Photo-degradation of the antimicrobial ciprofloxacin at high pH: Identification and biodegradability assessment of the primary by-products

Chemosphere 76 (2009) 487–493

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Photo-degradation of the antimicrobial ciprofloxacin at high pH: Identification and biodegradability assessment of the primary by-products Tibiriçá G. Vasconcelos a,b, Danielle M. Henriques a, Armin König b, Ayrton F. Martins a, Klaus Kümmerer b,* a b

Department of Chemistry of the Federal University of Santa Maria, 97105-900 Santa Maria-RS, Brazil Department of Environmental Health Sciences, University Medical Center Freiburg, Breisacherstraße 115b, D-79102 Freiburg, Germany

a r t i c l e

i n f o

Article history: Received 9 December 2008 Received in revised form 11 March 2009 Accepted 12 March 2009 Available online 17 April 2009 Keywords: Pharmaceuticals Photo-process Photo-products Biodegradability assessment

a b s t r a c t Photo-treatment for the removal of pharmaceuticals in effluents is a topic currently under discussion. In some countries effluents from hospitals are directly emitted into open ditches without any further treatment and with very little dilution. Under such circumstances photo-degradation in the environment can occur. However, photo-degradation does not necessarily end up with the complete mineralization of a chemical. Therefore, photo-product biodegradability and toxicity against environmental bacteria is of interest. Hospital effluents have often a pH around 9. Therefore, photo-oxidation (150 W medium-pressure Hg-lamp, batch reactor) of ciprofloxacin (CIP) was studied at pH 9. The primary elimination of CIP was monitored and structures of photo-products were assessed by liquid chromatography ion trap mass spectrometry (LC–MS/MS). Five compounds were identified as probable products of photo-defluorination, -decarboxylation and loss of the piperazine moiety. These photo-products were not biodegradable in the Closed Bottle test – OECD 301D. They did not affect Vibrio fisheri in the applied concentrations. Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction The presence of pharmaceuticals in the environment has attracted attention within the scientific community around the world. In the literature, many reports about the occurrence, fate, effects and risks of these compounds are available (Kümmerer, in press, 2009). Efforts to eliminate them from waste- and drinking water have been growing recently (Huber et al., 2005; Joss et al., 2005). Photo-oxidation is under discussion as a possible tool to remove active compounds through advanced sewage treatment. Additionally, compounds are directly emitted into water bodies in countries lacking effluent treatment. There, photolysis may also happen to some extent if the compounds are emitted into open ditches. However, little is known about the fate of the resulting photo-products. Very often it is argued that the photo-products are better biodegradable and less toxic than the parent compounds. However, data are lacking. Antibiotics are one of the most important groups of pharmaceuticals. They often have little to no biodegradability (Kümmerer et al., 2000; Alexy et al., 2004) and can have toxic effects on bacteria. They can also contribute to the development of resistant bacteria in aqueous systems (Kümmerer, 2009). Ciprofloxacin (CIP) is a broad-spectrum fluoroquinolone antimicrobial which is active against both gram-positive and gram-negative bacteria and is often used to treat human or animal bacterial infections. * Corresponding author. Tel.: +49 761 270 8235; fax: +49 761 270 8213. E-mail address: [email protected] (K. Kümmerer). 0045-6535/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2009.03.022

CIP has been found in hospital wastewater, sewage treatment plants and surface water (Golet et al., 2002a; Karthikeyan and Meyer, 2006; Vieno et al., 2007; Watkinson et al., 2007). In the literature it is described that specific conditions can lead to CIP environmental concentrations 5–20 000-fold higher than that reported usually (Martins et al., 2008). CIP can adsorb to sludge, sediment and soil (Golet et al., 2002b, 2003; Lindberg et al., 2006, 2007). Levels of CIP found in the environment can be a risk (Halling-Sørensen et al., 2000; Kümmerer et al., 2000; Lindberg et al., 2007). CIP can be degraded by photolysis (Burhenne et al., 1997; Pereira et al., 2007). Variations in pH and exposure time can result in the formation of different by-products via different pathways (Burhenne et al., 1997). Because of this photo-sensitivity, phototechnologies seem to be a promising tool in the treatment of wastewater containing fluoroquinolones such as CIP. In countries lacking appropriate effluent treatment, photo-degradation in open water could be an important attenuation mechanism. Photo-degradation is involved in the removal of CIP from natural aqueous compartments (Cardoza et al., 2005; Belden et al., 2007). However, very few investigations have addressed the fate of the photo-products in the environment. In this study, photo-induced oxidation of CIP and the nature of some photo-products formed under alkaline pH, which to date had not been investigated, were identified by liquid chromatography ion trap mass spectrometry (LC–MS/MS). Additionally, the biodegradability of CIP photo-transformation products was assessed by the Closed Bottle test (CBT; OECD 301D, 1992). Furthermore, the Vibrio fischeri test was applied in order to assess bacterial toxicity.

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2. Experimental 2.1. Chemicals All the chemicals used in this study were of analytical grade and were used without further purification. Ciprofloxacin (CIP) (CAS number 85721-33-1) was obtained from Fluka (Sigma–Aldrich, Steinheim, Germany). HPLC grade acetonitrile and methanol were purchased from Mallinckrodt Baker, Deventer, Netherlands. KOH, NH4Cl, formic acid, and o-H3PO4 were purchased from Merck, Darmstadt, Germany. 2.2. Kinetic study Aqueous solutions of CIP were adjusted to the desired pH with NH4Cl (0.050 mol L1 plus KOH 3 mol L1). The CIP concentration was 100 lg L1 (0.3  106 mol L1), which is similar to that found in wastewater (effluent of the Hospital of University of Santa Maria, Brazil; Martins et al, 2008). Samples were taken at different reaction times for the analysis of the CIP concentration as described below.

sion of a 10 lg mL1 standard at a flow rate of 4 lL min1 using a SGE syringe (500 lL). The trap parameters were set with a specific target mass of 332 m z1 and a maximum accumulation time of 200 ms at a scan range from 100 m z1 to 1000 m z1. The limit of detection (LOD) was 0.2 lg mL1, the limit of quantification (LOQ) was 0.1 lg mL1. The software used for the acquisition of the data was Bruker Daltonics esquire 5.1. 2.5. Biodegradability of photo-products The CBT is recommended as a first, simple test for the assessment of the biodegradability of organic compounds (OECD, 1992). It was performed with deionised water in the dark at room temperature (20 ± 1 °C), according to guidelines (OECD, 1992). The inoculum was taken from the effluent of a municipal waste water treatment plant (Abwasserzweckverband Breisgauer Bucht, Forchheim, Germany), filtered (Schleicher & Schuell, Dassel, Germany, order-no. 0851 1/2) and the first 200 mL were discarded (OECD, 1992). During the CBT, the three incubated samples (in replicate) were monitored on the 1st, 7th, 14th, and 28th day. 2.6. Bacterial toxicity of photo-products

2.3. Photo-degradation The experiments were conducted in a 2.5 L batch photo-reactor provided with magnetic stirring and a cooling system. A Heraeus (www.heraeus.com) 150 W medium-pressure mercury lamp was used with the aid of a quartz immersion tube (over all length 384 mm, immersion was 297 mm, main light emission at 41 mm from the lowest point). Prior to each experiment, the lamp was warmed up for 2 min. Since alkaline pH is often observed in hospital effluents, the pH of the solutions was adjusted to 9 with 0.1 M KOH. All experiments were performed at 30 °C and Milli-Q purified water was used to prepare all the solutions. In order to reach the adequate theoretical oxygen demand conditions for the Closed Bottle test (CBT), photo-processes were carried out using high CIP concentration 6 mg L1 and in large sampling volumes (2 L). Three experiments were run in order to get the necessary aliquots at three different time points (0, 2 and 4 min).

Samples collected at 0 and 4 min during the photo-degradability trial were used to assess the capacity of photo-processes to reduce bacterial toxicity with the luminescent bacterium V. fischeri. This method is one of the most widely employed in wastewater and environmental monitoring, and is based on the empirical observation of the decay of the light emitted by V. fischeri when stressed by toxic compounds. A modified method based on ISO/ CD 11348 (1994) and the Lumistox LCK 482 (Dr. Lange GmbH) was used. Data analysis and graphics were done using the software packages Excel and R (version 2.2.1), respectively. 3. Results and discussion The general objective of this study was to assess biodegradability and toxicity of photo-products of ciprofloxacin (CIP) solutions and to correlate this information with data obtained from the photo-product analysis.

2.4. LC–MS/MS analysis 3.1. Kinetic studies In order to monitor the CIP concentration within the CBT and to investigate its photo-products, LC–MS/MS was performed using an Agilent Series 1100 LC (Agilent Technologies, www.chem.agilent.com) equipped with two binary pumps, a degasser, a column oven, an auto sampler and a fluorescence detector (Agilent G 1321A, excitation at 278 nm and emission at 445 nm). A reverse phase column C18 (RP18 CC 125  4 mm Nucleodur 100-5) and a pre-column (RP18 CC 8  4 mm Nucleosil 100-5) were used (Macherey-Nagel, http://www.macherey-nagel.com). A gradient was used for the mobile phase (solvent A formic acid 0.1% in water v/v; solvent B acetonitrile) as follows: 1–40% of B (until 25 min), 40–1% of B (until 30 min). The flow rate was 0.5 mL min1, the injection volume was 25 lL, and the column temperature was maintained at 40 °C. No sample preparation was necessary. Chemstation software (Agilent Technologies) was used for instrument control. The MS-system consisted of an Esquire 3000 plus ion trap (Bruker Daltonics, http://www.bdal.com) with an orthogonal electrospray ionization source. The operating conditions of the source were: 500 V end plate, 3300 V capillary voltage, 30 psi nebulizer pressure, 12.00 L min1 dry gas flow at a dry temperature of 350 °C. The selected lens and block voltages were: 40 V skimmer, 115.9 V capillary exit, 12 V octopole one, 1.7 V octopole two, 37.2% trap driver, 150 V octopole reference and 5 V lens one and 60.0 V lens two. Tuning was performed for CIP by direct infu-

The photo-degradation results of CIP n = 3 were plotted as a function of irradiation time, and the data were assuming first-order degradation rate (Atkins and de Paula, 2006). ½C vs. irradiation time gave a straight line whose A plot of ln ½C0 slope was k, and the regression analysis gave a correlation coefficient (r2) higher than 0.999, which demonstrates the high validity of the assumed first order kinetics (Fig. 1). The results of Gagliano and McNamara (1996) showed that the kinetics of CIP photo-degradation depend on pH, and this was confirmed in our study. The fastest degradation (t1/2 0.15 h) was found at pH 7, which is very close to the iso-electric point of CIP (CIP is a zwitterion, i.e. both positively and negatively charged, but electrically neutral in total). The degradation at pH 5 (CIP is a cation) and pH 9 (CIP is an anion) was slower (t1/2 0.77 h, 0.38 h respectively). First order t1/2 of CIP photo-degradation was estimated as 1.14– 0.97 h via field mesocosm experiments (Burhenne et al., 1997) and 1.9 (quasi natural light source) and 46 h under an artificial light source (Cardoza et al., 2005). In other studies, a first order model resulted in a t1/2 of 2.9 h (Belden et al., 2007) and a pseudo-firstorder rate constant in 1.4  101 min1 (Pereira et al., 2007). Our results were in the range of these values and slightly lower at pH 9. In contrast, Burhenne et al. (1997) established a second-order reaction for the photo-degradation of CIP (t1/2 = 1.5 h).

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0

50

100

15 0

200

25 0

0.0 -0.5 -1.0

ln C/Co

-1.5 -2.0 -2.5 -3.0 y = -0.0153x - 0.0538

-3.5

R2 = 0.9993

-4.0 -4.5

time [s]

Fig. 1. Fist-order plot for the photo-induced oxidation of ciprofloxacin (CIP) in synthetic solution. 30 °C, pH 9, (NH4Cl 0.005 M, KOH 3 M), CIP concentration: 0.1 mg L1 (0.3  106 M).

3.2. Photo-product analysis Preliminary studies of photo-degradation showed that photoproducts were present in high concentrations at 2 and 4 min. Therefore, samples were collected before treatment and at these two times. The level of CIP applied in the photolysis trial is higher than what would be normally measured in wastewater. However, in the literature it is described that specific conditions can lead

6000

to CIP environmental concentrations 5–20 000-fold higher than that reported usually (Martins et al., 2008). The chromatographic behavior demonstrated that most compounds formed by photolysis up until 4 min have a polarity similar to CIP and it was therefore assumed that the procedure used for determination of CIP was adequate in order to identify these photo-products. Two fluorescence peaks, 1 and 2, close to and more polar than CIP i.e. with shorter retention time (RT) on the RP C18 column, first appeared during the photo-induced oxidation process. Peak 1 (RT = 7.5 min; RT of CIP = 8.4 min), was the first to be detected within the course of the photo-degradation trial, and the higher it was, the lower the CIP peak was indicating that this was the photo-product formed first. The second peak (RT = 7.2 min) arised later within the photo-treatment. In the further reaction sequence both peaks decreased until they finally disappeared. This indicates that the ring systems was still intact in these intermediates and was degraded later within the course of the reaction sequences. The mass spectra obtained for the irradiated samples (0, 2 and 4 min) at the retention times of peaks 1 and 2 are demonstrated in Fig. 2 and 3, respectively. With regard to peak 1 it should be pointed out that the peaks at m z1 306.5 and 217.3 increased until the 2 min irradiation, after which they then decreased. The m z1 306.5 (Fig. 2) has the same specific mass as 7-[(2-aminoethyl)amino]-6-fluoroquinoline (product 1, Fig. 4) formed by the loss of the piperazine ring. This pathway was widely reported in the literature as being the primary one for CIP photo-degradation under acidic conditions (Burhenne et al., 1997; Mella et al., 2001). Previous studies demonstrated that with longer irradiation times, compound 1 is further photo-degraded, resulting in 7-amino-6-fluoroquinoline (Burhenne et al., 1997). However, this species

+MS, 7.5min (#212)

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4000 2000 0

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328.4

268.4

217.3 235.3

288.5 306.5 328.4

250

300

357.2

350

455.5

400

450

500 m/z

Fig. 2. Mass spectra of irradiated synthetic samples of ciprofloxacin (CIP) at the same retention time as signal 1. Photo-induced oxidation conditions: 30 °C, pH 9 (without buffer). CIP concentration: 6 mg L1 (1.81  105 M).

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+MS, 7.2min (#204)

6000 4000 2000 193.2

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330.5

0 250

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399.4

350

475.0

400

450

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Fig. 3. Mass spectra of irradiated synthetic samples of ciprofloxacin (CIP) at the same retention time as signal 2. Photo-induced oxidation conditions: 30 °C, pH 9 (without buffer). CIP concentration: 6 mg L1 (1.81  105 M).

was not detected in the present study. Despite the fact that these compounds were preferentially found under acidic conditions, they have been observed to a very low extent at neutral pH (Mella et al., 2001), as well as in alkaline medium (pH 8.6 and 10.6; Torniainen et al., 1996). According to the authors, under such alkaline conditions several additional photo-degradation products were detected, corroborating our results. However, no consistent information was found regarding the mechanism of these reactions. Albini and Monti (2003) have suggested possible hydrogen

O

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O O

OH

N H

NH2

F

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N

N

Product 5 [M + H]+ = 288

CIP

O

N

HO

O

N

OH

NH2

N H

N

N

HN

O

HN Product 2 [M + H]+ = 330

O

O OH

O

F OH

N

Product 1 [M + H]+ = 306

N

abstraction either through some excited state or by hydroxyl radicals arising through the direct oxidation of water or the activation of residual dioxygen. Because an air-flushed solution was used in our study, the latter hypothesis must especially be considered here. Regarding peak 2, the m z1 288.5 and 268.4 demonstrated behavior similar to peak 1. Different pathways can be considered for m z1 288.5: The formed species could correspond to the loss of the fluorine atom at position 6 in addition to the break of the

O OH

N

HN Product 4 [M + H]+ = 330

Product 3 [M + H]+ = 288

Fig. 4. Putative photo-products of ciprofloxacin (CIP) identified by LC–MS analysis.

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piperazine ring forming compound 3 (Fig. 4) referred to above. Photo-degradation of CIP in the presence of an electron donor leads to electron transfer induced defluorination (Mella et al., 2001). This happens through quenching of the formed transient triplet state by an electron donor generating radical anion a (Fig. 5). The next step involves fluoride loss to form an aryl radical b. If, for example, phosphate is present, reduction of the radical does not take place, but instead a H-atom is abstracted from the side chain, and diradical d is formed. This species undergoes ring cleavage and water addition to give compound 2, and afterwards, compound 3 (Fig. 5; Mella et al., 2001). Compound 2 represents M + H+ = 330. A weak peak (m z1 330.5) was identified at the retention time of

O

O

F

O OH

N

288.5. Compound 2 was also formed at a low concentration during the irradiation of CIP in pure water (Mella et al., 2001). This would follow the well-known mechanism of hydrogen abstraction by hydroxyl radical. Taking into account the electrophilic character of hydroxyl radicals, the reaction between hydroxyl radicals and the species, either a, or b, must be considered. The product would be compound 4 (M + H+ = 330, Fig. 5). The saturation of the solution with air should be considered, since with an air-saturated solution a lower yield of the defluorination of norfloxacin to form the analogue of product 4 is found compared to an argon-flushed solution (Fasani et al., 1999). Fluoroquinolones in the triplet state

O SO3-.

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hv -e- -CO2

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SO3

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OH

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OH OH

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.

-H2PO4-

b

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Product 4

O

N HN

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SO3

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SO3-.

HN

N

O

O OH

N

N H +N

O

O

O OH

NH2 Product 2

N e

O OH

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O OH

H2O

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N d

c O

O

O

2-

O OH

HN

O

F

X.

CIP3 *

g

HO

N

HN

HN

N H

N

Product 3

Fig. 5. By-products formed and pathways followed during photo-degradation of ciprofloxacin (CIP) at different conditions that are already referred to in the literature.

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can be quenched by oxygen through energy transfer with the formation of singlet oxygen and electron transfer to form the superoxide anion and the fluoroquinolone cation radical (Albini and Monti, 2003). Another possibility is the direct decarboxylation of the CIP forming compound 5 (M + H+ = 288) shown in Fig. 4. As stated earlier, in the current investigation no other electrolyte or buffer was present and only KOH was added to adjust the pH to 9. Under such conditions, HO would work as an electron donor following the mechanisms discussed. Starting from there and taking into account the capacity of quinolones without a piperazine moiety to convert to a triplet state, one can also expect the formation of products 3 and 1. After partial degradation of the piperazine side chain and subsequent formation of product 1, the reductive defluorination results in product 3 (Fig. 6). This pathway also depends on the presence of HO and its capacity to act as an electron donor. Based on the LC–MS/MS analysis, the pathway shown in Fig. 6 should be the most likely to occur. At higher retention times, less polar fluorescent compounds are detected. They were formed after a longer irradiation time. However, the mass spectra of those compounds could not be interpreted with respect to their chemical structure. All the compounds discussed above are expected to be simply the first steps in fluoroquinolone photo-degradation, since the formation of more polar products (second step) and CO2 loss have been observed after longer irradiation times (Burhenne et al., 1997). This is probably what occurred in the current study, since the peaks of the fluorescent compounds disappeared after 30 min when we worked with CIP concentrations of 0.1 lg L1 (work in progress). Additionally, a less intense peak at m z1 330.5 was present. Despite the new conditions used in this investigation (pH 9, non-buffered), products and pathways suggested in the literature were considered (Fig. 4).

The CBT experiments were valid according to the test guidelines (results not shown). Samples which were irradiated exhibited no difference from those prior to treatment, demonstrating that the photo-process did not increase the ready biodegradability or toxicity against the bacteria present. The toxicity control (vessel containing both test mixture obtained from photolysis and readily biodegradable sodium acetate) did not indicate toxicity against the bacteria present. Sometimes, the presence of a secondary carbon source may improve biodegradability. However, that is not always the case. In this test the results for the biodegradation of the CIP photoproducts in the toxicity control did not differ markedly from the ones in the test vessel. Summarizing the CBT showed that the formed photo-products were not biodegradable not even in presence of a secondary ready biodegradable carbon source. 3.4. Toxicity assessment In this study the inhibition of acute bioluminescence in V. fischeri was used to assess the bacterial toxicity of the photoproducts. The test was considered to be valid as inhibition values between 40% and 60% after incubation time of 30 min were obtained for standard solutions (7.5% NaCl). CIP showed no effects against V. fisheri in a concentration up to 0.3 mg L1. Other studies have demonstrated that photolysis can lead to a loss in the antimicrobial activity of CIP (Phillips et al., 1990). The antimicrobial activity of fluoroquinolones is believed to derive from the quinolone moiety (Dodd et al., 2006). As all identified photo-products in this study preserved this part of the molecule, it could be expected that their activity against bacteria was still present. Earlier studies demonstrated that 5 mg L1 of CIP causes an inhibition effect of 28% to V. fischeri (Hernando et al., 2007). In our study (starting with 6 mg L1 of CIP) after 4 min of irradiation an effect against V. fischeri was not detectable. This finding indicates that the total toxicity of CIP still present and the photo-products already formed has been reduced, i.e. the toxicity of the photo-products is probably not higher than that of CIP itself. The concentrations used in this study were significantly higher than the ones normally measured in the environment. Therefore, it is open for discussion whether antimicrobial activity of CIP-photo-products in the environment is of importance. Although short term standard assays are recommended for the assessment of aquatic toxicity, their suitability for that purpose is controversial and will not always be adequate (Backhaus et al.,

3.3. Biodegradability of the photo-products The same samples analyzed by LC–MS/MS were submitted to the Closed Bottle test (CBT) in order to obtain biodegradability information about the by-products formed during the photo-process. Analysis of the photo-products from the current study (see Section 3.2) showed that only a substitution/loss of the fluorine atom, a decarboxylation reaction and partial break of the piperazine moiety occurred. The identified compounds were very similar to CIP. 36% and 20% of the initial concentration of CIP were found in solution after 2 and 4 min irradiation time, respectively.

O

O

F

NH2

O

.

HO- HO

OH

N H

Product 13*

N

O

F

NH2

OH

N H

N i

Product 1

O

O

O OH

NH2

N H

OH

+H+

N HO

.

HO-

O

NH2

N H

N j

Product 3 Fig. 6. Most probable by-products formed and pathway followed during photo-degradation of ciprofloxacin in the present study.

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1997; Backhaus and Grimme, 1999; Froehner et al., 2000; Kümmerer et al., 2004). In the specific case of the bioluminescence inhibition assay, the toxicity of substances that affect biosynthetic pathways supporting growth and reproduction can only be determined if the bioassay which is used covers an adequate time period of the cell cycle. Incubation times of 30 min or less are too short for this. In cases such as this, long term assays (e.g. 24 h) give more realistic results, whereas short term assays underestimate or even fail to detect the toxicity (Backhaus et al., 1997; Backhaus and Grimme, 1999; Kümmerer et al., 2004; Dodd et al., 2006). This is the case with most antibiotics. However, it is important to keep in mind that the aim of this study was only to roughly compare the toxicity of the parent compound and its photo-products as a very first step in understanding. More detailed studies, especially those applying different toxicity tests and longer irradiation times, will be necessary. A 24 h test with Vibrio fisheri is currently under development. 4. Conclusion In this study it was confirmed that primary photo-elimination of CIP is very rapid and that these kinetics depend on pH. Against wide-spread expectation, the photo-products of CIP were not biodegradable and may pose a risk to the environment. There may be different photo-products in the environment too as photo-type-II reactions may take place and turbidity of the water is higher. However, the products resulting from the first and second level photo-products in our test, that could not be identified here individually, were not readily biodegradable. Effects on bacteria have to be investigated more detailed. One cannot assume full detoxification of hospital effluents emitted directly into open ditches or surface water. Therefore, it is not only primary or secondary elimination by photo-degradation that is of interest but the degree of mineralization. Acknowledgements Dr. Jürgen Steck (University of Freiburg) generously supplied the photo-reactor. T.V. thanks the Brazilian Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq-CT/Hidro) and the German Academic Exchange Service (DAAD) for their sponsorship and financial support. References Albini, A., Monti, S., 2003. Photophysics and photochemistry of fluoroquinolones. Chem. Soc. Rev. 32, 238–250. Alexy, R., Kümpel, T., Kümmerer, K., 2004. Assessment of degradation of 18 antibiotics in the Closed Bottle test. Chemosphere 57, 505–512. Atkins, P.W., de Paula, J., 2006. Atkin‘s Physical Chemistry, eighth ed. Oxford University Press. Backhaus, T., Grimme, L.H., 1999. The toxicity of antibiotic agents to the luminescent bacterium Vibrio fischeri. Chemosphere 38, 3291–3301. Backhaus, T., Froehner, K., Altenburger, R., Grimme, L.H., 1997. Toxicity testing with Vibrio fischeri: a comparison between the long term (24 h) and the short term (30 min) bioassay. Chemosphere 35, 2925–2938. Belden, J.B., Maul, J.D., Lydy, M.J., 2007. Partitioning and photodegradation of ciprofloxacin in aqueous systems in the presence of organic matter. Chemosphere 66, 1390–1395. Burhenne, J., Ludwig, M., Nikoloudis, P., Spiteller, M., 1997. Photolytic degradation of fluoroquinolone carboxylic acids in aqueous solution. Primary photoproducts and half-lives. Environ. Sci. Pollut. Res. 4, 10–15. Cardoza, L.A., Knapp, C.W., Larive, C.K., Belden, J.B., Lydy, M., Graham, D.W., 2005. Factors affecting the fate of Ciprofloxacin in aquatic field systems. Water Air Soil Poll. 161, 383–398. Dodd, M.C., Buffle, M.-O., Gunten, U.V., 2006. Oxidation of antibacterial molecules by aqueous ozone: moiety-specific reaction kinetics and application to ozone-

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