ARTICLE IN PRESS

WAT E R R E S E A R C H

41 (2007) 4164 4176

Available at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/watres

Removal of antibiotics in conventional and advanced

wastewater treatment: Implications for environmental
discharge and wastewater recycling
A.J. Watkinsona,b,, E.J. Murbyc, S.D. Costanzoa
a
National Research Centre for Environmental Toxicology, University of Queensland, 39 Kessels Road, Coopers Plains, Brisbane,
Qld 4108, Australia
b
Cooperative Research Centre for Water Quality and Treatment, PMB 3, Salisbury, SA 5108, Australia
c
National Measurement Institute, 1 Suakin St, Pymble, Sydney, NSW 2073, Australia

ar t ic l e i n f o

abs tra ct

Article history:

Removal of 28 human and veterinary antibiotics was assessed in a conventional (activated

Received 5 June 2006

sludge) and advanced (microfiltration/reverse osmosis) wastewater treatment plant

Received in revised form

(WWTP) in Brisbane, Australia. The dominant antibiotics detected in wastewater influents

5 April 2007

were cephalexin (med. 4.6 mg L1, freq. 100%), ciprofloxacin (med. 3.8 mg L1, freq. 100%),

Accepted 9 April 2007

cefaclor (med. 0.5 mg L1, freq. 100%), sulphamethoxazole (med. 0.36 mg L1, freq. 100%) and

Available online 23 May 2007

trimethoprim (med. 0.34 mg L1, freq. 100%). Results indicated that both treatment plants

Keywords:

significantly reduced antibiotic concentrations with an average removal rate from the

Activated sludge

liquid phase of 92%. However, antibiotics were still detected in both effluents from the low-

Antibiotics
Environment

to-mid ng L1 range. Antibiotics detected in effluent from the activated sludge WWTP
included ciprofloxacin (med. 0.6 mg L1, freq. 100%), sulphamethoxazole (med. 0.27 mg L1,

Microfiltration

freq. 100%) lincomycin (med. 0.05 mg L1, freq. 100%) and trimethoprim (med. 0.05 mg L1,

Reverse osmosis

freq. 100%). Antibiotics identified in microfiltration/reverse osmosis product water included

Water

naladixic acid (med. 0.045 mg L1, freq. 100%), enrofloxacin (med. 0.01 mg L1, freq. 100%),
roxithromycin (med. 0.01 mg L1, freq. 100%), norfloxacin (med. 0.005 mg L1, freq. 100%),

Reuse
Recycling
WWTP

oleandomycin (med. 0.005 mg L1, freq. 100%), trimethoprim (med. 0.005 mg L1, freq. 100%),
tylosin (med. 0.001 mg L1, freq. 100%), and lincomycin (med. 0.001 mg L1, freq. 66%). Certain
traditional parameters, including nitrate concentration, conductivity and turbidity of the
effluent were assessed as predictors of total antibiotic concentration, however only
conductivity demonstrated any correlation with total antibiotic concentration (p 0.018,
r 0.7). There is currently a lack of information concerning the effects of these chemicals
to critically assess potential risks for environmental discharge and water recycling.
& 2007 Elsevier Ltd. All rights reserved.

1.

Introduction

Water is a precious commodity in Australia and its management is critical for preserving the future of this resource.

Despite being the driest continent on earth, Australia has one

of the highest water consumption rates per capita of any
country worldwide (OECD, 1999). Currently, 97% of urban
runoff and 86% of effluent discharge is not reused and is

Corresponding author. National Research Centre for Environmental Toxicology, University of Queensland, 39 Kessels Road, Coopers
Plains, Brisbane, Qld 4108, Australia. Tel.: +61 7 32749004; fax: +61 7 32749003.
E-mail address: a.watkinson@uq.edu.au (A.J. Watkinson).
0043-1354/$ - see front matter & 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.watres.2007.04.005

ARTICLE IN PRESS
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4 1 (200 7) 416 4 417 6

discharged into our rivers and coastal areas (CSIRO, 2005).

Initiatives such as the National Water Initiative (NWI, est.
2004), National Water Commission (NWC, est. 2004), Water
Smart Australia (est. 2006) and the Australian Water Conservation and Reuse Research Program (AWCRRP, est. 2000)
have been established to improve water management in this
country. Consequently, wastewater re-use is topical in
Australia, as water managers examine the possibilities for
utilising this resource for purposes such as irrigation,
aquaculture, indirect potable reuse (IPR) and even direct
potable reuse (DPR).
Current global water recycling practices directly reflect
surface water resources. For example, Windhoek (Namibia)
have exhausted all available surfaces water supplies and are
completely dependant on a direct potable reuse scheme
(Marsalek et al., 2002). In contrast, Canada having the 3rd
largest renewable water supply globally practices minimal
water recycling (Gleick, 1998). The reuse of wastewater has
twofold benefits through not only the reduction of discharged
effluent to the environment but also a potential reduction of
pressure on existing water resources. Public perception of
wastewater recycling is highly variable, and tends to decline
the closer its use is to the household as the chance of physical
contact increases (Toze, 2006). This is directly related to the
perceived risks associated with these types of waters and
their potential to contain pathogens, trace organics, heavy
metals, nutrients, endocrine-disrupting chemicals (EDCs) and
pharmaceutically active compounds (PhACs) such as antibiotics. The recycling of wastewater must be treated with
caution until we fully understand the potential negative
consequences for the reuse of water containing low concentrations of these emerging contaminants. For example, a
recent study has demonstrated the continued presence of
these compounds in soils irrigated with reclaimed water
(Kinney et al., 2006a, b). Research has demonstrated that even
at low concentrations, these emerging contaminants can
have detrimental effects. Wilson et al. (2003) demonstrated
that three of these chemicals may potentially influence both
the structure and the function of algal communities in stream
ecosystems receiving WWTP effluents. Additionally, Bistodeau et al. (2006) showed that nonylphenolethoxylate/
octylphenolethoxylate compounds, commonly used as surfactants, have an effect on the reproductive competence of
male fathead minnows, with some effects seen below
regulatory guidelines.
Until recently, antibiotics have received comparatively little
attention as pollutants in the aquatic environment which is
surprising considering that unlike many other pollutants,
antibiotics have a direct biological action on microbes. Many
of these antibiotics are not completely metabolised or
eliminated in the body and between 30% and 90% are excreted
unchanged into the waste system (Hirsch et al., 1999). Recent
studies (McArdell et al., 2003; Miao et al., 2004; Clara et al.,
2005; Batt et al., 2006a,b; Brown et al., 2006; Karthikeyan and
Meyer, 2006) have demonstrated that conventional WWTPs
often only partially remove selected drugs (2090%) demonstrating the potential for these compounds to be consistently
present in effluents. Certain metabolites present in WWTP
effluents can also be converted back to their parent form
upon entering surface waters (Ternes, 1998; Heberer, 2002).

4165

Effluents containing antibiotics are of concern as there is

potential to promote or maintain bacterial resistance and
disrupt key cycles/processes critical to aquatic ecology
(nitrification/denitrification) or crop (soil fertility) and animal
(rudimentary processes) production (Kummerer, 2004a, b;
Costanzo et al., 2005; Crane et al., 2006; Kinney et al.,
2006a, b). Biosolid components of WWTPs are an additional
concern through their ability to act as a sink for some PhACs,
including antibiotics (Kinney et al., 2006a, b; Xia et al., 2005).
These biosolids are regularly applied to crops and land further
exacerbating exposure to this group of chemicals. This has
major implications for both effluents discharged to the
aquatic environment and effluents used for wastewater
recycling.
Little is known of the risks associated with effluents containing trace pollutants such as antibiotics, although research in this
area is developing. Oetken and co-workers (2005) demonstrated
that the antiepileptic carbamazepine had a significant and
specific chronic effect against the oligochaete Chiromus at
environmentally relevant concentrations. This is further highlighted by Flaherty and Dodson (2005) who observed chronic
fluoxetine exposure at low concentration significantly increased
Daphnia fecundity. Furthermore, a mixture of fluoxetine and
clofibric acid caused significant mortality and deformities and
mixtures of three to five antibiotics elicited changes in Daphnia
sex ratio (Flaherty and Dodson, 2005). Similar results have been
reported (Brain et al., 2005; Hernando et al., 2006; Pomati et al.,
2006). Degradation productions of these chemicals can also be
considered as contaminants contributing to these complex
mixtures that are present. There is even some evidence that
these degradation products can be as active and/or toxic as their
parent (Halling Sorensen et al., 2002; Sengelov et al., 2003).
Current water quality and reuse guidelines in Australia do
not include antibiotics or PhACs in general (ANZECC,
2000a, b). While the presence of these compounds has been
well documented in the Northern Hemisphere (Heberer et al.,
1998; Ternes, 1998; Alder et al., 2001; Golet et al., 2002; Kolpin
et al., 2002; Giger et al., 2003; Loffler and Ternes, 2003; Miao et
al., 2004; Brown et al., 2006; Karthikeyan and Meyer, 2006),
there is a significant lack of data concerning their presence in
Australian effluents. Costanzo et al. (2005) reported on the
presence of three antibiotics (ciprofloxacin, norfloxacin and
cephalexin) in both sewage effluent and surface waters
downstream from a sewage discharge and Khan and Ongerth
have presented a number of fugacity model calculations of
selected PhACs (Khan and Ongerth, 2002, 2004). The object of
this study was to obtain data on the passage of selected antibiotics through a wastewater treatment plant (WWTP) utilising a conventional treatment process (activated sludge) and
an additional advanced treatment process (microfiltration/
reverse osmosis) and assess their potential for use in water
recycling.

2.

Experimental

2.1.

Sample collection and preparation

Target antibiotics were identified following review of current

Australian import practices (Watkinson and Costanzo, 2005)

ARTICLE IN PRESS
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41 (2007) 4164 4176

and cover the range of most commonly used antibiotic groups

(human and veterinary). As no antibiotics are produced
industrially in Australia, this import data can provide a
representative guide on the usage of antibiotics in this
country (Collignon, 1997). Details of the 27 chosen target
analytes, including average import volumes, are provided in
Table 1. Standards were obtained from Sigma Aldrich at
purities typically greater than 95%. Stock Solutions (200 mg/L)
were prepared in methanol (Mallinckrodt Chemicals ChromARs 99.9%) and stored at 18 1C, with the exception of
sulphasalazine which was made up to 20 mg/L to optimise
dissolution and the quinolones (200 mg/L) which were made
up using 1 mM sodium hydroxide (Sigma Aldrich 99.998%)
in methanol as described in Johnston et al. (2002). A mixed
standard was then prepared in methanol at concentrations
of either 0.25, 2.5 or 5 mg L1 depending on the compound
(see Table 1 for specifics). Calibration standards (at 6 points
in the range 037.5, 0375 or 0750 mg L1) were then prepared
by adding specific quantities of the mixed standard with
30 mL of internal standard and milli-Q water to 1 mL final
volume in glass chromatography vials. All glassware used
in this study was autoclaved and subsequently washed
with Acetone (BDH AnalaRs 99.5%), followed by methanol
(Mallinckrodt Chemicals ChromARs 99.9%) and finally
water purified using a Milli-Q system (Millipore, Bedford,
MA, USA). The internal standard consisted of D3-chloramphenicol (CIL, Andover 4 mg L1), D5-norfloxacin (WITEGA,
Berlin 4 mg L1), D3-nandrolone (NMI, Sydney 2.5 mg L1),
D4-sulphamethoxazole (TRC, Toronto 2.5 mg L1) and
D4-sulphathiazole (TRC, Toronto 2.5 mg L1) and were prepared in methanol.
The two WWTP systems (activated sludge and MF/RO)
investigated are in combination and together form the largest
WWTP in the region servicing approximately 700 000 people
with an average dry weather flow of 140 ML day1. This
treatment facility has been proposed for inclusion in a
regional wastewater reuse scheme and acts as a representative plant for the region. Influent flow characteristics of this
plant are domestically dominated (485%). However, this
proportion decreases substantially (to 57%) when analysing
biological oxygen demand (BOD) of influent, with a variety of
industrial effluents to note including food manufacture/
processing (15%), hospitality (10%), hospitals (3%), and motor
industry (3%) wastes. The MF/RO plant receives approximately 10% of effluent flow from the conventional plant for
supply to a local oil refinery. Fig. 1 illustrates operation of both
plants and highlights sampling locations within the plants.
Sludge retention time (SRT) of the bioreactor and hydraulic
retention times (HRT) are also indicated. Sampling was
conducted separately for each treatment train (conventional
and advanced) in early 2006. For conventional treatment
sampling, samples were collected to reflect HRTs of plant
stages with three time points included for all sampling points.
A further two time points were included for screened influent
and final settling tank (Fig. 1). Triplicate 200 mL grab samples
were collected at each sampling point in amber glass bottles
and stored on ice for transport back to the laboratory. Field
and laboratory blanks were also prepared using Milli-Q water.
In the laboratory, triplicate samples were composited, filtered
through 0.22 mm filters (MF-MilliporeTM) and acidified to pH 3

using H2SO4 (BDH AnalaRs 98%) as previously reported in the

literature (Hirsch et al., 1998; Lindsey et al., 2001; Kolpin et al.,
2002). Aliquots were extracted on Oasiss HLB 60 mg
(Waters, Andover, MA) SPE cartridges at a flow rate of
approximately 23 mL per minute using a vacuum extraction
manifold (VisiprepTM, Supelco, Bellefonte, PA). Following a 5%
methanol wash (3 mL) and 5 min air drying using the vacuum,
cartridges were eluted into test tubes using two separate
volumes of 2 mL methanol. Milli-Q water (500 mL) was
added to all eluants and sample volume was blown
down to 500 mL in a 45 1C water bath with nitrogen using a
TurboVap (Zymark, Hopkinton, MA). Sample eluants were
transferred to glass chromatography vials where 30 mL of
internal standard in methanol was added and made up to a
final volume of 1 mL with Milli-Q water. A Milli-Q blank was
also included to account for potential machine contamination and carry-over.

2.2.

LCMSMS Analysis

LCMSMS was employed for detection and quantification of

analytes using an Alliance 2695 (Waters, Andover, MA) HPLC
coupled to a Micromass Quattro LCZ triple quadrupole mass
spectrometer (Waters, Andover, MA). The mobile phase
contained a constant 0.1% formic acid (aqueous) with a
methanol gradient from 12% to 95% over 7 min. Separation
was achieved using a Synergi Hydro RP 50  2.0 mm, 4 mm
(Phenomenex, Torrance, CA) coupled to a Fusion RP
SecurityGuard 4.0  2 mm (Phenomenex, Torrance, CA). The
LC was coupled to the MS using electrospray in positive
ion mode. Multiple reaction monitoring (MRM) was
employed to identify and quantify the protonated product
ion [M+H]+ and MRM data for specific drugs can be found in
Table 1. For quantification, a first-order calibration curve was
used without weighting and including the origin. The
concentrations of target analytes were calculated using
commercial quantification software (Masslynx V4.0, Waters,
Andover, MA).
Suppression or enhancement of analyte responses by coextracted matrix material (matrix effect) is commonly
encountered in methods based on electrospray mass spectrometry. The results for antibiotics with an isotopically-labeled
internal standard are automatically corrected for the matrix
effect in each sample. As these effects are both sample and
analyte specific and isotopically-labelled analogues are not
available for many compounds, a standard addition into each
sample was employed to ascertain matrix effects on the
remaining drugs. Standard additions were performed by
individually transferring 450 mL from each of the 1 mL final
samples to a fresh vial and adding 50 mL of the mixed
antibiotic stock prepared for analytical standards. This was
equivalent to spiking the final extract at the same concentration as the 100 mL calibration standard. The matrix effect can
be calculated by dividing the analyte concentration found in
the standard addition sample by the concentration calculated
for the aqueous standard spiked at the same level. Where a
sample contained detectable levels of a particular analyte, the
concentration found in the sample (corrected for the slight
dilution in preparing standard additions) was subtracted from
the total concentration observed after standard addition.

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4 1 (200 7) 416 4 417 6

Table 1 Average import volume (t y1), instrument parameters, MRM data, and limits of detection for investigated
antibiotics
Ave. IV

Retention
time
(min)

Collision
energy

Cone
voltage

Parent
ion
(m/z)

Daughter
ion 1
(m/z)

Daughter
ion 2
(m/z)

LOD
(ng mL1)

102
8.7
26.7
12.4
18
4.7

1
1.618
2.384
6.997
7.409
7.943

20
25
10
15
15
25

15
15
21
35
15
40

365.9
367.9
348
334.9
351
435.9

114
106.1
158
217.1
160
178.1

208
174.1
106
91.1

220

14
18
8
8
16
2

Quinolones
Norfloxacin (NOR)
Ciprofloxacin (CIP)
Enrofloxacin (ENR)
Nalidixic acid (NAL)

3.6
5.5
0.7
0.4

4.005
4.161
4.848
7.487

23
16
16
28

28
40
40
28

320.2
332.2
360.2
233.1

233.1
314.2
316.2
187

276.2
288.1
245.1
104

2
2
1
1

Lincosamides
Lincomycin (LIN)
Clindamycin (CLI)

2.3
8.5

1.508
5.805

25
25

30
30

407
425/427a

126.1
126.1

359.2
126.1

1
1

41.9

15.4
3.5
3.9

6.63
6.874
6.743
7.287
7.371

20
20
38
19
25

25
25
45
29
20

734.2
716
916.5
837.3
772.2

576.4
558
772.5
679.4
158.1

158.2
158.2
174.2
158.2
586.3

1
1
1
1
1

56.6
4.3
17.1
7.2

2.9
4.021
5.775
6.085

20
20
20
20

15
20
20
20

460.9
444.9
478.9
445

426.1
410
444
428

201.1
154.1
154.1
154.1

6
5
9
4

29.7
86.2

12.434
12.576

51
51

59
59

773.3
693.3

265.2
461.2

431.2
675.3

1
1

27.1

6.165

25

20

712/475a

669.4

199.2

12

16.7
9.2
0.6

2.51
6.137
7.346

15
18
20

25
22
25

255.9
253.9
398.9

156
156.1
381

108
92.1
119

1
3
5

5.3

2.376

25

33

291.2

230.1

123.1

18

7.409

10

15

348.8

208.1

114.1

16

Antibiotic

ESI
positive
b-lactams
Amoxicillin (AMX)
Cefaclor (CEF)
Cephalexin (CEP)
Penicillin G (PNG)
Penicillin V (PNV)
Cloxacillin (CLO)

Macrolides
Erythromycin (ERY)
Erythromycin-H2O (E-H)
Tylosin (TYL)
Roxithromycin (ROX)
Oleandomycin (OLE)
Tetracyclines
Oxytetracycline (OTC)
Tetracycline (TET)
Chlortetracycline (CTC)
Doxycycline (DTC)
Polyether ionophores
Salinomycin (SAL)
Monensin (MON)
Polypeptides
Bacitracin (BAC)
Sulphonamides
Sulfathiazole (STZ)
Sulfamethoxazole (SMX)
Sulfasalazine (SSZ)
Others
Trimethoprim (TRI)
ESI Negative
b- lactams
Penicillin V (PNV)

Average import volume (t y1) 19922003, TGA (2003).

a
Used in pairing with secondary daughter ion.

This is summarised by the following equation:

Matrix Effect

Observed Concentration  Concentration in sample  0:9

.
Spiked Concentration

Results reported are corrected for the calculated matrix

effect. In cases where the matrix effect caused responses
below 10% of that expected, only a qualitative result is

reported. Only responses detected three times above the

average laboratory, field blank and milli-Q responses were
reported.

2.3.

Additional WWTP parameters

Online monitoring data was provided by WWTP operators for

the study period. Nitrate and ammonia concentrations

ARTICLE IN PRESS
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41 (2007) 4164 4176

Return Activated Sludge (RAS)

Inlet
Screens
ANA
Grit
Removal

Primary Settling
Tank (PST)
(5.5 hrs)

Bioreactor
(11 hrs)
SRT 12.5d

Final Settling
Tank (FST)
)
(9 hrs)

4
10% Flow 90% Flow Surface water
discharge
Cl
NH4

pH
adjustment

Antiscalant
6

Pump
5

Backwash
~30 mins+Cl

MF (2mins)
90% recovery

SMBS

BalanceTank
(15 mins)

RO (0.5 mins)
75% recovery

10

Storage
Ponds

Injection
1 Screened Influent (INF)

MF Feed (MFF)

8 Combined RO Feed (ROF)

2 Primary Settling Tank (PST)

MF Backwash (MFB)

9 Combined RO Concentrate (ROC)

3 Bioreactor (BRT)

Combined MFPermeate (MFP)

10 Combined RO Permeate (ROP)
ANA-Automated Nutrient Analysis

4 Final Settling Tank (FST)

Fig. 1 Process flow diagram for investigated WWTP with associated hydraulic retention times (HRT) of various treatment
components. Sampling points are identified.

measured post bioreactor (Fig. 1) were used to assess

conventional WWTP operational efficiency. For the advanced
plant, pH and feed conductivity was used to assess water
quality entering the plant, and three measurements of
conductivity (RO feed, RO concentrate and RO permeate) to
assess the operational efficiency of the RO membrane
(Fig. 1).

3.

Results and discussion

3.1.

Conventional treatment

Of the 28 investigated antibiotics, penicillin G, oleandomycin

and salinomycin were the only drugs not detected in any
samples within the conventional treatment plant (Table 2).
These drugs are primarily used for animal applications
(therapeutic or growth-promotion) in Australia (JETACAR,
1999), explaining their absence in domestic waste.
Cephalexin was the dominant antibiotic in the influent
(med. 4.6 mg L1, freq. 100%) followed by ciprofloxacin (med.
3.8 mg L1, freq. 100%), both of which in combination accounted for over 80% of the total median screened influent
concentration (Table 2). These drugs were followed by cefaclor
(med. 0.5 mg L1, freq. 100%), sulphamethoxazole (med.
0.36 mg L1, freq. 100%), trimethoprim (med. 0.34 mg L1, freq.
100%) and amoxicillin (med. 0.19 mg L1, freq. 100%). This is
not surprising given that all are in the top 10 most prescribed
antibiotics in Australia, with amoxicillin and cephalexin in

the top three (TGA, 2003). No other reference to the reporting

of cephalexin and cefaclor in WWTPs could be found, so it is
difficult to draw comparisons to other findings, however
sulphamethoxazole has been previously reported in WWTP
influents with average concentrations of 0.243 mg L1 (Miao
et al., 2004), 0.3 mg L1 (Karthikeyan and Meyer, 2006) and
1.09 mg L1 (Yang et al., 2005), indicating a similar range to
that found in this study and trimethoprim has been
reported at concentration ranging from 0.18 to 1 mg L1 in
New Mexico Influents (Brown et al., 2006). Reports of
amoxicillin are scarce, however (Andreozzi et al., 2004)
reported amoxicillin in Italian WWTP effluents at concentrations up to 0.12 mg L1.
Overall, the concentration of the investigated antibiotics
was reduced by an average of 87% in the liquid phase during
conventional treatment (Table 3). The majority of this
removal appeared to occur during biological treatment with
large reduction in the concentrations of both b-lactam and
quinolone drugs, which dominated the overall antibiotic
concentration in previous stages of the plant. b-lactams are
all related structurally though the presence of a b-lactam ring,
which is readily degraded through hydrolytic cleavage and
ultimately mineralised to CO2 and water (Hirsch et al., 1999).
However, low concentrations of a number of b-lactam drugs
were sporadically reported in effluent in this study, and
similarly amoxicillin has also been reported in some Italian
WWTP effluents (Andreozzi et al., 2004). Quinolones have
been reported to undergo photochemical (Boreen et al., 2003)
and thermal (Fasani et al., 1998) degradation and sorb

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4 1 (200 7) 416 4 417 6

Table 2 Median and maximum concentration of investigated antibiotics through conventional treatment
INF (n 5) (ng L1)

Post PST (n 3) (ng L1)

Pst BRT (n 3) (ng L1)

Post FST (n 5) (ng L1)

Med.

Max.

Med.

Max.

Med.

Max.

Med.

Max.

b-lactams
Amoxicillin
Cefaclor
Cephalexin
Penicillin G
Penicillin V
Cloxacillin

190
500
4600
nd
50
nd

280
980
5600
nd
160
320

230
680
3700
nd
5
nd

270
800
3900
nd
10
nd

nd
nd
nd
nd
10
nd

nd
nd
nd
nd
20
nd

nd
nd
nd
nd
30
nd

30
60
nd
nd
80
nd

Quinolones
Norfloxacin
Ciprofloxacin
Enrofloxacin
Nalidixic acid

170
3800
10
nd

210
4600
100
200

10
5000
10
nd

145
6900
20
nd

145
600
5
1

15
742
5
1

25
640
10
55

40
720
10

Lincosamides
Clindamycin
Lincomycin

2
60

5
80

2
55

5
70

5
40

5
50

5
50

5
60

Macrolides
Erythromycin
Erythromycin-H2O
Tylosin
Oleandomycin
Roxithromycin

deta
deta
nd
nd
nd

55
nd
18

deta
deta
nd
nd
nd

nd
nd
9

deta
deta
nd
nd
nd

20
nd
60

deta
deta
nd
nd
nd

65
nd
100

Tetracyclines
Oxytetracycline
Tetracycline
Chlorotetracycline
Doxycycline

nd
nd
nd
nd

nd
35
nd
65

nd
nd
nd
nd

nd
nd
nd
40

nd
nd
nd
nd

nd
20
nd
20

nd
nd
nd
nd

20
30
5
40

Polyether ionophores
Salinomycin
Monensin

nd
10

nd
190

nd
5

nd
10

nd
1

nd
1

nd
25

nd

Polypeptides
Bacitracin

nd

nd

nd

nd

nd

nd

nd

nd

Sulphonamides
Sulphamethoxazole
Sulphathiazole
Sulphasalazine

360
2
nd

500
40
60

480
nd
10

570
nd
15

185
nd
nd

200
nd
nd

270
nd
nd

320
5
10

Other
Trimethoprim

340

930

370

480

30

30

50

70

nd: not detected.

INF Influent, PST Primary Settling Tank, BRT Bioreactor, FST Final Settling Tank.
a
Detected, matrix effects prevented quantification.

significantly to soils and sludge (Golet et al., 2003), indicating

possible fates in this study. Mass balance studies have
demonstrated the main reservoir for fluoroquinolone antibiotics is sewage sludge (Giger et al., 2003). While ciprofloxacin was greatly reduced through treatment, it was still
present in all measured effluent at a median concentration of
0.64 mg L1. This is comparative to other studies where
ciprofloxacin has been reported between 0.13 and 0.42 mg L1
(Alder et al., 2001; Golet et al., 2001; Costanzo et al., 2005).
The presence of tetracycline drugs was comparatively low
compared to other studies (Table 3). Total tetracycline influent

concentrations of 1.9 mg L1 have been reported in the

literature (Yang and Carlson, 2003), at least an order of
magnitude greater than reported here. Tetracyclines have
been shown to form relatively stable complexes with
particulates and metal cations (Alexy et al., 2004), demonstrating a capacity to be more abundant in the sludge
component (Daughton and Ternes, 1999). Depending on the
aqueous pH, presence of cations and light exposure, a
number of tetracycline degradates will also form (Halling
Sorensen et al., 2002), many of which are highly soluble and
have been shown to be the more stable form in receiving

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41 (2007) 4164 4176

Table 3 Total concentration of major antibiotic groups and proportion removed through each process during
conventional treatment
INF

POST PST

POST BRT

POST FST

Overall
%PRa

Sub-total
(ng L1)

Sub-total (ng
L1)

%PRa

Sub-total
(ng L1)

%PRa

Sub-total
(ng L1)

%PRa

b-lactams
Quinolones
Lincosamides
Macrolides
Tetracyclines
Polyether
ionophores
Sulphonamides

5340
3980
62
detb
0
10

4615
5155
57
detb
0
5

14
30
8

50

10
616
45
detb
0
1

100
88
21

88

30
680
55
detb
0
2

200
10
21

234

99
83
11

81

362

490

35

185

62

270

46

25

Other
Overall

340
10093

370
10691

9
6

30
886

92
92

50
1087

67
23

85
89

Total concentrations are represented by the sums of the median values for each antibiotic.
INF Influent, PST Primary Settling Tank, BRT Bioreactor, FST Final Settling Tank.
a
Proportion removed of previous process.
b
Detected, matrix effects prevented quantification.

waters (Halling-Sorensen et al., 2003). The lack of a suitable

chelating agent, such as ethylenediaminetetraacetate (EDTA),
could also account for apparent lack of tetracycline drugs in
this study. Previous studies have shown the presence of such
agents to enhance recovery of these drugs (Heberer et al.,
1997; Lindsey et al., 2001; Miao et al., 2004), and even other
groups such as the quinolones (Miao et al., 2004).
The lincosamide and sulphonamide drugs were the least
affected by conventional treatment with an average removal
from the liquid phase of only 11% and 25%, respectively (Table
3). Little data have been presented on the presence of the
lincosamide antibiotics, however lincomycin has been reported in surface waters ranging from 0.03 to 0.25 mg L1
(Kolpin et al., 2002, 2004; Calamari et al., 2003) with no
source identified. Little is known of its stability and fate
with only Andreozzi et al. (2006) reporting long persistence
(T1/2 17602033 d) under solar radiation. Sulphonamide
drugs are frequently described in effluent (Alder et al., 2001;
Ashton et al., 2004, Glassmeyer and Shoemaker, 2005)
and environmental surface waters (Lindsey et al., 2001; Kolpin
et al., 2002; Yang and Carlson, 2003; Batt et al., 2006a, b; Brown
et al., 2006) indicating a general resilience to conventional
treatment. Carballa et al. (2004) reported degradation of 67%
for sulphamethoxazole and Perez and co-workers (2005) have
reported biodegradation in activated sludge (up to 74%),
however this was not as marked in this study.
Previous studies have reported the strong pH sensitivity of
erythromycin, resulting in the formation of a degraded
erythromycin product (erythromycin-H2O) through the loss
of a water molecule and the inability to detect the parent
erythromycin at pHo7 (Hirsch et al., 1999; Yang and Carlson,
2004). However, both compounds were detected throughout
this study but due to the lack of a suitable standard for
erythromycinH2O and the substantial matrix effects exerted
on both compounds, quantification was not feasible. Given

this particular antibiotic is the 2nd most prescribed antibiotic

in Australia (TGA, 2003), one might expect to find erythromycin at relative high concentrations in the WWTP effluent and
this analytical issue needs to be further addressed to fully
quantify the risk of these wastewaters to the environment
and in recycled water. Erythromycin-H2O has been reported in
WWTP effluent in concentrations ranging from 0.08 to
2.5 mg L1 (Hirsch et al., 1999; Alder et al., 2001; Yang and
Carlson, 2003) indicating the potential for this antibiotic to
pass through conventional treatment. Of the other macrolides investigated, none were detected with any frequency or
above relatively low concentrations.
Nitrate concentrations are used to assess the efficiency of
nutrient removal through bioreactors. Nitrate and ammonia
concentrations over the sampling period and the days
preceding and following this period are shown with respective total antibiotic concentrations in Fig. 2. Ammonia
concentrations did not vary through the study period, but
variations were seen in nitrate levels. Total antibiotic
concentration measured at online monitoring stations did
not correlate (po0.05, Spearmans rank correlation) with
ammonia (p 0.147, r 0.06), however the correlation with
nitrate (p 0.074, r 0.7) was approaching significance,
highlighting the need for further investigation of this
relationship and the potential for nitrate concentration to
predict total antibiotic concentration in treated effluents.
Given the limited availability of analysis for these compounds
and the associated high cost, a predictive tool could prove
very useful for water and environmental managers in
monitoring these chemicals.

3.2.

Advanced treatment

The MF/RO plant receives treated effluent from the conventional plant, where the majority of antibiotics have already

ARTICLE IN PRESS

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Sample Period

7
Nutrient Concentration (mgL-1)

4171

4 1 (200 7) 416 4 417 6

Ammonia
Nitrate
Antibiotics

6
5

1.1

1.0

4
0.9

3
2

0.8

1
0
0:00 12:00
4th February

0:00 12:00 0:00 12:00 0:00

5th February
6th February

12:00 0:00
7th February

Total Antibiotic Concentration (mgL-1)

WAT E R R E S E A R C H

0.7
12:00 0:00
8th February

Fig. 2 Nitrate and ammonia concentrations measured in bioreactor effluent showing associated total antibiotic
concentrations.

been removed from the liquid phase. Therefore, only the

more persistent drugs are likely to be present, creating a
challenge for removal. However, most investigated antibiotics
were identified at some stage in the advanced plant, with only
penicillin G, penicillin V, doxycycline and bacitracin not
detected in any samples (Table 4).
The microfiltration stage of the plant removed approximately 43% of total antibiotics from the liquid phase (Table 5).
This is most likely through the direct trapping of antibiotics,
which are evident in the backwash (MFB), although this is
difficult to discern as the filtrate from the microfiltration
(MFB) is used in the backwash, which also contains antibiotics. It would appear that the majority of this trapping
would be through the binding of the antibiotics to larger
particles (such as proteins or other organics) given pore size is
unlikely to trap these molecules. Additionally, the microfiltration feed (MFF) is chloraminated prior to filtration, which
could affect detection of these compounds in a number of
ways. Chloramine, a combination of chlorine and ammonia,
is a weaker oxidiser than chlorine but more stable in solution
(De Zuane, 1997). Little has been reported on the effects of
chloramination of antibiotics, but chlorine has been shown to
degrade a number of antibiotics in solution (Glassmeyer and
Shoemaker, 2005). In that study amoxycillin, cephalexin and
trimethoprim where converted to a range of different
products (both chlorinated and non-chlorinated) that would
not have been detected in this work because of their altered
mass spectra. The failure to detect the parent compound does
not preclude the presence of degradation products capable of
similar deleterious effects. Sulphamethoxazole, trimethroprim and erythromycin-H2O have all been shown to be
heavily oxidised with chlorination (Adams et al., 2002;
Westerhoff et al., 2005; Qiang et al., 2006), however all three
compounds were detected through the early stages of the
advanced plant, indicating little effect of chloramination in
this study. Chamberlain and Adams (2006) demonstrated that
removal of sulphonamides during chlorination is dependent
on pH, but only at higher pH, which unfortunately was not
determined in this study, while (Gibs et al., 2007; Qiang et al.,

2006) demonstrated complete oxidation of investigated sulphonamides with chlorine. They demonstrated that macrolides are little affected by chlorination and would also appear
to be the case with chloramination, as demonstrated in this
study, however, Gibs et al. (2007) demonstrated complete
oxidation of investigated macrolides with chlorine, highlighting the importance of exposure conditions for oxidation.
More detailed investigation of both pH and the effects of
chloramine within the plant are needed to fully address this
potential impact.
The RO membrane reduced the concentration of antibiotics
present in the RO feed (ROF) by approximately 94% (Table 5).
Only eight antibiotics were present in the RO permeate (ROP)
with naladixic acid the most prominent (med. 0.045 mg L1)
followed by enrofloxacin, roxithromycin, norfloxacin, oleandomycin, trimethoprim, tylosin and lincomycin (Table 4).
Adams et al. (2002) demonstrated that reverse osmosis was
effective in removing selected antibiotics, of which sulphathiazole and trimethoprim were also investigated here.
The sulphonamide drugs were not found above detection
limits after RO, however trimethoprim was still present in RO
permeate (ROP) in this study. Drewes et al. (2003) has
demonstrated total organic carbon (TOC) permeating through
RO membranes have molecular weights less than 500. Five of
the detected antibiotics in this study have relatively low
molecular weights (261443), however, the three macrolide
antibiotics have molecular weights (8141066) above this size
exclusion. There is much debate about the size exclusion
range for reverse osmosis membranes. Studies have shown
that rejection of organic compounds by reverse osmosis is
more dependent on molecular length and width, rather than
molecular weight (Van der Bruggen et al., 1999; Bellona et al.,
2004). Bellona et al. (2004) also identified certain key
membrane properties that affect membrane permeability
including pore size, surface charge, hydrophobicity/hydrophilicity (measured as contact angle), and surface morphology. In addition, feed water composition, such as pH, ionic
strength, temperature, hardness, and the presence of organic
matter, was also identified as having an influence on solute

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41 (2007) 4164 4176

Table 4 Median and maximum concentration of investigated antibiotics through MF/RO treatment
MFF (n 3)
(ng L1)

MFB (n 3)
(ng L1)

MFP (n 3)
(ng L1)

ROF (n 3)
(ng L1)

ROC (n 3)
(ng L1)

ROP (n 3)
(ng L1)

Med.

Max.

Med.

Max.

Med.

Max.

Med.

Max.

Med.

Max.

Med.

Max.

b-lactams
Amoxycillin
Cefaclor
Cephalexin
Penicillin G
Penicillin V
Cloxacillin

nd
70
55
nd
nd
nd

90
70
315
nd
nd
50

nd
nd
50
nd
nd
nd

nd
nd
145
nd
nd
nd

nd
nd
nd
nd
nd
nd

nd
nd
100
nd
nd
nd

nd
nd
nd
nd
nd
nd

190
335
445
nd
nd
nd

nd
nd
nd
nd
nd
nd

nd
nd
nd
nd
nd
nd

nd
nd
nd
nd
nd
nd

nd
nd
40
nd
nd
nd

Quinolones
Norfloxacin
Ciprofloxacin
Enrofloxacin
Nalidixic acid

160
110
40
330

355
155
40
590

70
150
25
115

70
240
40
220

50
120
60
60

190
170
240
260

30
70
40
100

495
105
50
360

120
430
nd
85

175
475
30
170

5
nd
10
45

15
nd
10
75

Lincosamides
Clindamycin
Lincomycin

1
10

10
40

5
5

15
45

nd
5

10
35

1
nd

5
75

nd
nd

1
nd

nd
1

5
1

Macrolides
Erythromycin
Erythromycin-H2O
Tylosin
Oleandomycin
Roxithromycin

deta
deta
20
20
140

deta
deta
40
190
175

deta
deta
10
20
150

deta
deta
10
20
170

deta
deta
1
5
80

deta
deta
10
25
125

deta
deta
1
5
100

deta
deta
70
90
130

deta
deta
5
10
150

deta
deta
10
45
340

deta
deta
1
5
10

deta
deta
5
30
15

Tetracyclines
Oxytetracycline
Tetracycline
Chlorotetracycline
Doxycycline

nd
nd
10
nd

nd
nd
35
nd

nd
nd
nd
nd

nd
nd
nd
nd

nd
nd
nd
nd

nd
nd
30
nd

nd
nd
nd
nd

40
40
50
nd

nd
nd
nd
nd

nd
nd
nd
nd

nd
nd
nd
nd

nd
nd
nd
nd

Polyether ionophores
Salinomycin
Monensin

5
40

130
190

20
55

65
55

nd
10

nd
20

nd
25

nd
30

nd
10

nd
15

nd
nd

25
15

Polypeptides
Bacitracin

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

Sulphonamides
Sulphamethoxazole
Sulphathiazole
Sulphasalazine

255
nd
40

333
nd
48

304
nd
48

372
nd
59

303
nd
45

445
nd
55

280
nd
35

295
35
55

230
nd
45

525
35
70

nd
nd
nd

nd
nd
nd

Other
Trimethoprim

80

145

105

420

45

85

80

280

215

270

10

nd: not detected.

MFF Microfiltration feed, MFB Mircofiltration backwash, MFP Microfiltration permeate, ROF Reserve osmosis feed, ROC Reverse
osmosis concentrate, ROP Reverse osmosis permeate.
a
Detected, matrix effects prevented quantification.

rejection (Bellona et al., 2004). There was very little variation

in ROF conductivity, however a slight increase in ROP
conductivity was seen corresponding with a drop in ROC
conductivity during the study period (Fig. 3). However, a
correlation (po0.05, Spearmans rank correlation) was identified between total antibiotic concentration and conductivity
(p 0.018, r 0.7). No correlation was identified with turbidity (p 0.333, r 0.5) and pH (p 0.313, r 0.5). A more
detailed assessment of these relationships is needed before
serious conclusions can be drawn.

3.3.

Risk for potential wastewater reuse

With increasing concern for water as a diminishing resource

in Australia, optimisation of our water resources is critical
for sustainability. Studies, including this one, investigating
the presence of these drugs in treated wastewater (Giger
et al., 2003; Andreozzi et al., 2004; Miao et al., 2004; Brown
et al., 2006) have identified certain antibiotics, mostly from
the sulphonamide (including trimethoprim), tetracycline,
quinolone and macrolide groups, however the potential

ARTICLE IN PRESS
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4173

4 1 (200 7) 416 4 417 6

Table 5 Total concentration of major antibiotic groups and proportion removed through each process during MF/RO
treatment
MFF

b-lactams
Quinolones
Lincosamides
Macrolides
Tetracyclines
Polyether
ionophores
Sulphonamides
Other
Overall

MFP

ROF

ROP

Overall
%PRa

Sub-total
(ng L1)

Sub-total
(ng L1)

%PRa

Sub-total (ng
L1)

Sub-total
(ng L1)

%PRa

125
640
11
180
10
45

0
290
5
86
0
10

100
55
56
52
100
78

0
240
1
106
0
25

0
60
1
16
0
0

75
0
85

100

100
91
91
91
100
100

295
80
1386

348
45
785

18
44
43

315
80
768

0
5
82

100
94
89

100
94
94

Total concentrations are represented by the sums of the median values for each antibiotic.
MFF Microfiltration feed, MFP Microfiltration permeate, ROF Reserve osmosis feed, ROP Reverse osmosis permeate.
a
Proportion removed of previous process.

100

8000
7000

90
6000
5000

80

4000
70

3000
2000
RO Feed
RO Concentrate
RO Permeate

1000
0
0:00

12:00
21th May

12:00

0:00

22th May

0:00

12:00

Permeate Conductivity (S cm-1)

Feed and Concentrate Conductivity (S cm-1)

Sample Period

60

50
0:00

23th May

Fig. 3 Conductivity measurements of the RO feed, concentrate and permeate.

risk created by their presence is still largely unknown.

The extensive range of active pharmaceutical ingredients
reported in effluents, combined with multiple modes of
action makes ecosystem risk assessment difficult. Generic
toxicity tests applied to all pharmaceuticals do not
provide sufficient information and, if applied, would
likely lead to bias and incorrect assumptions. A review by
Hernando et al. (2006) concluded that pharmaceuticals
considered of high risk in STP effluents were antibiotics
(erythromycin), anti-inflammatories (ibuprofen, naproxen,
diclofenac, ketoprofen), lipid regulating agents (gemfibrozil,
clofibric acid), b-blockers (propanolol, metoprolol) and antiepileptics (carbamazepime). While antibiotics do not appear
to represent a direct toxic risk, either environmentally
or in water reuse schemes, the potential role they may play

in the development or transfer of bacterial resistance is a

major concern.
Concentrations of antibiotics reported are at least three
orders of magnitude below reported inhibitory concentrations
(therapeutic concentration required to inhibit bacteria). A
review by Kummerer (2004a, b) concluded that the impact of
sub-inhibitory concentrations of antibiotics on the frequency
of resistance transfer/development/maintenance is questionable but the input of resistant bacteria into the environment
appears to be an important source of resistance. Knowledge
of sub-inhibitory effects of antibiotics on environmental
bacteria is scarce and contradictory (Kummerer, 2004b),
however there is a huge volume of evidence that antibiotic
resistance is already present in natural environments and
that it is exchanged between bacteria (Davison, 1999).

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41 (2007) 4164 4176

Synergistic or additive effects are another aspect of

environmental exposure assessment that requires better
understanding. In a study investigating the effects of a
complex mixture of therapeutic drugs on human embryonic
cells, an inhibition of cell differentiation was observed that
was not evident during exposure to the individual drugs
(Pomati et al., 2006). Furthermore, research has demonstrated
that mixtures of three to five antibiotics elicited changes in
Daphnia sex ratio (Flaherty and Dodson, 2005). Currently,
there is a critical lack of information to appropriately assess
the threat of antibiotics in wastewater reuse. This does not
only apply to the fate of antibiotics through different
treatment processes (e.g. reverse osmosis, ozonation, chlorination, sand filtration, etc.) but also their fate in potential
applications.

4.

Conclusion

This study has identified that while both conventional and

advanced treatment processes are efficient in removing
antibiotics from the liquid phase, antibiotics were still
detected in final effluents in the low mg L1 concentrations.
Other investigations have demonstrated the accumulation of
these compounds within the biosolids (Kinney et al., 2006a, b;
Xia et al., 2005), creating an alternative pathway for dissemination to the environment. Within the conventional
system, nitrate was identified as a potential indicator to
predict total antibiotic concentration in effluent and conductivity was identified as potential predictors within the
advanced system. However, a more detailed study is required
on these relationships before substantial conclusions can be
made in this area.
There is currently a lack of critical data to critically assess
the potential consequences of the presence of these antibiotics during environmental discharge or wastewater reuse.

Acknowledgements
The authors wish to acknowledge technical assistance
provided by Lesley Johnston, Masooma Trout and the staff
at the National Measurement Institute. This project was
supported through an ARC Linkage Grant (LP0453-708) and in
part by the Wastewater Program of the Cooperative Research
Centre for Water Quality and Treatment (Project number
666003). The use of trade, firm, or brand names in this
paper is for identification purposes only and does not
constitute endorsement by the National Research Centre for
Environmental Toxicology or the CRC for Water Quality and
Treatment.
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