulvoulis, N.; Lester, J. N. Water Res. 2002, 36, 5012-
5022.
[45] Pfluger, P.; Dietrich, D. R. In Pharmaceuticals in the environment;
Kümmerer, K., Ed.; Springer, 2001.
[46] Brain, R. A.; Johnson, D. J.; Richards, S. M.; Sanderson, H.; Sibley, P. K.;
Solomon, K. Environ. Toxicol. Chem. 2004, 23, 371-382.
[47] Schwartz, D. E.; Rieder, J. Chemotherapy 1970, 75, 337-355.
a **i "'*» n; "3
MC lit '*. (î
S 'V»** I ^4 S* tr V« fl
Chapter 5
Behavior in Wastewater Treatment
The elimination of sulfonamides, macrolides and trimethoprim from raw
wastewater was investigated in several municipal wastewater treatment plants.
Primary treatment provided no significant elimination for the investigated
substances. Similar eliminations were observed in the secondary treatment of
two conventional activated sludge (CAS) systems and a fixed-bed reactor
(FBR). Sulfamethoxazole, including the fraction present as N4-
acetylsulfamethoxazole, was eliminated by approximately 60% in comparison to
about 80% in a membrane bioreactor (MBR) independently of the solid
retention time (SRT). The elimination for macrolides and trimethoprim varied
significantly between the different sampling campaigns in the two CAS systems
and in the FBR. In the MBR, these analytes were eliminated up to 50% at SRT
of 16 ± 2 and 33 ± 3 days. Trimethoprim, clarithromycin and dehydro-
erythromycin showed a higher elimination of up to 90%> at a SRT of 60 - 80
days. One of the sand filters investigated led to a significant elimination of most
macrolides (17 - 23%) and trimethoprim (74 ± 14%), while no elimination was
observed in the other sand filter investigated.
Göbel, A., McArdell, CS., Joss, A., Siegrist, H., Giger, W.
Fate ofSulfonamides, Macrolides and Trimethoprim in Different Wastewater
Treatment Technologies
submitted to Environmental Science and Technology
Behavior 109
5.1 Introduction
Pharmaceuticals have been detected in various compartments of the aquatic
environment as a result of the substantial progress achieved by chemical
analysis. This has lead to an increasing interest in the assessment of fate,
environmental risk and potential regulations of these emerging contaminants,
mirrored by the large number of publications and reviews available, e.g. [1-9].
Within the large group of pharmaceuticals, antimicrobials are of special interest
because of their potential impact on the spread and maintenance of antimicrobial
resistance. Following consumption, the unchanged parent compound and
possible human metabolites are discharged to the sewers and, therefore, residual
concentrations mainly enter the aquatic environment after incomplete
elimination during municipal wastewater treatment.
In the last 40 years, wastewater treatment has continuously been amended to
fulfill the increasing requirements on the quality of the final effluents.[10]
However, the efficiency of distinct wastewater treatment processes, e.g.
conventional activated sludge treatment, fixed-bed reactors or membrane
bioreactors, for the elimination ofpharmaceuticals is mostly unknown.
Joss et al. studied the removal of estrogens in municipal wastewater treatment
processes showing a removal of > 90% for estrogens during activated sludge
treatment.1111 Similar efficiencies were observed for a fixed-bed reactor and a
membrane bioreactor operated with a sludge age of 30 days. The fate and
behavior of antimicrobial agents in wastewater treatment and the aquatic
environment has only been addressed in a few studies.[12"15] McArdell et al.
investigated macrolides in WWTP effluents and a receiving watershed and
observed no significant elimination of clarithromycin in the investigated river
stretch.[14] For fluoroquinolones however, Golet et al. reported removal
efficiencies between 48 and 66% in the same river.[12] Tn conventional
wastewater treatment fluoroquinolones are eliminated by 88 to 91%, with
sorption to sewage sludge being the main process responsible.[13] By comparing
in- and out-flowing loads of a German wastewater treatment plant an
elimination of 94 ± 4% was reported by Ternes et al. for sulfamethoxazole and
ranged between 30 and 61% for the investigated macrolides.[,5] No significant
elimination was observed for trimethoprim (18 ± 14%).
The occurrence and sorption behavior of sulfonamide and macrolide
antimicrobials in activated sludge treatment is discussed in Chapter 4 including
110 Chapter 5
mass flow studies. Sorption to activated sewage sludge was found to be ofminor
importance for these compounds, with estimated sorption constants (Kd)
between 114 and 460 L/kg. In general, compounds with a Kd < 500 L/kg are
eliminated by less than 10% through sorption on to activated sludge at an
average specific sludge production of 200 g/m3.[16]The main aim of this study was to compare the elimination of sulfonamides,
macrolides and trimethoprim in different wastewater technologies using
complete mass flow analyses, including sewage sludge measurements. By
elimination the combination of all processes involved, e.g. transformation and
sorption, is addressed. Two conventional activated sludge treatment plants, a
fixed-bed reactor and a membrane bioreactor pilot plant, operated at 3 different
sludge ages, are compared as well as two different sand filters as tertiary
treatment steps. The results obtained are discussed with regard to general
parameters, e.g. temperature, hydraulic retention time and solid retention time.
5.2 Experimental Section
5.2.1 Wastewater Treatment Plants
The wastewater treatment plant of Kloten-Opfikon (WWTP-K) treats 55 000
population equivalents (PE): the combined sewage of 25 900 residents and of an
unknown number of air traffic passengers in the catchment area (Figure 5.1).
The average inflow (dry weather) amounted to 16 500 m3/d. The main
wastewater characteristics are summarized in Table 5.1. Primary treatment
consists of a screen, an aerated grit removal tank, and a primary clarifier.
Approximately 60%> of the primary effluent passes an activated sludge treatment
system operated at a sludge age of 3 days and a hydraulic retention time of 5 h
(V = 2 500 m3) as a pre-treatment. The main conventional activated sludge
treatment (CAS-K) includes denitrification (V = 1 900 m3) and nitrification (V =
3 700 m3) with a solid retention time of 10 - 12 d. The hydraulic retention time
(HRT) including the secondary clarifier is -15 h. Phosphate is removed by
simultaneous precipitation with Fe3+ in secondary treatment. The effluent is
discharged to the receiving water after filtration in a discontinuously operated
two-layer sand filter (SF-K). SF-K consists of 8 compartments with a total
volume of 288 m3, which are filled with a 1.2 m thick layer of schistose material
(diameter 2-3 mm) and 0.4 m of silica sand (diameter 0.7 - 1.2 mm). The filters
Behavior 111
Table 5.1 Average wastewater characteristics of of the municipal wastewater
treatment plants Kloten-Opfikon (WWTP-K) andAltenrhein (WWTP-A)
WWTP-K WWTP-A
parameter raw tertiary raw tertiary
influent effluent influent effluent
PH 5.8 ±0.9 6.8 ±0.2 7.5 ±0.3 7.6 ±0.1
BOD5 [mg/L] 220 ± 67 2.2 ± 0.7 210 ±45 8.7 ±4.6
COD [mg/L] 360 ± 76 16±2 590 ±300 35 ± 10
Ntot [mg/L] 43 ±6 23 ±4 38 ±6 15 ±7.6
Ptot [mg/L] 5.5 ±0.8 0.4 ±0.1 6.7 ±1.4 0.5 ±0.2
are backwashed daily by pumping a combination of air and filtrate through the
filter bed for ~25 min. The secondary sludge of both units is recycled to the
primary clarifier together with the sand filter backwash, except for the first two
sampling campaigns (March 2002 and February 2003), where the excess sludge
of the main unit was directly transferred to sludge treatment.
The membrane bioreactor pilot plant (MBR, 100 PE) is operated in parallel to
CAS-K using primary effluent at a flow rate proportional to raw water influent
(HRT -13 h). The bioreactor consists of a stirred anaerobic compartment (V = 6
or 8 m3), denitrification (V = 4 m3) and nitrification (V = 6 m3). The solid
retention time was increased in-between sampling campaigns from 16 ± 2 over
33 ± 3 to 60 - 80 d (steady state operation for two to three sludge ages prior to
sampling). For the 60 - 80 d sludge age no steady state was achieved, but the
sludge age was steadily increased over 110 d from a solid retention time of
33 ± 3 d to 60 - 80 d. In the final aerobic compartment, the secondary effluent
(permeate) is generated by three different membrane filtration units with a
maximal flow rate of 1.3 m3/h each. For test purposes a microfiltration plate
membrane module (Kubota A50), and two ultrafiltration hollow-fibre modules
(Mitsubishi Aqua-RM and Zenon ZeeWeed 500-C) were run in parallel. The
respective nominal pore sizes were 0.4 pm for microfiltration and 0.1 pm and
0.04 pm, respectively, for ultrafiltration.
The wastewater treatment plant of Altenrhein (WWTP-A), located in the canton
St. Gall near the Austrian border, handles the combined sewage of 80 000 PE,
including 52 000 inhabitants (Figure 5.2). The average inflow (dry weather)
Figure
5.1Flowschemeofthemu
nicipalwastewater
treatmentplantKloten-Opf
ikon
(WWTP-K),showingsamplingpoints
forwastewaterandsl
udge
.A
conventionalactivatedsludge
system(CAS-K,55000PE)andamembrane
bioreactor(MBR,
100PE)
areoperatedinparallel
raw
influent
primary
influent
effluent
CAS-K
-40%
secondary
effluent
tertiary
effluent
grit
removal
tank
compositesample
pnmary
sludge
^t
grabsample
secondary
(exc
ess)
sludge
sand
filter
4flffi5£
o|/k
>o/o
MBR1J
secondaryeffluent
(permeat)
secondary
(exc
ess)
sludge
a
CAS-K
consistsofdenitrificationand
nitrificationcascadewithahydraulicretentiontimeof-15handa
solidretentiontimeof10
-
12
d.Approximately60%
oftheprimary
effluentpass
firs
tlyth
roug
han
additional
activated
slud
gesystem(CAS),
oper
ated
ata
hydr
auli
cretentiontimeof-5handasolidretentiontimeof3
d.
bMBR
consistsofananaerobictankanda
denitrificationand
nitrificationcascadewithahy
drau
licretentiontimeof-13
h.The
solid
retentiontimeswere16±2d,33±3dand60
-80dduring
there
spec
tive
samplingca
mpai
gns.
Figure
5.2Flowschemeofthemu
nicipalwastewater
treatmentplantAltenrhein(WWTP-A),
showingsamplingpointsfor
wastewaterand
slud
ge.A
conventionalactivatedsludge
treatmentplant(CAS-A,
40000PE)andfixed-bedreactor(FBR,
40000PE)
areop
erat
edinparallel
rawinfluent
screen
grit
removal
tank
^P
compositesample
^t
grabsample
primary
effluent
primary
sludge
O0
o
CAS-Aa
secondary
effluent
secondary
(exc
ess)
sludge
öono
öooo
"~T
Q__°_0__Q_^Q___o_-
I
FBRb
filter
backwash
secondary
clarifier
secondary
effluent
tertiary
effluent
sand
filter
a
CAS-A
consistsofdenitrificationand
nitrificationcascadewithahydraulicretentiontimeof-31handasolidretentiontimeof21
25
d.
FBR
consistsofadenitrificationandnitrificationzonewithahydraulicretentiontimeof-1
h.
114 Chapter 5
amounted to 21 000 m3/d. The main wastewater characteristics are summarized
in Table 5.1. Primary treatment consists of a screen, an aerated grit removal
tank, and a primary clarifier. Secondary treatment is performed in two parallel
operated treatment units: a conventional activated sludge (CAS-A) and a fixed-
bed reactor (FBR), receiving -50% of the primary effluent each. Both systems
are designed for nitrification and denitrification. Conventional activated sludge
treatment includes a denitrifying volume (anoxic, mixed) of 2 300 m~ and
nitrifying (aerobic) volume of 6 800 m3. The solid retention time in CAS-A
ranged between 21 d to 25 d, while no value can be given for the FBR. The
hydraulic retention time was -31 h for the CAS-A including the secondary
clarifier, whereas it ranges below 1 h for the FBR. The FBR consists of 8
Biostyr up-flow cells L17] filled with 3.6 mm Styrofoam beads as biofilm support
(V = 1 500 m3) and an anoxic zone (below) and an aerated zone (above the
aeration nozzles). Excess sludge is removed by daily backwash with secondary
effluent. The secondary effluents of both units are combined and treated by a
continuously operated one-layer sand filter (SF-A) before discharge. SF-A
consists of 8 up-flow filter units containing a 1.5 m high silica sand column with
a total volume of-360 m3. The sand is continuously cleaned by pumping it from
bottom to top with an average turnover time of 6 - 8 h. The secondary excess
sludge of the CAS-A and the FBR is recycled to the primary clarifier.
5.2.2 Sample Collection
Figures 5.1 and 5.2 give an overview of the chosen sample locations in both
wastewater treatment plants investigated. Sampling campaigns were performed
in March 2002, February 2003 and November 2003 at WWTP-K and in
September 2002 and March 2003 at WWTP-A. At both locations samples were
taken from the raw influent after the screen, from the primary effluent after
primary settling, from the secondary effluents after the respective biological
treatment and after sand filtration. In all cases flow proportional composite
samples were taken. For weekly sampling campaigns, 24 h composite samples
were mixed flow proportional to yield two samples during the week (combining
two and three days, respectively) and one during the weekend (combining
Saturday and Sunday). In February 2003, also 8 h composite samples were
collected for a period of 24 h. Additionally, grab samples were taken in these
three consecutive 8 h intervals from the anaerobic and anoxic compartment of
Behavior 115
the MBR and from the anoxic compartment of CAS-K. For sludge analysis grab
samples were taken from the aerobic compartment of the CAS-K, the CAS-A
and the MBR, respectively from the filter backwash in the case of the FBR.
After freeze-drying, they were combined for each plant and sampling campaign
to yield one weekly sample.
5.2.3 ChemicalAnalysis
Details on the methods applied to aqueous samples and for sludge analysis
including materials and reagents used are described in Chapter 2 and 3. Briefly,
aqueous samples were filtered (0.45-pm cellulose nitrate filters, Schleicher &
Schuell) and subsequently concentrated by solid phase extraction on polymeric
cartridges. Analysis is performed using reversed phase liquid chromatography
coupled to tandem electrospray mass spectrometry in the positive ionization
mode. Sample-based quantification limits depended on the analyte and the
sample matrix and ranged between 1 and 214 ng/L. Sludge samples were filtered
(glass fiber filters, GF8, Whatman) and the solid fraction was freeze-dried. The
investigated compounds were extracted using pressurized liquid extraction with
water:methanol (1:1) as extraction solvent. The extracts were diluted and
analyzed with the same method as aqueous samples. The limits of quantification
for activated sludge varied between 3 and 41 pg/kg.
5.2.4 Calculated Elimination
Average daily loads were used in all cases unless stated otherwise. They were
calculated from the weekly loads being the sum of the loads determined in the
two or three day sampling intervals (Chapter 4). Elimination (weight
percentage) resulted from comparing the loads entering a specific treatment step
to the sum of all loads leaving the treatment step with the aqueous phase.
Elimination therefore combines sorption onto excess sludge and transformation
due to biological or chemical processes.
The sorbed fraction in the excess sludge was calculated from the measured
concentration in the weekly sludge samples and the specific sludge production
of the secondary treatment. It was assessed using the measured chemical oxygen
demand in the primary effluent and general correlations according to literature
data.[18] During the performed sampling campaigns the average excess sludge
116 Chapter 5
production ranged between 0.1 and 0.2 g/L. The specific sludge production was
used, since the daily withdrawn amount of excess sludge is related mainly to
operational criteria. In this manner, only the amount of newly produced sludge
in activated sludge treatment is taken into account for elimination through
sorption. Since the solid retention times is significantly higher than the hydraulic
retention time, the activated sludge in the reactor (-3 g/L) was considered to be
already in equilibrium with the dissolved fraction. This issue is discussed in
detail by Joss et al.[19]
The highest sorption coefficient obtained for secondary sludge was also used to
estimate the respective loads on primary sludge, since sorption coefficients were
not available for primary sludge. Because of the different sludge characteristics
of primary sludge compared to secondary sludge, the results for primary sludge
have to be regarded as rough estimates. This was considered by assigning a
relative uncertainty of 50% to the used values.
Elimination over the sand filter was calculated correspondingly to secondary
treatment, but no distinction between transformation and sorption was made.
However, sorption to solid particles during sand filtrations is assumed to be
negligible for the investigated compounds due to the small additional sludge
production in the filter.
The uncertainty of the calculated elimination was estimated using error
propagation from the relative measurement uncertainty of the respective in and
out flowing load. A correct calculation of the uncertainty including all factors is
not possible due to the inter-relation of the individual parameters and the small
amount of data sets available. The resulting uncertainty stated for a weekly
sampling campaign, do therefore only include the impact of measurement
uncertainties and do not mirror possible daily variations.
5.3 Results and Discussion
Complete mass balances were performed for sulfonamides, macrolides and
trimethoprim to assess their distribution in wastewater treatment and the
importance of individual treatment steps on the overall removal. The results
obtained for the individual weekly sampling campaigns are used to evaluate
possible differences in elimination due to changing treatment conditions, e.g.
solid retention time and temperature. The uncertainty connected to these values
represents only a calculated measurement uncertainty. To assess the behavior
Behavior 117
and fate of the investigated antimicrobials in a specific treatment step over time,
the elimination observed in different sampling campaigns was combined (e.g. in
Figure 5.3 and 5.6).
5.3.7 Primary Treatment
The elimination of the investigated antimicrobials observed in primary treatment
is generally low (Table 5.2). Varying results were obtained for sulfapyridine,
mirrored in a high standard deviation, while negative eliminations were found
for sulfamethoxazole. This is probably caused by the simultaneous presence of
de-conjugable substances, e.g. human metabolites, of these compounds in the
raw influent (Chapter 4). In accordance with the increase in sulfamethoxazole
loads, a slight elimination was observed for its main human metabolite, AT-
acetylsulfamethoxazole, in primary treatment (9 - 21%). In the case of the
investigated macrolides and trimethoprim an elimination of up to 33% was
observed. Due to the high uncertainty connected with the calculation of the
sorbed fractions in primary treatment as well as with the measurement of such
complex samples, this elimination has to be considered as not significant.
5.3.2 Secondary Treatment
The behavior of the investigated antimicrobials was investigated in two
conventional activated sludge systems and a fixed-bed reactor (Table 5.3 and
Figure 5.3). Additionally, weekly sampling campaigns were performed on a
membrane bioreactor operated at three different solid retention times (Figure
5.4). The observed eliminations combine the reduction due to sorption and
transformation processes. However, the share of elimination caused by sorption
to secondary excess sludge generally ranged below 6%> for all compounds
investigated. Compared to the overall uncertainty connected with elimination
studies, sorption to activated sludge seems to be of minor importance for the
investigated sulfonamides, macrolides and trimethoprim. No distinction between
the processes responsible for the observed elimination rates is therefore made in
the following.
Reported negative elimination results from an observed increase of loads from
the inflow to the outflow of the respective treatment step - mainly observed for
sulfapyridine and sulfamethoxazole. In some cases more than twice the
118 Chapter 5
Table 5.2 Elimination ofsulfonamides, macrolides and trimethoprim in primary
wastewater treatment
compounda
acronym CASRN elimination
(%)b
range
sulfapyridine SPY 144-83-2 -29-20
sulfamethoxazole SMX 723-46-6 -21--5
A^-acetylsulfamethoxazole N4AcSMX9-21
SMX±N4AcSMXc0-9
trimethoprim TRI 738-70-5 -13-31
azithromycin AZI 83905-01-5 10-33
erythromycin ERY-H20 114-07-8 -8-4
clarithromycin CLA 81103-11-9 11-14
roxithromycin ROX 80214-83-1 3-9
a
Sulfadiazine (CASRN 68-35-9), sulfathiazole (CASRN 72-14-0) and sulfamethazine
(CASRN 57-68-1) were not at all or very rarely detected.
b
Range of the results obtained in February 2003 and November 2003 at WWTP-K and
March 2203 at WWTP-A. No raw influent was sampled in the other two sampling
campaigns. Negative values result from an observed increase of loads from inflow to
outflow of the respective treatment step.cSulfamethoxazole load, including the amount present as A^-acetylsulfamethoxazole.
inflowing load was detected for these compounds in the respective secondary
effluents of the conventional activated sludge systems and the fixed-bed reactor
(Table 5.3). For both compounds a very inconsistent picture is obtained with
elimination rates ranging between +72 and -104% for sulfapyridine and ±60 and
-138% for sulfamethoxazole. This can be explained by the presence of
substances, e.g. human metabolites, in the inflow, which are subsequently
transformed to sulfapyridine and sulfamethoxazole during biological treatment
(Chapter 4). Neither sulfasalazine, the administered pharmaceuticals containing
sulfapyridine, nor its main human metabolite, A^-acetylsulfapyridine, were
included in this study. The results obtained, however, strongly suggest the
presence of one or both of them in the influent and a possible transformation to
sulfapyridine in biological treatment.
In the case of sulfamethoxazole the fate of the main human metabolite,
Table
5.3Eliminationofsu
lfon
amides
,macrolidesand
trimethoprim
intwo
conventionalactivatedsludge
systemsanda
fixed-bedreactor
CAS-Ka
CAS-A
a
FBR
a
solidretentiontime
(d)
12
12
10
25
21
--
wastewatertemperature(°C)
14
12
16
19
12
19
12
compound
March
2002
February
2003
elimination(%)b
November
September
2003
2002
March
2003
September
2002
March
2003
SPY
-74±66c
-16±45
-107±8
49±5
72±5
52±5
41±9
SMX
-107±8
9±3
-79±7
-138±15
60±3
-61±
10
29±4
N4AcSMX
94±2
87±1
90±1
96±2
85±1
81±1
89±1
SMX+N4AcSMX
50±3
53±1
-1±3
61±3
76±1
60±2
67±2
TRI
3±5
-1±6
14±5
20±
11
-40±20
17±
11
12±11
AZI
d-26±8
-18±7
55±4
22±11
30±6
-13±10
ERY-H20
6±4
-14±4
-22±4
-6±8
-9±8
7±7
-13±8
CLA
9±4
-45±7
-7±5
4±7
20±6
5.6±6
14±6
ROX
18±4
38±3
-18±6
38±5
5±8
35±6
4±8
aCAS-K=conventionalactivatedsl
udge
system
atthemu
nici
palwastewatertreatmentpl
antKl
oten
-Opf
ikon
,Switerland.
CAS-A=conventionalactivatedsl
udge
system
atthemu
nici
palwastewatertreatmentplan
tAl
tenr
hein
,Switzerland
FBR:
fixed-bedreactoratthemunicipa
lwastewatertreatmentplantAltenrhein,Switzerland.
b
Nega
tive
valuesresultfromanobservedincreaseofloadsfrominflowtooutflowoftherespective
treatment
step.
c
Unce
rtai
ntyestimatedfromrelativemeasurementuncertaintyofinandoutfl
owin
gload.
dNo
resultsavailablebecauseofanalytic
alinterferences.
120 Chapter 5
A^-acetylsulfamethoxazole, was also investigated. A high elimination rate of up
to 96% was observed for this compound in the two conventional activated
sludge systems and the fixed-bed reactor (Table 5.3). The possible
transformation of A^-acetylsulfamethoxazole to sulfamethoxazole and a
simultaneous elimination of sulfamethoxazole itself during biological treatment
probably lead to the observed high variability of sulfamethoxazole elimination.
Taking the fraction present as A^-acetylsulfamethoxazole into account, an
average reduction of sulfamethoxazole to relative residual loads of 32 to 49%
was found in the conventional activated sludge systems and the fixed-bed
reactor (Figure 5.3). In the case of CAS-K only the results from the first two
sampling campaigns were taken into account. In the third sampling campaign in
November 2003 no significant elimination was observed for the
sulfamethoxazole load including the fraction present as A^-acetyl-
sulfamethoxazole, in contrast to March 2002 and February 2003 (Table 5.3). No
explanation could be found comparing wastewater temperature or known
operational parameters, e.g. solid retention time.
In the membrane bioreactor an elimination ofA^-acetylsulfamethoxazole of over
95 ± 2% was detected independent of the prevailing solid retention time (Figure
5.4). For sulfapyridine and sulfamethoxazole similar elimination rates were
observed in all three sampling campaigns. The average relative residual loads
measured were 46 ± 5% and 63 ± 1%, respectively. In contrast to the other
treatment technologies investigated, no increase in sulfapyridine and
sulfamethoxazole loads was observed from the influent to the effluent of the
membrane bioreactor. These findings suggest an effective simultaneous
elimination of these compounds during biological treatment in the membrane
bioreactor. Subsequently, a slightly increased average elimination of 78 ± 10%
was observed for sulfamethoxazole, including the fraction present as 1\-
acetylsulfamethoxazole, in the membrane bioreactor (Figure 5.4) compared to
conventional activated sludge treatment and the fixed-bed reactor (Figure 5.3).
For trimethoprim only a slight elimination of up to 20% was observed in
conventional activated sludge treatment and the fixed-bed reactor, not taking the
deviating result of -40 ± 20 obtained in March 2003 into account. Varying
results, including negative elimination, were obtained for the investigated
macrolides, with no obvious pattern between the sampling campaigns (Table
5.3). The presence of de-conjugable metabolites, however, seems unlikely for
macrolides.f20] Since they are mainly excreted with bile and feces, the load
Figure
5.3Relativeresidualloadsofsu
lfon
amid
es,macrolidesandtrimethoprim
intheef
flue
ntof
twoconventionalactivated
sludge
systemsandafi
xed-
bed
reactora
SPY
SMX
N4AcSMX
SMX
+
N4AcSMX
TRI
AZI
ERY-H20
CLA
ROX
aErrorbarsrepresentthe
standard
deviationfrom
threesamplingcampaigns(CAS-K)and
therangeoftwo
values(CAS-A,FBR),
resp
ecti
vely
.
Figure
5.4
Relative
residual
loadsofsu
lfon
amid
es,
macrolidesand
trimethopr
iminamembrane
bioreactoroperated
at
differentsolidretention
timesa
150
:,
,—.
N4AcSMX
SMX
TRI
AZI
ERY-H20
CLA
ROX
+
N4AcSMX
aErrorbarsrepresenttheuncertaintiesestimatedfromrelativemeasurementuncertaintiesofinandoutfl
owin
gload.
bNo
resultsavailablebecauseofanalyt
ical
interferences.
Behavior 123
entering biological treatment may be underestimated taking only the dissolved
fraction and sorption to the suspended solids into account. With this, the amount
enclosed in feces particles, which may be released during biological treatment,
is neglected. It may, together with possible differences in the sludge
composition, lead to the observed variations in the elimination of macrolides.
The average relative residual loads observed for macrolides after the two
conventional activated sludge systems and the fixed-bed reactor, ranged between
78 and 122%, taking the observed inter-campaign variations into account
(Figure 5.3).
As for the sulfonamides, no increase in loads was observed for macrolides and
trimethoprim in the membrane bioreactor (Figure 5.4). Additionally, elimination
observed for these compounds in the membrane bioreactor tends to be higher
compared to conventional activated sludge treatment and the fixed-bed reactor
(Figure 5.3). At solid retention times comparable to those of the other
technologies investigated, i.e. 16 ± 2 and 33 ± 3 d, similar relative residuals
were obtained in the membrane bioreactor. They ranged between 39 and 72%
for trimethoprim and the investigated macrolides, except azithromycin (96%).
Since sorption to excess sludge is of minor importance, operational differences
between the investigated treatment technologies, e.g. concerning the sludge
retention and redox conditions (additional anaerobic compartment), may cause
the observed differences. They may have an effect on certain wastewater and
sludge characteristics as well as on the biodiversity of the microbial flora
present.
5.3.3 Solid Retention Time
The solid retention time describes the mean residence time of the biomass within
the system. It is positively correlated with the concentration of the biomass if the
volume of the system is constant. Increased solid retention times allow the
enrichment of slow growing bacteria and may therefore lead to a broader
microbial flora potentially resulting in extended physiological capabilities.
Additionally, it leads to an increase of the inert fraction of the sludge via the
accumulation of decay products and inorganic material, and a decrease of
observed sludge production per volume of wastewater, caused by higher sludge
decay.
124 Chapter 5
A possible influence on the elimination of sulfonamides, macrolides and
trimethoprim were investigated in a membrane bioreactor operated at three
different sludge ages (Figure 5.4). In the beginning (March 2002) a solid
retention time of 16 ± 2 d, comparable to that of conventional activated sludge
systems, was investigated. For the second sampling campaign (February 2003) it
was increased to 33 ± 3 d, representing a solid retention time usually applied in
membrane filtration systems. Additionally, the impact of a high sludge age (60 -
80 d) on the elimination of the selected compounds was investigated (November
2003).
No dependence of the elimination on the solid retention time could be seen for
the selected sulfonamides including A^-acetylsulfamethoxazole in the membrane
bioreactor. For trimethoprim and most of the investigated macrolide
antimicrobials a similar elimination was observed at a solid retention time of 16
± 2 and 33 ± 3 d, respectively. However, a two to three times higher reduction
was seen for these compounds (except roxithromycin) at a solid retention time
of 60 - 80 d compared to the lower solid retention times. It ranges between 87
and 90% for trimethoprim, dehydro-erythromycin and clarithromycin and
amounts to 25% for azithromycin in this last sampling campaign. In the case of
roxithromycin, the observed elimination increases from 39% at a solid retention
time of 16 ± 2 days to -60% at the two higher solid retention times.
Similar average eliminations were obtained for all compounds investigated in
both conventional activated sludge systems independently of the solid retention
time, which ranged between 21 - 25 d in CAS-A and between 10 - 12 d in CAS-
K (Figure 5.3). Also in literature, no dependence of the elimination on the solid
retention time was reported for sulfamethoxazole and roxithromycin in
laboratory scale experiments,12'1 which confirms the observations made in the
MBR. An elimination of 70 to 90% was reported for sulfamethoxazole at
varying solid retention times between 1 d and 35 d. For roxithromycin no
significant elimination (9%) was observed at a solid retention time of 1 d, while
it ranged between 39 and 66%> at higher solid retention times.
5.3.4 Substrate Dependencies
The partial elimination observed for sulfonamides in the MBR independent of
the solid retention time (Figure 5.4), suggests a correlation of the elimination
with the substrate concentration in the influent for these compounds, i.e. with
Behavior 125
the ratio of a specific substrate to the sulfonamide concentration. This is
supported by the fact that no further elimination of sulfonamides is observed
during sand filtration as described below.
For trimethoprim and the investigated macrolides (except roxithromycin) the
results obtained in the membrane bioreactor indicate that the transformation of
these compounds may be inversely correlated to the sludge loading. By sludge
loading the ratio of substrate input to the amount of biomass present is assessed.
Since the reactor volume is constant, higher solid retention times result in
increased sludge concentrations, and therefore in a reduced sludge loading. The
combination of high solid retention times and reduced sludge loading may cause
an increase in the biodiversity of the active biomass, which may have an
influence on the elimination of compounds undergoing co-metabolism, as
assumed for antimicrobials.
In the case of A^-acetylsulfamethoxazole an almost complete elimination was
observed in all systems investigated and seems to occur under all treatment
conditions investigated. This may suggest a transformation by a widely available
biological reaction or even by abiotic processes.
However, in all cases direct experimental evidence, e.g. from batch experiments,
would be necessary to confirm possible substrate dependencies of the
eliminations observed for the investigated antimicrobials in biological treatment.
5.3.5 Anaerobic Compartment
Another factor that may influence the elimination of sulfonamide and macrolide
antimicrobials observed in the membrane bioreactor may be the additional
anaerobic compartment not present in the investigated conventional activated
sludge treatment plants. Therefore the elimination of the selected compounds in
the different redox compartments was investigated. The concentrations in the
inflow and the outflow of the respective cascade used for biological treatment
were measured in 8 h composite samples. Grab samples were taken from the
anaerobic and anoxic compartment of the membrane bioreactor operated at a
solid retention time of 33 ± 3 d and from the anoxic compartment of CAS-K in
the same 8 h intervals. From this, expected concentrations, assuming no
elimination in the respective compartment, were estimated. The calculated
concentrations, assuming no elimination, were than compared to the
concentrations measured to investigate a possible elimination under the given
Figure5.5Infl
uenc
eofdifférentredoxconditionsonthetr
ansf
orma
tion
ofsulfonamideandmacrolide
antimicrobialsa
300
250
200
150
O) f
100
O
50
anoxicco
mpar
tmen
tCAS-K
Daerobiccr
jrrp
artm
ertCAS-K
anaerobiccorrpartmertM3R
Bano>dccorrpartmert
h/BR
DaerobiccorrpartmentM3R
SPY
SMX
N4ACSMX
SMX
+
N4AcSMX
TRI
AZI
ERY-H20
CLA
ROX
aErrorbarsrepresentthestandarddeviationoftheresultsfromthree8hsa
mpli
ngintervals.
bComparisonofestimatedconcentrations(c
alcu
late
dassumingnoeliminationinthere
spective
compartment)andmeasured
concentrationsingrab
samplesfromtheanaerobic,
anoxicandaerobiccompartmentsofthemembranebioreactor(MBR)andthe
aerobicandanoxiccompartmentsoftheconventionalactivatedsludge
system(CAS-K)
atWWTP
Klot
en-O
pfik
on.
Behavior 127
redox conditions. Figure 5.5 summarizes the results obtained combining all three
8 h intervals. They have to be regarded with caution, however, since only grab
samples were used.
For AAacetylsulfamethoxazole a significantly lower concentrations than
estimated assuming no elimination, were measured in all compartments,
suggesting a transformation of this human metabolite under all redox conditions
given. For sulfamethoxazole itself, the measured concentrations were higher
than those estimated assuming no elimination, in the anaerobic and anoxic
compartments and lower than expected in the aerobic compartments.
Sulfamethoxazole hence seems to be fransformed mainly under aerobic
conditions. The observed accumulation in the other compartments may be
explained by the postulated transformation of /V-acetylsulfamethoxazole to
sulfamethoxazole. A pattern similar to sulfamethoxazole was found for
sulfapyridine, suggesting the presence of retransformable substances in the
influent of biological treatment also in this case.
No significant differences between estimated concentrations assuming no
elimination and measured concentrations were obtained for trimethoprim,
azithromycin and dehydro-erythromycin. For clarithromycin and roxithromycin
a tendency to lower concentrations measured than those estimated assuming no
elimination was observed in the anaerobic and anoxic compartment of the
membrane bioreactor. The combination of anaerobic and anoxic treatment may
therefore have an impact on the elimination of specific compounds, e.g.
macrolide antimicrobials. However, further experiments, e.g. using composite
samples or laboratory scale experiments, would be necessary to confirm the
observed behavior.
5.3.6 Wastewater Temperature
Sampling campaigns were performed at different times of the year resulting in
different wastewater temperatures. In the case of WWTP-A, sampling was
performed in March 2003 and in September 2002, resulting in an increase of the
wastewater temperature from 12 °C to 19 °C. Generally, no correlation between
the water temperature and the respective elimination was observed for the
investigated compounds (Table 5.3). A tendency to higher elimination with
increased water temperature could be seen for azithromycin and roxithromycin.
However, one has to be careful in interpreting this tendency, since variations at
128 Chapter 5
similar temperatures but different sampling times are in the same range.
Additionally, it is unclear, whether temperature dependencies as commonly
observed for biological treatment,1181 also apply to the transformation of
antimicrobials, or micropollutants in general.
5.3.7 Hydraulic Retention Time
Although the hydraulic retention time in the fixed-bed reactor ranged below 1 h,
a similar elimination as in conventional activated sludge treatment with a
hydraulic retention time of up to 31 h was observed (Figure 5.3). The results
indicate that the lower hydraulic retention time in the fixed-bed reactor is
approximately compensated by a higher bioactive sludge concentration per
reactor volume. In the FBR this is caused by the regular discharge of fast
growing heterotrophs and inert material with the backwash of the filter bed. In
general, it seems that for a given influent composition the elimination of
antimicrobials is not directly related to the hydraulic retention time but to a
similar efficiency in nutrient removal. Another possible factor increasing the
elimination of antimicrobials, if assuming a pseudo-first-order-kinetic depending
on the substance concentration as shown for estrogens/111 may be the strict plug
flow regime in the filter bed. It may lead to higher momentary concentrations of
the antimicrobials compared to the concentration in the fully mixed reactors in
conventional activated sludge treatment.
5.3.8 Sand Filtration
Two sand filters used for tertiary treatment - SF-K in WWTP Kloten Opfikon
and SF-A in WWTP Altenrhein - were investigated with respect to their removal
efficiency for sulfonamides, macrolides and trimethoprim (Figure 5.6).
In all five sampling campaigns higher loads of sulfapyridine were detected in the
tertiary effluents compared to the respective influent loads of the sand filters,
resulting in an average increase of 28 ± 17% for sulfapyridine during sand
filtration. It can be inferred that significant amounts of possible sulfapyridine
precursors, e.g. de-conjugable human metabolites, pass through biological
treatment and are transformed to sulfapyridine during sand flltration. Since only
sulfapyridine itself was analyzed in this study no information on a potential
Figure
5.6Relativeresidualloadofsu
lfon
amid
es,
macrolidesandtrimethoprim
inthe
effluent
oftwosandfiltersusedas
tert
iary
treatmenta
SPY
SMX
TRI
AZI
ERY-H20
CLA
ROX
N4AcSMX
a
Errorbarsrepresentthestandarddeviationfromthreesamplingcampaigns
forthesand
filt
eratWWTP-K
(SF-K)andtherangeoftwo
valuesforthesand
filter
atWWTP-A
(SF-A),re
spec
tive
ly.
130 Chapter 5
simultaneous transformation of the parent compound itself on the sand filter is
available.
In the case of sulfamethoxazole the main human metabolite, A^-acetyl-
sulfamethoxazole, was included in the investigation. However, only very small
concentrations of this compound were detected in secondary effluents due to the
high transformation efficiency of biological treatment for V-acetyl-
sulfamethoxazole. The total sulfamethoxazole load, including the amount
present as //-acetylsulfamethoxazole, did not significantly change during sand
flltration (Figure 5.6). Combining all sampling campaigns, no significant change
of the azithromycin load was observed, however high variations were observed
between sampling campaigns, mirrored by the large error bars.
For dehydro-erythromycin, clarithromycin and roxithromycin comparable loads
were detected in the inflow and outflow of one of the sand filters (SF-A), while
a significant reduction was observed for these compounds on the other sand
filter (SF-K). The average elimination amounts to 20 ± 5% for dehydro-
erythromycin, 17 ± 9% for clarithromycin and 23 ± 4% for roxithromycin during
tertiary treatment with SF-K, located at the WWTP Kloten-Opfikon. A poor
elimination of 15 ± 10% was detected for trimethoprim on the sand filter of
WWTP Altenrhein (SF-A). In the case of WWTP-K, sand flltration (SF-K) is
the main step responsible for a reduction of the trimethoprim load with an
average elimination of 74 ± 14%.
Due to the small amount of suspended solids produced in the sand filter, the
effect of sorption can be neglected for the investigated compounds. The results
therefore suggest a highly effective and diverse biofilm present on the sand
particles of SF-K. Additionally, it is noteworthy that an elimination on SF-K is
mainly seen for those compounds also showing an increased elimination in the
membrane bioreactor operated at a high sludge age, i.e. trimethoprim, dehydro-
erythromycin and clarithromycin.
An elimination between 56 - 70% on SF-K was observed for nonylphenolic
compounds by Wettstein.[22] From laboratory scale studies, the observed
elimination for these substances was assigned to the biological activity of the
sand filter material and not to a catalytic or reactive effective of the sand itself.
Even though both sand filters investigated were operated at a hydraulic retention
time of ~ 25 min and have similar hydraulic loads per surface of biofilm,
significant differences were observed in the elimination of certain
antimicrobials, especially trimethoprim. One possible explanation may be the
Behavior 131
different availability of oxygen during the passage of the two filters. The
secondary effluent of WWTP-K is aerated to oxygen saturation (aerated
flocculation reactor) prior to sand filtration, while the secondary effluents at
WWTP-A directly enters tertiary treatment. Further on, the water entering SF-A
shows a higher biological oxygen demand (BOD5) of 10 - 20 mg/L compared to
~3 mg/L in the case of SF-K. This may lead to additional oxygen consumption
during the sand filter passage. The resulting oxygen limitations in SF-A
compared to SF-K may result in the observed differences in the elimination of
certain antimicrobials. Another possible explanation could lay in the design and
operation of the two sand filters investigated. While SF-A is regenerated
continuously, by turning over the sand bed, only a daily backwash of SF-K is
performed. Together with the oxygen availability, this may result in a more
diverse and stable microbial community in SF-K. Additionally, a possible
formation of specific zones may be more predominant in a two-layer filter
disturbed only periodically (SF-K), compared to a continuously regenerated one-
layer filter (SF-A).
5.4 Conclusions
For selected macrolides and trimethoprim elimination seems to correlate with
increasing solid retention time and, hence, with reduced substrate loading. Both
parameters may lead to an increased biodiversity of the active biomass, resulting
in a broader range of degradation pathways available. For sulfonamides a
positive correlation of the observed elimination to the organic substrate
concentration appears to exist. In the case of sand filtration, used as tertiary
treatment, elimination efficiencies were observed for selected macrolides and
trimethoprim and gave the impression to depend on operational parameters of
the sand filter, e.g. oxygen limitations. The correlations observed for the
elimination of the selected antimicrobials in field studies should be verified
under more controlled conditions, i.e. in batch experiments.
Even though the removal in wastewater treatment may be optimized for certain
compounds, e.g. by an increased sludge age or an additional tertiary treatment
step, the overall elimination of sulfonamide and macrolide antimicrobials in
wastewater treatment was shown to be incomplete. Therefore, residual amounts
of these emerging contaminants are continuously discharged to receiving surface
waters. Our ongoing research aims at investigating the ozonation of wastewater
132 Chapter 5
effluents as a possible technique to further reduce the antimicrobial load
entering the aquatic environment. Additionally, no knowledge on the
transformation products formed in wastewater treatment is available. The latter
topic needs to be investigated further, especially considering the environmental
risk assessment for these compounds.
Acknowledgements
Abbott GmbH (Wiesbaden, Germany) is acknowledged for supplying
clarithromycin and Pfizer AG (Zurich, Switzerland) for supplying azithromycin.
Partial financial support came from the EU project POSEIDON (EVK1-CT-
2000-00047)[23] and the EAWAG project on human-use antibiotics
(HUMABRA) within the framework of the National Research Program on
antibiotic resistance funded by the Swiss National Science Foundation/241 We
would also like to thank the Swiss Agency for the Environment, Forestry and
Landscape, the Swiss cantons of Aargau, Basel Land, Bern, Luzern,
Schaffhausen, Schwyz, St. Gallen, Thurgau, Ticino, Zurich and the WWTPs of
Kloten-Opfikon and Altenrhein for additional financial support. We thank the
technical staff of the WWTP Kloten Opfikon and the WWTP Altenrhein for
their assistance during sampling. For helpful comments on the manuscript we
acknowledge M.Suter and T.Ternes.
5.5 Literature cited
[1 ] Stan, H. J.; Heberer, T. Analusis 1997, 25, M20-M23.
[2] Halling-Sorensen, B.; Nors Nielsen, S.; Lanzky, P. F.; Ingerslev, F.; Holten
Lützenhßft, H. C; Jorgensen, S. E. Chemosphere 1998, 36, 357-393.
[3] Daughton, C. D.; Ternes, T. A. Environ. Health Perspect. 1999,107, 907-
938.
[4] Kümmerer, K. Pharmaceuticals in the environment: Source, fate, effects
and risks; Springer: Berlin, Heidelberg, New York, 2001.
[5] Erickson, B. E. Environ. Sei. Technol. 2002, 36, 140-144.
[6] Heberer, T. Toxicol. Lett. 2002, 131, 5-17.
[7] Diaz-Cruz, M. S.; Lopez de Aida, M. J.; Barcelo, D. Trends Anal. Chem.
2003,22,340-351.
Behavior 133
8] Giger, W.; Alder, A. C; Golet, E. M.; Kohler, H.-P. E.; McArdell, C. S.;
Molnar, E.; Siegrist, H. R.; Suter, M. J.-F. Chimia 2003, 57, 485-491.
9] Boxall, A. B. A.; Fogg, L. A.; Blackwell, P. A.; Kay, P.; Pemberton, E. J.;
Croxford, A. Reviews in Environmental Contamination and Toxicology
2004,180,1-91.
10] Ternes, T. A.; Joss, A.; Siegrist, H. Environ. Sei. Technol. 2004, 38, 392A-
399A.
11] Joss, A.; Andersen, H. R.; Ternes, T. A.; Richie, P. R.; Siegrist, H. R.
Environ. Sei. Technol. 2004, 38, 3047-3055.
12] Golet, E. M.; Alder, A. C; Giger, W. Environ. Sei. Technol. 2002, 36,
3645-3651.
13] Golet, E. M.; Xifra, L; Siegrist, H. R.; Alder, A. C; Giger, W. Environ. Sei.
Technol. 2003, 37, 3243-3249.
14] McArdell, C. S.; Molnar, E.; Suter, M. J.-F.; Giger, W. Environ. Sei.
Technol. 2003, 37, 5479-5486.
15] Ternes, T. A., Habilitation Thesis, Mainz, 2000.
16] Ternes, T. A.; Herrmann, N.; Bonerz, M.; Knacker, T.; Siegrist, H.; Joss,
A. Water Res. 2004, 38, 4075-4084.
17] Rogalla, F.; Lamouche, A.; Specht, W.; Kleiber, W. Water Sei. & Technol.
1994, 29, 207-216.
18] ATV-DVWK Fachausschuss, "Arbeitsblatt ATV-DVKW-A 131," ISBN 3-
933707-41-2, 2000.
19] Joss, A.; Keller, E.; Göbel, A.; Alder, A. C; McArdell, C. S.; Siegrist, H.
submitted to Water Research.
20] Bryskier, A. J.; Butzler, J.-P.; Neu, H. C; Tulkens, P. M. Macrolides;
Arnette Blackwell: Paris, 1993.
21] Clara, M.; Strenn. B.; Gans, O.; Kreuzinger, N. In Water Resources
Management II; Brebbia, C. A., Ed.; WIT Press: Southampton, UK, 2003;
Vol. ISBN 1-85318-967-4, pp 227-236.
22] Wettstein, F. "Auftreten und Verhalten von Nonylphenoxyessigsäure und
weiteren Nonylphenolverbindungen in der Abwasserreinigung," PhD
Thesis No. 15315, ETH Zurich, 2004.
23] http://www.eu-poseidon.com.
24] http://www.nrp49.ch/pages/.
c
L'a ^u*^
Chapter 6
Ozonation
Sulfonamide and macrolide antimicrobials are generally not completely
eliminated during wastewater treatment. To further reduce the residual amounts
being discharged to receiving waters, the ozonation of wastewater effluents was
investigated. Extensive oxidation of sulfonamide and macrolide antimicrobials
was achieved at an ozone dose of only 2 mg/L, resulting in a reduction of the
respective loads by over 90%. A reduced reactivity towards ozone was observed
for acetylated sulfonamides, e.g. the human metabolite A^-acetyl-
sulfamethoxazole, illustrating the significance of the aniline moiety in
sulfonamides as the main site of ozone attack. The amount of suspended solids
in the wastewater, varying between 3 and 22 mg/L, had no significant impact on
the oxidation efficiencies for all investigated compounds. As expected,
variations in oxidation efficiencies occurred in relation to the pH of the
wastewater, since the rate constants of the compounds as well as the ozone
stability are pH dependent.
partly published in
Huber, M.M., Göbel, A., Joss, A., Hermann, N., Löffler, D., McArdell, CS.,
Ried, A., Siegrist, H., Ternes, T.A., von Gunten, U.
Oxidation of Pharmaceuticals during the Ozonation of Municipal Wastewater
Effluents: A Pilot Study
Environmental Science and Technology, 2005, in press.
Ozonation 137
6.1 Introduction
In recent years, various studies have reported the occurrence of a large number
of pharmaceuticals in the aquatic environment/1"31 Even though the detected
concentration levels are typically in the ng/L to pg/L range, it cannot be
excluded that molecules designed to be biologically active, affect sensitive
aquatic organisms even at such low concentrations. Immediate effects caused by
pharmaceuticals may be subtle and difficult to detect, but nevertheless could
lead to important long-term consequences in aquatic ecosystems/41 After
consumption, pharmaceuticals are excreted from the human body in the
unchanged form or in the form of human metabolites. In developed countries,
wastewater is usually treated in wastewater treatment plants before it is
discharged into receiving surface waters. Municipal wastewater is therefore the
major source of human pharmaceuticals in the aquatic environment. Since it is
highly unrealistic to prohibit or limit the consumption of any pharmaceuticals,
the improvement of wastewater treatment is an effective option to diminish the
release of these compounds into the aquatic environment. Among the various
classes of pharmaceuticals, antimicrobials are of special interest due to the
potential spread and maintenance of antibacterial resistance, especially in human
pathogens. Based on annual consumption data in Switzerland, the most
important antimicrobial classes applied in human medicine are the ß-lactams
(17.5 t/a), sulfonamides (5.7 t/a), macrolides (4.3 t/a) and fluoroquinolones
(3.9 t/a).[5"7] While ß-lactams seem to be hydrolyzed shortly after excretion,
representatives of the other three classes have been detected in wastewater
effluents/8121 The occurrence and fate of fluoroquinolones in the environment
has been intensively studied by Golet et al.,[13'14] while the occurrence and fate of
sulfonamides, macrolides and trimethoprim is the focus of this dissertation.
They are, as shown in the previous chapters, removed to varying extents in
wastewater treatment. Elimination efficiencies proved to be strongly compound
dependent and also varied with the treatment technology investigated. However,
incomplete elimination in wastewater treatment was observed in all cases.
Residual concentrations of these emerging contaminants are therefore constantly
discharged to the aquatic environment. Advanced treatment technologies would
be necessary to achieve a further removal of sulfonamides, macrolides and
trimethoprim from wastewater treatment plant effluents. Ozonation has shown a
high potential for the oxidation of pharmaceuticals in drinking water[,5'16] and
138 Chapter 6
wastewater/ J In wastewater, ozone doses ranging from 5 to 15 mg/L led to a
complete disappearance of most of the pharmaceuticals except iodinated X-ray
contrast media. Also the antimicrobials investigated were eliminated to below
the limit of quantification by an ozone concentration of 5 mg/L, which equals
elimination rates between 76 and 92%.
The aim of the present study was to investigate the removal of selected
sulfonamide and macrolide antimicrobials, including two sulfonamide
metabolites, and trimethoprim, from wastewater effluents by ozonation. To be
able to determine removal of 95 - 99%, the selected antimicrobials were spiked
to the wastewater. By applying comparatively low ozone doses ranging from 0.5
to 5 mg/L, we aimed at finding minimum doses required for the removal of the
selected pharmaceutical classes and to gain a better insight into the ozonation
process. To investigate the influence of particles, three effluents with different
concentrations of suspended solids were treated in the pilot plant. The secondary
wastewater effluents used exhibit a substantially lower dissolved organic carbon
(DOC) concentration than the wastewater investigated by Ternes et al/17] Such
conditions are more representative for Switzerland, due to the higher dilution of
wastewater in Switzerland by e.g. extraneous wasters.
6.2 Experimental Section
6.2.1 Ozonation Pilot Plant
The pilot plant consists of two ozonation columns operated in series with an
active reactor volume of 140 liter each and a filling level of 4.8 m (0.193 m
nominal inner diameter, 5.2 m total height). A flow scheme of the plant is given
in Figure 6.1. The water enters the plant on the top of column 1, operated in the
downstream mode and leaves the plant at the top of column 2, operated in the
upstream mode. Tracer experiments with a salt spike showed a slightly better
plug flow behavior in the second column as compared to the first. Modeling the
reactor volume as a series of 3 and 4 fully mixed compartments with comparable
total volume, could best simulate the salinity profile at the outflow of column 1
and 2, respectively. With a flow rate of 2 ± 0.1 m3/h, the total hydraulic retention
time amounts to 4.2 ± 0.2 min in each column. The ozone was continuously
supplied by an ozone generator (Ozomatic SWO 200) fed with technical oxygen.
Ozone containing gas was applied at the bottom of column 1 at a flow of
Ozonation 139
200 ±10 L/h, resulting in a counter flow of water and gas bubbles. Ozone
concentrations in the feed and off gas were measured with a UV ozone monitor
(BMT 936 Vent, 0.1-50 g/m3). By adjusting the power input of the ozone
generator, the desired ozone concentrations were obtained. The respective
concentrations yielded transferred ozone doses of 0.5, 1, 2.5, 3.5 and 5 mg/L in
wastewater. Transfer efficiencies were > 98%. No ozone was applied to column
2, serving as a reaction vessel for the dissolved ozone. The residual ozone
concentration was measured at the interface between column 1 and 2 (C (O3-
Cl)) and at the outflow of column 2 (C (03-C2)).
Table 6.1 Set up ofthe ozonation pilot plant used
column 1 column 2
6.2.2 Wastewater Effluents
The pilot plant was operated on site of the municipal wastewater treatment plant
(WWTP-K) in Kloten-Opfikon, Switzerland, located near the international
airport of Zurich. The combined sewage of 55 000 population equivalents (PE)
is treated using a conventional activated sludge system. A pilot-scale membrane
bioreactor (100 PE) is operated in parallel. A detailed description of the plant
140 Chapter 6
and the treatment technologies applied are given in Chapter 5. For the ozonation
experiments the secondary effluent of the conventional activated sludge system
(CAS) and the final effluent of the membrane pilot plant (MBR) were used. The
respective water was continuously pumped into a 300 L tub, mechanically
mixed, and subsequently fed to the pilot plant. To increase the total amount of
suspended solids (TSS) by -15 mg/L (CAS±TSS), sludge from the conventional
activated sludge system was continuously added to the inflow of the tub in one
experiment. The resulting water quality parameters of the 3 different matrices
investigated are summarized in Table 6.1.
Table 6.1 Average water quality parameters of the three wastewater effluents
investigateda
effluent pH T DOC COD TSS alkalinity
[°C] [mg/L] [mg/L] [mg/L] [mM]
CAS 7.0 ±0.1 16±1 7.7 ±0.5 29 ±3 7±2 3.1 ±0.1
CAS±TSS 6.9 ±0.1 15 ±1 7.0 ±0.5 41 ±1 22 ±2 3.2 ±0.2
MBR 7.5 ±0.1 17 ±1 6.6 ±0.2 22 ±2 3±2 5.4 ±0.2
aErrors represent one standard deviation.
6.2.3 Spiking of Wastewater Effluents
An aqueous solution of the investigated antimicrobials (Figure 6.2 and 6.3) was
prepared and continuously added to the respective wastewater effluent prior to
entering the mixing tub. It was taken care that acetone residuals from primary
stock solutions were low enough not to influence the ozonation process. A -500
fold dilution of the spiking solution with wastewater effluent resulted in a final
concentration of ~2 pg/L for all antimicrobials expect for A^-acetyl-
sulfamethazine (~0.5 pg/L). Due to the limited commercial availability of
azithromycin at the time, this compound was not spiked. Also A^-acetyl-
sulfamethoxazole, the human metabolite of sulfamethoxazole, was not spiked to
eliminate a possible influence on the sulfamethoxazole concentration. A^-acetyl-
sulfamethazine, the analogous metabolite of sulfamethazine, a sulfonamide
primarily used in veterinary medicine, was spiked instead as a model substance
for A^-acetylsulfamethoxazole. Sulfamethazine itself, however,
Ozonation 141
Figure 6.2 Chemical structures ofsulfonamide antimicrobials and trimethoprim
andproposed main sites ofozone attack
o,
r~
h\_/o
-s-
II0
R2
trimethoprim
(TRI)
sulfadiazine
(SDZ)
/ sulfathiazole
\ (STZ)
sulfapyridine
(SPY)
sulfamethoxazole
(SMX)
A^-acetyl^ sulfamethoxazole *
(N4AcSMX)
sulfamethazine *
(SMZ)
A^-acetylsulfamethazine
(N4AcSMZ)
Rl
H
H
H
H
COCH
H
COCH3
R2
N=\
-O
—i J
N=\
-o
-O
. HX
CH3
CH,
* Not spiked, but present in wastewater samples.
was not added. The actual concentration of all antimicrobials investigated, was
determined in each matrix and for each ozone dose applied, in the inflow
(C (In)) and the outflow (C (Out)) of the ozonation pilot plant.
6.2.4 Sample Collection and Chemical Analysis
Samples were taken at the inflow of column 1, the interface between the two
columns and at the outflow of column 2 (Figure 6.1). Dissolved ozone
concentrations were determined on the respective sampling days in the later two
samples, using the indigo method/181 The detection limit was 0.05 mg/L. For the
determination of antimicrobials, 100 mL of inflow and 250 mL of outflow were
taken, adjusted to pH 4, and enriched unfiltered using solid phase extraction on
142 Chapter 6
Figure 6.3 Chemical structure of macrolide antimicrobials andproposed main
site ofozone attack
\/N
03
,.nl«*
azithromycin
H°V/^OH \>*
erythromycin
(ERY)
clarithromycin
(CLA)
roxithromycin
(ROX)
H3C,
CH3
Ri
H
CH3
H
R2
0
O
/V^N/
aPH3
dehydro-erythromycin
(ERY-H20)
1 y
V\
o'y^Z-^H
Not spiked, but present in wastewater samples.
Oasis HLB (Waters) cartridges on the sampling day. The dried cartridges were
then frozen and transported to the laboratory, where they were eluted within one
week. Measurement was performed using reversed-phase liquid chromatography
coupled to electrospray positive tandem mass spectrometry. Duplicate analysis
was performed in all cases. Details on the analytical method used, including
materials and reagents, are given in Chapter 2.
For method validation the accuracy of the method and the sample-based limit of
quantification (LOQ) was determined in the investigated sample matrices. The
accuracy was determined by recovery studies in each wastewater matrix for the
inflow and the outflow after a reaction with 0.5 and 5 mg/L of ozone,
respectively. To non-spiked wastewater, used for the recovery studies, 100 and
200 ng of analytes, respectively, were added prior to extraction (n = 4). The
calculated amount of antimicrobials minus the amount already present before
Ozonation 143
spiking (n = 2) was then compared to the spiked amount. Additionally recovery
rates were verified in spiked wastewater treated with the selected ozone doses
(0.5 - 5 mg/L), by adding 250 ng of analytes prior to extraction to the respective
outflow samples (n = 2). The sample-based limits of quantification were defined
as those concentrations in a sample matrix resulting in a signal with signal-to-
noise (S/N) ratios of 10. The concentration corresponding to the defined S/N
was determined by scaling down, using the measured concentration in the
samples and the assigned S/N of the peak - assuming a linear correlation
through zero. Table 6.2 summarizes the obtained results for method validation in
each wastewater matrix. A^-acetylsulfamethoxazole was accidentally not
included in the recovery studies. Since an isotope labeled standard, A^-acetyl-sulfamethoxazole-û?5 was used, a relative recovery of 100 ± 10% was assumed
for the quantification of this compound. No results are given for sulfamethazine,
which was not spiked to the wastewater and also not detected in any of the non-
spiked samples. Therefore no method validation was performed for this
compound.
6.2.5 Calculation ofRelative Residual
To compensate differences in the input concentrations, the outflow
concentrations are reported as relative values. Therefore, relative residual
concentrations after the application of different ozone doses were calculated by
comparing the measured concentration in the inflow to the respective
concentration in the outflow of the ozonation pilot plant. The average of
duplicate analysis is used in all cases. The respective uncertainty of the relative
residuals was estimated by combining the uncertainty of the in- and out-flowing
concentration. Therefore the square root of twice the square of the measurement
uncertainty was used as relative uncertainty of the residual percentage. The
standard deviation of the recovery studies was used as an estimation of the
measurement uncertainty of the investigated antimicrobials in the three matrices
(Table 6.2).
Table
6.2Methodvalidationparametersforsu
lfon
amid
es,macrolidesandtrimet
hoprim
inwastewater
effluents
compound
relativerecovery(%)
average±SD(n=18-22)
CAS
CAS+TSS
MBR
sample-basedLOQ
(ng/
L)
average(r
ange
)(n=
22)
CAS
CAS±TSS
MBR
SDZ
109±4
105±3
107±4
68
(8-
252)
50(9
-186)
69
(10
-
287)
STZ
102±2
100±3
102±5
83(11-261)
104(17-431)
135(18-534)
SPY
117±6
107±9
112±
18
114(56-135)
77(12-383)
77
(9-
507)
SMX
106±9
99±6
104±5
34
(6-
169)
42(5-163)
39
(4-
209)
N4AcSMX
a
(100±10)
(100±10)
(100
±10)
38(10-145)
51(27-134)
49(18-169)
N4AcSMZ
b118±
11
122±8
121±
11
16(4-43)
25
(6-
87)
18(4-71)
TRIC
98±15
59±15
81±17
7(2-21)
16(2-91)
17
(2-
126)
AZI
114±13
165±25
103±13
3(0
.2-
7)3
(0.6
-
12)
3(0.2
-
8)
ERY-H20
103±
14
118±18
93±16
9(0.1-34)
11(0.4-41)
11(1.4-36)
CLA
99±13
114±11
91±15
10(0.1-67)
12
(1-
47)
8(0.1-53)
ROX
128±9
143±20
128±17
7(0.3
-
30)
5(0.3
-
26)
6(0.3
-
32)
a
N4AcSMXwas
acci
dent
ally
notincludedintherecovery
studies.Estimatedvaluesaregi
veninbrackets.
bA^-acetylsulfamethoxazole-wasusedassurrogatestandardforN4AcSMZ,
fortheothercompounds
refertoChapter
2.
c
InthecaseofTRI,only
therecoveriesfromno
n-sp
iked
samp
leswereconsidered(n=
12).
Ozonation 145
6.3 Results and Discussion
6.3.1 Spiking of Wastewater Effluents
After spiking, the concentrations in the inflow to the ozonation pilot plant were
determined at the same time as the concentrations after the respective ozone
treatment. By comparing the measured concentration in the inflow to the spiked
amount, the spiking procedure and the chemical analysis can be examined. In
the case of azithromycin and //-acetyl sulfamethoxazole, which were not spiked,
the measured concentrations are normalized to the average concentration
measured to assess the general variability. Within one wastewater matrix the
concentrations measured in the spiked wastewater showed a very consistent
picture, generally with a variation of less than ± 30%. The concentrations
measured, also agreed well (± 30%.) with the spiked amounts, as shown for the
five different inflows of the ozonation pilot plant from the experiments with
secondary effluent of the conventional activated sludge system (Figure 6.4). In
the case of dehydro-erythromycin and roxithromycin the measured
concentrations were generally higher than those theoretically spiked, reaching
values of up to 150%. Great variations, however, were observed for
trimethoprim within and between the experiments. This is caused by the high
concentration spiked, which was outside of the linear range of the calibration.
Therefore only the results obtained from non-spiked samples during recovery
studies are discussed in this study in the case oftrimethoprim.
Generally, the spiking procedure proved to be successful and led to reproducible
concentrations in the inflow. However, the results also illustrate the importance
of also measuring respective inflowing concentrations.
6.3.2 Oxidation Efficiencies
In Figure 6.5 the relative residual concentrations of the investigated
sulfonamides, macrolides and trimethoprim are given as a function of the ozone
dose applied to the spiked secondary effluent of conventional activated sludge
treatment. Additionally the results for A^-acetylsulfamethoxazole, the main
human metabolite of sulfamethoxazole, and TA^-acetylsulfamethazine, the
analogous metabolite of the veterinary sulfonamide sulfamethazine, are given.
In the case of trimethoprim results from the recovery studies in non-spiked
Figure
6.4Comparisonoftheoreticalandmeasuredantimicrobialconcentration
inspiked
secondary
effl
uent
(CAS)
atfi
ve
differenttimepointsa
SDZ
STZ
SPY
SMX
N4AcSMZ
TRI
AZIb
ERY-H20
CLA
ROX
N4AcSMXb
aErrorbarrepresenttherangeofduplicatean
alys
es.
b
Compared
toanaverageenvironmentalco
ncen
trat
ion,
sincenotsp
iked.
Table
6.3Residualozone
concentrationsmeasured
inwastewater
effl
uent
streatedwithselectedozonedoses
afte
rthefirst
(CI)
andthesecondcolumn(C2)
oftheozonationpi
lotplant
appl
ied
CAS
(Mo)
a
ozonedose
Cl
C2
5mg/L
2.5
0.7
3.5mg/L
1.4
0.3
2mg/L
-
lmg/L
-
0.5mg/L
-
residualozoneconcentration(mg/L)
CAS
CAS±TSS
CI
1.8
0.14
<0.05
<0.05
C2
0.6
<0.05
<0.05
<0.05
CI
C2
MBR
CI
C2
1.6
0.2
1.7
0.34
0.6
<0.05
0.7
<0.05
0.08
<0.05
<0.05
<0.05
<0.05
<0.05
<0.05
<0.05
<0.05
<0.05
<0.05
<0.05
Due
tothehigh
erdilutionofthewastewater,
resultsobtainedonMonday
aregi
vense
parately
fromthoseonotherweekdays.
148 Chapter 6
matrix are shown, and therefore results are only available for ozone doses of 0.5
and 5 mg/L.
Efficient oxidation (> 90 %) was observed for all antimicrobials at ozone doses
above 2 mg/L. These findings correlate well with the measured ozone residuals
(Table 6.3). For ozone doses below 2 mg/L no significant ozone residuals (<
0.05 mg/L) were detected at the outlet of the first column, indicating that ozone
concentrations in the bulk liquid are presumably close to zero. The observed
reaction of antimicrobials with ozone must therefore mainly take place in the
liquid film surrounding the gas bubbles. Under these conditions, the
antimicrobials have to compete with reactive wastewater components for ozone.
Consequently, even sulfonamide and macrolide antimicrobials with very high
reaction rate constants for ozone/15J are only partly oxidized. From the results
the initial ozone demand of the investigated wastewater was estimated to be ~2
mg/L, since dissolved ozone could be measured in the effluent of column 1 at
higher ozone doses.
As expected, similar oxidation patterns were observed for all sulfonamide and
macrolide representatives (Figure 6.5). Within these classes, compounds are
structurally very similar and it can be assumed that ozone attack takes place on
the same functional groups. The reactive functional group in macrolides and
sulfonamides are the tertiary amino group and the aniline moiety, respectively
(Figure 6.2 and 6.3). Since the chemical environment of these reactive moieties
is in most cases quite similar within one class, it can also be assumed that the
rate constants for the reaction with ozone must be very similar. Therefore also
the oxidation efficiency for all compounds of a class should be comparable.
Consequently, a very concise picture was obtained for the four macrolides -
greater variations were observed for the four sulfonamides investigated, which
might be caused by one of the following reasons: On one hand, it cannot be
excluded that in the case of sulfathiazole the thiazole moiety is more reactive to
ozone than the aniline moiety. On the other hand, the speciation of the
sulfonamides at the given pH may influence their reactivity towards ozone. The
pKa of the /»-amino group ranges between 5.7 (sulfamethoxazole) and 8.4
(sulfapyridine) for the investigated sulfonamides. Consequently,
sulfamethoxazole is present in its anionic and sulfapyridine in its neutral form,
whereas the remaining sulfonamides are present as a mixture of both species.
Anionic species can be many times more reactive towards ozone than their
neutral equivalents - the observed variation in the reactivity of the sulfonamides
Ozonation 149
is therefore surprisingly low. A possible explanation is that the higher electron
density on the acidic nitrogen reflected by a higher pKa, extents to the adjacent
moieties, making them significantly more reactive towards ozone. The reactivity
of the neutral form of sulfapyridine seems, therefore, to be as high as that of the
anion of sulfamethoxazole. Consequently the reasonable agreement in the
oxidation pattern may be a coincidence in the case of the sulfonamides. In
general, significant differences in the extent of parent compound oxidation have
to be expected when the compared compounds exhibit different speciation under
the investigated conditions. In the case of the acetylated sulfonamide
metabolites, the ozone reactive moiety is protected by an acetyl group.
Therefore, its reactivity to ozone is considerably reduced and the oxidation
efficiencies much lower compared to the respective parent compounds. At an
ozone dose of 2 mg/L, for example, only -45% of the human metabolite
A^-acetylsulfamethoxazole were removed by oxidation with ozone, while a
reduction by ~98%> was observed for sulfamethoxazole itself. However, only
small amounts of /V-acetylsulfamethoxazole can be detected in secondary
effluents due to the effective transformation of this compound in biological
treatment (see Chapter 4 and 5)
6.3.3 Influence ofthe Wastewater Matrix
Suspended solids
The present study was performed with three different wastewater effluents. The
water quality parameters of the investigated wastewater effluents are
summarized in Table 6.1. One main objective thereby was to investigate the
effect of suspended solids on the oxidation efficiencies of sulfonamide and
macrolide antimicrobials. The effluent of the membrane bioreactor (MBR)
represents a wastewater practically free of suspended solids, due to the small
pore size (< 0.4 pm) of the membranes. With the secondary effluent of the
conventional activated sludge system (CAS) an average effluent quality was
investigated. By fortifying this effluent with activated sludge (CAS±TSS) an
activated-sludge process with sub optimal clarification was simulated. In Figure
6.6 the relative residual concentrations in all three wastewater effluents are
given as a function of the applied ozone dosage for three selected compounds.
Results are shown for the most predominant representative of the sulfonamides,
150 Chapter 6
Figure 6.5 Relative residual concentrations ofsulfonamides (A), macrolides (B),
sulfonamide metabolites and trimethoprim (C) in CAS effluent for ozone doses
rangingfrom 0.5 to 5 mg/L
05mg/L H"lmg/L a2mg/L o35mg/L ö5mg/L
-100
o
I 75
o
II
I 50
D
e 25
ro
p
___(A)
pB
rii-L
i LSDZ STZ SPY SMX
AZI ERY-H20 CLA ROX
N4AcSMX N4AcSMZ
3
In the case of TRI the results from non-spiked samples are shown.
Ozonation 151
sulfamethoxazole, and of the macrolide antimicrobials, clarithromycin. Similar
patterns were observed for the other representatives of the two classes
investigated. Additionally, results from the main human metabolite of
sulfamethoxazole, A^-acetylsulfamethoxazole, are given. Overall the differences
in oxidation efficiencies are relatively small between the different effluents and
no significant trend can be observed. The amount of suspended solids in the
treated effluents is therefore of minor importance for the oxidation efficiencies
of sulfonamide and macrolide antimicrobials with ozone. This demonstrates
that, especially for low doses, ozone is consumed by dissolved components of
the wastewater before it reaches the sludge particles.
pH dependence
For the macrolides, e.g. clarithromycin, slightly better oxidation efficiencies
were observed for the effluent of the membrane bioreactor at low ozone doses
compared to the other two effluent matrices investigated. This can be explained
by the pH dependent reactivity of these compounds with ozone.tl5J In the case of
macrolides the neutral form is the most reactive species. With a pKa of -9 for
the tertiary amine group, the most predominant form present in the investigated
wastewater effluents, however, is the protonated form. Therefore the reactivity
of these compounds is higher at the increased pH of-7.5 in the effluent of the
membrane bioreactor compared to -7.0 in the other two effluents investigated.
In the case of the sulfonamides the dissociated form is the most reactive species,
present at a pH above the respective pKa of the /?-amino group. However, no
strong dependence of the reactivity with ozone on the speciation can be
observed as discussed above. Significantly lower oxidation efficiencies were
even observed for sulfonamides in the effluent of the membrane bioreactor at
ozone doses below 2 mg/L compared to the other two effluents investigated.
This effect can be attributed to a slightly faster decay of ozone caused by the
higher pH in the effluent of the membrane bioreactor. As a result no ozone
residuals were detected in this matrix at the outlet of column 1 after an ozone
application of 2 mg/L ozone (Table 6.3). Therefore less ozone was available for
the reaction with antimicrobials in the effluent of the membrane bioreactor
compared to the other two wastewater effluents investigated. As a result, also
significantly lower oxidation efficiencies were measured for macrolides in
effluent of the membrane bioreactor at an ozone dose of 2 mg/L (Figure 6.6).
152 Chapter 6
Acetylated sulfonamides
Ozone doses above 2 mg/L resulted in a complete oxidation of sulfonamide and
macrolide antimicrobials in all three matrices. In the case of sulfonamide
metabolites, e.g. A^-acetylsulfamethoxazole, a reduced reactivity with ozone is
observed, due to the protection of the reactive group by an acetyl group. In this
case, the direct reaction with ozone is less important at low ozone doses and the
oxidation with OH radicals becomes the predominant reaction. Therefore low
oxidation efficiencies are observed for A^-acetylsulfamethoxazole at ozone
doses between 0.5 and 2 mg/L that only slightly increase with increased ozone
dose applied. However, the oxidation efficiencies significantly increase at ozone
doses of 3.5 and 5 mg/L. In these cases, also significant residuals of dissolved
ozone were measured in the outflow of column 1, making the reaction with
ozone itself more relevant.
Residual ozone concentration
Additionally, the influence of the wastewater matrix was investigated on the
amount of residual ozone present. After an ozone application of 3.5 or 5 mg/L,
similar ozone residuals were measured at the outlet of column 1 in all three
wastewater effluents investigated. An exception are those ozone concentrations
measured on Monday in the secondary effluent of the conventional activated
sludge treatment, which were higher than those measured on Tuesday and in the
other wastewater matrices on weekdays. This difference can be explained by the
stronger dilution of the wastewater on the weekend, still present on Monday
morning. Generally it can be stated that the influence of suspended solids on the
dissolved ozone concentration is low after the first column. On the other hand,
ozone residuals at the outlet of the second column seem to be influenced by the
pH and the amount of suspended solids of the wastewater matrix. Therefore,
lower ozone residuals were measured after an application of 5 mg/L ozone in the
effluent of the membrane bioreactor (pH) and the fortified effluent of the
conventional activated sludge treatment (TSS). The fact that ozone residuals
measured in the effluent of the conventional activated sludge treatment are
higher than those in the particle free effluent of the membrane bioreactor
indicates that the pH difference is more important than the amount of suspended
Ozonation 153
Figure 6.6 Relative residual concentrations ofsulfamethoxazole, clarithromycin
and N4-acetylsulfamethoxazole in three wastewater effluents as afunction ofthe
ozone dose applied
I CAS iCAS+TSS qMBR
0.5 mg/L 03 1 mg/L 03 2 mg/L 03 3.5 mg/L 03 5 mg/L 03
0.5 mg/L 03 1 mg/L 03 2 mg/L 03 3.5 mg/L 03 5 mg/L 03
0.5 mg/L 03 1 mg/L 03 2 mg/L 03 3.5 mg/L 03 5 mg/L 03
154 Chapter 6
solids in this case. Similar conclusions can be drawn for the simultaneous
disinfection efficiencies by ozone treatment in wastewater effluents, since
disinfection strongly depends on the exposure to ozone itself.1-191
6.3.4 Comparison ofSpiked and Non-SpikedSamples
To be able to determine up to 95 - 99% removal of sulfonamide and macrolide
antimicrobials during ozonation, the selected compounds were spiked to the
wastewater matrices (0.5-2 pg/L). Thereby native concentrations were increased
by a factor of 2 to 420, depending on the antimicrobial compound. Additionally,
the reactivity of compounds not usually present in the investigated wastewater
samples (e.g. SDZ, STZ, N4AcSMZ) could also be investigated. Additionally,
relative recovery studies were performed in untreated wastewater and after the
application of 0.5 and 5 mg/L ozone, respectively, to verify the accuracy of the
analytical method. For the recovery studies non-spiked wastewater was used
and, consequently, only contained antimicrobials already present in the
wastewater. By comparing the concentrations measured in the untreated
wastewaters to those measured in the ozone treated samples, relative residual
amounts can be calculated for the two ozone doses applied in the recovery
studies. All samples of one matrix were taken on the same day within 4 hours
and measured in duplicate analysis. This provides the opportunity to verify that
experiments conducted with spiked antimicrobials yield similar results as
experiments performed at native concentrations. Additionally, the impact of
variations in the wastewater quality in between different days can be
investigated in the case of the non-spiked compounds. In Figure 6.7, the results
for macrolide and sulfonamide antimicrobials obtained at an ozone dose of 0.5
mg/L from the spiked experiments are plotted versus the results from the
recovery studies (non-spiked). The data points close to the solid line with a slope
of one indicate comparable behavior of the compounds. In both experiments, no
residual amounts of the investigated antimicrobials could be detected after an
application of 5 mg/L ozone. The result obtained in the spiked and non-spiked
experiments correlate very well. Significant differences were, however, detected
for the macrolide antimicrobials in the effluent ofthe membrane bioreactor. This
may be explained by a high turbidity observed in the respective wastewater on
the day ofthe recovery experiments. The cause ofthis turbidity in the membrane
effluent could not be identified, but may result in the observed differences
Ozonation 155
concerning the reaction of macrolides with ozone, probably due to sorption
effects.
Figure 6.7 Comparison of relative residual concentrations obtained in spiked
and non-spiked samples at an ozone dose of0.5 mg/L
0 20 40 60 80 100
relative residual obtained in non-spiked samples (%)
6.3.5 Conclusions
Ozonation of wastewater effluents was shown to be a potential tool for the
removal of sulfonamide and macrolide antimicrobials. An ozone dose of 2 mg/L
leads to an almost complete oxidation (> 90%) of the investigated
antimicrobials. The amount of suspended solids present in the wastewater
effluents showed no significant influence on the observed oxidation efficiencies.
The pH of the wastewater effluent, however, influenced the oxidation
efficiencies, due to the pH dependent stability of ozone. Additionally, pH
differences can affect the reaction rate constants of specific compounds with
ozone.
During the ozonation of wastewater only a partial oxidation of pharmaceuticals
is achieved and therefore could yield biologically still active oxidation products.
156 Chapter 6
Ozonation generally increases the number of polar functional groups and
thereby the overall polarity of the substance. However, in the case of
sulfonamide and macrolide antimicrobials the main sites of ozone attack (Figure
6.2 and 6.3) are also crucial for their effectiveness as antimicrobial agents.
Their ozonation products should therefore be less reactive and not further
promote the spread and maintenance of antibacterial resistance. For other
pharmaceuticals, recent studies on 17a-ethinylestradiol[21] and carbamazepine[have shown that partial oxidation was sufficient to significantly reduce
pharmacological activity and toxicity, respectively.
6.4 References cited
[I] Ternes, T. A. Water Res. 1998, 32, 3245-3260.
[2] Kolpin, D. W.; Furlong, E. T.; Meyer, M. T.; Thurman, E. M.; Zaugg, S.
D.; Buxton, H. T. Environ. Sei. Technol. 2002, 36, 1202-1211.
[3] Heberer, T. Toxicol. Lett. 2002,131, 5-17.
[4] Daughton, C. D.; Ternes, T. A. Environ. Health Perspect. 1999, 107, 907-
938.
[5] Annual Report; Swiss Importers of Antibiotics (TSA): Berne, Switzerland,
1998.
[6] Pharmaceuticals Sold in Switzerland; Swiss Market Statistics, 1999.
[7] Antibiotics used in Veterinary Medicine; Swiss Federal Office for
Agriculture (BLW): Berne, Switzerland, 2001.
[8] Hirsch, R.; Ternes, T. A.; Haberer, K.; Mehlich, A.; Ballwanz, F.; Kratz,
K.-L. J. Chromatogr., A 1998, 815, 213-223.
[9] Hartig, C; Storm, T.; Jekel, M. J. Chromatogr., A 1999, 854, 163-173.
[10] McArdell, C. S.; Molnar, E.; Suter, M. J.-F.; Giger, W. Environ. Sei.
Technol. 2003, 37, 5479-5486.
[II] Giger, W.; Aider, A. C; Golet, E. M.; Kohler, H.-P. E.; McArdell, C. S.;
Molnar, E.; Siegrist, H. R.; Suter, M. J.-F. Chimia 2003, 57, 485-491.
[12] Vanderford, B. J.; Pearson, R. A.; Rexing, D. J.; Snyder, S. Anal. Chem.
2003, 75, 6265-6274.
[13] Golet, E. M.; Aider, A. C; Giger, W. Environ. Sei. Technol. 2002, 36,
3645-3651.
[14] Golet, E. M.; Xifra, L; Siegrist, H. R.; Aider, A. C; Giger, W. Environ. Sei.
Technol. 2003, 37, 3243-3249.
i
Ozonation157
[15] Huber, M. M.; Canonica, S.; Park, G.-Y.; von Gunten, U. Environ. Sei.
Technol. 2003, 37, 1016-1024.
[16] Ternes, T. A.; Meisenheimer, M.; McDowell, D.; Sacher, F.; Brauch, H.-J.;
Haist-Gulde, B.; Preuss, G.; Wilme, U.; Zulei-Seibert, N. Environ. Sei.
Technol. 2002, 36, 3855-3863.
[17] Ternes, T. A.; Stüber, J.; Herrmann, N.; McDowell, D.; Ried, A.;
Kampmann, M.; Teiser, B. Water Res. 2003, 37, 1976-1982.
[18] Bader, H.; Hoigne, J. Water Res. 1981,17, 185-194.
[19] von Gunten, U. Water Res. 2003, 37, 1469-1487.
[20] Walsh, C. Antibiotics: actions, origins, resistance; ASM Press:
Washington, DC, 2003.
[21] Huber, M. M.; Ternes, T. A.; von Gunten, U. Environ. Sei. Technol. ASAP.
[22] McDowell, D.; Huber, M. M.; Wagner, M.; von Gunten, U.; Ternes, T. A.
Environ. Sei. Technol. in preparation.
! Blar
Chapter 7
Conclusions and Outlook
Seite Lee,, ;
Blank lc ' i
Conclusions and Outlook 161
Wastewater treatment is the main exposure route for human used
pharmaceuticals to the environment. The principle aim of this work was to
increase the knowledge on the occurrence and fate of sulfonamides, macrolides
and trimethoprim in wastewater treatment. Different treatment steps in
conventional wastewater treatment and newer techniques, i.e. a fixed bed reactor
and a membrane bioreactor, were investigated. The main achievements and
some general conclusions are summarized in the following paragraphs:
(1) Specific and reliable analytical methods were developed and validated for
the trace determination of sulfonamides, macrolides and trimethoprim in
wastewater and sewage sludge. These methods are suitable to
comprehensively investigate the occurrence, behavior, and fate of the
selected compounds in wastewater treatment from the raw influent to the
final effluent. With a few adaptations these analytical methods can also be
applied to other environmental matrices, e.g. hospital wastewater effluents,
surface and ground waters.
(2) This study provides an example for the environmental exposure assessment
of human used pharmaceuticals. The importance of composite samples was
illustrated by investigating daily profiles of the selected antimicrobials. The
loads entering wastewater treatment were shown to correlate reasonably well
with the loads estimated from available consumption data and to vary with
season. Additionally, it proved to be of crucial importance to include human
metabolites or other compounds that may be transformed to the active
pharmaceutical ingredient, in fate and behavior studies. This was
exemplified in this study by including A^-acetylsulfamethoxazole, the main
human metabolite of sulfamethoxazole.
(3) Mass flow analyses showed that sorption to sewage sludge plays a minor
role (< 10%) in the elimination of sulfonamides, macrolides and
trimethoprim in wastewater treatment. The predicted sorption coefficients to
activated sludge generally ranged below 500 L/kg for all substances
investigated.
162 Chapter 7
(4) Incomplete removal was observed for all investigated sulfonamide and
macrolide antimicrobials as well as for trimethoprim, in wastewater
treatment. The observed elimination strongly differed for each compound
class and the various treatment steps.
(5) For selected macrolides and trimethoprim elimination seems to be correlated
with increasing solid retention time and, hence, with reduced substrate
loading. Both parameters may lead to an increased biodiversity of the active
biomass, resulting in a broader range of degradation pathways available. For
sulfonamides a positive correlation of the observed elimination to the
organic substrate concentration seems to exist. In the case of sand filtration,
used as tertiary treatment, elimination efficiencies were observed for selected
macrolides and trimethoprim and appeared to depend on operational
parameters ofthe sand filter, e.g. oxygen limitations.
(6) Independent on the amount of suspended solids, sulfonamides, macrolides
and trimethoprim are efficiently oxidized (> 90%) in wastewater effluents by
ozone doses above 2 mg/L. Ozonation, thus, proves to be an efficient
technique to eliminate residual antimicrobials from wastewater, e.g. if
advisable for the receiving ambient water or for infiltration.
Overall, none of the technologies investigated for secondary wastewater
treatment led to an efficient removal (> 90%) of all investigated sulfonamides,
macrolides and trimethoprim. For this purpose, other techniques, e.g. post-
ozonation, were necessary. Residual concentrations of these emerging
contaminants therefore continuously reach ambient waters. Based on the
knowledge available today, however, adverse effects in the environment seem
unlikely.
The behavior and fate observed strongly depend on the compound investigated,
with similar results obtained within the group of sulfonamides and macrolides,
respectively. However, no correlations could be made between the elimination
in wastewater treatment and the molecular structure of the investigated
compounds. Consequently, no extrapolations of the results to other
pharmaceuticals are possible.
Conclusions and Outlook 163
Based on the results from this study the following additional aspects would be of
interest:
(1) In this study only A^-acetylsulfamethoxazole, as main human metabolite of
sulfamethoxazole, was included. By considering additional metabolites, e.g.
A^-acetylsulfapyridine, the occurrence and behavior of the selected
antimicrobials could be assessed to a larger extent. Additionally, to fully
investigate their fate in wastewater treatment, including ozonation, the
identification of transformation products would be necessary. These
transformation products should be included in the environmental risk
assessment ofthe respective compounds.
(2) Well designed laboratory studies are needed to elucidate significant fate
processes such as biotransformation and the cleavage of adducts. In
particular, the results obtained in this study strongly indicate the
transformation of A^-acetylsulfonamides to the respective parent compound
in wastewater treatment. However, laboratory scale studies under controlled
conditions would be necessary to verify this hypothesis.
(3) The dependences of the elimination of antimicrobials on specific parameters,
such as substrate concentration, solid retention time or redox conditions (e.g.
denitrifying and anaerob), should be further investigated.
(4) Based on the obtained data, the importance of municipal wastewater
treatment plants as exposure route for sulfonamide and macrolide
antimicrobials to the aquatic environment was shown. Further studies in
hospital effluents would be needed to assess their importance as point
sources.
(5) Profound studies on the ecotoxicological effects of sulfonamide and
macrolide antimicrobials in the environment, especially caused by the
constant exposure to low doses and complex mixtures, are needed to
evaluate the overall risk associated with the discharged residual
concentrations of these substances. Additionally, the impact of the low
concentrations in wastewater treatment on the spread and maintenance of
antimicrobial resistance needs to be further investigated.
oei .A
Vielen Dank...
..an alle, genannt oder ungenannt, die auf verschiedenste Weise zum Entstehen
dieser Arbeit beigetragen haben!
..an Walter Giger für die Leitung der Doktorarbeit, für die vielen Hinweise aus
seinem jahrelangen Erfahrungsschatz und das Vertrauen in mich und meine
Arbeit.
..an Christa McArdell für die ausgezeichnete Betreuung. Vor allem dafür, dass
sie immer Zeit für meine Fragen und das perfekte Maß zwischen Freiraum und
Unterstützung gefunden hat.
..an Hansruedi Siegrist und Thomas Ternes für die Übernahme des Korreferates,
insbesondere für die Diskussion und Ratschläge beim Publizieren der
Ergebnisse.
..an Elvira Keller für das Teilen von Labor und aller anfallenden Freude und
Frust - fachlich sowie privat. Die vielen Diskussionen, sowie ihre fachliche und
moralische Unterstützung waren eine große Hilfe für mich.
..an Angela Thomsen für ihr Interesse am Thema dieser Arbeit und für die
hervorragende Arbeit im Bereich der Schlammanalytik. Sie ist ein wichtiger
Baustein dieser Arbeit.
..an Adriano Joss für den unermüdlichen Enthusiasmus und die gute
Zusammenarbeit in praktischen sowie theoretischen Dingen. Gut, dass interne
Telefongespräche nichts kosten!
.. an Thomas Ternes für die ausgezeichnete Leitung des EU-Projektes
POSEIDON, das mir optimale Bedingungen fur eine spannende Dissertation
geboten hat. Allen Teilnehmern des Projekts, insbesondere Marc Huber, danke
ich für die gute Zusammenarbeit über fachliche, sprachliche und kulturelle
Grenzen hinweg.
..an alle „TSQ User" für die gute Zusammenarbeit beim Probleme lösen,
Termine verteilen und unerklärliche Phänomene verstehen. Insbesondere danke
ich Marc Suter und René Schönenberger für ihre Hilfe.
..an die Mitarbeiter der Kläranlagen Kloten-Opfikon und Altenrhein für die
Unterstützung bei der Probenahme, sowie bei Fragen und Sonderwünschen.
..an Bert Reinold und Bruno Tona (ETH) für die geduldige statistische Beratung
bei einer so kleinen Datenmenge.
..an alle ehemaligen und jetzigen CHPler für die tolle Gruppenatmosphäre und
die intensive Hilfe beim Einleben an der EAWAG. „Apéro" ist ein Wort, dessen
Bedeutung ich durch Euch gelernt habe.
..an die ganze EAWAG für die vielen offenen Türen und die unglaubliche
Bereitschaft mit Rat und Tat unkompliziert zu helfen. Dies hat die Zeit an der
EAWAG unvergesslich gemacht.
..an alle Mitdoktoranden/innen für den guten Austausch und die vielseitige
gemeinsame Freizeitgestaltung.
..an meine Familie für ihre Liebe und unermüdliche Unterstützung auf meinem
bisherigen Lebensweg. Ohne Euch wäre dies nie möglich gewesen.
..an Bert dafür, dass er all die Gedanken an die Arbeit ertragen hat, die mich
nach Hause begleitet haben.
Curriculum Vitae
Anke Göbel
geboren am 14.03.1975 in Köln, Deutschland
1981 - 1985 Städtische Katholische Grundschule, Köln-Porz, Deutschland
1985 - 1994 Stadtgymnasium Köln-Porz, Deutschland
17.06.1994 Abitur
1994 - 1998 Studium der Lebensmittelchemie an der Rheinischen
Friedrich-Wilhelms-Universität Bonn, Deutschland
03.12.1998 Erste staatliche Prüfung für Lebensmittelchemiker
1999 Forschungspraktikum an der
University of California, Davis, USA
1999 - 2000 Praktische Ausbildung am Chemischen Landes- und Staatlichen
Veterinäruntersuchungsamt Münster, Deutschland
15.11.2000 Zweite staatliche Prüfung für Lebensmittelchemiker
2001 - 2004 Dissertation an der Eidgenössischen Anstalt für
Wasserversorgung, Abwasserreinigung und Gewässerschutz
(EAWAG) und der Eidgenössischen Technischen Hochschule
(ETH), Zürich, Schweiz