Marine Pollution Bulletin 51 (2005) 218–223

www.elsevier.com/locate/marpolbul

Ecosystem response to antibiotics entering the aquatic environment

a,*
Simon D. Costanzo , John Murby b, John Bates c

a
National Research Centre for Environmental Toxicology, University of Queensland, 39, Kessels Road, Coopers Plains, Brisbane 4108, QLD, Australia
b
Australian Government Analytical Laboratories, 1, Suakin Street, Pymble, Sydney 2073, NSW, Australia
c
Queensland Health Scientific Services, 39, Kessels Road, Coopers Plains, Brisbane 4108, QLD, Australia

Abstract

Awareness of antibiotics in wastewaters and aquatic ecosystems is growing as investigations into alternate pollutants increase and
analytical techniques for detecting these chemicals improve. The presence of three antibiotics (ciprofloxacin, norfloxacin and cepha-
lexin) was evaluated in both sewage effluent and environmental waters downstream from a sewage discharge. Bacteria cultured from
the sewage bioreactor and receiving waters were tested for resistance against six antibiotics (ciprofloxacin, tetracycline, ampicillin,
trimethoprim, erythromycin and trimethoprim/sulphamethoxazole) and effects of short term exposure (24 h) to antibiotics on bac-
terial denitrification rates were examined. Antibiotics were detected entering the sewage treatment plant with varying levels of
removal during the treatment process. Antibiotics were also detected in effluent entering receiving waters and detectable 500 m from
the source. Among the bacteria cultured from the sewage bioreactor, resistance was displayed against all six antibiotics tested and
bacteria cultured from receiving waters were resistant against two of the antibiotics tested. Rates of denitrification were observed to
decrease in response to some antibiotics and not to others, though this was only observed at concentrations exceeding those likely to
be found in the environment. Findings from this preliminary research have indicated that antibiotics are entering our aquatic sys-
tems and pose a potential threat to ecosystem function and potentially human health.
Ó 2004 Elsevier Ltd. All rights reserved.

Keywords: Antibiotics; Bacteria; Denitrification; Resistance; Pollution; Aquatic

1. Introduction that unlike many other pollutants to the aquatic envi-

Substantial attention has been directed to improve
the health of our waterways in recent decades. This
has largely been in response to public concern regarding
visible signs of environmental degradation (e.g., algal
outbreaks associated with nutrient over-enrichment)
(Costanzo et al., 2001; Smith et al., 1999; Carpenter
et al., 1998). Despite the widespread use of antibiotics as
medicines and animal growth promoters (>700,000 kg/yr
in Australia—(JETACAR, 1999)), these chemicals have
received comparatively little attention as pollutants in
the aquatic environment. This is surprising considering
*
Corresponding author. Tel.: +61 7 3274 9016; fax: +61 7 3274
9003.
E-mail address: s.costanzo@uq.edu.au (S.D. Costanzo).
ronment, antibiotics have a direct biological action on
microbes (Waksman, 1947), and in many cases are con-
tinually emitted to the environment thereby acting as
persistent pollutants even if some have high degradation
rates.
Antibiotics released into the aquatic environment are
of concern for the following reasons: (i) contamination
of raw, treated and recycled water used for drinking,
irrigation and recreation; (ii) potential to accelerate wide-
spread bacterial resistance to antibiotics; and (iii) nega-
tive effect on important ecosystem bacteria (through
death or inhibition). Between 30% and 90% of an admin-
istered dose of most antibiotics to humans and animals
are excreted in the urine as the active substance (Rang
et al., 1999). Antibiotics are predominantly water soluble
and enter the aquatic environment through sewage

0025-326X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.marpolbul.2004.10.038
 S.D. Costanzo et al. / Marine Pollution Bulletin 51 (2005) 218–223
systems following consumption and excretion by humans
and via effluent from farms, abattoirs and landfills
(Daughton and Ternes, 1999). The volume of antibiotics
used in livestock is greater than in humans, though they
are used predominantly for different purposes, i.e.,
growth promotion (Collignon, 1999).
Recent findings in the US and Germany have found
in excess of 15 different types of antibiotics in streams
receiving urban and industrial wastewaters and/or run-
off from agricultural practices (Ternes et al., 2002; Kol-
pin et al., 2002). This diversity is likely to be an
underestimate considering many antibiotics were not
investigated and methods for sample preparation and
extraction are still under development.
Widespread use of antibiotics, since their inception as
‘‘Wonder Drugs’’ has been the cause of much debate due
to the deleterious effects of their overuse and misuse
(JETACAR, 1999). Resistance of virulent bacteria in
the environment to widely used medical and veterinary
prescribed antibiotics poses a potential hazard to hu-
mans and livestock exposed to and infected by these
bacteria. Also at risk are aquatic ecosystems, which
are largely controlled by, and dependent upon, micro-
bial organisms for a suite of crucial processes (e.g., deni-
trification), associations (e.g., nitrogen fixation) and
services (e.g., organic breakdown), all of which can
potentially be hindered by antibiotic substances. Similar
effects are possible in sewage treatment facilities that
have adopted bacteria within their structure to perform
wastewater treatment functions.
This paper presents preliminary data on antibiotic
concentrations in local waterways of south east Queens-
land, Australia, and demonstrates potential impacts that
these compounds may have on ecosystem bacteria and
function.
2. Materials and methods
2.1. Field collection and antibiotic analysis
Surface water samples (500 ml) were collected from
within and downstream of a large sewage treatment
plant in Brisbane, Australia. Water was stored in solvent
washed bottles and transported on ice to the laboratory.
Samples were filtered through 0.22 lm sterile filters and
pH adjusted to 3 with H2SO4. Samples were then passed
through solid phase cartridges (OASIS HLB 60 mg
3 cc—conditioned with 5 ml methanol and 5 ml deionised
water) for extraction of the target analytes (Kolpin
et al., 2002; Lindsey et al., 2001). Following extraction,
target analytes (ciprofloxacin, norfloxacin and cepha-
lexin) were eluted with methanol and blown down with
N2 gas to 1 ml aliquots. Samples were analysed by high
performance liquid chromatography (HPLC) with elec-
trospray tandem mass spectrometric detection (Micro-
219
mass Quattro LCZ). Confidence in the identity of
analytes detected was maximised by monitoring two
molecular transitions specific to each target analyte
and assuring their presence in the correct ratio at the
appropriate HPLC retention time.
2.2. Antibiotic resistance testing
The NCCLS Disc Susceptibility Test was employed to
assess bacterial resistance to antibiotics (NCCLS, 2003).
This involved placing a series of antibiotic-impregnated
discs (impregnated with (i) ciprofloxacin (5 lg), (ii) tetra-
cycline (30 lg), (iii) ampicillin (10 lg), (iv) trimethoprim
(5 lg), (v) erythromycin (5 lg) and (vi) trimethoprim/sul-
phamethoxazole (25 lg)) on a plate inoculated with bac-
teria collected from both a sewage treatment plant
bioreactor and a local waterway receiving sewage. Two
methods were used to prepare bacteria for sensitivity test-
ing. Samples collected from the sewage treatment plant
contained enough bacteria to allow direct plating of
the sample without pre-concentrating. Initially, water
samples were spread directly onto Mueller–Hinton Agar
(Oxoid), dried and incubated for 24 h at 36 °C. Differ-
ent individual colonies were then selected and plated
out as monocultures for sensitivity testing. Difficulty
was observed in applying the same technique to water
samples from the creek. Therefore, samples were concen-
trated via filtration of 100 ml samples, 24 h incubation of
the filter paper on horse blood agar and then individual
colony selection and plating out for resistance testing.
Bacteria were identified using the Microbact 24E identifi-
cation test kit. Plates were incubated for 24 h at 36 °C to
allow growth of the bacteria and time for the antibiotics
to diffuse into the agar. The base medium selected for this
test was Mueller–Hinton Agar (Oxoid) because of its
international recognition for antimicrobial susceptibility
testing. Interpretation of results was carried out using the
National Committee for Clinical Laboratory Standards
(NCCLS) guidelines. All strains showing ‘‘resistant’’ or
‘‘intermediate’’ behaviour were classified as ‘‘resistant’’,
otherwise ‘‘sensitive’’.
2.3. Impact on ecologically important bacteria—
denitrifying bacteria
Denitrification by sediment-borne bacteria was meas-
ured using the acetylene blockage technique based on
the interference of acetylene with nitrous oxide (N2O)
metabolism (Sorensen, 1978; Yoshinari et al., 1977).
Sediment samples from the aquatic environment were
collected at low tide using a 60 ml stoppered corer.
The top 2 cm of two collected samples were extruded
directly into a 250 ml rubber sealed Schott bottle (three
replicates) with 40 ml of water collected from the
site. Bottles were then spiked with KNO3 (2 mM) as a
substrate for denitrification and treatments received
220 S.D. Costanzo et al. / Marine Pollution Bulletin 51 (2005) 218–223

respective solutions of antibiotics at varying concentra- Table 2

tions. Cores from each location were incubated anaero- Bacterial resistance to antibiotics in a sewage treatment plant
bically by purging the bottles with nitrogen gas (N2). A Antibiotic E. coli 1 E. coli 2 E. coli 3 Unknown
portion of the headspace (25 ml) was removed via syr- Erythromycin R R R R
inge and replaced with acetylene gas. Ampicillin S R R R
This procedure was performed twice comparing: (i) a Tetracycline S S R R
Trimethroprim R S S S
dose response experiment conducted with ciprofloxacin Ciprofloxacin R S S S
at 0 lg/l, 0.1 lg/l, 10 lg/l and 1000 lg/l and (ii) an exper- Tri/sulphamethoxazole S S S R
iment investigating the effects of different antibiotics R = resistant; S = sensitive.
(amoxicillin/clavulanic acid, erythromycin, clarithro-
mycin, amoxicillin and ciprofloxacin) all at 1000 lg/l to
a control with no added antibiotics. of Escherichia coli and an unidentifiable bacterium. All
bacteria were resistant to at least two of the six antibio-
tics applied, the unknown bacteria resistant to four
3. Results (Table 2). Not one antibiotic caused inhibition of
growth to all bacteria, with resistance and sensitivities
3.1. Antibiotic concentrations varying between bacteria and antibiotics. Predominant
resistance was observed to erythromycin in which all
Antibiotics were detected at every site examined from bacteria displayed resistance, followed by ampicillin
sewage influent to 500 m downstream of the point of (resistance in three bacteria), tetracycline (resistance
treated sewage discharge (Table 1). While ciprofloxacin, in two bacteria) and ciprofloxacin, trimethoprim and
norfloxacin and cephalexin were identified in the sam- trimethoprim/sulphamethoxazole (resistance in one bac-
ples with a very high confidence, the quantities listed terium for each).
in Table 1 are only indicative as factors such as matrix Two types of bacteria were isolated from waters
suppression and recovery losses were not corrected for obtained from the local creek for sensitivity testing com-
during analysis. Concentrations typically decreased at prising a strain of E. coli and Xanthomonas maltophilia.
consecutive sites from the influent to the furthermost Resistance was again observed in responses to erythro-
site away from the discharge site. Analysis of sewage mycin (X. maltophilia) and ampicillin (E. coli) but no
influent showed cephalexin to be in highest concentra- resistance was detected in response to the other four
tions (at least 2000 ng/l), followed by ciprofloxacin. antibiotics tested (Table 3).
The concentration of the latter was little changed at
the next stage of sewage treatment (bioreactor) and a 3.3. Impact on ecologically important bacteria—
similar level of norfloxacin was found, while cephalexin denitrifying bacteria
decreased throughout treatment to be at levels compa-
rable to ciprofloxacin and norfloxacin in the effluent Denitrification rates obtained from the initial dose re-
(Table 1). All antibiotics were detectable at 50 m from sponse experiment conducted with ciprofloxacin found
the site of discharge, whereas cephalexin was the only no significant difference between treatments and controls
antibiotic detectable at 500 m downstream (Table 1). (Fig. 1). Significant depressions in denitrification rates
were observed in certain treatments from the second
3.2. Bacterial sensitivity testing experiment investigating the effects of different antibio-
tics at 1000 lg/l concentrations. Mean denitrification
Four types of bacteria were isolated from the bioreac- rates were significantly lower than the control in treat-
tor of the sewage treatment plant including three strains ments spiked with erythromycin, clarithromycin (p <
0.05) and amoxicillin (p < 0.001) (Fig. 2).
Table 1
Indicative concentration of antibiotics at different stages of sewage
treatment and at locations in receiving waters (mean of two
determinations) Table 3
Bacterial resistance to antibiotics in receiving waters
Site Ciprofloxacin Norfloxacin Cephalexin
(ng/l) (ng/l) (ng/l) Antibiotic E. coli X. maltophilia
Influent 90.0 NT 2000.0 Erythromycin S R
Bioreactor 154.8 192.0 316.4 Ampicillin R S
Effluent 132.2 210.0 78.2 Tetracycline S S
0 m Downstream 41.5 80.0 15.4 Trimethroprim S S
50 m Downstream 23.0 32.0 20.4 Ciprofloxacin S S
500 m Downstream ND ND 26.8 Tri/sulphamethoxazole S S
NT = not tested; ND = not detected. R = resistant; S = sensitive.
 S.D. Costanzo et al. / Marine Pollution Bulletin 51 (2005) 218–223 221

10000 in sewage effluent indicating high removal rates, prima-

Denitrification rate

rily via biodegradation (Khan and Ongerth, 2004). This

(mol N m-2 h-1)

8000
drug did, however, demonstrate persistence in receiving
6000
waters due to its detection at 500 m from the source. Via-
4000 bility of certain antibiotics in different matrices have
2000 been investigated and found to behave very differently
to varied environmental processes, with degradation
0
rates from days to non-degradable (Halling-Sorensen
0 0.1 10 1000
et al., 1998). This type of information is important for
Ciprofloxacin (µg/L) enhanced management of use and disposal of these
Fig. 1. Denitrification rates in response to varied concentrations of the
chemicals in the environment.
antibiotic ciprofloxacin. No significant difference in any treatments in The ever-increasing issue of bacterial resistance was
comparison to control. upheld in this study with isolation of bacteria from sew-
age and receiving waters resistant to antibiotics. Antibi-
10000 otic resistance of bacteria in the natural environment
Denitrification rate

8000 has been reported in the literature, particularly in re-

(mol N m -2 h-1)

6000 gions prone to pollution containing antibiotics (Smith

4000 et al., 1994; Chelossi et al., 2003; Boon, 1992; Fujita
*
2000
* * * et al., 1993; Huys et al., 2000), though resistance has
0 *
been observed in unpolluted sites (Boon, 1992; Boon
l

n
in

n
in
id
tro

in n /

illi

ci

and Cattanach, 1999; Jones et al., 1986; Chelossi et al.,

yc

yc
ac

xa
on

ul illi

yc
om

om
ic
va c

flo
ox
C

la xy

hr

hr

2003). Multi-resistance is another feature commonly

ro
Am
C o

yt

rit

ip
Am

Er

la

C
C

identified in investigations parallel to this study (Che-

Treatment (1000 µg/L)
lossi et al., 2003; Fujita et al., 1993; Tendencia and de
Fig. 2. Mean denitrification rates in response to varied antibiotic types la Pena, 2001; Pillai et al., 1997; Hall, 1997). Three fac-
at 1000 lg/l. * indicates significantly different from the control at tors have been recognised as contributing to the devel-
p < 0.05; *** indicates significantly different from the control at
p < 0.001.
opment and spread of resistance: mutation in common
genes that thereby extend their spectrum of resistance,
transfer of resistant genes among diverse micro-organ-
4. Discussion isms (e.g., plasmids), and increases in selective pressures
that enhance the development of resistant organisms
Antibiotic compounds were found entering local (i.e., antibiotics in pollution entering waterways)
waterways and do appear to be capable of influencing (Halling-Sorensen et al., 1998). Although acquisition
biotic processes such as denitrification and bacterial of antibiotic resistance was not determined in this study,
resistance in these receiving environments. Concentra- research has suggested that plasmid borne resistance
tions of antibiotics identified in this study (low lg/l) genes are the most prolific mechanism of resistance
were comparable to concentrations found elsewhere in transfer between bacteria (Olsen, 1999), more so in
the world (Kolpin et al., 2002; Hirsch et al., 1999). How- eutrophic environments due to elevated carbon concen-
ever, their environmental impacts have largely not been trations and, therefore, increased bacterial activity
reported and are mostly not understood. The presence (Davison, 1999). It is by this mechanism that many fae-
of resistant bacteria and depressed denitrification rates cal bacteria (resistant to antibiotics in the gut of humans
found in this study is highly relevant from environmen- or animals) reaching the aquatic environment can dis-
tal and public health perspectives, particularly as antibi- tribute resistant genes to the native bacterial population.
otics are used and disposed of widely in most countries This has potential to create a widespread reservoir of
around the world. This is of particular concern in devel- genetic material containing resistant traits that may
oped countries that have good access to these drugs and remain within the environment long after the source of
widespread sewage systems that act as conduits for these antibiotic pollution has gone.
drugs to make their way to the aquatic ecosystem. The effects of antibiotics on denitrification do suggest
Concentrations of antibiotics examined in sewage in that antibiotics can have deleterious impacts on denitri-
this study do reflect general antibiotic usage patterns fying bacteria. It is intuitive that they would affect
within Australia (JETACAR, 1999), however, the level Gram-negative denitrifying bacteria (i.e., Nitrosomonas,
of removal via treatment and levels within the environ- etc.) as all the antibiotics used in this study have actions
ment were variable for each. For example, of the three on Gram-negative bacteria. However, not all antibiotics
antibiotics investigated, cephalexin (second most pre- tested in this study depressed denitrification rates signif-
scribed antibiotic in Australia) was found in highest icantly from the control (i.e., amoxicillin/clavulanic acid
concentrations in sewage influent, though was lowest and ciprofloxacin). For ciprofloxacin, this is likely
222 S.D. Costanzo et al. / Marine Pollution Bulletin 51 (2005) 218–223
explained by the antagonistic effect that seawater cations
(namely magnesium and sodium) can have on certain
antibiotics such as ciprofloxacin (a quinolone) resulting
in precipitation of the antibiotic and loss of bioavailabil-
ity (Smith, 1989, 1990; Barnes et al., 1995), however, this
is not found to occur with amoxicillin (a penicillin) (Bar-
nes et al., 1995) and our findings are as yet unexplained.
Other evidence does exist regarding the impact of antibio-
tics on nitrogen cycling bacteria including widespread
anecdotal evidence from the aquarium industry. This
industry relies heavily on nitrogen processing bacteria
in biological filters to maintain good water quality
which have been found to be inhibited following antibio-
tic additions aimed at curing fish infections. Research
has also shown negative effects of antibiotics on nitro-
gen cycling processes within sewage treatment facilities
(Gomez et al., 1996; Campos et al., 2001), yet there is
an obvious lack of research on bacteria involved in envi-
ronmental nitrogen cycling. More importantly, it is the
effects of long term and/or synergistic exposure of deni-
trifying bacteria to the low concentrations of antibiotics
that are being observed in the environment that requires
further assessment.
In summary, it is evident from this and other research
that antibiotics and resistant bacteria are entering our
local waterways and have potential to influence biotic
processes. The key concern with these findings is the risk
associated with the transfer of resistant genes from
harmless bacteria to pathogenic bacteria and on to hu-
mans interacting with the aquatic environment. It is
impossible and impractical to halt usage of antimicro-
bial agents within our environments, though manage-
ment of their overuse and disposal within catchments
is likely to be on environmental management agendas
in the near future.
Acknowledgments
We thank Gino Micalizzi at Queensland Health Sci-
entific Services for his fine efforts in conducting and
teaching bacterial sensitivity tests; Tamara Ivastinovic
for sample preparation and Keith Barr from Brisbane
Water for assistance in sample collection. Thanks also
to Rene Diocares for assistance with gas chromatogra-
phy and Andrew Watkinson for reviewing and com-
menting on this manuscript.
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