FACTORS AFFECTING THE FATE OF CIPROFLOXACIN

IN AQUATIC FIELD SYSTEMS

L. A. CARDOZA1 , C. W. KNAPP2 , C. K. LARIVE1 , J. B. BELDEN3 , M. LYDY3

and D. W. GRAHAM2,∗
1
Department of Chemistry, University of Kansas, Lawrence, KS 66045; 2Department of Civil,
Environmental and Architectural Engineering, University of Kansas, Lawrence, KS 66045;
3
Fisheries and Illinois Aquaculture Center and Department of Zoology; Southern Illinois University,
Carbondale, IL 62901
(∗author for correspondence, e-mail: dwgraham@ku.edu, Fax: 785-864-5379, Tel: 785-864-2945)

(Received 12 October 2003; accepted 26 June 2004)

Abstract. Ciprofloxacin (cipro) is a broad-spectrum antibiotic used in human and veterinary medicine
that is readily transported into the environment via domestic wastewaters and through direct runoff.
Although factors governing cipro fate are becoming understood, an integrated evaluation of disappear-
ance mechanisms in aquatic systems has not been performed. Here we examined cipro disappearance
rate in surface waters using both laboratory and field systems under different light, and dissolved
(DOC) and particulate organic carbon (POC) conditions to determine when photodegradation versus
adsorption dominates cipro fate. Initial laboratory experiments showed that cipro rapidly photode-
graded (t1/2 ∼ 1.5 h) with numerous photodegradation products being noted when POC levels were low.
However, even moderate water column POC levels resulted in reduced photodegradation (no break-
down products detected) and soluble cipro disappearance rates were accelerated. 14 C-ciprofloxacin
studies confirmed significant adsorption onto aquatic POC (K OC values of 13,900 to 20,500 L/kg at
neutral pH). In contrast, a follow-up mesocosm-scale field study using low POC water showed that
photodegradation could also dominate cipro fate. In conclusion, both adsorption and photodegrada-
tion strongly influence cipro fate in aquatic systems, although the dominant mechanism appears to
depend upon the ambient POC level.

Keywords: aquatic systems, ciprofloxacin, field mesocosm, fluoroquinolone antibiotics, particulate

organic carbon, photodegradation

1. Introduction

Fluoroquinolones (FQs) are a class of synthetically produced antibiotics that pos-

sess broad-spectrum antibacterial properties. The environmental fate of such an-
tibacterial agents is important because of their continued use in both human and
veterinary medicine, and their potential for migration into the environment and
the possible development of resistance in environmental pathogens (Kidwai et al.,
1998; Yoshida et al., 1993; Halling-Sørensen et al., 2000; Alonso et al., 2001). The
fate of FQ compounds is of particular significance because FQs are often “drugs
of last resort” in medicine and they have been shown to potentially be genotoxic in
hospital wastewaters (Hartmann et al., 1998). Ciprofloxacin (Figure 1), a common
Water, Air, and Soil Pollution 161: 383–398, 2005.

C 2005 Springer. Printed in the Netherlands.
384 L. A. CARDOZA ET AL.

Figure 1. Structure of ciprofloxacin.

FQ, is especially interesting because it is used therapeutically in many critical appli-

cations such as the treatment of Anthrax infections, and it is a primary degradation
product of enrofloxacin, which is used worldwide in aquaculture and agricultural
applications.
Although our understanding of factors that govern ciprofloxacin (cipro) fate in
the environment is improving, an integrated evaluation of fate-driving mechanisms
that incorporates both laboratory and field-testing has not been performed. Pre-
vious work has indicated that various mechanisms conditionally influence cipro
fate in the environment, including photodegradation (Hidalgo et al., 1993; Yoshida
et al., 1993; Tornianen et al., 1996; Burhenne et al., 1997a,b; Torniainen et al.,
1997a,b; Araki and Kitaoka, 1998; Hartmann et al., 1998; Schmitt-Kopplin et al.,
1999; Mella et al., 2001; Lam et al., 2003), adsorption onto particles (Nowara
et al., 1997; Golet et al., 2003), and biotransformation (Gau et al., 1986; Wetzstein
et al., 1999; Parshikov et al., 2001). Particularly extensive information exists on
the photosensitivity of cipro largely based on observed effects during therapeutic
use; however, most results are from laboratory experiments that do not necessarily
reflect environmental exposures. Regardless, cipro is highly photodegradable with
half-lives ranging from as low as 5 min to about 1.5 h in different assessments
(Burhenne et al., 1997a; Lam et al., 2003). Further, many factors have been found
to affect photodegradation, including light intensity, pH, cipro and phosphate (TP)
levels, and the presence of organic compounds (Torniainen et al., 1997b; Schmitt-
Kopplin et al., 1999; Lam et al., 2003), although unified conclusions on fate have
not been made.
Ciprofloxacin readily adsorbs onto particles in solution (Nowara et al., 1997;
Tolls et al., 2001), although direct adsorption studies on particulate organic carbon
(POC) have been limited. In fact, Golet et al. (2003), the only previous field-scale
report on cipro fate in environmental samples, attributed absorption and settling
as the primary fate-driving mechanism for cipro in Swiss river waters. They con-
cluded that while photodegradation probably played a role in cipro fate, it was
likely small compared with adsorption. Biodegradation of cipro also has been
noted in pure fungal cultures (Wetzstein et al., 1999; Parshikov et al., 2001),
although reactions appear to be slow and complete mineralization has not been
noted.
 CIPROFLOXACIN FATE IN AQUATIC SYSTEMS 385

In summary, results suggest that cipro can be photodegraded, conditionally

biodegraded, and can adsorb onto organic solids, but dominant mechanisms in any
system appear to be environment sensitive and questions still remain about which
mechanism(s) is most important in different natural systems. In particular, the possi-
bility of photodegradation versus adsorption is a key question because photodegra-
dation appears to eliminate cipro, whereas adsorption only appears to partition cipro
onto particulate matter and little is known about cipro fate after adsorption.
In this work, we assessed the fate of cipro under varying light exposures, organic
particle densities, and water chemistries using both laboratory and field systems
to assess under what conditions each mechanism will likely be more important.
Laboratory experiments compared adsorption and photodegradation of cipro under
different light sources, and in the presence of different types and levels of dissolved
organic carbon (DOC) and POC. A field mesocosm experiment was subsequently
performed to assess the validity of laboratory results under quasi-natural field con-
ditions (Graham et al., 1999; Ensz et al., 2003).

2. Materials and Methods

2.1. MATERIALS

Ciprofloxacin was purchased from Serological Proteins Inc. (Kankakee, IL). HPLC-
grade water with resistance greater than 18 M
was obtained using a Labconco
Water Pro PS purification system (Kansas City, MO). Ammonium hydroxide,
formic acid (90%) and HPLC-grade methanol were purchased from Fisher Sci-
entific (Springfield, NJ). Trifluoroacetic acid (TFA), nalidixic acid, and flumequine
were purchased from Sigma (St. Louis, MO). The OmnisolvTM acetonitrile (MeCN)
used in the LC/MS analysis was purchased from EM Science (Gibbstown, NJ). The
14
C-Cipro was obtained from Bayer AG (Wuppertal, Germany) and had a specific
activity of 6.96 MBq/mg. After purification (via thin layer chromatography), the
working purity of the 14 C-Cipro was >98%.

2.2. LABORATORY EXPERIMENTAL CONDITIONS

Three bench-scale experiments were performed to assess the affect of light source,
DOC level, and POC level on the fate of cipro in aquatic systems. These experiments
were performed in 3-L glass beakers maintained in a temperature-controlled room
(20 ◦ C) under different light sources, depending upon the experiment (100% Philips
Econo-watt 34-W fluorescent shop lights; 33% Vita-light UVA-UVB bulbs +66%
Philips fluorescent bulbs or “reptile” lamps). Light intensity levels were identical
among all experiments, with intensities employed being typical of ∼2–3 meters
from the surface of a mesotrophic lake. All beakers were provided SaranTM wrap
lids to minimize evaporative losses and the beaker solutions were completely mixed
at ∼200 RPM using magnetic stirrers with insulated bases.
386 L. A. CARDOZA ET AL.

The water used in the laboratory experiments was obtained from the tank meso-
cosms that were used for subsequent field experiments. These waters originated
from a protected storage reservoir located at the Nelson Environmental Study Area
(NESA) north of Lawrence, Kansas, and had no previous contact with surface
contamination. Although source-water conditions varied slightly among experi-
ments, conditions were routinely monitored to compare conditions among exper-
iments. All laboratory experiments were initiated by the addition of a pre-mixed
aqueous cipro stock solution that was added to reach a target concentration of
250 µg L−1 .
The first experiment assessed the effects of various light sources on cipro dis-
appearance rate. All treatments were performed in duplicate and comprised of
non-sterile light-exposed beakers (with both light sources), pre-sterilized (via auto-
claving) and non-sterile “dark” controls, and pre-sterilized light-exposed beakers.
Early results indicated that “reptile” lamps most closely resembled natural light;
therefore follow-up experiments that assessed the effect of DOC (from ∼5.0
to 38 mg C/L) and POC (from ∼1.0 to 18 mg C/L) on aqueous phase cipro
fate used the “reptile” lamp system. The design of these latter experiments in-
cluded dark, light-exposed, and pre-sterilized control beakers, as required by each
experiment.
For the experiments on POC effects, special water preparation was required.
Approximately 15 L of field mesocosm water was collected and pooled from the
tank mesocosms. It was subsequently centrifuged (Sorvall RC-5B), pelleted, and
re-suspended as a concentrate in 50 mL of deionized water. The POC level in
the concentrate was measured using a Dohrmann [DC-80] carbon analyzer and
appropriate volumes were distributed to each test beaker to achieve targeted POC
levels for each treatment (in duplicate). Final POC levels in each beaker were then
re-analyzed to verify achieved POC levels.
The following analytical procedures were used in all experiments. Prior to
cipro addition, pH and dissolved oxygen (DO) levels were determined using
Accumet and YSI probes, respectively. Duplicate 100-mL water samples were
also collected for analyses of total nitrogen (TN), TP, DOC, and total organic
carbon (TOC) levels. POC was calculated as the difference between measured
TOC and DOC values. After cipro addition, duplicate 50-mL samples were im-
mediately collected to determine initial cipro levels in each beaker. Between
6 and 16 samples were then collected over time for quantifying cipro levels
(the sample number differed among experiments). At least three parallel de-
terminations of pH, DO, TN, TP, DOC, and POC were performed over each
experiment. All sampling employed 50-mL wide-tipped glass pipettes. Sam-
ples for routine chemistry were stored at 4 ◦ C prior to analysis, whereas sam-
ples for cipro were filtered immediately using 0.7-µm GF/F filters (Whatman)
and temporarily retained in amber glass bottles before extraction within 1 h of
sampling.
 CIPROFLOXACIN FATE IN AQUATIC SYSTEMS 387

2.3. FIELD EXPERIMENT – MESOCOSMS AND EXPERIMENT PREPARATION

Six mesocosms were used for the field experiment in this study and were identical
to those described in Ensz et al. (2003). The basic units were cylindrical, flat-
bottomed fiberglass tanks, 3.2 m in diameter and 1.4 m deep, that were filled with
∼11 m3 of uncontaminated water from an adjacent protected reservoir at NESA.
The tanks were arranged in two rows of three in a single pond that was filled to
∼0.25 m of the tank brim to insulate tank waters from sudden variations in air
temperature. The only manipulation to the tanks, other than the inclusion of lids
(see below), was the placement of three 39 × 53 cm plastic sediment-containing
trays at the bottom of each tank to provide a microbial seed for tank waters. Meso-
cosm water conditions were monitored three times over the 2 weeks prior to cipro
addition to determine ambient water chemistry conditions prior to starting the
experiment.
On October 6, 2002, cipro was added to all six tanks as a pre-mixed 1.0 L
aqueous emulsion in pond water (in the dark) to target concentration of 25 µg L−1 .
Simultaneous to rapid dispersal with paddles, black vinyl plastic lids with PVC
hoop-frames were rapidly placed over 3 of the 6 tanks. The black lids reduced light
intensities by >99% compared with the open tanks as described previously (Ensz
et al., 2003). After placement of the lids and the addition of cipro, 12 and 8 samples
were collected for cipro and water chemistry analyses, respectively, over the 12-day
monitoring period. After 12 days, the lids were removed from the lidded tanks to
assess the rate of cipro disappearance in the previously covered units.
Sample collection was performed using dedicated 1.5 m length, 25 mm diameter
PVC samplers, equipped with a check valve at the bottom (Graham et al., 1999).
Similar to the laboratory studies, pH, DO, TN, TP, DOC, and POC were monitored in
the mesocosms. In field sampling, however, DO, pH, and temperature measurements
were made and averaged from three water depths (0.3 m, 0.7 m and 1.2 m) using
a Water Checker Field Monitor (Horiba Instruments). Light intensities also were
routinely measured (LiCor probe #LI185A) at each mid-day just below the water
surface and ∼0.25 m from the tank bottoms. Water samples for cipro and other
analysis were collected and stored on ice in the dark using acid-washed amber
glass bottles equipped with Teflon-lined caps prior to returning to the laboratory
for analysis.
Samples for cipro analysis were extracted immediately upon return to the labora-
tory, whereas water quality samples (typically 1 L) were divided into two fractions.
One hundred mL of unfiltered water was retained for TN, TP, and TOC analy-
sis, whereas the remaining 900 mL was separated and filtered through pre-rinsed
Whatman GF/F glass-fiber filters (particle retention of >0.7 µm) for DOC and
other testing. TN and TP were always analyzed within 5 h of sampling, whereas
TOC and DOC samples were acidified to pH 2 with 85% phosphoric acid, stored
at 4 ◦ C, and processed within 72 h.
388 L. A. CARDOZA ET AL.

14
2.4. C-CIPROFLOXACIN ADSORPTION ASSESSMENT

14
C-Ciprofloxacin was used to directly assess cipro adsorption rates onto different
particulates present in the laboratory and field experiments. Adsorption tests were
performed on two POC sources; “Particulate 1” from the first beaker study and “Par-
ticulate 2” from the field mesocosm experiment. Adsorption rates for Particulate 2
were assessed at pH 3.0 and 7.3.
For each experiment, POC levels were initially measured and 10-mL suspen-
sions at differing concentrations were transferred to a series of 20 mL amber vials
in triplicate. 14 C-Cipro and analytical grade cipro were then added to each vial with
a 10-µL volume of methanol carrier, resulting in cipro levels of 12,800 dpm/ml and
25 µg/L, respectively. All experiments were performed at 22 ◦ C under cool white
fluorescent lighting filtered to remove UV wavelengths. After cipro addition, the
samples were agitated at 125 RPM for 24 h on an orbital shaker table. After 24 h,
vial contents were filtered through a 25 mm Versapor
R
filter (0.45 µm, supported
acrylic copolymer, Pall Gilman, New York, NY, U.S.A.) to separate POC from
the water phase and the amount of radioactivity in the residual water was deter-
mined by placing a 2 mL fraction into a scintillation cocktail (Scintisafe Plus 50%,
Fisher Scientific). Radioactivity in the water was quantified by liquid scintillation
(LSC; Packard 1900TR Liquid Scintillation Analyzer, Packard Instrument Com-
pany, Meriden, CT U.S.A.), whereas radioactivity in the POC was determined by
measuring the filters. The filters were prepared for analysis by backflushing with
2 mL of 1:1 methanol:phosphate buffer (0.25 mM, pH 3) to collect loose particles,
which were then combined with the filter and counted using LSC.
For verification, water samples were also analyzed for cipro throughout each
experiment using HPLC to determine whether radioactivity in the water was likely
attributable to cipro. Separations used an Agilent 1100 series HPLC equipped with
a binary pump, a Zorbax
R
C18 column (4.6 × 150 mm, 5 µm), and a fluorescence
detector (278 nm emission and 445 nm excitation). The initial mobile phase con-
sisted of 93% phosphate buffer (0.25 mM, pH 3), and 7% acetonitrile that was held
for 2 min and then ramped to 75% acetonitrile over 8 min.

2.5. SAMPLE ANALYSIS

2.5.1. Ciprofloxacin Sample Preparation and Extraction

All water samples for cipro analysis were placed in 125-mL amber bottles after
filtration. For laboratory samples, the 50-mL aliquots were spiked with 97.4 µL of
formic acid and 503.2 µL of the surrogate standard flumequine (9,920 µg/L) prior
to solid-phase extraction (SPE). For field samples, 498.1 mL was transferred to
1000-mL amber bottles, which was then supplemented with 1006.4 µL of formic
acid and 503.2 µL of the surrogate standard flumequine (9,920 µg/L) prior to
SPE. Due to expected interference from aquatic natural organic matter, a two-step
 CIPROFLOXACIN FATE IN AQUATIC SYSTEMS 389

SPE method was employed. Cipro and the surrogate standard flumequine were
concentrated using 3 mL Sep-pak C-18 phase SPE cartridges (Waters, Milford,
MA) and the matrix from the natural water was reduced using 100 mg (1-mL)
Fisherbrand strong anion exchange (SAX) cartridges (Fisher Scientific, Springfield,
NJ). The C-18 and SAX cartridges were both conditioned prior to use by washing
with methanol followed by 0.2% formic acid.
Typically, one C-18 SPE cartridge was used per beaker sample in the laboratory
experiments, whereas two C-18 cartridges were used for field samples because of
larger sample volumes. In both cases, the C-18 cartridge(s) were stacked on top of
the SAX cartridge and sample was applied to cartridges in series at a flow rate of ∼1
to 1.5 mL/min. After loading, the cartridges were washed with 3 mL of 0.1% formic
acid and allowed to dry for 15–20 min under vacuum. Cartridges were then eluted
into 5 mL centrifuge tubes with 3 mL of a solution containing a mixture of 0.1%
formic acid (10%) and methanol (90%). Samples were spiked with 198.1 µL of the
internal standard nalidixic acid (49,608 µg/L), vortexed to obtain a homogeneous
mixture, and evaporated to approximately 300 µL in a 40 ◦ C water bath under a
stream of nitrogen.
Two separate recovery trials with eight replicate samples were performed to
assess whether sample volumes used in the beaker and field experiments affected the
separation efficiency. SPE recovery was 94 ± 3% for 50 µg/L cipro in 100 mL lab
water, whereas cipro recovery of 20 µg/L in 500 mL natural water was 104 ± 11%
as compared the internal standard, naldixic acid.

2.5.2. Liquid Chromatography – Diode Array Detection/Mass

Spectrometric Analysis
Cipro was analyzed and quantified using an HP1100 Series HPLC (Agilent, Palo
Alto, CA) equipped with diode array and mass spectrometric detectors. The sepa-
rations were performed on a Supelco Discovery HS C-18 column (Bellefonte, PA)
with dimensions of 4.6 mm ID × 15 cm with 3 µm particles at a flow rate of 0.5
mL/min. The HPLC solvents were (A) 0.1% TFA(aq) adjusted to pH 3 with NH4 OH
and (B) MeCN. The binary gradient used was as follows: initial composition B of
15% increased to 35% over 10 min. and then to 65% over 24 min where the com-
position remained isocratic for an additional 4 min. The column was allowed to
equilibrate for 15 min. between samples at the initial gradient conditions. Cipro,
nalidixic acid, and flumequine eluted with following retention times 11.2, 21.6 and
22.3 min, respectively.
The ESI-MSD spectra were acquired in positive ion mode using an Agilent
1946B MSD (Palo Alto, CA). The fragmentor was set at 70 V with an applied
capillary voltage of 3000 V and a nebulizer pressure of 50 psig. The instrument
was operated in scan mode from 100 to 450 m/z at rate of 0.1 s/scan. The nitrogen
drying gas was operated at 350 ◦ C and a flow rate of 10.0 L/min. Quantitation
was achieved using the diode array detector with the chromatographic peak area
for ciprofloxacin measured at 270 nm and the peak areas for the flumequine and
390 L. A. CARDOZA ET AL.

naldixic acid standards measured at 285 nm. Qualitative identification of several

cipro breakdown products was carried out during the LC/UV-MS analysis. Standard
solutions were prepared in water and stored at 4 ◦ C in amber bottles, containing
4,890–98,697 µg/L cipro, 50,068 µg/L flumequine, and 50,471 µg/L nalidixic acid.

2.5.3. Other Chemical Analysis

DOC and TOC were determined using a Dohrmann Organic Carbon Analyzer. TP
and TN, respectively, were analyzed spectrophotometrically (Shimadzu UV-160)
following wet digestion with potassium persulfate (Prepas and Rigler, 1982) and
alkaline persulfate digestion (APHA et al., 1999; Solorzano and Sharp, 1980).

2.6. DATA ANALYSIS

2.6.1. Estimation of Mean Water Conditions in the Different Flasks or Mesocosms

The estimated mean water conditions in each flask or tank were based on all samples
collected over the course of the experiment. Data from the replicate mesocosms
(per treatment) or flasks were combined to produce overall descriptive means for
each condition. The 95% confidence intervals (C.I.) were calculated from standard
errors associated with each mean. All statistical analyses used SPSSTM Version 8.0
(SPSS-Inc., 1998).

2.6.2. Estimation of Ciprofloxacin Decay Rates and Half-lives

Curve-fitting techniques were used to estimate ciprofloxacin decay rates in each
treatment. Cipro decay rate coefficients were estimated for each test unit using a
first-order exponential decay model and SPSSTM Version 8.0 software based on the
detected cipro levels over time. The estimated mean decay rates for each treatment
were calculated based on cumulative data from all units under each condition and
half-lives were calculated directly from estimated decay rate coefficients.

2.6.3. Estimation of Adsorption Partition Coefficients

The adsorption partition coefficient was calculated by dividing the concentration
of cipro bound to the particulate matter by the concentration of cipro in the test
water. All calculations were performed based on the radioactivity distribution at
24 h after the initiation of the test. Preliminary studies indicated that this length of
time was sufficient to allow the system to reach steady state, while still adequately
short such that cipro degradation was not significant.

3. Results

3.1. LABORATORY-SCALE CIPROFLOXACIN FATE STUDIES

The flask experiments assessed cipro fate as a function of sterility, light supply
source, and ambient DOC and POC levels in aquatic systems. Water conditions
 CIPROFLOXACIN FATE IN AQUATIC SYSTEMS 391

Figure 2. Attenuation of ciprofloxacin exposed to different light sources. The symbols
,•, and 
represent laboratory fluorescent light, reptile lamps (emitting 33% UVA and 8% UVA to simulate
natural sunlight), and dark conditions, respectively. Each point represents the mean of duplicate
beakers; error bars refer to the range of measured ciprofloxacin in the two beakers.

were similar among experiments with mean pH and DO ranging from 7.5 to 8.6
and 6.7 to 8.1 mg/L, respectively, and temperature constant at 20 ◦ C. The average
light intensity at mid-depth in the flasks was 470 µE m−2 s−1 (±12), which is
roughly equivalent to light intensities at ∼2 to 3 m depth in a mesotrophic mid-
latitude lake (Kirk, 1994). Figure 2 summarizes the effect of light source on the
cipro degradation rate. In general, the cipro disappearance rate was rapid under
quasi-natural reptile lamps (mean half-life (t1/2 ) ∼ 1.9 h; R 2 = 0.99, p < 0.025),
slower under indoor fluorescent lighting (mean t1/2 ∼ 46 h; R 2 = 0.98, p < 0.025),
and was functionally nil in the “dark” over 100 h. Pre-sterilization did not affect the
rate of cipro disappearance (data not shown), suggesting that light effects dominated
cipro fate in these systems. POC levels were low in this first experiment to ensure
that the effects of adsorption were minimized.
The influence of DOC level on cipro fate is shown in Figure 3. The beakers
were retained in the dark prior to light exposure to test the effect of ambient DOC
level on parent cipro disappearance rate; however, minimal cipro loss was noted
over 70 h. When light was provided, cipro rapidly disappeared in all beakers with
a typical t1/2 of 4.6 ± 0.4 h (among the four DOC conditions) hours. No significant
difference in t1/2 was noted among DOC levels, ranging from 5.36 to 38.13 mg-C/L.
Slightly reduced cipro levels were seen in all beakers just before light was provided,
especially in the two higher DOC beaker treatments; however, this reduction was
also noted in duplicate beakers that were not provided light (data not shown).
A number of transformation products, shown in Figure 4, were noted in the first
two laboratory light-exposure studies that were typical of cipro photodegradation.
392 L. A. CARDOZA ET AL.

Figure 3. Ciprofloxacin attenuation patterns without and with light exposure as a function of DOC
concentration. Beakers maintained in the dark for 72 h and were then exposed to reptile light (emitting
33% UVA and 8% UVA to simulate natural sunlight), where , , •, and  represent DOC levels of
5.36, 11.2, 19.4 and 38.1 mg-C/L, respectively.

Figure 4. Typical presumptive photodegradation products of ciprofloxacin in aquatic systems.

Observed molecular mass [M + H]+ peaks included 263 (Burhenne et al., 1997a,b;
Torniainen et al., 1997a,b; Schmitt-Kopplin et al., 1999), 306 (Burhenne et al.,
1997a,b; Torniainen et al., 1997a,b; Schmitt-Kopplin et al., 1999), 360 (Burhenne
et al., 1997a,b) 330 (Mella et al., 2001), and 288 (Mella et al., 2001). Trans-
formations resulting from the decomposition of the piperazine ring were most
prevalent, including ring cleavages and functional group substitutions or loss. Low
concentrations of presumptive breakdown products were occasionally noted in the
darkened units; however, product production rates were very low.
 CIPROFLOXACIN FATE IN AQUATIC SYSTEMS 393

Figure 5. Adsorption of ciprofloxacin onto suspended biosolid particulates in complete darkness,

where
represents DOC of 5.36 mg-C/L and POC < 1.0 mg-C/L;  represents DOC of 11.2 mg-C/L
and POC < 1.0 mg-C/L;  represents DOC and POC of 2.94 and 14.60 mg-C/L, respectively, and •
represents DOC and POC of 8.13 and 16.52 mg-C/L, respectively.

The final laboratory experiment, which is summarized in Figure 5, describes the

effect of POC level on aqueous-phase cipro concentrations. Four water conditions,
with varied DOC and POC levels, were assessed in the dark. Figure 5 shows that
elevated POC, independent of DOC level or light, resulted in the rapid disappearance
of soluble phase cipro. Soluble cipro levels dropped substantially after 15 min of
exposure to particles, a rate more rapid than the highest observed rate for cipro
photodegration under similar aquatic conditions and no degradation products were
observed.

14
3.2. C-CIPROFLOXACIN ADSORPTION STUDIES ON ORGANIC PARTICULATES

To assess actual cipro adsorption tendencies onto aquatic organic solids, 14 C-

ciprofloxacin adsorption assays were performed on two particulates associated with
the laboratory and field experiments. Table I shows that two particulate sources had
similar K OC values at mid-pH levels, ranging from 13,900 to 20,500 L/kg. However,
K OC values were ∼5 times greater at pH 3.0 (77,500 to 97,300 L/kg), suggesting
that cipro adsorbs more readily onto aquatic POC under acidic conditions. These
results are similar to the previously reported K OC value of 61,000 L/kg determined
with an acidic (∼pH 5.0) organic clay soil (Nowara et al., 1997).

3.3. MESCOSM-SCALE CIPROFLOXACIN FATE STUDY

The field mesocosm experiment was similar to laboratory work except a lower cipro
concentration was employed (25 µg/L). Figure 6 shows that cipro level dropped very
394 L. A. CARDOZA ET AL.

TABLE I
Effects of particulate source, concentration, and pH on adsorption of ciprofloxacin onto aquatic
biosolids
Particulate 1 Particulate 2 Particulate 2
Source 7.8 7.3 3.0
pH
POC (mg-C/L) 1.0 5.8 1.0 8.9 2.0 7.1

% in water 97.1 (2.4)a 89.6 (0.3) 88.6 (2.0) 81.9 (1.8) 73.8 (2.1) 56.6 (1.8)
% Bound to OMP 4.3 (1.0) 13.2 (0.4) 5.7 (0.2) 12.9 (1.0) 18.9 (0.7) 36.4 (0.9)
Mass balance 96.2 (2.3) 98.1 (0.2) 94.3 (1.9) 94.9 (1.7) 92.7 (2.4) 92.9 (0.9)
K OC, L/kg 19800 20300 20500 13900 97300 77500
(9400) (800) (1500) (1400) (4400) (4500)
a
95% confidence interval.

Figure 6. (A) Attenuation of ciprofloxacin in mesocosm tanks exposed to sunlight and under dark
conditions. The three symbols •, , and  correspond to the three lidded (dark) mesocosm tanks,
and  and  correspond to the light-exposed units. For the light exposed tanks, the concentration
measured for all the samples after the first time point were at or below the limits of detection. (B) The
attenuation of ciprofloxacin in the dark tanks ( and ) when exposed to natural light after removing
the lids.

rapidly in the open tanks (3,400–3,900 µE m−2 s−1 atmospheric light intensities
were present during initial hours of the experiment) with cipro levels ∼10 µg
L−1 after 1.1 h and not detectable after 5 h (Figure 6A). In contrast, cipro was
relatively persistent in the covered tanks with concentrations slowly dropping to
between 13 and 20 µg L−1 over the first 120 h. After approximately 120 h, routine
cipro analysis detected an anomalous sudden drop in cipro level in one of the three
 CIPROFLOXACIN FATE IN AQUATIC SYSTEMS 395

covered units. However, upon investigating this result, it was discovered that the lid
had blown off this tank the previous afternoon and it had been replaced by one of
the field crew without informing others. As a result, the previously “covered” unit
was temporarily exposed to light (estimated to be about 8 h), which presumably
allowed photodegradation to occur thus explaining the sudden drop in cipro level
in the unit. This unit was not monitored further as a “covered” tank because very
little residual cipro was present after this event.
Because initial disappearance rates were so rapid and to generate more quantita-
tive data on cipro half-life under natural light, the lids were intentionally removed
from the remaining covered tanks after about 290 h. Figure 6B shows the very
rapid degradation of cipro upon exposure to light. Atmospheric light levels ranged
between 1275 and 2700 µE m−2 s−1 over that day and first-order half-lives were
estimated as 1.14 and 0.97 h for the two units.
Water chemistry conditions in the six mesocosms were similar regardless of
whether they were open or covered. DOC and POC levels were always low (e.g.,
POC < 1.0 mg-C/L), pH was ∼8.5, DO ranged from 6.6 to 7.4 mg/L, and secondary
nutrient levels were typical of lower mesotrophic conditions (e.g., TP ∼8 to 13 µg/L
and TN ∼0.32 to 0.48 mg/L). The only major difference between the open and
covered tanks was the ambient light intensities through the water column as intended
in the experimental design. The average midday light levels were 1200 (±20, 95%
CI) and 21 µE m−2 s−1 (±3, 95% CI) for the open and covered units, respectively.
Water temperatures decreased over time in all units (ranging from ∼21 to ∼16 ◦ C,
this was an autumn experiment); however, no significant difference in temperature
was noted between the open and covered units.

4. Discussion

The fate of fluoroquinolone antibiotics, such as ciprofloxacin, in aquatic systems

appears to be dominated by adsorption and photodegradation reactions. Although
biological mechanisms have been insinuated in laboratory studies (Wetzstein et al.,
1999; Parshikov et al., 2001), such reactions appear to be very slow or require
very specific environmental conditions to be practically relevant. Therefore, the
key question relative to cipro in aquatic systems is whether adsorption versus pho-
todegradation is more important under a given set of environmental conditions.
Golet et al. (2003) concluded in their studies of Swiss rivers that adsorption was
more significant for cipro, although this conclusion was based on deductive logic
rather than direct measurement under field conditions. Results shown in Table I
indicate that adsorption reactions are rapid and significant between cipro and POC;
however, Figures 2, 3, and 6 also suggest that photodegradation is important, espe-
cially when POC levels are low. Therefore, both mechanisms appear to be condi-
tionally significant to cipro fate in aquatic systems. Furthermore, Figure 4 indicates
that signature photodegradation products are clearly apparent when light-related
degradation is observed.
396 L. A. CARDOZA ET AL.

To assess the dominant mechanism under quasi-natural field conditions, we per-

formed a controlled field experiment (Figure 6). The results strongly suggest that
photodegradation dominated cipro fate in these systems, although these waters had
very low POC levels. Regardless, the significant result here is that both photodegra-
dation and adsorption can be conditionally important in different natural waters,
probably being regulated by light intensity and source, POC level, and other factors
like pH, which Table I suggests can be important to adsorption. Therefore, the con-
trolling mechanism of cipro fate in aquatic systems appears to vary from scenario
to scenario.
Although this might not seem important, especially given that both mechanisms
result in rapid disappearance of cipro from the aqueous phase, the mechanism that
dominates is not simply an academic issue. Although adsorption onto POC is rapid
and appears to control fate when particles are present, there is no indication that
adsorption destroys cipro, i.e., adsorption onto solids may only be a transient step in
the transport of intact cipro through the environment. In contrast, photodegradation
appears to completely destroy cipro, suggesting that this mechanism would be
preferred for long-term destruction of cipro in the environment.

5. Conclusions and Practical Implications

This study used laboratory and field systems to assess dominant mechanisms af-
fecting the fate of ciprofloxacin in aquatic systems. Results suggest that cipro
is highly photosensitive and also readily adsorbs onto organic solids. There-
fore, residual soluble ciprofloxacin is unlikely in most aquatic systems. How-
ever, whether photodegradation versus adsorption dominates any fate scenario
is a practically significant issue. Photodegradation destroys cipro (most degra-
dation products appear also to be photodegradable), whereas adsorption only ap-
pears to change its phase. As a result, an important issue for future study is de-
termining the fate of cipro that is adsorbed onto solids, especially its potential
for photodegradation and desorption, which are likely keys to determining its
ultimate fate in aquatic systems. We conclude that additional work on the fate
of adsorbed cipro is especially needed to assess its impact on the environment
and, in turn, perform better risk assessments on the effects of cipro in aquatic
systems.

Acknowledgments

This research was supported by U.S. Environmental Protection Agency STAR Grant
No. R82900801-0. The authors would like to thank Tess Lane, Jackie Miller, Roric
Moore-Jansen, F. Jerry deNoyelles Jr., and Andrew Ensz who assisted in support
fieldwork and laboratory analyses.
 CIPROFLOXACIN FATE IN AQUATIC SYSTEMS 397

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