Journal of Hazardous Materials 198 (2011) 22–30

Contents lists available at SciVerse ScienceDirect

Journal of Hazardous Materials

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

Biodegradation of ciprofloxacin in water and soil and its effects on the

microbial communities
Cristobal Girardi a,∗ , Josephine Greve b , Marc Lamshöft c , Ingo Fetzer d , Anja Miltner a , Andreas Schäffer e ,
Matthias Kästner a
a
UFZ – Helmholtz Centre for Environmental Research, Department of Environmental Biotechnology, Permoserstraße 15, 04318 Leipzig, Germany
b
Minnesota State University, Mankato, MN 56001 8400, USA
c
Institute of Environmental Research (INFU), TU Dortmund University, Otto-Hahn-Str. 6, NRW 44221 Dortmund, Germany
d
UFZ – Helmholtz Centre for Environmental Research, Department of Environmental Microbiology, Permoserstraße 15, 04318 Leipzig, Germany
e
Department of Environmental Biology and Chemodynamics, Institute for Environmental Research (Biology V), RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany

a r t i c l e i n f o a b s t r a c t

Article history: While antibiotics are frequently found in the environment, their biodegradability and ecotoxicological
Received 29 April 2011 effects are not well understood. Ciprofloxacin inhibits active and growing microorganisms and therefore
Received in revised form can represent an important risk for the environment, especially for soil microbial ecology and microbial
27 September 2011
ecosystem services. We investigated the biodegradation of 14 C-ciprofloxacin in water and soil following
Accepted 2 October 2011
OECD tests (301B, 307) to compare its fate in both systems. Ciprofloxacin is recalcitrant to biodegradation
Available online 10 October 2011
and transformation in the aqueous system. However, some mineralisation was observed in soil. The
lower bioavailability of ciprofloxacin seems to reduce the compound’s toxicity against microorganisms
Keywords:
Ciprofloxacin
and allows its biodegradation. Moreover, ciprofloxacin strongly inhibits the microbial activities in both
Radiotracer systems. Higher inhibition was observed in water than in soil and although its antimicrobial potency
Biodegradation is reduced by sorption and aging in soil, ciprofloxacin remains biologically active over time. Therefore
Bioavailability sorption does not completely eliminate the effects of this compound.
Toxicity © 2011 Elsevier B.V. All rights reserved.

1. Introduction water to agricultural land [6–9]. Therefore, antibiotics enter the

soil, but their fate and effects on this ecosystem are unknown
Antibiotics are designed to be refractory to biodegradation and [2].
to act effectively even at low doses. Recently, the concern about CIP concentrations in the environment range from ng L−1 to
potential ecological impacts of synthetic antibiotics increased mg L−1 . Larsson et al. [10] reported concentrations up to 31 mg L−1
because they may inhibit key environmental processes mediated in the effluents of a wastewater treatment plant (WWTP) for
by microorganisms like nutrient regeneration, carbon and nitrogen pharmaceutical industries in India. During wastewater treatment,
cycles and pollutant degradation [1]. 80–90% of CIP is removed via sorption to sludge, which stabi-
The most widely prescribed fluoroquinolone antibiotic is lizes the substance [2]. Therefore, digested sludge contains CIP
ciprofloxacin (CIP) [2], which is active against a broad spectrum (around 3 mg kg−1 ; [5]). In soil, reported concentrations range from
of Gram-negative and Gram-positive bacteria [3]. It is frequently 0.37 mg kg−1 to 0.40 mg kg−1 [11,12], underlining the ecotoxicolog-
detected in the environment and proven to be genotoxic [4]. ical relevance of CIP in soil. CIP is not readily biodegradable [4]. It
Also, it is the main metabolite of enrofloxacin, a commonly used also strongly sorbs to soil [2,13], mostly by cation exchange [14].
veterinary fluoroquinolone [2]. Of the administered dose of CIP Therefore, the soil can act as a reservoir of this and other antibiotics
in humans, 45–62% is excreted unmetabolised via urine and [15].
15–25% via faeces [5]. Thus, CIP can reach the environment by The effects of CIP on microbial communities in wastewater,
sewage, discharges from sewage treatment plants, leaching from streamwater and marine and salt marsh sediment were studied
landfills, its release from pharmaceutical industries, livestock activ- thoroughly [4,16–18]. It reduced algal diversity at environmentally
ities and application of sewage sludge, manure or treated waste relevant concentrations [19]. However, nothing is known about its
effects on soil microbial communities [2], and standardised stud-
ies on its degradation in soil, e.g. OECD 307 tests, are missing. This
∗ Corresponding author. Tel.: +49 341 235 1753; fax: +49 341 235 1471. information is needed to estimate the fate of these compounds and
E-mail address: [email protected] (C. Girardi). to perform accurate risk assessments.

0304-3894/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.jhazmat.2011.10.004
 C. Girardi et al. / Journal of Hazardous Materials 198 (2011) 22–30 23

Exposure of bacteria in the environment may contribute to local wastewater treatment plant (Klärwerk Rosental, Leipzig,
spreading antibiotic resistance to pathogens [7]. Fluoroquinolone Germany). Two different incubations were performed: (1) soil with
resistant Campylobacter jejuni was found in poultry husbandry [20]. [2-14 C] ciprofloxacin, (2) sterilised soil (autoclaved 3 times on 3
Furthermore, antibiotics in sewage can inhibit the microbiota of consecutive days) with [2-14 C] ciprofloxacin (sterile control). The
WWTP [21,22] and thus reduce the waste water treatment effi- radiolabeled mixture was initially added to 10% of the soil. Then
ciency. Composting is used to degrade organic contaminants such the spiked soil was thoroughly mixed with the remaining 90% with
as pesticides, PAHs and PCBs in sewage sludge before its applica- a pastry blending machine. The final concentration of ciprofloxacin
tion to soils [23]. However, the effectiveness of this process and the was 20 mg kg−1 of soil (60% of WHC); the radioactivity added was
fate of the antibiotics themselves remain unclear. 10 kBq per system. Twenty grams of soil were incubated in 500 mL
We hypothesise that CIP is not degraded in water or soil and Schott bottles in the dark, at 60% WHC and at 20 ◦ C for 90 days. Sam-
that it can pose a risk for the environment. Therefore, the aims ples were flushed with air every 3 days and the CO2 produced was
of this study were (1) to directly compare the biodegradation of trapped in two NaOH traps as described above. The bottles were
radiolabeled CIP in water and in soil following the OECD tests 301B destructively sampled after 17, 32, 60 and 93 days.
and 307 [24,25] in order to produce a database for extrapolating
biodegradation data obtained in water-based tests to soil; (2) to 2.4. Extractable and non-extractable residues in soil
obtain a mechanistic overview of CIP’s biodegradation and (3) to
elucidate the potential effects of this antibiotic on soil microbes 2.4.1. Soil extractions by ASE
and their activities. Five grams of soil were mixed in a 33 mL stainless steel extrac-
tion cell with HydromatrixTM (Varian Inc., Santa Clara, USA) and
2. Materials and methods extracted with a mixture of 63% ethyl acetate, 25% methanol and 3%
ammonium hydroxide using an ASE 200 accelerated solvent extrac-
2.1. Chemicals and soil material tion system (Dionex, Sunnyvale, USA) at the following operating
conditions: extraction temperature, 100 ◦ C; extraction pressure,
All chemicals were analytical or reagent grade obtained from 120 bar; preheating period, 5 min; static extraction period, 30 min;
VWR (Darmstadt, Germany) or Sigma–Aldrich (Munich, Germany) number of extraction cycles, 5; solvent flush, 50% of the cell vol-
if not specified otherwise. Ciprofloxacin hydrochloride (99% purity) ume; and nitrogen purge, 120 s. A subsample of the extract was
was purchased from Biotrend Chemicals (Zurich, Switzerland), [2- removed for 14 C analysis and the remaining sample was diluted
14 C] ciprofloxacin (radiochemical purity 99.4%; specific activity with MilliQ water until <5% solvent content for purification and
20 mCi mmol−1 ) from Hartmann Analytic GmbH (Braunschweig, chemical analysis.
Germany), sodium acetate-U-14 C (≥98 atom% 14 C, 50 mCi mmol−1 )
from Biotrend GmbH (Cologne, Germany). 2.4.2. Non-extractable residues
The soil experiments were performed with soil samples (21% In order to determine the initial total radioactivity in soil and
clay, 68% silt, 11% sand, TOC 2.1%, total N 0.17%, pH 6.6 and the 14 C in non-extractable residues after extraction by ASE, soil
water holding capacity 37.5%) from the A horizon of a Haplic samples were combusted in a biooxidizer (Biological oxidizer OX
Chernozem from the agricultural long-term experiment “Statischer 500, Zinsser Analytic, Frankfurt, Germany; [27]). The CO2 produced
Düngungsversuch” (Bad Lauchstädt, Germany). The plot has been during combustion was trapped in Oxysolve 400 (Zinsser Analytic)
fertilised with farmyard manure (30 t/ha) every second year since and analysed by liquid scintillation counting (LSC) with a Wallac
1902 [26]. 1414 scintillation counter (Perkin Elmer Wallac GmbH, Freiburg,
Germany).
2.2. Incubations in mineral medium
2.5. Radioactivity measurements
Four different incubations were performed to test the
biodegradability in aqueous systems according to the OECD guide- The radioactivity in liquid samples (NaOH traps, MM, SS, and soil
line 301B [24]: (1) standard mineral medium (MM) with [2-14 C] extracts) was determined by LSC after addition of Ultima GoldTM
ciprofloxacin; (2) sterilised MM with [2-14 C] ciprofloxacin (ster- scintillation cocktail (Perkin Elmer).
ile control to account for abiotic degradation processes); (3) MM
with 14 C-acetate (positive control); and (4) MM with 14 C-acetate 2.6. Chemical analyses
and unlabelled CIP (inhibition test). Each bottle was inoculated
with diluted fresh activated sludge (10 mg L−1 of suspended solids Filtered MM samples were analysed by thin layer
[SS], sterilised by autoclaving for the sterile control) from a chromatography (TLC) on silica plates developed with a
municipal wastewater treatment plant (Klärwerk Rosental, Leipzig, dichloromethane–methanol–2-propanol–25% NH3 (3:3:5:2)
Germany). The final concentration of ciprofloxacin or acetate was mixture [28]. The radioactivity on the TLC plates was determined
20 mg L−1 , the radioactivity added was 10 kBq per system. 300 mL with a Linear TLC Analyser LB284 (Berthold GmbH & Co KG, Bad
of the spiked MM were incubated in 500 mL Schott bottles in Wildbad, Germany; [27]).
the dark at 20 ◦ C for 29 days. Samples were periodically flushed The diluted soil extracts were acidified to pH 3 and puri-
with air to provide O2 . The gas leaving the bottles was passed fied by solid phase extraction (SPE; [13]). Samples were passed
through 1 M NaOH (2 × 20 mL) to trap 14 CO2 . The bottles were through a 500 mg anion-exchange cartridge (Waters, Taunton,
destructively sampled after 12 and 29 days. Samples were filtered USA), stacked on top of a 500 mg hydrophilic–lipophilic balance
through 0.22 ␮m cellulose filters to determine the radioactivity in cartridge (Waters) and eluted with methanol/NH3 6%. The samples
the medium and SS. were concentrated under nitrogen and resuspended for analysis.
The extracts were analysed by reversed phase liquid
2.3. Soil incubation experiments chromatography–tandem mass spectrometry (LC–MS/MS) with a
Thermo Fisher Surveyor HPLC-system (Thermo Fisher Scientific,
Biodegradation experiments in soil were based on the OECD Waltham, USA) equipped with a Luna PFP(2) column (150 × 2 mm,
guideline 307 [25]. The soil was sieved to 2 mm and amended 3 ␮m particle size; Phenomenex, Aschaffenburg, Germany). Ten
with stabilised sludge at 1.8 g kg−1 soil (dry weight) from a microliters samples were separated with the following gradient
24 C. Girardi et al. / Journal of Hazardous Materials 198 (2011) 22–30

Table 1
SRM data, retention time, LOD and LOQ of CIP.

Compound Retention Precursor Product Collision LOD (␮g L−1 ) LOQ (␮g L−1 )
time (min) ion (m/z) ions (m/z) energy (eV)

CIP 19.07 332.1 314.1 −19 5 15

332.1 288.0 −17

program: 90% A (solvent A: 1 mM ammonium acetate and 0.1% 2.10. Data analysis, mass balance and statistical analysis
HCOOH in water) for 2 min, linear gradient to 50% A over 23 min,
and to 100% within the next 1 min. Subsequently, the column was All results are presented as means of triplicate experiments with
rinsed with 100% B (solvent B: 0.1% HCOOH in methanol) for 5 min, standard deviation. Mineralisation and radioactivity in medium or
and then the system was returned to 90% A within 1 min where soil, SS and extractable and bound residues were quantified on each
it was held for 5 min before the next run was started. The mobile sampling date. The recovery was calculated to set up a complete
phase flow rate was 0.3 mL min−1 ; the column temperature was mass balance.
26 ◦ C. The mass spectra were acquired using a TSQ Quantum Ultra To visualise the changes caused by CIP on the soil microbial
AM mass spectrometer (Thermo Fisher) with a HESI-II ion source communities, non-metric multidimensional scaling analysis (MDS)
operating in positive mode. Nitrogen was both the drying and were performed using the Bray–Curtis distance and Jaccard index
the nebulizer gas, and argon (1.5 bar) was the collision gas. The measure on the T-RFLP data [34]. A two-way PERMANOVA was used
capillary temperature for the TSQ Quantum was 250 ◦ C, and the to test between groups and ANOSIM within treatment differences
vaporizer temperature 350 ◦ C. The MS/MS parameters (tube lens, of the T-RFLP data results.
collision energy) were optimized in continuous flow mode for Due to the unequal number of replicates for biotic and abiotic
maximum sensitivity for product ions, and the two most sensitive incubations, the Kruskal–Wallis test was used to identify differ-
SRM (selected reaction monitoring) transitions were determined ences in mineralisation, non-extractable residues (NER), ER, soil
for each molecule (for instrument parameters and SRM data for respiration and metabolite formation. For soil respiration data, 95%
CIP, see Table 1). confidence intervals were calculated based on results from tripli-
The main metabolites were identified by ESI-HR-MS with an cates, assuming a balanced normal distribution. Differences were
LTQ-Orbitrap Spectrometer (Thermo Fisher) according to [28]. regarded statistically significant for all tests if p < 0.05. Statistical
tests were conducted with the software packages PAST [35] and R
[36].
2.7. Toxicity study in pure culture

The EC50 for Pseudomonas putida mt-2 (a soil isolate) in pure 3. Results
culture was determined as described by [29].
3.1. Biodegradation study in aqueous and soil systems

2.8. Inhibition studies in soil 3.1.1. Mass balance

No mineralisation was observed in the aqueous system over
Soil incubations with ciprofloxacin hydrochloride at different 29 days of incubation (Fig. 1) and only the parent compound was
concentrations (0 mg L−1 , 0.2 mg L−1 , 2 mg L−1 and 20 mg L−1 ) were detected by TLC analysis at that time (Fig. S1). Consistently, the
performed in a Sapromat® E BOD Measuring Unit (H + P Labortech- radioactivity in the MM remained high. Radioactivity in SS was
nik, Oberschleissheim, Germany) to study the effects of CIP on soil similar for biotic and abiotic incubations (Table S1). CIP is therefore
respiration and soil microbial community structure (by terminal recalcitrant to degradation under the OECD 301 test conditions.
restriction fragment length polymorphism [T-RFLP] analysis using In soil, however, some mineralisation (0.9% of the added CIP
universal primers for the 16S rRNA). The samples were incubated after 93 days; p < 0.05) was observed (Fig. 1). The contribution of
for 113 days, but soil respiration was only recorded through day 77. biotic and abiotic processes was approximately equal. Until day
6 the degradation rate was relatively high (around 0.03% day−1 ),
thereafter, it was low but constant (0.008% day−1 ).
2.9. DNA extraction, T-RFLP analyses and detection of resistance The extractability by ASE decreased over time. On day 0, 39% and
genes 46% of the initial 14 C amount was extracted in the biotic and abiotic
incubations, respectively (Fig. 2). The extractability decreased to
Total DNA was extracted from 0.5 g of soil using the UltraClean® 12% and 15% after 93 days.
Soil DNA Isolation Kit (MO BIO Laboratories, Carlsbad, USA). 16S
rDNA was amplified by PCR with the primers 27f [30] and 1378R
[31]. The reaction conditions for the 25 ␮L reaction (Hot Start Taq
PCR master mix, Qiagen) were: 15 min at 95 ◦ C, 32 cycles of 30 s at
94 ◦ C, 30 s at 52 ◦ C, 1.2 min at 72 ◦ C, and a final extension for 10 min
at 72 ◦ C. PCR products were purified using the QIAquick® PCR Purifi-
cation Kit (Qiagen, Hilden, Germany), and quantified by a NanoDrop
ND-100 device (NanoDrop Technologies. USA). The PCR products
were digested with MspI (Fermentas, St. Leon-Rot, Germany) and
the resulting T-RFs were analysed [32].
Resistance genes qnrA, qnrB and qnrS were amplified with the
same PCR reagents and appropriate primers [33]. The reaction con-
ditions for the 25 ␮L reaction were: 10 min at 95 ◦ C, 35 cycles of
Fig. 1. Mineralisation of [2-14 C]-ciprofloxacin in mineral medium and soil. (䊉) Soil,
1 min s at 94 ◦ C, 1 min s at 52 ◦ C, 45 s at 72 ◦ C, and a final extension () sterile soil and () mineral medium. Percentages refer to the total radioactivity
at 72 ◦ C for 10 min. PCR products were purified as described above. applied. Standard deviations are not visible if smaller than the symbols.
 C. Girardi et al. / Journal of Hazardous Materials 198 (2011) 22–30 25

3
A 110

Soil respiration rates (mg O2 /kg.day)

100
% of initially applied C

2.5
14

90
80
2
70
60
50 1.5
40
30 1
20
10 0.5
0
0 10 20 30 40 50 60 70 80 90 100
0
Days
B 110
0 10 20 30 40 50 60 70 80
Days
100
% of initially applied C
14

90
Fig. 3. Soil respiration rates in the inhibition test. (䊉) 0 mg kg−1 CIP, (♦) 0.2 mg kg−1
80 CIP, () 2 mg kg−1 CIP, (x) 20 mg kg−1 CIP.
70
60
50 aqueous system, but also decreased with time. After 2 days, cumu-
40 lative soil respiration was inhibited by approximately 70% at all
30 three CIP concentrations, as opposed to only roughly 35% at the
20 end of the experiment (Table 4).
10 Microbial activity was thus strongly inhibited both in aqueous
0 systems and soil. Even though concentrations in the soil solution
0 10 20 30 40 50 60 70 80 90 100
were much higher than in MM (Table 4), it seems that CIP was more
Days toxic in aqueous media than in soil. One explanation might be the
higher diversity of microorganisms in soil. More important, how-
Fig. 2. Degradation of [2-14 C]-ciprofloxacin in soil under biotic (A) and abiotic con-
ditions (B). (䊉) Mineralisation, () extractable amount, () non-extractable residues ever, seems to be the reduced bioavailability of CIP in soil, which
and (♦) recovery. Standard deviations are not visible if smaller than the symbols. potentially reduces the toxicity of this compound. Moreover, in
the concentration range studied, CIP toxicity did not depend on
its concentration (Fig. S3); this suggests that the maximum effect
Non-extractable residues on day 0 accounted for 57% and 54%
was already obtained with the lowest concentration studied. The
of the applied radioactivity for biotic and abiotic systems, respec-
decrease of inhibition in soil over time can be explained by the aging
tively, and over time increased to 88% (biotic) and 81% (abiotic)
of the compound and by the adaptation of microorganisms.
of the initial 14 C amount on day 93. NER formation slowed down
The reduction of soil respiration rates by CIP was most evident
after 30 days, but increased steadily until day 93 (Fig. 2). CIP thus
during the first month, whereas later respiration rates of CIP treat-
strongly sorbs to soil and aging increases NER formation. Overall,
ments were similar to the controls (Fig. 3). Ciprofloxacin mainly
the extractability of ciprofloxacin-derived radioactivity and NER
reduced the microbial activity at the beginning of the experiment,
were not statistically different between the biotic and abiotic sys-
because it is a biostatic compound that targets growing microor-
tems (p > 0.05). Total recoveries ranged from 93 to 101% (Table S2).
ganisms.
T-RFLP analyses were applied to study the effects of CIP on the
3.1.2. Ciprofloxacin and its metabolites soil bacterial community. MDS analyses of soil bacterial commu-
Extractable CIP in the soil declined over time in both biotic and nities (Fig. 4) revealed a shift in both microbial abundance and
abiotic incubations (Table 2), consistent with 14 C extractability. microbial diversity after the application of CIP. The relevant factors
This decline was more pronounced in biotic incubations (10.5% driving this change were CIP and time. Statistical analysis revealed
of initially extracted CIP on day 93) than in abiotic incubations that these two factors were uncorrelated (p = 0.431), demonstrating
(25.2%; p < 0.05). Two known metabolites of ciprofloxacin [37] (F9, that both factors were acting independently. The samples clustered
F6; Tables 2 and 3) and one unknown, M311, of m/z 311, were into four groups. Two of them comprised the controls and the other
found in low amounts at all the sampling times (including time 0). two the CIP treatments at the early (days 3, 14 and 29) and late (days
Unfortunately, the information obtained from the analysis was not 65 and 113) stages of incubation. The microbial community sta-
sufficient to propose a chemical structure for M311. The amounts of bilised after 65 days of incubation. PERMANOVA analysis confirmed
F6 were similar in both experiments (p > 0.05) and the amount of F9 a significant difference (p < 0.001) between control and CIP treat-
slightly more abundant under biotic conditions (Table 2; p < 0.05). ments. However, the difference between the three CIP treatments
was not significant (p = 0.67), as confirmed by ANOSIM analysis.
3.2. Induced effects on the sludge and soil microbial community Although the effect of CIP on microbial communities was evident,
no clear concentration effect was detectable, which is consistent
3.2.1. Inhibition tests in MM and soil with the effect on soil respiration.
To test the effect of CIP on the general microbial activity in aque- Moreover, when Jaccard index was used instead of Bray–Curtis
ous systems, the inhibition of acetate mineralisation by CIP was for the analysis of the changes in the community, significant
analysed. Without CIP, acetate mineralisation started immediately, differences were also found between the control and the treat-
and, after 29 days, 70% of the acetate was mineralised. In the pres- ments (data not shown), which indicates a shift in species
ence of CIP, acetate was slowly degraded after a 5-day lag phase composition.
(Fig. S2). At the end of the experiment, mineralisation was inhibited
by 75% compared to the control without CIP (Table 4). 3.2.2. EC50 for bacteria (Pseudomonas putida)
Soil respiration was used as an indicator of microbial activity. The inhibition study of CIP in pure culture of P. putida mt-2
The inhibition of soil respiration by CIP was lower than in the revealed an EC50 of 0.25 mg L−1 (Fig. 5). At 1 mg L−1 , growth was
26 C. Girardi et al. / Journal of Hazardous Materials 198 (2011) 22–30

Table 2
Ciprofloxacin and metabolites relative abundance (F6 and F9) in purified soil extracts.

Time (days) % of initial 14 C2 -ciprofloxacina

CIP F6 F9

Biotic 0 100 (±13.1) 4.70 (±0.918) 3.28 (±0.501).

17 38.66 (±0.982) 6.49 (±1.26) 13.84 (±6.10)
32 46.5 (±13.1) 1.85 (±0.144) 3.01 (±0.260)
60 20.76 (±1.30) 1.48 (±0.245) 3.23 (±0.867)
93 10.46 (±2.87) 0.674 (±0.177) 4.43 (±2.73)

Abiotic 0 100 (±5.82) 8.12 (±2.63) 4.14 (±0.488)

32 44.3 (±3.38) 1.51 (±0.464) 0.916 (±0.087)
93 25.17 (±7.65) 0.854 (±0.043) 0.793 (±0.043)

Values in brackets (±) represent the standard deviation of the average of triplicates.
a
100% corresponds to initially measured amount of ciprofloxacin in the time 0 extract.

Table 3
Accurate masses of [M + H]+ and chemical structures of ciprofloxacin (332 m/z) and metabolites F9, 7-Amino-1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-3-
quinolinecarboxylic acid (263 m/z); F6, 1-cyclopropyl-7-(1-piperazinyl)-6-fluoro-1,4-dihydro-8-hydroxy-4-oxo-3-quinolinecarboxylic acid (348 m/z) and M311 (311 m/z)
in soil (structures from Wetzstein et al. [37]).

Compound [M+H]+ [m/z] (experimental) [M+H]+ [m/z] (theoretical) Calculated formula Chemical structure

CIP 332.14042 332.14050 C17 H19 N3 O3 F

F9 263.08260 263.08265 C13 H12 N2 O3 F

F6 348.13540 348.13541 C17 H19 N3 O4 F

M311 311.12787 Not available C14 H13 O4 N3 Not determined

completely inhibited. In contrast to these results, soil microor- 3.2.3. Ciprofloxacin resistance genes in soil
ganisms were still active at this concentration and acetate was To assess the adaptation of soil microbiota to CIP, samples from
mineralised at a much higher concentration in the OECD inhibition different incubation times were tested for CIP resistance genes
test (Fig. S2 and Table 4). One explanation for this difference is the (qnrA, qnrB, qnrS). The genes, qnrA and qnrB, were not detected
higher microbial diversity in activated sludge and soil compared to in any of the samples (Fig. 6). Gene qnrS was not detected in either
the pure culture. Additionally, the fraction of CIP adsorbed to sludge the control (non-amended) or in incubations with CIP after 3 days.
(10% of initial 14 C; Table S1) and soil (up to 88%, Table S2) could also In contrast, it was detected in samples with 20 mg kg−1 CIP from
contribute to the reduced toxicity against microorganisms. days 14, 29 and 65; in samples with 0.2 mg kg−1 CIP from day 29;

Table 4
Microbial activity inhibition in soil and water at different concentrations and times.

Time (days) % of inhibition (compared to control)

Soil MM
−1 −1 −1
0.2 mg kg CIP 2 mg kg CIP 20 mg kg a
CIP 20 mg L−1 CIP

2 70.9 (±12.0) 69.1 (±3.78) 71.5 (±15.6) 99.1 (±4.03)

7 56.4 (±3.02) 56.7 (±3.77) 56.8 (±3.23) 87.8 (±0.81)
12 45.9 (±1.19) 47.0 (±2.14) 48.3 (±1.33) 86.8 (±1.7)
20 46.9 (±4.14) 49.0 (±4.78) 50.4 (±7.99) 79.4c
Endb 33.1 (±1.49) 36.8 (±4.06) n.a. 74.9 (±11.2)

n.a.: not assessed.

Values in brackets (±) represent the standard deviation of the average of triplicates.
a
The concentration in the soil solution corresponded to 45.8 mg L−1 and 12.5 mg L−1 on days 0 and 77, respectively.
b
End of the experiment was on day 29 for water and on day 77 for soil.
c
Estimated by linear interpolation.
 C. Girardi et al. / Journal of Hazardous Materials 198 (2011) 22–30 27

Fig. 4. T-RFLP analysis of bacterial 16S rRNA from bacteria in soil. Non-Metric Mul-
tidimensional Scaling plot using Bray-Curtis similarity measure of the bacterial
communities after 3, 14, 29, 65 and 113 days of incubation with different concen-
trations of ciprofloxacin. () 0 mg kg−1 CIP, (♦) 0.2 mg kg−1 CIP, () 2 mg kg−1 CIP,
() 20 mg kg−1 CIP. The closer two communities are in the plot, the more similar
they are. Groups of triplicates are connected by polygons.

and in samples with 2 mg kg−1 after 65 and 113 days of incubation.

The low intensity of the qnrS bands indicates a low copy number of
this gene in soil.

Fig. 6. PCR of CIP resistance genes qnrA (580 bp), qnrB (264 bp) and qnrS (428 bp)
4. Discussion
from soil incubations with ciprofloxacin. Agarose gel electrophoresis (2%). Lanes: 1
and 13, Molecular size marker; 2, 3 and 4, qnrB, qnrA, qnrS respectively from CIP
Our data provides reliable information from OECD degradation 20 mg kg−1 day 14; 5, 6 and 7, qnrB, qnrA, qnrS respectively from CIP 0.2 mg kg−1
tests to estimate the fate of CIP in aqueous media [24] and in a day 29; 8, 9, 10; qnrB, qnrA, qnrS respectively from CIP 20 mg kg−1 day 29; 14, 15
typical agricultural soil [25]. Based on a combined approach of CIP and 16, qnrB, qnrA, qnrS respectively CIP 2 mg kg−1 day 65; 17, 18 and 19, qnrB,
qnrA, qnrS respectively from CIP 20 mg kg−1 day 65; 20, 21, 22, qnrB, qnrA, qnrS
degradation data and its induced effects on microbial communi-
respectively from CIP 2 mg kg−1 day 113; 25, 26, 27 qnrB, qnrA, qnrS respectively
ties, we evaluated the risk of this antibiotic for the environment. from CIP 20 mg kg−1 day 3; 28, 29 qnrA, qnrS from non spiked soil day 3; 30, 31, 32
We obtained a general overview on the degradation process, quan- qnrB, qnrA, qnrS from non spiked soil day 113; 11, 23 and 33 qnrS in resistant strain
tified mineralisation and carbon distribution during degradation, (positive control); 12, 24 and 34 no DNA (negative control).
and analysed metabolites and the effects of CIP on microbial com-
munities.
4.1. Ready biodegradability and fate in soil

No degradation was observed in water because CIP is highly

toxic and inhibits the microbial activity. It is resistant to abiotic
degradation reactions such as hydrolysis [8]. Therefore, CIP is recal-
citrant to degradation under ready biodegradability test conditions.
In the biotic soil systems, however, 0.9% of the added CIP was
mineralised after 93 days. Thus, contrary to the well-accepted rule
that degradation of antibiotics is hampered by fixation to the soil
matrix [8], more ciprofloxacin was degraded in soil than in water
under the used conditions. Sorption to soil particles may have
reduced the bioavailability and thus the effective toxicity of CIP
[38]. An association of the bioactive functionalities to soil exchange
sites is particularly efficient in this respect [39]. The biotic miner-
alisation may be performed by fungi [37], archaea, yeasts [40] or
CIP-resistant bacteria.
Similar low mineralisation levels (0.49–0.58%) were reported
for 4 mg kg−1 sarafloxacin (another fluorquinolone) after 80 days
Fig. 5. Effect of CIP on the growth of Pseudomonas putida mt-2 in pure culture. of incubation in various soils [41]. The authors attributed the low
28 C. Girardi et al. / Journal of Hazardous Materials 198 (2011) 22–30

mineralisation to the strong sorption and the resulting low bioavail- Bioavailability influences the effects of antibiotics on the soil
ability, but did not test inhibition of microbial activity. Our results microbial community. The presence of multivalent cations was
indicate that mineralisation was low mainly because the compound reported to inhibit the antimicrobial potential of fluoroquinolones
is toxic. [48]. This may partly explain the lower toxicity in soil, even though
Although CIP can be degraded, its strong sorption to soil, par- the concentration of CIP in the soil solution was higher than in
ticularly to humic acids (see Supplementary Material S4), makes the mineral medium. Furthermore, some soil microorganisms are
it highly persistent, while abiotic and microbial degradation are naturally tolerant towards antibiotics [49], such as some pseu-
less important. In our experiment, NER formation was similar in domonades [50], while others can acquire resistance. In addition,
biotic and abiotic incubations, suggesting that it occurred mainly the large microbial diversity in soil may be responsible for the
abiotically. This is consistent with the low mineralisation under weaker effects of antibiotics in soil than in water [51]. The antibiotic
biotic conditions. The decline of extractable CIP is thus mainly does not target Archaea and fungi and many soil bacteria are dor-
governed by sorption and formation of NER. The metabolites we mant and thus not sensitive to bacteriostatic antibiotics [52]. All
found at day 0 have to be formed by fast abiotic reactions with these reasons may account for the difference in toxicity towards
soil components, which is consistent with the relatively high ini- “active” activated sludge bacteria and soil bacteria.
tial abiotic mineralisation rate (Fig. 1). The unknown metabolite Ciprofloxacin inhibited the indigenous microbial activity in soil
M311 was also detected during the biodegradation of norfloxacin (Fig. 3), in particular the growing or active bacteria. This is consis-
by white-rot fungi (Prieto et al., personal communication), and tent with its bacteriostatic mechanism of action, which inhibits the
thus can be regarded as a degradation product of CIP. The detected DNA gyrases involved in DNA replication, recombination and tran-
metabolites F6 and F9 were reported to be produced by the brown scription [53]. The results highlight the important potential impacts
rot fungus Gloeophyllum striatum [37]. In our incubations, degra- of CIP on microbial ecosystem services, such as nutrient recycling.
dation pathways mediated by hydroxyl radical attack [37] were Although readily sorbed to the soil matrix, CIP was already fully
most common. Metabolite F9 can either be formed by the loss effective at the lowest concentration employed, which is similar to
of the piperazine moiety or be formed by photodegradation [42], what was described for sulfadiazine [54]. Since microorganisms are
which can proceed rapidly in surface water [43]. Additionally, living attached to soil particles, the CIP sorbed on soil surfaces may
sarafloxacin was degraded abiotically immediately after contact to be still bioavailable and thus toxic for microorganisms. Another
soil by surface-catalyzed hydrolysis or by oxidation resulting in a explanation can be the fact that the maximum effect on target bac-
polar transformation product [41]. teria was already obtained with the lowest concentration studied.
CIP proved to be extensively degraded by soil fungi [37,44], Presumably, the microorganisms inhabiting our soil have a lower
but these results were obtained under special artificial conditions EC50 than P. putida mt-2. Therefore, it would be interesting to know
(i.e. one fungal species degrading CIP in pure culture with optimal more about the effect of lower concentrations, even those below the
growth conditions) and may not be relevant for the real environ- detection limit of chemical analysis (5 ␮g kg−1 , Table 1).
ment. Golet et al. [5] proposed an initial phase of biodegradation
followed by long term persistence in soil. They explained this by 4.3. Implications for environmental risk assessment of
the aging of CIP residues in soil or by reaching the biodegradation ciprofloxacin
concentration threshold, but they did not consider the toxicity of
CIP and how it inhibits soil microbial activity. Predicted environmental concentration for CIP in soil indi-
cated the need for assessing its environmental fate and effects
[5]. The present study was conducted using environmentally rel-
4.2. Toxicity and bioavailability evant CIP concentrations [10–12]. It provided strong evidence for
a high persistence of CIP in both our aqueous system and in soil,
In general, toxicity was higher in water than in soil. Sorption of and demonstrated the negative effects of fluoroquinolones on soil
toxicants is one of the main mechanisms controlling toxicity via and water ecosystems. These results contradict with the previ-
reduction of bioavailability [38]. Toxicity in soil declines with time ous assessments of low persistence and low ecological risk of CIP
because of aging and transformation to less toxic molecules. Sorp- [37,45].
tion and desorption of compounds in soil systems play key roles for The EC50 of CIP varies over a wide range. EC50 of 0.006 mg L−1
their environmental fate, even though sorption of CIP to soil does and 0.61 mg L−1 were reported for sewage sludge bacteria
not completely inactivate this compound. [46,55]. The EC50 for Microcystis aeruginosa (cyanobacteria) was
Ciprofloxacin persists even after stabilisation of activated sludge 0.005 mg L−1 but 2.97 mg L−1 for Selenastrum capricornutum (algae;
under methanogenic conditions [5]. However, it may lose its antibi- [55]). Moreover, at relevant environmental concentrations [10,11]
otic potential under such conditions [16]. We detected some CIP has phytotoxic effects on the aquatic plant Lemna gibba EC25
transformation products in soil. According to Wetzstein et al. 271 ␮g L−1 ; [56]. These results are in the same range as the EC50
[37,45], CIP loses its antibacterial activity by defluorination, decar- 0.25 mg L−1 . We determined for P. putida mt-2 and reported CIP
boxylation, hydroxylation or oxidation of the amine moiety. These concentrations in soil [12]. Therefore, according to the European
processes, however, are unlikely to occur in our soil. In general, the legislation [57] CIP can be classified as “very toxic to aquatic organ-
decline in the antimicrobial potential was slow and incomplete as isms” (EC50 below 1 mg L−1 ) and “toxic to soil organisms”. These
previously reported for sludge [46]. Possible reasons are incomplete results and the strong inhibition of the soil microbial activity, as
transformation of the molecule and the stability of the fluorine sub- well as the induced shift in the microbial community abundance
stituent at the aromatic C-6 position [47], which is crucial for the and composition reported here, underline the strong antibacterial
antibiotic potency [2]. power of CIP and its hazardous consequences on the environment.
Enrofloxacin labelled in the piperazine moiety or the carboxyl Moreover, CIP derived NER apparently are still toxic to soil bac-
group, which are suggested as good indicators for the antibi- teria. Our results agree with a life cycle assessment of pollutants
otic activity and degradability of the compound, was extensively in waste water, which showed that CIP contributes significantly to
degraded [37,45]. Our conservative approach, using the label in one ecotoxicity in terrestrial and freshwater systems [58].
of the most stable carbon positions of the molecule, contradicted Moreover, the qnrS CIP resistance gene appeared after 14 days
this extensive degradation and provided consistent results of both of exposure, independent of the CIP concentrations studied. Resis-
low degradation and inactivation of the antibiotic. tance development is promoted by continuous exposure of bacteria
 C. Girardi et al. / Journal of Hazardous Materials 198 (2011) 22–30 29

to concentrations below therapeutic levels [2]. This is exactly what [5] E.M. Golet, I. Xifra, H. Siegrist, A.C. Alder, W. Giger, Environmental exposure
occurs in soil, where due to the reduced bioavailability, bacteria are assessment of fluoroquinolone antibacterial agents from sewage to soil, Envi-
ron. Sci. Technol. 37 (2003) 3243–3249.
exposed to lower effective concentrations of CIP. Therefore soils can [6] A.B.A. Boxall, D.W. Kolpin, B. Halling-Sørensen, J. Tolls, Peer reviewed: are vet-
be an important source of resistant bacteria that can transfer the erinary medicines causing environmental risks? Environ. Sci. Technol 37 (2003)
corresponding genes to other bacteria living in ground or drinking 286A–294A.
[7] C.G. Daughton, T.A. Ternes, Pharmaceuticals and personal care products in the
water. Under appropriate conditions, these genes can eventually be environment: agents of subtle change? Environ. Health Perspect. 107 (1999)
transferred to pathogenic microorganisms [59]. 907–938.
Consequently, our knowledge on the fate and effects of pharma- [8] S. Thiele-Bruhn, Pharmaceutical antibiotic compounds in soils – a review, J.
Plant Nutr. Soil Sci. 166 (2003) 145–167.
ceuticals in the environment and their degradation products must
[9] E. Topp, S.C. Monteiro, A. Beck, B.B. Coelho, A.B.A. Boxall, P.W. Duenk, S.
be improved for proper risk assessment, e.g. for the normally occur- Kleywegt, D.R. Lapen, M. Payne, L. Sabourin, H. Li, C.D. Metcalfe, Runoff of phar-
ring mixtures of pharmaceuticals which have stronger effects than maceuticals and personal care products following application of biosolids to an
agricultural field, Sci. Total Environ. 396 (2008) 52–59.
single compounds [60,61]. Improved strategies to remediate sludge
[10] D.G. Larsson, C. de Pedro, N. Paxeus, Effluent from drug manufactures con-
and manure contaminated with antibiotics or to restrict their appli- tains extremely high levels of pharmaceuticals, J. Hazard. Mater. 148 (2007)
cation to agricultural fields are needed to avoid the input of these 751–755.
compounds into the soil ecosystem. [11] E.M. Golet, A. Strehler, A.C. Alder, W. Giger, Determination of fluoroquinolone
antibacterial agents in sewage sludge and sludge-treated soil using accelerated
solvent extraction followed by solid-phase extraction, Anal. Chem. 74 (2002)
5. Conclusion 5455–5462.
[12] E. Martínez-Carballo, C. González-Barreiro, S. Scharf, O. Gans, Environmental
monitoring study of selected veterinary antibiotics in animal manure and soils
This work contributes to the proper environmental risk assess- in Austria, Environ. Pollut. 148 (2007) 570–579.
ment of fluoroquinolone antibiotics, for which insufficient and [13] M. Uslu, A. Yediler, I. Balcıoğlu, S. Schulte-Hostede, Analysis and sorption behav-
ior of fluoroquinolones in solid matrices, Water Air Soil Pollut. 190 (2008)
contradictory data on their environmental fate and effects are avail- 55–63.
able. We clearly demonstrated that CIP is persistent and affects [14] D. Vasudevan, G.L. Bruland, B.S. Torrance, V.G. Upchurch, A.A. MacKay, pH-
the microbial communities and activities in soil. Therefore, much dependent ciprofloxacin sorption to soils: interaction mechanisms and soil
factors influencing sorption, Geoderma 151 (2009) 68.
more attention has to be given to antibiotic contamination in soil,
[15] S.J. Rooklidge, Environmental antimicrobial contamination from terraccumu-
which often has been neglected. Furthermore, we conclude that for lation and diffuse pollution pathways, Sci. Total Environ. 325 (2004) 1–13.
toxic compounds in soil, reduced bioavailability results in reduced [16] A.L. Cordova-Kreylos, K.M. Scow, Effects of ciprofloxacin on salt marsh sediment
microbial communities, ISME J. 1 (2007) 585–595.
effective toxicity and higher biodegradation compared to aqueous
[17] J.D. Maul, L.J. Schuler, J.B. Belden, M.R. Whiles, M.J. Lydy, Effects of the antibiotic
systems. However, even if soil has a buffering capacity against toxic ciprofloxacin on stream microbial communities and detritivorous macroinver-
compounds, it does not inhibit their antimicrobial activity com- tebrates, Environ. Toxicol. Chem. 25 (2006) 1598–1606.
pletely. Compound dissipation with low or without mineralisation [18] J. Näslund, J.E. Hedman, C. Agestrand, Effects of the antibiotic ciprofloxacin
on the bacterial community structure and degradation of pyrene in marine
indicates abiotic NER formation, which may contain toxic parent sediment, Aquat. Toxicol. 90 (2008) 223–227.
compound and/or its primary metabolites. [19] B.A. Wilson, V.H. Smith, F. Denoyelles, C.K. Larive, Effects of three pharma-
Fluoroquinolones have significant effects on environmental pro- ceutical and personal care products on natural freshwater algal assemblages,
Environ. Sci. Technol. 37 (2003) 1713–1719.
cesses and ecosystems services. The effects of these compounds on [20] P.N. Gaunt, L.J.V. Piddock, Ciprofloxacin resistant Campylobacter spp. in
specific soil processes such as nutrient or carbon cycles, and the humans: an epidemiological and laboratory study, J. Antimicrob. Chemother.
adaptation of the microbial communities to continuous applica- 37 (1996) 747–757.
[21] A. Al-Ahmad, F.D. Daschner, K. Kümmerer, Biodegradability of cefotiam,
tion of these compounds with manure or sewage sludge still need ciprofloxacin, meropenem, penicillin G, and sulfamethoxazole and inhibition
to be studied. of waste water bacteria, Arch. Environ. Contam. Toxicol. 37 (1999) 158–163.
[22] B. Halling-Sørensen, Inhibition of aerobic growth and nitrification of bacteria
in sewage sludge by antibacterial agents, Arch. Environ. Contam. Toxicol. 40
Acknowledgements (2001) 451–460.
[23] K. Xia, A. Bhandari, K. Das, G. Pillar, Occurrence and fate of pharmaceuticals and
The study was supported by the European Commission fund- personal care products (PPCPs) in biosolids, J. Environ. Qual. 34 (2005) 91–104.
[24] OECD, OECD Guidelines for Testing of Chemicals, Guideline 301B: Ready
ing the RAISEBIO project (Contract: MEST-CT-2005-020984) under Biodegradability – CO2 Evolution Test, OECD, Paris, 1992.
Human Resources and Mobility Activity (6th Framework pro- [25] OECD, OECD Guideline for Testing of Chemicals, OECD 307: Aerobic and Anaer-
gramme) in particular the fellowship of C. Girardi. The stay of obic Transformation in Soil, OECD, Paris, 2002.
[26] N. Blair, R.D. Faulkner, A.R. Till, M. Korschens, E. Schulz, Long-term management
J. Greve at the UFZ was funded by the DAAD-RISE program. The impacts on soil C, N and physical fertility: Part II: Bad Lauchstadt static and
authors thank Paula Martinez, Kerstin Ethner and Hermann Heip- extreme FYM experiments, Soil Tillage Res. 91 (2006) 39–47.
ieper for their contribution in the experimental setup and analyses. [27] M. Weiß, R. Geyer, T. Günther, M. Kaestner, Fate and stability of 14C-labeled
2,4,6-trinitrotoluene in contaminated soil following microbial bioremediation
processes, Environ. Toxicol. Chem. 23 (2004) 2049–2060.
Appendix A. Supplementary data [28] P. Sukul, M. Lamshoft, S. Kusari, S. Zuhlke, M. Spiteller, Metabolism and excre-
tion kinetics of C-14-labeled and non-labeled difloxacin in pigs after oral
administration, and antimicrobial activity of manure containing difloxacin and
Supplementary data associated with this article can be found, in its metabolites, Environ. Res. 109 (2009) 225–231.
the online version, at doi:10.1016/j.jhazmat.2011.10.004. [29] H.J. Heipieper, B. Loffeld, H. Keweloh, J.A.M. de Bont, The cis/trans isomeri-
sation of unsaturated fatty acids in Pseudomonas putida S12: an indicator
for environmental stress due to organic compounds, Chemosphere 30 (1995)
References 1041–1051.
[30] D. Lane, 16s/23S rRNA sequencing, in: M.G.E. Stackebrandt (Ed.), Nucleic Acid
[1] J. Ollivier, K. Kleineidam, R. Reichel, S. Thiele-Bruhn, A. Kotzerke, R. Kindler, Techniques in Bacterial Systematics, Wiley, Chichester, UK, 1991, pp. 11–175.
B.-M. Wilke, M. Schloter, Effect of sulfadiazine-contaminated pig manure on [31] H. Heuer, M. Krsek, P. Baker, K. Smalla, E. Wellington, Analysis of actinomycete
abundance of genes and transcripts involved in nitrogen transformation in the communities by specific amplification of genes encoding 16S rRNA and gel-
root-rhizosphere complexes of maize and clover, Appl. Environ. Microbiol. 76 electrophoretic separation in denaturing gradients, Appl. Environ. Microbiol.
(2010) 7903–7909. 63 (1997) 3233–3241.
[2] Y. Picó, V. Andreu, Fluoroquinolones in soil—risks and challenges, Anal. Bioanal. [32] P. Bombach, A. Chatzinotas, T.R. Neu, M. Kästner, T. Lueders, C. Vogt, Enrichment
Chem. 387 (2007) 1287–1299. and characterization of a sulfate-reducing toluene-degrading microbial consor-
[3] R. Davis, A. Markham, J.A. Balfour, Ciprofloxacin – an updated review of its phar- tium by combining in situ microcosms and stable isotope probing techniques,
macology, therapeutic efficacy and tolerability, Drugs 51 (1996) 1019–1074. FEMS Microbiol. Ecol. 71 (2010) 237–246.
[4] K. Kummerer, A. Al-Ahmad, V. Mersch-Sundermann, Biodegradability of some [33] V. Cattoir, L. Poirel, V. Rotimi, C.-J. Soussy, P. Nordmann, Multiplex PCR for detec-
antibiotics, elimination of the genotoxicity and affection of wastewater bacteria tion of plasmid-mediated quinolone resistance qnr genes in ESBL-producing
in a simple test, Chemosphere 40 (2000) 701–710. enterobacterial isolates, J. Antimicrob. Chemother. 60 (2007) 394–397.
30 C. Girardi et al. / Journal of Hazardous Materials 198 (2011) 22–30

[34] G. Rees, D. Baldwin, G. Watson, S. Perryman, D. Nielsen, Ordination and sig- [47] C. Murphy, B. Clark, J. Amadio, Metabolism of fluoroorganic compounds in
nificance testing of microbial community composition derived from terminal microorganisms: impacts for the environment and the production of fine chem-
restriction fragment length polymorphisms: application of multivariate statis- icals, Appl. Microbiol. Biotechnol. 84 (2009) 617–629.
tics, Antonie van Leeuwenhoek 86 (2004) 339–347. [48] A.J.H. Marshall, L.J.V. Piddock, Interaction of divalent cations, quinolones and
[35] Ø. Hammer, D. Harper, P. Ryan, PAST: paleontological statistics software pack- bacteria, J. Antimicrob. Chemother. 34 (1994) 465–483.
age for education and data analysis, Palaeontologia Electronica 4 (2001) art. [49] N. Esiobu, L. Armenta, J. Ike, Antibiotic resistance in soil and water environ-
4. ments, Int. J. Environ. Health Res. 12 (2002) 133–144.
[36] R Development Core Team, R A language and environment for statistical com- [50] N.R. Krieg, J.G. Holt, Bergey’s Manual of Systematic Bacteriology, 1st edn.,
puting, R Foundation for Statistical Computing, Vienna, Austria, 2011. Williams & Wilkins, Baltimore, 1984.
[37] H.-G. Wetzstein, M. Stadler, H.-V. Tichy, A. Dalhoff, W. Karl, Degradation of [51] K. Schauss, A. Focks, H. Heuer, A. Kotzerke, H. Schmitt, S. Thiele-Bruhn, K. Smalla,
ciprofloxacin by basidiomycetes and identification of metabolites generated B.-M. Wilke, M. Matthies, W. Amelung, J. Klasmeier, M. Schloter, Analysis, fate
by the brown rot fungus Gloeophyllum striatum, Appl. Environ. Microbiol. 65 and effects of the antibiotic sulfadiazine in soil ecosystems, Trends Anal Chem.
(1999) 1556–1563. 28 (2009) 612–618.
[38] G. Welp, G.W. Brümmer, Effects of organic pollutants on soil microbial activity: [52] S. Thiele-Bruhn, I.-C. Beck, Effects of sulfonamide and tetracycline antibiotics
the influence of sorption, solubility, and speciation, Ecotoxicol. Environ. Saf. 43 on soil microbial activity and microbial biomass, Chemosphere 59 (2005) 457.
(1999) 83–90. [53] R. Moore, B. Beckthold, S. Wong, A. Kureishi, L. Bryan, Nucleotide sequence
[39] S. Thiele, Adsorption of the antibiotic pharmaceutical compound sulfapyridine of the gyrA gene and characterization of ciprofloxacin-resistant mutants of
by a long-term differently fertilized loess Chernozem, J. Plant Nutr. Soil Sci. 163 Helicobacter pylori, Antimicrob. Agents Chemother. 39 (1995) 107–111.
(2000) 589–594. [54] A. Kotzerke, S. Sharma, K. Schauss, H. Heuer, S. Thiele-Bruhn, K. Smalla, B.-M.
[40] Y. Chen, J.P.N. Rosazza, C.P. Reese, H.Y. Chang, M.A. Nowakowski, J.P. Kiplinger, Wilke, M. Schloter, Alterations in soil microbial activity and N-transformation
Microbial models of soil metabolism: biotransformations of danofloxacin, J. Ind. processes due to sulfadiazine loads in pig-manure, Environ. Pollut. 153 (2008)
Microbiol. Biotechnol. 19 (1997) 378–384. 315–322.
[41] J.R. Marengo, R.A. Kok, K. O’Brien, R.R. Velagaleti, J.M. Stamm, Aerobic biodegra- [55] B. Halling-Sørensen, H.-C.H. Lützhøft, H.R. Andersen, F. Ingerslev, Environmen-
dation of (14C)-sarafloxacin hydrochloride in soil, Environ. Toxicol. Chem. 16 tal risk assessment of antibiotics: comparison of mecillinam, trimethoprim and
(1997) 462–471. ciprofloxacin, J. Antimicrob. Chemother. 46 (2000) 53–58.
[42] P. Calza, C. Medana, F. Carbone, V. Giancotti, C. Baiocchi, Characteriza- [56] R.A. Brain, D.J. Johnson, S.M. Richards, H. Sanderson, P.K. Sibley, K.R. Solomon,
tion of intermediate compounds formed upon photoinduced degradation Effects of 25 pharmaceutical compounds to Lemna gibba using a seven-day
of quinolones by high-performance liquid chromatography/high-resolution static-renewal test, Environ. Toxicol. Chem. 23 (2004) 371–382.
multiple-stage mass spectrometry, Rapid Commun. Mass Spectrom. 22 (2008) [57] European Commission, Commission Directive 93/21/EEC of 27 April 1993
1533–1552. adapting to technical progress for the 18th time Council Directive 67/548/EEC
[43] M.W. Lam, K. Tantuco, S.A. Mabury, PhotoFate: a new approach in accounting on the approximation of the laws, regulations and administrative provisions
for the contribution of indirect photolysis of pesticides and pharmaceuticals in relating to the classification, packaging and labelling of dangerous substances,
surface waters, Environ. Sci. Technol. 37 (2003) 899–907. in Off. J. Eur. 1993.
[44] I.A. Parshikov, J.P. Freeman, J.O.J. Lay, R.D. Beger, A.J. Williams, J.B. Sutherland, [58] I. Muñoz, M. José Gómez, A. Molina-Díaz, M.A.J. Huijbregts, A.R. Fernández-Alba,
Regioselective transformation of ciprofloxacin to N-acetylciprofloxacin by the E. García-Calvo, Ranking potential impacts of priority and emerging pollutants
fungus Mucor ramannianus, FEMS Microbiol. Lett. 177 (1999) 131–135. in urban wastewater through life cycle impact assessment, Chemosphere 74
[45] H.-G. Wetzstein, J. Schneider, W. Karl, Comparative biotransformation of flu- (2008) 37–44.
oroquinolone antibiotics in matrices of agricultural relevance, in: Veterinary [59] J.L. Martinez, Environmental pollution by antibiotics and by antibiotic resis-
Pharmaceuticals in the Environment, American Chemical Society, Washington, tance determinants, Environ. Pollut. 157 (2009) 2893–2902.
DC, 2009, pp. 67–91. [60] T. Backhaus, M. Scholze, L.H. Grimme, The single substance and mixture toxicity
[46] B. Halling-Sørensen, G. Sengeløv, F. Ingerslev, L.B. Jensen, Reduced antimi- of quinolones to the bioluminescent bacterium Vibrio fischeri, Aquat. Toxicol.
crobial potencies of oxytetracycline, tylosin, sulfadiazin, streptomycin, 49 (2000) 49–61.
ciprofloxacin, and olaquindox due to environmental processes, Arch. Environ. [61] M. Cleuvers, Aquatic ecotoxicity of pharmaceuticals including the assessment
Contam. Toxicol. 44 (2003) 0007–0016. of combination effects, Toxicol. Lett. 142 (2003) 185–194.