Effects and Risks of Pharmaceuticals in the Environment

Dissertation Thesis in Environmental Chemistry

Radka Zounková

2010

Masaryk University, Faculty of Science, RECETOX – Research Centre for Toxic Compounds in the Environment

Brno, Czech Republic

Supervisor: Assoc. Prof. Blahoslav Maršálek, Ph.D.

Supervisor – specialist: Assoc. Prof. LudˇekBláha, Ph.D.

Bibliographic identiﬁcation

Author: Radka Zounková

Title of dissertation: Effects and Risks of Pharmaceuti- cals in the Environment

Title of dissertation (in Czech): Úˇcinkya rizika léˇcivv životním prostˇredí

Ph.D. study program: Chemistry

Specialization: Environmental Chemistry

Supervisor: Assoc. Prof. Blahoslav Maršálek, Ph.D.

Supervisor - specialist: Assoc. Prof. LudˇekBláha, Ph.D.

Year of defence: 2010

Keywords: pharmaceuticals, metabolites, ecotoxicity, genotoxicity, ecotoxicological bioassays, risk assessment

Keywords (in Czech): léˇciva, metabolity, ekotoxicita, genotoxicita, ekotoxikologické biotesty, analýza rizik ⃝c Radka Zounková, Masaryk University, 2010 Acknowledgements

I would like to thank my supervisor Assoc. Prof. Blahoslav Maršálek, Ph.D. and especially my supervisor - specialist Assoc. Prof. LudˇekBláha, Ph.D. for all the support, advice, motivation and help during my Ph.D. studies. Thanks belong also to Mgr. Klára Hilscherová, Ph.D. for her help at the beginning of my work. I would like to express my gratitude to my colleagues and friends from the RECETOX for their help, advice, and answers to my questions, as well as my colleagues from IHU in Aachen and AQUAbase project-mates for the opportunity to work in such an inspiring and motivating environment. Further I would like to thank Ondráš Pˇribyla,without whom this dissertation would have never been written, my sister Danka, Jakub Mareˇcek, and Petr Tobola, without whom this dissertation would have never looked like it does. Last but not least, I wish to thank to the FRVŠ fund of the Ministry of Education, Youth and Sports and the Marie-Curie Actions Programme of the European Commission for their ﬁnancial support.

Abstract

Pharmaceuticals, diagnostics, and other products of the pharmaceutical industry are widely perceived as helpful. However, when enter the environment, it may not be so. On the contrary, they may have effects as any other chemical compounds polluting the environment. Pharmaceuticals have been developed to have specific effects on living organisms, i.e. humans or animals. As pollutants, they may well affect the environment in similar, but often unforeseen ways. The presented work describes using of classic aquatic bioassays and genotoxicity assays in order to evaluate the risks associated with two groups of pharmaceuticals – veterinary antimicrobials and cytostatics (antineoplastics), and their metabolites. Antimicrobials are used in the treatment of infectious diseases. They aim at killing or controlling growth of bacteria and other micro organisms. Cytostatics are used in cancer treatment. They target rapidly growing eukaryotic cells, which form tumours. Both groups of pharmaceuticals can thus affect the growth and metabolism of cells, which form all the living organisms. These effects may manifest on different levels of ecosystem. This thesis investigates acute and chronic effects – immobilisation, growth inhibition, inhibition of reproduction – and genotoxicity. Within the frame of the three studies, bioassays involving representatives of all main trophic levels of an ecosystem – producers, consumers, and decomposers – were used. Genotoxicity was studied using both prokaryotic and eukaryotic models. Cytostatics are entering the environment in relatively small amounts mainly as more or less changed metabolic products of oncological patients. Some cytostatics, indeed, do not undergo any metabolic reactions at all. With respect to their high toxicity and the mode of action targeted on DNA, they are considered one of the priority groups of pharmaceuticals as far as the effects on ecosystem are concerned. 5-fluorouracil can be highlighted as the most toxic compound, but most tested compounds showed significant effects in all bioassays used. 5-fluorouracil and doxorubicin exhibited the highest genotoxicity, but significant genotoxicity of all tested compounds except cyclophosphamide was confirmed. The human metabolites of cytostatics elicited generally lower or no toxicity in tested concentrations. Only the metabolite of 5-fluorouracil reached values comparable with the toxicity of the less toxic parent compounds. Veterinary antimicrobials enter the environment in considerably higher amounts than cytostatics. This is true especially for substances used for prevention and treatment of fish infections in aquacultures. Results of the complex assessment confirmed that these compounds may have severe effects in aquatic ecosystems. Both compounds tested – oxytetracycine and flumequine – elicited significant effects in most tests. Flumequine elicited markedly higher toxicity in the tests with D. magna and genotoxicity. Risk assessment based on the results of the present dissertation seems to indicate significant risks for both veterinary antimicrobials and highly toxic cytostatic compounds. In summary, this work contributes to the environmental research of pharmaceuticals as an example of micropollutants with a specific mode of action. Compounds used in high quantities may represent a significant risk for the environment, especially when considering their potential long-term effects. Abstrakt (abstract in Czech)

Léky, diagnostické látky a další produkty farmaceutického pr ˚umyslujsou vˇetšinouchápány jako látky zdraví prospˇešné,v životním prostˇredítomu tak však není. Naopak, mohou p ˚usobitjako jakékoli jiné chemické látky, které životní prostˇredízneˇciš´tují. Prvotním úˇcelemlék ˚uje definovaným zp ˚usobemp ˚usobitna živý organismus – tedy ˇclovˇekanebo zvíˇre.Problém je však v tom, že podobný nebo jiný úˇcinekmohou mít léky i poté, co se dostanou do životmího prostˇredí. Pˇredkládanápráce popisuje použití klasických ekotoxikologických biotest ˚ua test ˚ugenotoxicity pro hodnocení úˇcink˚ua rizik dvou skupin lék ˚u– veterinárních antibiotik a cytostatik – a jejich metabolit ˚uv životním prostˇre- dí. Antibiotika se používají k léˇcbˇeinfekˇcníchonemocnˇení,jejich úˇcinekje zamˇeˇrenna mikroorganismy, pˇredevšímbakterie, a to tak, že jejich r ˚ust omezují nebo je zabíjejí. Cytostatika se používají k léˇcbˇerakoviny, jejich úˇcinekje zamˇeˇrenna rychle rostoucí eukaryotické buˇnky, z nichž jsou tvoˇre- ny nádory. Obˇedvˇeskupiny léˇcivtedy mohou ovlivˇnovatr ˚usta metabo- lické pochody bunˇek,z kterých jsou tvoˇrenyvšechny živé organismy, a tento úˇcinekse m ˚užeprojevit na r ˚uznýchúrovních ekosystému. Použité metody zahrnují studium akutních i chronických úˇcink˚u– imobilizaci, inhibici r ˚ustu,inhibici reprodukce – a genotoxicity. V rámci tˇrístudií byly použity testy zahrnují zástupce všech trofických úrovní ekosystému – pro- ducenty, konzumenty i destruenty. Genotoxicita byla studována na proka- ryotickém i eukaryotickém modelu. Cytostatika se do životního prostˇredídostávají v relativnˇemalém množ- ství, a to pˇrevážnˇejako více ˇcíménˇezpracované produkty metabolismu onkologických pacient ˚u;nˇekterácytostatika ovšem nepodstupují metabo- lické reakce v ˚ubec. Vzhledem k vysoké toxicitˇea mechanismu úˇcinku, který je cílen na DNA, jsou považována za jednu z prioritních skupin léˇciv co se týˇcep ˚usobenína ekosystém. Témˇeˇrvšechny testované látky vykazovaly významné úˇcinkyve všech použitých testech. Za nejtoxiˇctˇejšílátku m ˚užemeoznaˇcit5-fluorouracil. Výsledky test ˚ugenotoxicity potvrdily výz- namnou genotoxicitu u všech testovaných látek kromˇecyklofosfamidu. Nej- vyšší genotoxicitu prokázaly 5-fluorouracil a doxorubicin. Testované hu- mánní metabolity cytostatik vykazovaly všeobecnˇenižší nebo žádnou toxicitu v testovaných koncentracích. Pouze metabolit 5-fluorouracilu dosaho- val hodnot srovnatelných s toxicitou ménˇetoxických p ˚uvodníchlátek. Veterinární antibiotika ˇciantimikrobní látky se dostávají do životního prostˇredív ˇrádovˇevˇetšíchmnožstvích. Zvláštˇelátky používané v aqua- kulturách pro prevenci a léˇceníinfekcí ryb jsou aplikovány v podstatných množstvích pˇrímodo vody – pˇredevšímjako medikované krmné smˇesi. Výsledky komplexního testování potvrdily, že tyto látky mohou mít zá- važné úˇcinkyve vodních ekosystémech. Obˇetestované antimikrobní látky používané v aquakulturách – oxytetracyklin a flumequin – vykazovaly výz- namné úˇcinkytémˇeˇrve všech použitých testech. Flumequin vykazoval výraznˇevyšší toxicitu v testech s D. magna a genotoxicitu. Výpoˇctya odhady koeficientu rizika založené na výsledcích pˇredklá- dané práce naznaˇcujívýznamné riziko pro veterinární antibiotika i cytostatika. Pˇredkládanápráce pˇrispívásvými výsledky k výzkumu farmak jakožto mikropolutant ˚use specifickým mechanismem úˇcinku.Takové látky používané ve velkých množstvích mohou pˇredstavovatvýznamné riziko pro životní prostˇredízejména vzhledem ke svým možným dlouhodobým úˇcink˚um. List of abbreviations

5-FU 5-fluorouracil ADaM Aachener Daphnien Medium AF assessment factor APCI atmospheric pressure chemical ionization araU uracil-1-β-D-arabinofuranoside – metabolite of cytarabine ATC Anatomical-Therapeutic-Chemical classification CAS chemical abstract service CIS cisplatin CNI Czech Normalisation Institute CP cyclophosphamide CYT cytarabine DAD diode array detector dFdU 2’,2’-difluorodeoxyuridine – metabolite of gemcitabine DHF dihydrofolic acid DIN Deutsches Institut für Normung DNA deoxyribonucleic acid DOX doxorubicin EC50 half maximal effective concentration – concentration of a toxicant which induces a response halfway between the baseline and maximum ECD electron-capture detector ELISA enzyme-linked immunosorbent assay EMEA European Medicines Agency ESI electrospray ionization ETP etoposide FBAL α-fluoro-β-alanine – metabolite of 5-fluorouracil FETAX frog embryo teratogenesis assay – Xenopus FLU flumequine GC-MS gas chromatography with mass spectrometry detection GemC gemcitabine GFP green fluorescent protein GSA GreenScreen Assay HILIC hydrophilic interaction chromatography IC0 concentration of a toxicant which induces no inhibition ICP-OES inductively coupled plasma optical emission spectrometry IF induction factor IFA ifosfamide ISO International Organisation for Standardisation LC-MS liquid chromatography with mass spectrometry detection LC-MS/MS liquid chromatography with tandem mass spectrometry detection LOEC the lowest observable effect concentration MEC measured environmental concentration MGC minimal genotoxic concentration MIC minimum inhibitory concentration MS mass spectrometry OECD Organisation for Economic Co-operation and Develop- ment OTC oxytetracycline PABA para-aminobenzoic acid PEC predicted environmental concentration PNEC predicted no-effect concentration QSAR the quantitative structure activity relationship RNA ribonucleic acid RQ risk quotient RWTH Rheinisch-Westfälische Technische Hochschule STP sewage treatment plant THF tetrahydrofolic acid UV ultraviolet Contents

1 Aims of the present study ...... 19 2 Introduction ...... 21 2.1 Pharmaceuticals in the environment ...... 21 2.1.1 Sources and occurrence of pharmaceuticals in the environment ...... 22 2.1.2 Environmental fate of pharmaceuticals ...... 26 2.1.3 Effects of pharmaceuticals in the environment . . . . 27 2.1.4 Environmental risks of pharmaceuticals ...... 29 2.2 Cytostatics ...... 30 2.2.1 Pharmacology of cytostatics - mode of action . . . . . 31 2.2.2 Pharmacokinetics of cytostatic drugs ...... 33 2.2.3 Sources and occurrence of cytostatics in the environment ...... 34 2.2.4 Fate of cytostatics in the environment ...... 35 2.2.5 Effects of cytostatics in the environment ...... 36 2.2.6 Cytotoxic drugs studied in the present work . . . . . 38 2.3 The issue of antimicrobials in the environment ...... 38 2.3.1 Mechanism of action of antimicrobial drugs . . . . . 39 2.3.2 Sources and occurrence of antimicrobials in the environment ...... 41 2.3.3 Environmental fate of antimicrobials ...... 42 2.3.4 Ecotoxic and genotoxic effects of antimicrobials . . . 43 2.3.5 Veterinary antimicrobials ...... 44 2.3.6 Investigated aquaculture antimicrobials ...... 46 3 Materials and methods ...... 49 3.1 Tested compounds ...... 49 3.1.1 Cytostatics ...... 49 3.1.2 Veterinary antimicrobials ...... 51 3.2 Ecotoxicological bioassays and chemical analyses ...... 54 3.3 Data analysis ...... 61 3.4 Risk characterisation ...... 61 4 Results and discussion ...... 63

13 4.1 Cytostatics ...... 63 4.1.1 Ecotoxicity and genotoxicity assessment of cytostatic pharmaceuticals (Paper I) ...... 63 4.1.2 Ecotoxicity and genotoxicity assessment of cytotoxic antineoplastic drugs and their metabolites (Paper II) 69 4.2 Complex evaluation of ecotoxicity and genotoxicity of antimicrobials oxytetracycline and ﬂumequine widely used in aquaculture ...... 78 4.3 Environmental relevance of data obtained in experimental laboratory studies ...... 84 4.3.1 Cytostatic agents ...... 84 4.3.2 Aquaculture antimicrobials ...... 85 5 General discussion and conclusions ...... 87

14 List of original articles

Paper I

Zounková, R., Odráška, P., Doležalová, L., Hilscherová, K., Maršálek, B., and Bláha, L. (2007): Ecotoxicity and Genotoxicity Assessment of Cytostatic Pharmaceuticals. Environmental Toxicology and Chemistry 26(10): 2208-2214

Paper II

Zounková, R., Kovalová, L., Bláha, L., Dott, W.: Ecotoxicity and Genotoxicity Assessment of Cytotoxic Antineoplastic Drugs and their Metabolites. Chemosphere (accepted with moderate revision)

Paper III

Zounková, R., Klimešová, Z., Nepejchalová, L., Hilscherová, K., Bláha, L.: Complex Evaluation of Ecotoxicity and Genotoxicity of Antimicrobials Oxytetracycline and Flumequine used in Aquaculture (submitted to Environmental Toxicology and Chemistry)

15 16 The author’s contribution to the articles

Paper I

Radka Zounková participated in the experimental design and carried out a part of bioassays. She evaluated the data, interpreted the results and wrote the manuscript.

Paper II

Radka Zounková proposed the project and the experimental design, carried out all assessments, evaluated the data and interpreted the results. She wrote the manuscript and ﬁnalized the entire article.

Paper III

Radka Zounková participated in the experimental design and supervised the practical carrying out of the assessments. She evaluated the data, interpreted the results, wrote the manuscript and ﬁnalized the entire article.

17 18 Chapter 1 Aims of the present study

The impact of pharmaceuticals on the environment has become one of the hot topics in environmentally focused conferences recently. This work contributes to the fast developing ﬁeld of ecotoxicology by performing tests for selected pharmaceuticals on all trophic levels of the ecosystem and by assessing their genotoxicity. This study initiated investigation of pharmaceuticals and their effects in the aquatic environment at Masaryk University, Faculty of Science’s Research Centre for Toxic Compounds in the Environ- ment (RECETOX). The major hypothesis of the work was following: Pharmaceuticals are toxic compounds with speciﬁc modes of action. As they enter the environment, pharmaceuticals have adverse effects on different levels of the aquatic ecosystem. The following tasks were to be carried out:

• To promote further use of methods of aquatic ecotoxicology in study- ing the effects of pharmaceuticals in the aquatic environment

• To obtain primary information about ecotoxicity and genotoxicity of selected cytostatic pharmaceuticals

• To obtain detailed data of the potential ecotoxicological impact of selected veterinary antibiotics

• To perform screening of toxicity and genotoxicity of waste waters from a potential source of pharmaceuticals (data not presented)

Chapter 2 Introduction

2.1 Pharmaceuticals in the environment

Issues of environmental contamination have attracted much attention recently. Man-made chemical substances, in general, and pharmaceuticals, in particular, clearly present a risk to the environment. Considering the common perception of pharmaceuticals as "healthy", it is easy to disregard or underestimate their potentially adverse effects on the environment. The focus on contamination by pharmaceutical products such as human and veterinary medicaments, X-ray contrast media, and substances used in cos- metic preparations, hence seems particularly important (Kummerer 2001a, 2001b). More than 100,000 new chemical compounds were developed and synthesised in the twentieth century. They have various uses in households, industries, and agriculture. All substances newly synthesised in the Euro- pean Union since late 1970s have been registered. Since 1980s, it has been compulsory to assess their potential impact on the environment. Interest- ingly, pharmaceuticals, which have the obvious ecotoxicological potential, were not subject to the obligatory environmental risk assessment until recently (Kummerer, 2001b). There are a number of features rendering pharmaceuticals as an inter- esting object of ecotoxicological studies. Pharmaceuticals and their metabolites often are:

• biologically active

• hydrophilic,

• not readily biodegradable.

It may seem evident that pharmaceuticals have been developed to have an effect on living organisms. In many cases, which include antibiotics, antifungal agents and antiparasitics, or antineoplastic, the intended effects

21 2. INTRODUCTION are destructive. Due to their speciﬁc mode of action and the fact that these compounds are intentionally designed to affect humans, mammals or other vertebrates, residues of pharmaceuticals could pose more of a risk to human health than pesticides, which are created to affect weeds, fungi and invertebrates (Cleuvers, 2003). Effects of pharmaceuticals are not limited, however, only to higher organisms, as is evidenced in all ecosystems, passing various levels through the food chain. The fact that pharmaceuticals are often hydrophilic is due to their typically low molecular weight, as well as to the metabolic conversion to the substances easily eliminable by urine. The hydrophilic nature, in turn, makes pharmaceuticals mobile in aqueous solutions. These features alone should make pharmaceuticals prominent in ecotoxicology (Kummerer, 2001b). The level of public concern about effects of chemicals on the environment often correlates ﬁrmly with their human toxicity and the possibility of human exposure. There are three main routes of entry of pharmaceuticals into the human body:

• through drinking water, potentially contaminated with pharmaceuticals in ground- and surface waters,

• through ﬁeld crops, due to use of dung and liquid manure as fertilizers,

• by direct consumption of animal tissues, potentially contaminated with veterinary pharmaceuticals.

Once pharmaceutical residues enter human body, they may have adverse effects on human health.

2.1.1 Sources and occurrence of pharmaceuticals in the environment Potential sources of environmental pollution by pharmaceutical products include emissions during production, transport and storage, further improper disposal of unused preparatives, and excretion of metabolites or non-metabolized residues via urine or feces. Considering the requirements of good laboratory practice and often high prices of active compounds, one may expect emissions from the production to be very low. High emissions are thus likely only in the case of an accident (Kummerer, 2001b). Nevertheless, some authors still mention pharmaceutical manufacturers as an important source of environmental contamination. Organic compounds originating from the waste in the pharmaceutical

22 2. INTRODUCTION industry were found in the down gradient of a landfill (Halling-Sorensen et al., 1998). Sulfonamides and oxytetracycline were found in the surface water, and even in the ground water influenced by various pharmaceutical production facilities (Hirsch et al., 1999). This pathway, however, represents only point discharges affecting a limited area, whereas the main intake results from permanent use in human and veterinary medication (Hirsch et al., 1999). No information concerning emissions during transport and storage seem to be available in the literature (Kummerer, 2001b). Concerning disposal, the same instructions are applied to pharmaceuticals as to any other dangerous chemical compounds (Czech Act No. 185/2001 Coll., On Waste and on the Amendment to some Other Laws). In the An- nex No. 2 of this law, a list of dangerous properties of waste is stated, from which pharmaceuticals may show following properties: oxidation capabil- ity, toxicity, carcinogenicity, teratogenicity, mutagenicity, ability to release dangerous compounds to the environment during disposal, and ecotoxicity. In the Annex No. 5 (The List of Components Making Waste Dangerous) human or veterinary pharmaceuticals are listed under the code C33 (Sb.z., 2001). After administration and metabolic conversion inside organism, pharmaceuticals are excreted from the body into the environment. Some drugs are metabolized completely, others only in part. Some substances, e.g. X-ray contrast media, are excreted unchanged (Kummerer, 2001b). Most medical substances are metabolized to phase I or phase II metabolites (Fig. 2.1). (Halling-Sorensen et al., 1998). Phase I reactions usually involve oxidation, reduction or hydrolysis, and their products are often more reactive and sometimes more toxic than the original drug. Phase II reactions involve conjugation, which normally results in inactive compounds. Metabolism changes the physical-chemical properties of the substance and renders the metabolites more water soluble than the parent compound, in order to make them easily eliminable in urine (Halling-Sorensen et al., 1998). Some of the excreted metabolites can be cleaved easily, and the original active compound can then be released into the aquatic environment (Heberer, 2002). For instance, glucuronide of chloramphenicol has been reported to be converted to chloramphenicol in liquid manure (Hirsch et al., 1999). Numerous studies found drugs and their metabolites in municipal sewage, sewage treatment plants, sewage treatment plants effluents, surface water, soil, ground water, and consequently drinking water. First studies described the presence of lipid lowering agents. More recently, other groups of drugs were also found in environmental samples, including antibiotics, hormones, non-steroidal anti-inflammatory drugs, anticonvulsants,

23 2. INTRODUCTION

PHASE I Metabolites

PARENT COMPOUND

PHASE II Metabolites

More water soluble substance

Figure 2.1: An overview of the metabolism of parent compound into phase I and phase II metabolites. Solid lines indicate a transformation into a more water soluble compound. Dotted lines indicate a reactivation of the phase II metabolites into a less water soluble compound (Undertaken from Halling-Sorensen et al., 1998).

and other compounds used in high doses, as well as diagnostic aids, and disinfectants. Improper disposal of medicaments, "down the household drains", in particular, result in pharmaceuticals’ deposition at sewage treatment plants (STP). The subsequent elimination in STPs is often incomplete (Hirsch et al., 1999). If the drugs are not completely degraded in STPs, they may enter the aquatic environment and eventually reach groundwater and drinking water (Fig. 2.2) (Kummerer, 2001b). Similarly, drugs used in animal husbandry for veterinary purposes or as growth promoters are discharged into the environment through dung, liquid manure, and waste water. These substances may ultimately enter groundwater via soil after application of dung, liquid manure or sewage sludge as fertilizers (Kum- merer, 2001b). Drugs used in aquaculture also enter the aquatic environment by application directly into the water or as medicated food pellets (Hirsch et al., 1999). The most important factor of environmental pollution by pharmaceuticals is that although the amounts released into the environment are quite small, they are released over a long period and continuously (Mompelat et al., 2009). Various authors assessed pharmaceuticals in different matrices by different approaches. Heberer (2002) reports narrowly the presence of particular groups of pharmaceuticals in different environments. The review of

24 2. INTRODUCTION

Figure 2.2: Overview of sources and distribution of pharmaceuticals in the environment (undertaken from Diaz-Cruz et al., 2003).

Jones et al. (2001) lists tables of maximum concentrations of single agents from different groups of pharmaceuticals detected in surface water, groundwater and drinking water. Kummerer (2001a) also displays a brief table of concentrations found in waste water, surface water, ground and drinking water. More comprehensive tables may be found in review of Halling- Sorensen at al. (1998) and in the recent review of Mompelat et al. (2009). Concentrations of pharmaceuticals are mostly reported in the high ng/L to l’g/L range in the surface waters, high l’g/L range in STP efﬂuents and low ng/L in groundwater. Concentrations in drinking water were in low ng/L range or under limit of detection (Jones et al., 2001; Kummerer, 2001b). Although concentrations might seem low, they are of ecotoxicological concern as discussed below. Examples of detected concentrations of selected pharmaceuticals in surface and waste water are presented in Table 2.1. The analytical techniques most frequently used in contemporary ecotoxicological studies of pharmaceuticals are liquid chromatography with mass spectrometry detection (LC-MS) or tandem mass spectrometry (LC- MS/MS). Electrospray ionization (ESI) is the most frequently used inter- face, as it allows for the analysis of polar compounds. For less polar compounds, atmospheric pressure chemical ionization (APCI) is used. Tandem mass spectrometry enables a very good selectivity and sensitivity in trace

25 2. INTRODUCTION analysis of environmental pollutants. For identification and quantification, analytes do not have to be perfectly separated as when using the diode array detector (DAD). Furthermore, derivatization is not needed as it often is at other technique frequently used - gas chromatography with mass spectrometry detection (GC-MS) (de Alda et al., 2003). Non-steroidal anti- inflammatory drugs, such as diclofenac and ibuprofen, and lipid lowering agents (fibrates) can be determined after derivatization by GC-MS. On the contrary, analgesics, β-blockers, broncholytics, and certain cytostatics (ifosfamide, cyclophosphamide), iodinated X-ray media, and antibiotics were determined by HPLC-ESI-MS/MS with different ways to preliminary treatment of samples (Sacher et al., 2001). Another efficient, highly sensitive, and rather simple method for determination of some pharmaceuticals is ELISA - enzyme-linked immunosorbent assay (Deng et al., 2003).

2.1.2 Environmental fate of pharmaceuticals

Once in the environment, residues of pharmaceuticals and their metabolites can be transported, and either decomposed or stay unchanged for a long time. As pharmaceuticals are generally polar, water soluble and low volatile molecules, they tend to be diluted in surface water up to trace level (l’g/L to ng/L) and transported fast across long distances (Kummerer, 2001b; Mompelat et al., 2009). Passages of pharmaceutical residues are illustrated in Fig. 2.2. The elimination of organic compounds is a result of different processes, both biotic and non-biotic. Biodegradation by bacteria or fungi and non-biotic processes such as hydrolysis, photolysis, oxidation, and reduction are particularly important (Kummerer, 2009). Another potential attenuation factor is the adsorption on suspended solids, sediments, and dissolved organic matter. Pharmaceuticals sorbed on organic matter can form a "reservoir", and they may be released to the environment long after the original sorption. Pharmaceuticals are also prone to photodegradation, either direct by the solar absorption, or indirect by radicals, which are generated by the solar irradiation of photosensitizers such as nitrate, humic acids etc. (Mompelat et al., 2009; Buerge et al., 2006). Some compounds like fluoxetine, diclofenac, diazepam, fenofibrate, and fluoroquinolones are reported to be photoreac- tive substances, whereas some antibiotics, e.g. tetracycline, cannot be pho- todegraded (Mompelat et al., 2009). Biodegradation of pharmaceuticals is supposed to be insignificant in surface- and groundwaters. Biodegradation tests of antibiotics using OECD standards have shown that their biodegradation rates are low, with the ex-

26 2. INTRODUCTION ception of penicillin (Alexy et al., 2004). Details of biodegradability of pharmaceuticals, in general, are provided by Halling-Sorensen et al. (1998). The persistence and biodegradability of antibiotics and cytostatic compounds, which were investigated in detail in the present work, are also discussed below.

2.1.3 Effects of pharmaceuticals in the environment

Ways of entering the environment and fate of pharmaceutical compounds and their metabolites are similar to other environmental pollutants, and thus effects are also similar, in principle, to the effects of other contaminants. This includes the inhibition of enzymatic activities, competition with natural ligands or substrates, interferences with regulatory pathways, disruption of redox potential, disruption of membrane gradients, induction of stress proteins, endocrine disruption, teratogenity, immunotoxicity and others (Blaha, 2002). In contrast to most industrial chemicals, which often have a nonspecific narcotic effect, pharmaceuticals often elicit specific effects at lower concentrations. However, the mechanism of action of pharmaceutical compounds in the environment has not been understood well enough, so far (Cleuvers, 2003). As most pharmaceuticals are designed to affect mammalian physiology, it is not known what effects they may have on organisms such as invertebrates, plants or protozoa. Our understanding of the principles of their environmental toxicity is, generally, insufficient. Even current regulatory guidance only requires pharmaceuticals to undergo standard acute ecotoxicity tests (Jones et al., 2001). Particular modes of action may be understood on the basis of the mechanisms of intended and side effects in target organism (human or animal) and batteries of specific ecotoxicological tests. For the research in this area, new and more suitable test methods need to be developed (Sorensen, 2002). Seiler (2002) asked whether respective hazards of pharmaceuticals could be deduced from their pharmacodynamic properties, and whether there could indeed be relevant risks at (very low) environmental concentrations observed. A number of examples of species-specificities were discussed (Seiler, 2002). The conclusion is that pharmacodynamic effects, classified as secondary and considered irrelevant for the therapeutic activity in humans, might potentially play a major role in other (non-mammalian) organisms, and vice versa. Of course, when the non-target organism has similar target structure of metabolic and regulatory pathways, it is likely that the mechanism of action will be similar. One of the shared target structures

27 2. INTRODUCTION is DNA, and many pharmaceuticals target their effect on DNA structures or metabolic pathways connected with the synthesis of DNA. Those compounds will, possibly, have the effect across all levels of an ecosystem but the effects, however, may not become apparent for a very long time (Jones et al., 2001). For example, several studies provide evidence for genotoxic activity of hospital wastewater contaminated by a mixture of chemicals including pharmaceuticals (Giuliani et al., 1996; Jolibois et al., 2003). Cleuvers (2003) investigated whether any specific mode of action is expressed during acute ecotoxicological tests with pharmaceuticals (clofibric acid, diclofenac, ibuprofen, carbamazepin). The quantitative structure activity relationship (QSAR) for mechanism of non-polar narcosis was used for estimation of EC50 values. The results were compared with the values obtained from ecotoxicity tests (D. magna acute test and algal growth inhibition test). The measured values were higher than the estimated ones, in all cases. This could be explained by the tested compounds’ not eliciting any other specific mode of action (Cleuvers, 2003). However, considering that both tests used in the study of Cleuvers et al. reflect acute toxicity, possibility of any specific modes of action to be expressed during longer exposition cannot be excluded. A number of studies were performed to establish the effects of certain classes of pharmaceuticals on particular organisms such as Daphnia magna (Cleuvers, 2003; Hernando et al., 2004), Acartia tonsa (Lanzky & Halling- Sorensen, 1997), Danio rerio (Henschel et al., 1997; Lanzky & Halling-Sorensen, 1997), Lemna minor (Cleuvers, 2003), Desmodesmus subspicatus (Cleuvers, 2003), Pseudokirchneriella subcapitata (Lanzky & Halling-Sorensen, 1997), Chlo- rella sp. (Lanzky & Halling-Sorensen, 1997), natural freshwater algal assemblages (Wilson et al., 2003), Vibrio fischeri (Backhaus et al., 1997; Backhaus & Grimme, 1999; Henschel et al., 1997), sewage sludge bacteria (Kummerer et al., 2004), Tetrahymena pyriformis (Henschel et al., 1997), and many more. Also, various mixtures of pharmaceutical have been investigated, as pharmaceuticals commonly occur as mixtures in the environment. Various effects have been documented ranging from the antagonistic concept (mixtures with flumequine in the test with algae) (Christensen, A.M. et al., 2006), the concept of independent actions (e.g. carbamazepine and clofibrinic acid in algae test) and the concept of concentration additive (clofibrinic acid and carbamazepin in D. magna acute test) to the synergistic (e.g. diclofenac and ibuprofen in D. magna acute test) (Cleuvers, 2003). Examples of effects of selected pharmaceuticals are presented in Table 2.1. More information about the effects of pharmaceuticals on organisms, performed tests and test methods is available in the book by Kummerer

28 2. INTRODUCTION

(2001b) and in other reviews (Boxall et al., 2004; Halling-Sorensen et al., 1998; Jones et al., 2001).

Table 2.1: Examples of detected concentrations of selected pharmaceuticals in surface and waste water and their effects in ecotoxicological bioassays - acute immobilisation test with Daphnia magna, growth inhibition test with Desmodesmus subspicatus and Lemna minor.

∗ ∗∗ Substance concentration (µg/L) EC50(µg/L) surface water waste water Daphnia Desmodesmus Lemna Cloﬁbrinic acid (lipid regulator) 0,55 1,6 72 115 12,5 Carbamazepine (antiepileptic) 1,1 6,3 >100 74 25,5 Ibuprofen (antiphlogistic) 0,53 3,4 108 315 22 Diclofenac (antiphlogistic) 1,3 2,1 68 72 7,5 Naproxen (antiphlogistic) 0,39 0,52 174 >320 24,2 Captopril (ACE inhibitor) - - >100 168 25 Metformin (antidiabetic) - - 64 >320 110 Propranolol (betablocker) 0,59 0,29 7,5 5,8 114 Metoprolol (betablocker) 2,2 2,2 >100 7,3 >320 ∗(Ternes et al., 2001) ∗∗(Cleuvers, 2003)

2.1.4 Environmental risks of pharmaceuticals

The adverse effects of pharmaceuticals’ ingestion by drinking water seem to be negligible in humans. The maximum intake within the life span of a human, estimated at 2 L of drinking water per day over the period of 70 years, is far below the dosage used in therapies (Christensen, F.M., 1998). But the problem how to extrapolate data from high dosage in a short term therapy to low dosage long term ingestion remains an open question. Furthermore, the risk assessment has been conducted only for single substances, so far, rather than mixtures (Kummerer, 2001b). While the emissions from production, transport and storage of pharmaceuticals are mostly very low, the highest emissions are expected during and after their application. The most dangerous for the environment will, therefore, be those pharmaceuticals, which are used in the highest doses, such as veterinary drugs, and drugs used in-mass and repetitively. Some compounds are carcinogenic, mutagenic or reproductive-toxic. Beside those, the following groups of drugs and diagnostic aids may deserve a special attention (Kummerer, 2001b):

29 2. INTRODUCTION

• cytostatic agents - because of their carcinogenic, mutagenic or embryotoxic properties,

• antibiotics and desinfectants - because of their bacterial toxicity and the potential for forming resistance and disturb environmental bacterial communities,

• chlorine-releasing compounds used as disinfectants and bleaching agents or diagnostics like iodinated X-ray contrast media - because they contribute to the overall loads of halogenated organic compounds into the environment,

• heavy metals - such as disinfectants and preservatives containing mer- cury, cytostatics containing platinum or contrast media containing gadolinium.

Other compounds such as analgesics, sedatives, therapeutic agents of the cardiovascular system, lipid lowering agents, and oral contraceptives, deserve special attention with respect to the amounts in which they are used. From veterinary pharmaceuticals, especially medicaments against infectious and invasive diseases are worth of attention. Antibiotics, in particular, are used also as growth promoters, antiectoparasitics, and hormones. Apart from toxicity and amount potentially released into the environment, the element of environmental persistence is also important. At persistent compounds, the potential of long-term activity is increased and the multiple contamination of an ecosystem is possible (Kummerer, 2001b).

2.2 Cytostatics

Anticancer chemoterapeutics (cytostatics, antineoplastics) have a number of features which make them an important subject of study in Environ- mental Science. In comparison with other pharmaceuticals, the annual consumption of cytostatics is low. However, regarding their potential effects in the environment, they are considered very important. Cytostatics may represent a risk in the environment due to their speciﬁc pharmacologic properties. Their carcinogenicity, mutagenicity and embryotoxicity is well documented (Kummerer, 2001a). Research of this dissertation thesis focused on detailed ecotoxicological characterization of selected widely used cytostatic compounds. Cytostatics are used in lower quantities than other pharmaceuticals. For example, 0.9 million packings of all cytostatic drugs were used in the Czech

30 2. INTRODUCTION

Republic in 2009, whereas for analgesics and antibiotics these values were 31 million and 12 million, respectively1. Nevertheless, according to the costs, cytostatics range among the top pharmaceuticals sold in the Czech Republic.

2.2.1 Pharmacology of cytostatics - mode of action Simply given, cytostatics are pharmaceuticals used in cancer therapy. Anti- cancer chemotherapy belongs to the basic methods of treatment for tumour diseases (Suchopár, 1997), alongside surgical interventions and radiother- apy. Most anticancer chemotherapeutic drugs work by impairing mitosis - cell division (Fig. 2.3), effectively targeting the fast dividing cells of a tumour. This, however, inﬂuences also those tissues, which have naturally high rates of mitosis, such as hair bulbs, enteric mucosa, immune system, and bone marrow (Lüllmann, 1994). Some cytostatics cause cells to undergo controlled cell death - apoptosis. Overall, the aim is to magnify the effects towards fast dividing cells of a tumour, while minimizing impacts on other tissues. The basic modes of action of cytostatic pharmaceuticals are as follows (Klener, 2002; Suchopár, 1997):

1. Inhibition of nucleic acid synthesis, eventually protein synthesis by

• direct blocking of biosynthetic reactions • incorporation of cytostatic compound into the nucleotide which leads to synthesis of "false" non-functional DNA (antimetabolites) • inhibition of metabolic reaction by the method of negative feed- back

2. Alteration of structure and function of nucleic acids by

• alkylation of electronegative groups of nucleic acids (alkylating agents) • intercalating between base pairs of the DNA strand, thus pre- venting the replication (anthracyclines) • inhibition of topoisomerase enzyme which is responsible for coiling of DNA

1 Available on-line on: http://www.sukl.cz/4-ctvrtleti-a-za-cely-rok-2009

31 2. INTRODUCTION

• ﬁssuring of the molecule of DNA (some topoisomerase inhibitors, bleomycin)

3. Alteration of microtubules (vinca alcaliods - vincristin, vinblastin; taxanes - paclitaxel, docetaxel)

4. Combination of previous modes of action or indeﬁnite mode of action.

Figure 2.3: Mechanisms of action of cytostatic agents (according to http://www.elmhurst.edu/∼chm/vchembook/655cancer.html).

According to the mode of action and chemical structure, cytostatics have been divided into following groups (Klener, 2002; Suchopár, 1997) (in brack- ets, there is the code according to ATC - Anatomical-Therapeutic-Chemical classiﬁcation2):

1. Alkylating agents (L01A)

• Nitrogen mustards - cyclophosphamide, ifosfamide, mechlorethamine, chlorambucil, melphalan • Ethylendiamins (aziridins) - thiotepa • Alkyl sulfonates - busulfan

2Available on-line on http://www.whocc.no/

32 2. INTRODUCTION

• Nitrosoureas - carmustine, lomustine, streptozocin • Alkylating-like derivatives of platinum - cisplatin, carboplatin • Nonclasiccal alkylating agents - procarbazine, dacarbazine, mitomycin C

2. Antimetabolites (L01B)

• Antifolates (folic acid analoques) - methotrexate • Purine analoques - azathioprin, mercaptopurine, thioguanine, fludarabine, cladribine • Pyrimidine analoques - 5-fluorouracil, cytarabine, floxuridine, gemcitabine • Ribonucleotide reductase inhibitors - hydroxycarbamide (hydroxyurea) • Aminoacid analogues

3. Plant alcaloids and other natural products (L01C)

• Vinca alcaloids and analogues - vincristine, vinblastine, vindesine • Podophyllotoxin derivatives - etoposide, teniposide • Colchicine derivatives - demecolcine • Taxanes - paclitaxel, docetaxel

4. Cytotoxic antibiotics and related substances (L01D)

• Actinomycines - actinomycin D (dactinomycin) • Anthracyclines and related substances - daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone

5. Other antineoplastic agents

• Platinum compounds, methylhydrazines, monoclonal antibodies, protein kinase inhibitors etc.

2.2.2 Pharmacokinetics of cytostatic drugs Pharmacokinetics of pharmaceuticals, in general, and cytostatics, in particular, involves their resorption, distribution in organism, biotransformation, and excretion (Klener, 2002). From the ecotoxicological point of view, biotransformation is the most important step because it determines in what

33 2. INTRODUCTION form cytostatic will be excreted out of the organism and in what form they will enter the environment. Metabolic transformations of pharmaceuticals, generally, lead to their inactivation, detoxiﬁcation, and elimination. This, however, is not a strict rule. For example, the action of cyclophosphamide depends on its metabolic transformation resulting in its becoming an active metabolite (Klener, 2002). The biotransformation changes not only the effectiveness (and toxicity) of a compound, but also its physical-chemical properties. Metabolites are generally more polar, more water soluble, and therefore more mobile in aquatic environment, than the original pharmaceuticals. Anticancer chemotherapeutics and their metabolites are excreted mainly by kidneys and the liver (Klener, 2002). Some compounds are metabolized only to a small extent and are excreted as active substances. For example, approximately 14-53 % of administered dose of cyclophosphamide and ifosfamide are excreted by urine non-metabolized (Steger-Hartmann et al., 1996). Some metabolites might be more biologically active than the original pharmaceuticals (Al-Ahmad & Kummerer, 2001).

2.2.3 Sources and occurrence of cytostatics in the environment

Currently, residues of cytostatics almost exclusively originate from hospital applications (Heberer, 2002), but increasing amounts are being prescribed by practitioners for out-patient treatment. Some of the amounts administered in hospitals are excreted at home into municipal sewage by out- patients (Kummerer, 2001a). The concentrations of cytostatic drugs in hospital sewage may hence be up to the low l’g/L level (Steger-Hartmann et al., 1997). In effluents from those municipal STPs receiving hospital effluents, cytostatics have been found at trace concentrations mostly at the low ng/L level (Steger-Hartmann et al., 1996; Kummerer & Al-Ahmad, 1997; Ternes, 1998). So far, cytostatics have not been detected in surface waters. An- ticipated annual average concentrations in surface waters are presumably below 1 ng/L (Kummerer, 2001a). There is, in general, little information about concentrations of cytostatics in the environment available. A brief outline of the details available is presented in Table 2.2. Number of methods was developed for environmental analysis of cytostatic drugs. These include HPLC with DAD (diode-array detector) and fluorescence detection using gradient elution for separation of particular compounds (Kiffmeyer et al., 1998) or HPLC-MS/MS with - for example - hydrophilic interaction chromatography (HILIC) stationary phase (Kovalo- va et al., 2009). Other methods include gas chromatography coupled with

34 2. INTRODUCTION

Table 2.2: Concentrations of cytostatic pharmaceuticals in different waste water samples - summary of the literature data.

Compound Concentration detected Matrix Reference Cyclophosphamide 146 ng/L hospital effluent (Steger-Hartmann et al., 1996) 0.019 – 4.5 µg/L hospital effluent (Steger-Hartmann et al., 1996) < 6 – 143 ng/L STP influent (Steger-Hartmann et al., 1996) 7 – 15 ng/L STP effluent (Steger-Hartmann et al., 1996)

Ifosfamide 24 ng/L hospital effluent (Steger-Hartmann et al., 1996) up to 1914 ng/L hospital effluent (Kummerer et al., 1997) up to 29 ng/L STP influent (Kummerer et al., 1997) up to 43 ng/L STP effluent (Kummerer et al., 1997)

5-fluorouracil 27 ng/L hospital effluent (Kovalova et al., 2009) 20 – 122 µg/L hospital effluent (Mahnik et al., 2004) up to 4.0 µg/L hospital effluent (Mullot et al., 2009)

Gemcitabine 38 ng/L hospital efﬂuent (Kovalova et al., 2009) dFdU 840 ng/L hospital efﬂuent (Kovalova et al., 2009)

Doxorubicin 0.1 – 0.5 µg/L hospital efﬂuent (Mahnik et al., 2004) Epirubicin 0.1 – 1.4 µg/L hospital efﬂuent (Mahnik et al., 2004)

electron-capture detector (ECD) or MS (Steger-Hartmann et al., 1996) or determination of platinum-based cytostatics by inductively coupled plasma optical emission spectrometry (ICP-OES) (Komendova-Vlasankova, 2001) or highly sensitive method of adsorptive voltammetry (Kummerer et al., 1999).

2.2.4 Fate of cytostatics in the environment

Limited information on the degradation or persistence of cytostatics in the environment is available. The few studies available unambiguously demon- strate cytostatics’ low biodegradability and high environmental stability, leading to the long term persistence of cytostatics (Halling-Sorensen et al., 1998). The low degradability was experimentally proved using Zahn-Wel- lens test and closed bottle test. In experiments with vinca alkaloids, it was up to 30 % in 28 days (Al-Ahmad & Kummerer, 2001). During biodegradation of methotrexate, formation of a toxic and persistent degradation product 7-OH-methotrexat was shown. Cisplatin, cyclophosphamide and

35 2. INTRODUCTION ifosfamide were shown not to be biodegradable, whereas cytarabine and 5-ﬂuorouracil were biodegraded in different magnitudes (Kiffmeyer et al., 1998; Steger-Hartmann et al., 1997). In the study of Kummerer and Al Ahmad (1997), 5-ﬂuorouracil was shown not degradable. Cytarabine and gemcitabine were biodegraded only partially, posing a risk to the environment. The high persistence of these compounds results in their passing to the STPs non-changed and having long-lasting effects in the environment.

2.2.5 Effects of cytostatics in the environment The ecotoxicity of cytostatic pharmaceuticals has been studied only to a small extent. Only a limited number of studies are available on the effects in short-term ecotoxicological tests and no information about potential chronic impact on organisms. Considering that organisms are exposed continually to cytostatics as well as to other persistent pollutants, chronic tests are believed to be more suitable for risk analysis. Details of acute toxicity are available for methotrexat (Henschel et al., 1997) and 5-ﬂuorouracil (Backhaus & Grimme, 1999). FETAX test with frog embryos was performed with methotrexat (Bantle et al., 1994), and values calculated using QSAR (quantitative structure-activity relationship) were published for cyclophosphamide and ifosfamide (Sanderson et al., 2003). Ecotoxicity data on cytostatics found in the literature are summarized in Table 2.3. Al Ahmad and Kummerer (2001) examined also effects on waste water bacteria Pseu- domonas putida, however no toxicity against this bacteria was proved. As discussed, cytostatics are of special concern because of their mode of action, which is very often aimed at changes of the DNA. Generally, genotoxicity is necessary to be considered also in other organisms, e.g. bacteria directly in bioﬁlms in STPs, in aquatic or soil environment and in river sediments. The introduction of genotoxic compounds into the environment is generally considered as a very important risk factor - DNA damage in non-target native organisms may lead to severe changes in the ecosystem (Connell, 1999).

36 2. INTRODUCTION

Table 2.3: Summary of the literature on the ecotoxicity data of cytostatics.

Compound Bioassay Toxicity Reference

Cyclophposphamide Scenedesmus subspicatus 72-h growth EC50 = 11 mg/L (QSAR) (Sanderson et al., 2003) inhibition

Daphnia magna 48-h immobilization EC50 = 1795 mg/L (QSAR) (Sanderson et al., 2003)

Brachydanio rerio embryos 48-h EC50 = 70 mg/L (QSAR) (Sanderson et al., 2003) mortality

Ifosfamide Scenedesmus subspicatus 72-h growth EC50 = 11 mg/L (QSAR) (Sanderson et al., 2003) inhibition

Daphnia magna 48-h immobilization EC50 = 1795 mg/L (QSAR) (Sanderson et al., 2003)

Brachydanio rerio embryos 48-h mortality EC50 = 140 mg/L (QSAR) (Sanderson et al., 2003)

Methotrexate Scenedesmus subspicatus 72-h growth EC50 = 192 mg/L (QSAR) (Sanderson et al., 2003) inhibition

EC50 = 260 mg/L (Henschel et al., 1997)

Daphnia magna 48-h immobilization EC50 = 651 mg/L (QSAR) (Sanderson et al., 2003)

EC50 > 1000 mg/L (Henschel et al., 1997) 5 Brachydanio rerio embryos 48-h mortality EC50 = 3.83 × 10 mg/L (Sanderson et al., 2003) (QSAR)

EC50 = 85 mg/L (Henschel et al., 1997)

Tetrahymena pyriformis 48-h growth EC50 = 45 mg/L (Henschel et al., 1997) inhibition

Vibrio ﬁscheri 30-min luminescence EC50 = 1220 mg/L (Henschel et al., 1997) inhibition

Xenopus laevis 96-h malformations EC50 = 0.015 mg/L (Bantle et al., 1994)

5-ﬂuorouracil Pimephales promelas 120-h growth EC50 = 400 mg/L, (DeYoung et al., 1996) inhibition LOEC = 20 mg/L

Vibrio ﬁscheri luminescence 24-h EC50 = 0.122 mg/L (Backhaus & inhibition Grimme, 1999) Pseudomonas putida 16-h growth IC0 > 128 mg/L (Kummerer & inhibition Al-Ahmad, 1997) Cytarabime Pseudomonas putida 16-h growth IC0 > 128 mg/L (Kummerer & inhibition Al-Ahmad, 1997) Gemcitabine Pseudomonas putida 16-h growth IC0 > 128 mg/L (Kummerer & inhibition Al-Ahmad, 1997)

Cladribine Daphnia magna 48-h immobilization EC50 = 233 mg/L (FDA-CDER, 1996)

Thiotepa Daphnia magna 48-h immobilization EC50 = 546 mg/L (FDA-CDER, 1996)

37 2. INTRODUCTION

Scarce experimental studies (Giuliani et al., 1996; Jolibois et al., 2003) have proved significant genotoxicity in hospital effluent samples both above and underneath a STP. Genotoxicity in bacterial ecotoxicological biotests has been studied with only a few pure cytostatics. For instance cisplatin and mitomycin C were shown to have high induction of SOS system without growth inhibition in umuC test (Giuliani et al., 1996). Within the validation study of a genotoxicity test using the DNA repair reporter assay with yeast cells Saccharomyces cerevisiae (GreenScreen assay), several cytostatics have been tested. Bleomycin, daunorubicin and hydroxyurea showed high induction, cisplatin, mitomycin C and etoposide showed medium induction and chlorambucil, actionomycin D and vinblastin showed no induction. The results were compared to the results of Ames test, in which bleomycin, daunorubicin, hydroxyurea, cisplatin, mitomycin C and chlorambucil showed medium induction and etoposide, actionomycin D and vinblastin showed induction not sufficient to be considered genotoxic (Cahill et al., 2004).

2.2.6 Cytotoxic drugs studied in the present work The compounds of our interest have been selected based on the quantity of their use. Unfortunately, the data concerning consumption of cytostatic drugs are a subject of the Act on the Protection of Personal Data. As a con- sequence, it is not easy to find consumption of particular compounds. Nev- ertheless, direct consultations with hospital pharmacies and study of literature resulted in selection of the following important groups of cytostatics - cyclophosphamide, 5-fluorouracil, cisplatin, doxorubicin and etoposide. In the second part of investigation, we focused on a group of pyrimidine analoques 5-fluorouracil, cytarabine, gemcitabine and their metabolites. More details on the studied chemicals are available in the Materials and Methods section of this dissertation thesis. Major objective of the investigation was to derive new ecotoxicity and genotoxicity data for a set of widely used cytotoxic drugs, and try to evaluate potential environmental risks associated with these compounds.

2.3 The issue of antimicrobials in the environment

Antibiotics are indisputably a highly important group of pharmaceuticals (Hirsch et al., 1999). Generally, antibiotics are substances which kill or inhibit growth of microorganisms. The ﬁrst antibiotics were of natural origin, produced by microorganisms. Currently, some antibiotics are purely syn-

38 2. INTRODUCTION thetic or produced by chemical modification of compounds of natural origin. The base is, however, still natural. The term "antibiotic" now refers to substances with antibacterial, and sometimes anti-fungal or anti-parasitic (Kummerer, 2009), but not anti-viral activity. A distinct group of antimicrobial pharmaceuticals are antimicrobial chemotherapeutics, which have been created through purely synthetic means. The expression "chemother- apeutical" refers to compounds used for the treatment of disease which kill cells, specifically microorganisms or cancer cells (Kummerer, 2009). "Anti- microbials" are hence a group of pharmaceuticals which are active against microorganisms (all bacteria, fungi, and microbial parasites) and both antibiotics and antimicrobial chemotherapeutics belong to this group. Never- theless, the term "antibiotics" is very often used for all antimicrobial compounds in the context of environmental science. Antimicrobials can be classified in various ways, most notably according to their chemical structure and mechanism of action. There are a number of sub-groups, including β-lactams, tetracyclines, macrolides, glyco- sides, linkosamides (antibiotics); quinolones, sulfonamides (chemotherapeutics) and others (Kummerer, 2009). Particular groups of antimicrobials differ markedly from each other in their physical-chemical properties and modes of action. Outside of the grouping by their chemical structure or mode of action, antimicrobials can be divided into two big groups according to their effects on microorganisms - bacteriostatic (impairing bacterial growth), and bactericidal (causing bacterial death). Finally, antimicrobials can be classified by their target specificity to broad-spectrum, which affect a wide range of bacteria, and narrow-spectrum, which target particular types of bacteria, such as Gram-positive or Gram-negative. Antimicrobials are used for the treatment of various infections in humans and for the treatment and prevention of bacterial diseases and as growth promoters in veterinary medicine (Hirsch et al., 1999; Boxall et al., 2004).

2.3.1 Mechanism of action of antimicrobial drugs

Antimicrobials employ many different mechanisms of action (Fig. 2.3). Pos- sible targets of antimicrobials’ action are the cell wall, cell membrane, protein synthesis, nucleic acid synthesis or the cell metabolism.3

3Available on-line on: http://ezinearticles.com/?Antibiotics-And-The-Mode-Of-Action&id=1193644, http://www.tufts.edu/med/apua/Miscellaneous/mechanisms.html, http://en.wikipedia.org/wiki/List_of_antibiotics;

39 2. INTRODUCTION

• β-lactams (penicillins and cefalosporins) disrupt the synthesis of the peptidoglycan and thus inhibit the cell wall synthesis;

• Aminoglycosides and tetracyclines inhibit the protein synthesis by binding to the 30S subunit of bacterial ribosome and inhibiting the translocation of peptidyl-tRNA;

• Macrolides and chloramphenicol inhibit the protein synthesis by (ir- reversibly) binding to the 50S ribosomal subunit;

• Rifampicin inhibits the RNA synthesis by inhibiting the DNA-dependent RNA polymerase;

• Quinolones block the DNA synthesis by inhibiting the DNA-gyrase enzyme.

• Polymyxins disrupt the integrity and structure of cell membrane;

• Sulfonamides block the cell metabolism by inhibiting enzymes, which are necessary in the biosynthesis of folic acid, which is in turn necessary in the formation and maintenance of a bacterial cell.

According to the ATC classiﬁcation (Anatomical-Therapeutic-Chemical) antimicrobials belong to the J01 group - Antibacterials for systemic use and can be divided into these subgroups:

• Tetracyclines

• Amphenicols

• Beta-lactam antibacterials, Penicillins

• Other beta-lactam antibacterials

• Sulfonamides and Trimethoprim

• Macrolides, Lincosamides and Streptogramins

• Aminoglycoside antibacterials

• Quinolone antibacterials

• Combinations of antibacterials

• Other antibacterials (Suchopár, 1997)

40 2. INTRODUCTION

Other ATC groups including antimicrobial pharmaceuticals are e.g. J02 with Antimycotics, J04 with Antimycobacterials, and many other subgroups4.

Figure 2.4: Mechanisms of action of antimicrobials agents (available on-line on http://microblog.me.uk/ wp-content/uploads/Antibiotic_actions.jpg)

2.3.2 Sources and occurrence of antimicrobials in the environment Antimicrobials can enter the environment in the same ways as the other pharmaceuticals. After the administration, they may be metabolized and excreted via urine or feces. Approximately 70 % of the consumed amount of antibiotics is excreted unchanged (Kummerer & Henninger, 2003). They enter municipal sewage and sewage treatment plants but large fraction is not degraded in STP, and ultimately enters the environment (Halling-Sorensen et al., 1998; Kummerer, 2001b; Zuccato et al., 2000), as discussed in Chap- ter 2.1.1 and illustrated in Fig. 2.2. The reason why polar antibiotics may not be eliminated effectively stems from the fact that their elimination relies largely on the activated sludge, which is partly mediated through hy- drophobic interactions (Hirsch et al., 1999). Veterinary antimicrobials are an important source of antimicrobials in

4available on-line on http://www.whocc.no/atc_ddd_index/

41 2. INTRODUCTION the environment. They enter the environment through a number of pathways (Boxall et al., 2004). Antibiotics are used also in plant agriculture, mainly to control certain bacterial diseases of high-value fruit, vegetables, and ornamental plants. Antimicrobials applied to plants account for less than 0.5 % of total antibiotic use in the USA. To be used in agriculture, however, antibiotics need to be tolerant to oxidation, UV irradiation, rain- fall, and high temperatures, which are, worryingly, the features of the most awkward pollutants. Emissions from plant pro duction were regarded as of minor importance. Recently, however, it has been established that significant concentrations of single compounds can be found in effluents in some Asian countries (Kummerer, 2009). More than 30 different antibiotic substances have already been found in soil, sewage influent and effluent samples, in surface waters and even in ground and drinking water (Kemper, 2008). A well-arranged table of concentrations of antibiotics measured in the aquatic environment is presented in the review of Kummerer (2009). Kemper (2008) presents a summary of concentrations of antimicrobials found in soil and in water. Tetracyclines and sulphonamides were found in the highest concentrations in soil (tetracycline up to 900 l’g/kg, sulphadimidine up to 11 l’g/kg), tetracyclines, sufphonamides and macrolides in water (oxytetracycline up to 32 l’g/L, sulphadiazine up to 4.1 l’g/L, lincomycin up to 21 l’g/L) (Kemper, 2008). In general, concentrations were found to be in the higher l’g/L range in hospital effluents, in the lower l’g/L range in municipal waste water, and in higher and lower l’g/L range in different source waters, ground water and sea water in a harbour (Kummerer, 2009). Once in the environment, antibiotic efficiency depends on the physical-chemical properties and a variety of environmental factors.

2.3.3 Environmental fate of antimicrobials

Biodegradation of antimicrobials highly depends on the environmental conditions. Most antimicrobials are not biodegradable under aerobic conditions (Al-Ahmad et al., 1999; Ingerslev & Halling-Sorensen, 2001; Kum- merer et al., 2000; Thiele-Bruhn, 2003). Some do not well biodegrade even in anaerobic conditions, although some substances were at least partly degradable (Samuelsen et al., 1994; Samuelsen et al., 1991; Thiele-Bruhn, 2003). In general, antimicrobials are considered to be rather persistent. For example oxytetracycline was found in concentrations up to 4.9 mg/kg in bottom deposits from ﬁsh farms. This concentration was capable of causing antimicrobial effects up to 12 weeks after administration (Halling-Sorensen

42 2. INTRODUCTION et al., 1998). Hektoen et al. (1995) studied the persistence of more antimicrobials in marine sediments. Oxytetracycline, oxolinic acid, ﬂumequine and saraﬂoxacin were found to be very persistent with calculated half-life of more than 300 days in deeper layers of the sediment. Sulfadiazine and trimethoprim were less persistent. Florfenicol was subject of biodegradation with half-life of 4.5 days. The review of Halling-Sorensen et al. (1998) gives an overview of the present knowledge of the fate of medical substances in the environment, and includes antimicrobials.

2.3.4 Ecotoxic and genotoxic effects of antimicrobials

Although bacteria, fungi and microalgae are the organisms primarily affected by antibiotics (Kummerer, 2009), they may have effect on all trophic levels in the ecosystem. In general, effects on bacteria and microalgae are found to be 2-3 orders of magnitude below the toxic values for higher trophic levels (Halling-Sorensen, 2000; Halling-Sorensen et al., 2002; Migliore et al., 2000; Park & Choi, 2008; Robinson et al., 2005; Wollenberger et al., 2000). In some studies, algae and cyanobacteria, in particular, demonstrated higher sensitivity than other species (Lutzhoft et al., 1999). It is unlikely that concentrations of antimicrobials would be high enough to affect higher organisms directly. Nevertheless, if bacterial populations were altered, the feeding of microbivores like mites and nematodes may be signiﬁcantly affected and this could propagate the effects in the food chain (Beare et al., 1992). Most antimicrobials work preferentially against either Gram positive or Gram negative bacteria. This can lead to changes in the very complex bacterial communities, as well as to inaccuracy in toxicity testing when an inappropriate testing organism is chosen (Kümmerer, 2009). Antimicrobials have the potential to affect the microbial communities in sewage systems, which may seriously affect the organic matter degradation. Substances not eliminated in the sewage treatment plant may reach surface water affecting organisms of different trophic levels (Kummerer, 2009). Halling-Sorensen et al. (1998) shows a comprehensive table of effects of mainly used pharmaceuticals in which also antimicrobials are included. Limited data on the chronic toxicity of antibacterials to crustaceans are also available with acute to chronic ratio in the range from 2.2 to 16 (Wollen- berger et al., 2000). Further issue to be considered when evaluating ecotoxicity of antimicrobials is the duration of exposure. Acute tests seem to be inappropriate for assessing the effects of antimicrobials on bacteria. As they have speciﬁc

43 2. INTRODUCTION modes of action, impact frequently becomes evident only upon an extended incubation period (Kummerer, 2009). Many studies have shown that the chronic exposure of bacteria to antimicrobials is more important than the acute (Backhaus et al., 1997; Backhaus & Grimme, 1999; Kummerer et al., 2004). There has been an increasing interest in genotoxicological effects. The potential genotoxicity and mutagenicity of antibiotics were studied using SOS-chromotest and Ames test (Isidori et al., 2005). Waste water samples from a hospital were analyzed using umuC test (Giuliani et al., 1996). Geno- toxicity of antibiotics ciprofloxacin, ofloxacin and metronidazole in waste water and their biodegradation, and thus elimination of their genotoxicity was investigated by Kummerer et al. (2000). Some authors also suggested that genotoxicity of hospital waste waters could be caused by fluoroquinolones rather than antineoplastic drugs (Hartmann et al., 1998). The mode of action of these compounds in connection with genotoxicity will be discussed below. Antibiotic residues in the environment are suspected to induce resistances in bacterial strains (Hirsch et al., 1999; Halling-Sorensen et al., 1998). Other authors mention that we currently do not know whether their presence in the environment contributes to the spread of resistance (Kummerer, 2003). Assuming that they do have an effect on resistance development, the initiation of resistance is promoted by continuing a sublethal dosage of antibiotics (Hirsch et al., 1999). Three factors contribute to development and spread of resistance: mutations in bacterial DNA, transfers of resistance genes among diverse microorganisms (Hirsch et al., 1999), and a selective pressure that enhances the development of resistant organisms (Halling- Sorensen et al., 1998). Transfers of resistance genes among diverse microorganisms are of importance, as the genetic code for resistance is often placed on R-plasmids which can be transferred between bacteria. Some antibiotics can provoke the formation of cross- and multiple resistances (Hirsch et al., 1999). The development of resistant bacteria may represent a risk to humans and animals as more and more infections can no longer be treated with the presently known antimicrobials (Hirsch et al., 1999; Kemper, 2008). A brief table of species of bacteria showing resistance is to be found in the review of Nicole Kemper (2008).

2.3.5 Veterinary antimicrobials

A speciﬁc part of the research conducted in this dissertation focused on veterinary antimicrobials, which are widely used to treat infectious diseases,

44 2. INTRODUCTION to protect the health of animals and as growth promoters to improve their growth rates. Veterinary antimicrobials and their metabolites may be released into the environment either directly, for example when used in fish farms, or indirectly, during the use of manure or slurry to land as fertilizer (Boxall et al., 2004). Livestock and poultry are often farmed in large numbers, and it is therefore necessary to treat the entire flock or herd with the antimicrobials. Thus, the amounts used are very different between human and veterinary antimicrobials. Estimated antibiotic consumption worldwide is between 100,000 and 200,000 tons per year. In 1996, about 10,200 tons of antibiotics were used in EU, out of which approximately 50 % were applied in veterinary medicine and as growth promoters. The most widely used antimicrobials in the UK and Netherlands in 1990s were tetracyclines, followed by sulfonamides, β-lactams, macrolides, aminoglycosides and fluoroquinolones (Boxall et al., 2004). About 200 tons of antimicrobial agents are administered annually in Denmark, from that approximately 10 tons in fish farming (Wollenberger et al., 2000) (Halling-Sorensen et al., 1998). In 1999 there were a total of 13216 tons of antibiotics in the EU and Switzerland, 35 % of which was applied in veterinary medicine (Kummerer, 2009). A very important source of antimicrobials into the environment is related to the dissipation of dung and liquid manure on fields as fertilizers. Drugs may contaminate the soil column or be leached into surface water bodies or even groundwater (Hirsch et al., 1999). In addition, antibiotics are extensively used in aquaculture to treat bacterial infection in intensive fish farming. Antibacterial agents are distributed directly to the water as feed additives, mostly as medicated feed pellets (Lutzhoft et al., 1999) (Wollen- berger et al., 2000), or by simple addition to water (Hirsch et al., 1999). Veterinary antimicrobials have been measured in several environmental matrices, e.g. in sediment and biota around fish farms, in soil fertilised with manure, in streams, rivers and overland flow water (Kolpin et al., 2002; Tamtam et al., 2008; Kay et al., 2005). A comprehensive review of environmental monitoring data for veterinary medicines including antibiotics is available (Boxall et al., 2004). An overview of environmental concentrations of two veterinary antimicrobials reported in the literature is presented in Table 2.4. A number of studies investigating ecotoxicity of veterinary antibacterial agents were published. Toxicity to most aquatic species is generally in the mg/L range. Boxall et al. (2004) present very comprehensive tables of aquatic and terrestrial ecotoxicity data for a range of veterinary medicines including antimicrobials.

45 2. INTRODUCTION

2.3.6 Investigated aquaculture antimicrobials Two veterinary antimicrobial agents were investigated in depth - oxytetracycline and flumequine. In Italy, monitoring of these two veterinary antimicrobials was indicated as a priority for possible environmental side effects of aquaculture (Lalumera et al., 2004). They are used in high quantities also in the Czech Republic. Both oxytetracycline and flumequine are very persistent in sediment (more than 300 days in the depth 5-7 cm, or 151 and 60 days, respectively, in surface sediment) (Hektoen et al., 1995). They have been found in soil, sediment under the fish farms, in biota near fish farms, in surface water and even in ground water. Concentrations of these two antimicrobials found in different environmental matrices as well as ecotoxic effects reported in the literature are summarized in Tables 2.4 and 2.5. More details on the studied chemicals are available in the Materials and Methods section of this dissertation thesis. Besides the ecotoxicity studies of cytostatic drugs, the second objective of the present dissertation was to provide detailed and comparable investigation into ecotoxicity of these widely used aquaculture antimicrobials, which may represent direct hazard for the aquatic environment.

Table 2.4: Concentrations of oxytetracycline and ﬂumequine in different environmental matrices.

Compound Concentration detected Matrix Country Reference Oxytetracycline 0.34 µg/L surface water USA (Kolpin et al., 2002) 2.0 ng/L to 68 µg/L surface water Japan (Matsui et al., 2008) up to 71.7 µg/L overland flow water UK (Kay et al., 2005) 0.19 µg/L groundwater Germany Hamscher et al. (2000a) in (Boxall et al., 2004) 246.3 µg/kg fish farm sediment Italy (Lalumera et al., 2004) 1516 µg/kg fish farm sediment Czech Republic (Nepejchalova et al., 2008) 8.6 4.5 µg/kg soil Germany Hamscher et al. (2000a) in (Boxall et al., 2004) 305 µg/kg soil UK Boxall et al. (2005) in (Kemper, 2008) 0.05–1.3 µg/g biota – wild fish Norway (Bjorklund et al., 1990) 0.1–3.8 µg/g biota – crustaceans USA (Capone et al., 1996)

Flumequine 32 ng/L surface water France (Tamtam et al., 2008) < 10 ng/L surface and groundwater The Netherlands (Pozo et al., 2006) and Spain 578.8 µg/kg ﬁsh farm sediment Italy (Lalumera et al., 2004) 0.1–3.8 µg/g biota – crustaceans USA (Capone et al., 1996)

46 2. INTRODUCTION

Table 2.5: Effects of oxytetracycline and ﬂumequine in ecotoxicological biotests.

Bioassay Endpoint Toxicity Reference

Oxytetracycline

Activated sludge bacteria growth 48-h EC50 0.08 (0.06–0.1) µg/L (Halling-Sorensen inhibition et al., 2002) Pseudomonas sp. 24-h MIC 1.0 0.0 µg/L (Halling-Sorensen et al., 2002)

V. ﬁsheri luminescence inhibition 30-min EC50 64.50 (47.2–88.3) µg/L (Isidori et al., 2005) 120 (112–130) µg/L (Lalumera et al., 2004)

15-min IC50 87.0 (50.8–148.9) µg/L (Park & Choi, 2008)

Microcystis aeruginosa growth 7-d EC50 0.21 (0.18–0.25) µg/L (Lutzhoft et al., 1999) inhibition

Chlorella vulgaris growth 48-h EC50 6.4 (4.9–8.4) µg/L (Pro et al., 2003) inhibition

P. subcapitata growth inhibition 72-h EC50 4.5 (2.3–86) µg/L (Lutzhoft et al., 1999) 0.342 (0.321–0.364) µg/L (Eguchi et al., 2004) 0.17 (0.11–0.25) µg/L (Isidori et al., 2005)

Rhodomonas salina growth 72-h EC50 1.6 (0.4–6.1) µg/L (Lutzhoft et al., 1999) inhibition

Lemna minor growth inhibition 168-h EC50 4.92 (3.6–6.8) µg/L (Pro et al., 2003)

D. magna immobilization 48-h EC50 22.64 (17.19–29.81) µg/L (Isidori et al., 2005) 621.2 (437.71–804.8) µg/L (Park & Choi, 2008) 48-h LOEC 100 µg/L (Wollenberger et al., 2000)

C. dubia immobilization 48-h EC50 18.65 (15.96–21.79) µg/L (Isidori et al., 2005)

D. magna reproduction 21-d EC50 46.2 µg/L (Wollenberger et al., 2000)

Brachionus calyciﬂorus population 48-h EC50 1.87 (1.19–2.96) µg/L (Isidori et al., 2005) growth inhibition

C. dubia population growth 7-d EC50 0.18 (0.11–0.26) µg/L (Isidori et al., 2005) inhibition

Flumequine

V. ﬁsheri luminescence inhibition 30-min EC50 12.7 (12.0–13.5) (Lalumera et al., 2004)

Microcystis aeruginosa growth 7-d EC50 0.16 (0.066–0.38) µg/L (Lutzhoft et al., 1999) inhibition

5-d EC50 1.96 (1.76–2.16) µg/L (Robinson et al., 2005)

P. subcapitata growth inhibition 72-h EC50 5.0 (1.6–16) µg/L (Lutzhoft et al., 1999) 5.0 (4.8–5.2) µg/L (Robinson et al., 2005)

Rhodomonas salina growth 72-h EC50 18 (10–31) µg/L (Lutzhoft et al., 1999) inhibition

L. minor growth inhibition 7-d EC50 2.47 (1.65–3.3) µg/L (Robinson et al., 2005)

Atemia salina mortality (ArToxKit) 72-h EC50 96.0 (39–240) µg/L (Migliore et al., 1997)

Chapter 3 Materials and methods

3.1 Tested compounds

3.1.1 Cytostatics

In the first study, five cytostatic compounds were analysed: cisplatin, cyclophosphamide, doxorubicin, etoposide, and 5-fluorouracil. All samples were obtained as leftovers from the Masaryk Memorial Cancer Institute’s hospital pharmacy in Brno, the Czech Republic. The cytostatics were received in the original packaging that remained of on-demand prepared therapeutic infusions. All compounds were originally dissolved in buffered saline solution (isotonic solution for human infusions), with the exception of etoposide, where ethanol was used as carrier, up to 1% v/v. Subse- quently, the solutions were stored at lowered temperature for up to one week, before being diluted with the corresponding test media. Appropri- ate solvent controls were tested in all instances. In the second study, 5-fluorouracil, cytarabine and their metabolites α-fluoro-β-alanine and uracil-1-β-D-arabinofuranoside were purchased as standards for chemical analysis from Sigma-Aldrich (Seelze, Germany). Gemcitabine hydrochloride and its metabolite 2’,2’-difluorodeoxyuridine were kindly provided by the Eli-Lilly Research Laboratories (Indianapolis, IN, USA). Concentrated stock solutions 1000 µg/L in Milli-Q water were prepared for all compounds, stored in a refrigerator for up to three weeks, and subsequently diluted further in the corresponding test media. All operations carried out with cytostatics were in compliance with safety standards for dealing with cytostatics used in the hospital pharmacy. All operations were conducted in a safety hood or laminar flow box, appropriate protective means were used. All disposable instruments and materials that were in contact with tested compounds were treated as a hazardous waste. All tests were carried out in closed boxes and/or disposable sealing foil was used as a waterproof cover for microplates. Cisplatin (CIS) is an inorganic complex formed containing an atom of

49 3. MATERIALS AND METHODS platinum. Intracellularly, highly reactive charged platinum complexes are formed. These complexes inhibit DNA through covalent binding to DNA leading to intrastrand, interstrand, and protein cross-linking of DNA, leading to apoptosis1. Cyclophosphamide (CP) is an inactive cyclic phosphoramide ester of mechlorethamine. It is transformed via hepatic and intracellular enzymes to active alkylating metabolites such as 4-hydroxycyclophosphamide, al- dophosphamide, acrolein and phosphoramide mustard. CP causes prevention of cell division primarilly by cross-linking DNA strands. It is considered to be cell cycle phase-nonspecific2. Doxorubicin (DOX) is an anthracycline antibiotic produced by the fun- gus Streptomyces peucetius. It damages DNA by intercalation (non-covalent binding to DNA) of the anthracycline portion, metal ion chelation, or by generation of free radicals. Doxorubicin has also been shown to inhibit DNA topoisomerase II which is critical to DNA function. Cytotoxic activity is cell cycle phase-nonspecific3. Etoposide (ETP) is a semisynthetic podophyllotoxin derived from the root of Podophyllum peltatum (the May apple or mandrake). Etoposide causes DNA damage through inhibition of topoisomerase II, making single- strand breaks in DNA and activation of oxidation-reduction reactions to produce derivatives that bind directly to DNA. Topoisomerase II is necessary for normal cellular function and DNA replication. Etoposide is cell cycle phase-specific with predominant activity occurring in late S phase and G24. 5-fluorouracil (5-FU) is a fluorinated pyrimidine which is metabolized intracellularly to its active form fluorouridine monophosphate. The active form inhibits DNA synthesis by inhibiting thymidylate synthetase and the normal production of thymidine. 5-FU takes effect specific in the S-phase of the cell cycle5. Cytarabine (CYT) is metabolized intracellularly into its active triphosphate form (cytosine arabinoside triphosphate), which competes with de- oxycytidine triphosphate, the physiologic substrate of DNA polymerase. This metabolite damages DNA by multiple mechanisms including the inhibition of DNA polymerase or incorporation into DNA. Cytotoxicity is

1Available on-line on http://www.cancercare.on.ca/pdfdrugs/cisplati.pdf 2Available on-line on http://www.cancercare.on.ca/pdfdrugs/cyclopho.pdf 3Available on-line on http://www.cancercare.on.ca/pdfdrugs/doxorub.pdf 4Available on-line on http://www.cancercare.on.ca/pdfdrugs/etoposi.pdf 5Available on-line on http://www.cancercare.on.ca/pdfdrugs/ﬂuorou.pdf

50 3. MATERIALS AND METHODS highly specific for the S phase of the cell cycle6. Gemcitabine (GemC) is a pyrimidine antimetabolite related to cytarabine. It exhibits cell phase specificity, primarily killing cells undergoing DNA synthesis (S-phase) and also blocking the progression of cells through the G1/S-phase boundary. Gemcitabine is a pro-drug and is metabolized intracellularly to the active diphosphate (dFdCDP) and triphosphate (dFd- CTP) nucleosides. The cytotoxic effects of gemcitabine are exerted through dFdCDP-assisted incorporation of dFdCTP into DNA, resulting in inhibition of DNA synthesis and induction of apoptosis7. Chemical structures of all tested cytostatic compounds, their CAS numbers and ATC classification codes, together with the structures and CAS numbers of tested metabolites are listed in Table 3.1.

3.1.2 Veterinary antimicrobials

The samples of veterinary antimicrobials ﬂumequine and oxytetracycline hydrochloride used in the third study were obtained from the Institute for State Control of Veterinary Biologicals and Medicines in Brno, the Czech Republic. Oxytetracycline hydrochloride (OTC) is a tetracycline broad-spectrum antibiotic with bacteriostatic action against various gram-positive and gram- negative bacteria. It is produced by Streptomyces spp. fungi. The mode of action relies on reversible binding to the bacterial 30S ribosomal subunit of the microbial ribosomes, and thus inhibiting protein synthesis. Oxyte- tracycline is the most widely used antimicrobial agent in the treatment of bacterial ﬁsh diseases (Rigos et al., 2003).

Table 3.1: Description and structures of the studied antineoplastic drugs and their metabolites. CAS no. — Chemical Abstract Services number, ATC code — the code in accordance with the Anatomical- Therapeutic-Chemical Classiﬁcation System (available on-line on http://www.whocc.no/atc_ddd_index/)

6Available on-line on http://www.cancercare.on.ca/pdfdrugs/cytarabi.pdf 7Available on-line on http://www.cancercare.on.ca/pdfdrugs/gemcitab.pdf

51 3. MATERIALS AND METHODS

Compound (producer); CAS no./ATC code Structure Description Cisplatin Cytostatic - alkylating agent NH3 (Platidiam, Pliva-Lachema a.s.); NH Cl Pt 3 15663-27-1 / L01XA01 Cl

Cyclophosphamide Cytostatic - alkylating agent O N(CH2CH2Cl)2 (Endoxan, Baxter Oncology); P O 50-18-0 / L01AA01 NH

Doxorubicin O OH O Cytostatic - intercalator (Doxorubicin-Teva 0.2%, Teva OH Pharmaceuticals); OH 23214-92-8 / L01DB01

OMe O OH O NH2

O OH

CH3

Etoposide Cytostatic - topoisomeraze H C H (Etoposide-Teva, Teva 3 II inhibitor O Pharmaceuticals); HO O 33419-42-0 / L01CB01 HO O

O H O O O O

OMe OMe OH

5-ﬂuorouracil H Cytostatic - antimetabolite, N O (LA-FU, Pliva-Lachema pyrimidine analogue a.s./Sigma-Aldrich); NH F 51-21-8 / L01BC02 O

52 3. MATERIALS AND METHODS

Cytarabine N Cytostatic - antimetabolite, (Sigma-Aldrich); pyrimidine analogue N 147-94-4 / L01BC01 O N

O O

Gemcitabine O Cytostatic - antimetabolite, (Eli-Lilly Research pyrimidine analogue N Laboratoires); 95058-81-4 / L01BC05 O N F O F

O O

α-ﬂuoro-β-alanine (FBAL) O Metabolite of (Sigma-Aldrich); F 5-ﬂuorouracil 3821-81-6 O

2’,2’-diﬂuorodeoxyuridine O Metabolite of cytarabine (dFdU) N (Eli-Lilly Research Laboratoires); O N

114248-23-6 O O

O O

uracil-1-β-D- N Metabolite of gemcitabine arabinofuranoside (araU) N (Sigma-Aldrich); 3083-77-0 O N F O F

O O

Flumequine (FLU) is a quinolone broad-spectrum antibacterial agent with bactericidal action especially against gram-negative bacteria, widely

53 3. MATERIALS AND METHODS used in intensive aquaculture. The mode of action of quinolones is inhibition of bacterial growth by interfering with the enzyme DNA-gyrase (topoisomerase II), which is essential for coiling and uncoiling of DNA. Flume- quine thus terminates the normal DNA synthesis (Samuelsen, 2006). Chemical structures of the compounds, their CAS numbers and ATC classiﬁcation codes are listed in Table 3.2.

Table 3.2: Description and structures of the studied antimicrobial drugs. CAS no. — Chemical Abstract Services number, ATCvet code — the code in accordance with the Anatomical-Therapeutic-Chemical Clas- siﬁcation System for veterinary medicines (available on-line on http://www.whocc.no/atcvet/atcvet_index/)

Compound CAS no./ATCvet code Structure Description Oxytetracycline Tetracycline H3C CH3 H C OH OH N hydrochlorid 3 H H antibiotic OH 2058-46-0 / QJ01AA06 ClH

NH2 OH OH O OH O O

Flumequine Fluoroquinolone CH3 42835-25-6 / QJ01MB07 antibacterial N

OH F O O

3.2 Ecotoxicological bioassays and chemical analyses

Separate trophic levels in an ecosystem differ from each other in the way of obtaining energy and matter for living and their metabolism is different. As the toxic compounds in the environment target their effects on different metabolic processes, they may manifest differently in separate trophic levels of an ecosystem. Hence, it is important to employ organisms belong- ing to different trophic levels of an ecosystem. The major trophic levels of an ecosystem are as follows: producers, i.e. plants and green algae, consumers, i.e. animals, and decomposers, i.e. fungi and bacteria. Ecotoxico- logical bioassays have been carried out using representatives of all major

54 3. MATERIALS AND METHODS trophic levels of an ecosystem. For all bioassays, experiments included initial testing of a wide range of concentrations (1:10 dilutions). If necessary, more detailed assessment around the 50% effective concentration (EC50) followed. All assays were repeated independently at least three times.

Algal growth inhibition test In the algal growth inhibition test, cultures of selected green algae in the exponential phase of growth are exposed to various concentrations of the tested substance over several generations. The incubation time of the test solutions is 72 hours, during which the cell density in each solution is measured. The growth inhibition in relation to the control culture is determined. The purpose of this test is to determine the effects of a substance on the growth of a unicellular green algal species (OECD, 2002a). The algal growth inhibition test was performed according to the Czech norm (CNI, 1995a) based on the European standard EN ISO 8692:1989 (ISO, 1989b) using unicellular alga Pseudokirchneriella subcapitata. This assay was performed in modified and miniaturized version using 96-well microplates, as described in the article of Rojickova et al. (1998). 50% ZBB medium was used as a culture and test medium, prepared by mixing of Zehnder Z-medium and Bristol modified Bold (BB) medium8 and distilled water in the ratio 1:1:2. Cells in the exponential growth phase were used for the test with the initial cell density adjusted to 105 cells/mL. The cell suspension was mixed in the test wells with medium and the solution of the studied compound in five or six different concentrations. The incubation run at 24 ◦ C under continuous light. Growth was evaluated as the change of light absorbance at the beginning and the end of the exposure. Measurements were carried out every 24 hours (using Tecan GENios or BioTek Absorbance Microplate Reader). Light absorbance was measured at 680nm, which corresponds to the amount of chlorophyll. Total duration of the test was 96 hours when the growth inhibition was evaluated. During the second study with cytostatic pharmaceuticals, the algal growth- inhibition test was conducted according to the European standard EN ISO 8692:1989 (ISO, 1989b) and the German standard DIN 38412 Part 9 (DIN, 1993) using green alga Desmodesmus subspicatus. The assay was performed ◦ in 24-well microplates. The plates were incubated at 23 C under continuous light and discontinuous shaking (30 min 400 rpm, 15 min break). The fluorescence of chlorophyll was measured using Tecan SPECTRAFluor Plus Microplate Reader in the beginning of the exposure and then every 24

8Available on-line on http://www.butbn.cas.cz/ccala/index.php

55 3. MATERIALS AND METHODS hours. The absorbance at 650 nm was measured at the end of the test. The total duration of the bioassay was 72 hours. The average growth rates were calculated at the end of the test and the growth inhibition was evaluated.

Growth inhibition test with Lemna minor Plants of duckweed (Lemna minor) are allowed to grow in different concentrations of the tested substance for the period of seven days. The objective of the test is to quantify the effects on vegetative growth based on the assessments of frond number and, eventually, other endpoints (total frond area, dry weight or fresh weight). To quantify the effects, growth in the test solutions is compared with that of the controls (OECD, 2002b). Growth inhibition test with Lemna minor was performed according to the European standard (ISO, 2001) and the OECD standard (OECD, 2002b). The test was performed in 50 ml polystyrene vessels, test medium was prepared as described in the ISO norm (ISO, 2001). Five fronds, pre-cultivated one week prior to the start of the test were put into each vessel on the beginning of the test. Six concentrations in triplicates plus the controls have ◦ been tested. Test vessels have been cultivated at 24 ˛a2 C under continual light. Total duration of the exposure was 7 days. The growth inhibition was evaluated as the change of the total frond number in comparison to the control at the end of the test.

Acute immobilisation test with Daphnia magna Newly hatched animals of Daphnia magna are exposed to the test substance added to the test medium at a range of concentrations for 48 hours. The purpose of this test is to quantify the effect of a substance on the mobility of Daphnia in fresh water. The immobilization is evaluated as the number of immobile individuals in comparison to the control (OECD, 2004). Acute immobilisation test with Daphnia magna was conducted according to the Czech standard (CNI, 1997) which is identical with European Stan- dard EN ISO 6341:1996 (ISO, 1989a). The test was performed in polystyrene multiwell plates. Test medium was prepared, as described in the standard. Juveniles of D. magna obtained from the continuous laboratory culture were used. Newly hatched animals (less than 24 hours old), actively swimming, obtained from the continuous laboratory culture were used. Twenty animals for each tested concentration and the control were divided into four testing wells, each containing ﬁve animals in 10 ml of the medium. Five ◦ particular concentrations were tested. The test was run at 18–20 C under the photoperiod 16:8 hours light/dark. Total duration of the exposure was 48 hours. Immobilised organisms were counted, i.e. animals not able to

56 3. MATERIALS AND METHODS swim within 15 seconds after gentle agitation of the test container, after 24 hours of the test, and at the end of the test. The results were expressed as the percentage of the numbers of the exposed and control animals immobilised. During the second study with cytostatic pharmaceuticals, acute immobilisation test with D. magna was conducted according to the European Standard EN ISO 6341:1996 (ISO, 1989a) with some modiﬁcations. For the breed, as well for the test, we have used the ADaM medium (Aachener Daphnien Medium) imitating natural fresh water, described in the article of Klüttgen et al. (1994). For the test, glass beakers were used, and the ◦ cultivation run at 20 C in the dark.

Daphnia magna reproduction test Young Daphnia, less than 24 hours old at the start of the test, are exposed to the range of concentrations of the tested substance over a period of 21 days. The primary objective of the test is to assess the effect of chemicals on the reproductive output of Daphnia magna. The total number of living offspring produced per parent animal alive at the end of the test is assessed. The number of offspring of the animals exposed to the test substance is compared to that of the controls (OECD, 1998). Daphnia magna reproduction test was performed according to the OECD TG 211 Standard (OECD, 1998) and the Czech Standard (CNI, 2001). The test was performed in 50 ml polystyrene vessels, test medium M4 was prepared as described in the Czech Standard. Juveniles of D. magna, only females, less than 24 hours old, obtained from the continuous laboratory culture were used. Ten animals for each concentration and control were divided into the test vessels, each animal separately in 50 ml of M4 medium containing a speciﬁc concentration of the tested compound. Experiments ◦ were run at temperatures 21 ± 1 C and photo-period 16:8 light/dark. Total duration of the exposure was 3 weeks. Organisms were fed with mixture of green algae three times a week. Simultaneously, the medium was changed and offspring produced by parent animals was counted and removed. Sur- vival of parent animals and numbers of offspring alive were evaluated and expressed as the percentage of control. Juveniles produced by the adults that have died during the test were excluded from the calculations. During the second study with cytostatic pharmaceuticals, the reproduction test with Daphnia magna was conducted in accordance with the OECD TG 211 standard (OECD, 1998) with modiﬁcations. As in the acute test, the ADaM medium (Klüttgen et al., 1994) was used. The test was performed in glass beakers placed in translucent plastic boxes. Daphnids were fed with

57 3. MATERIALS AND METHODS the suspension of green alga Desmodesmus subspicatus.

Pseudomonas putida growth inhibition test In the Pseudomonas putida growth inhibition test, cultures of bacteria P.putida in the exponential phase of growth are exposed to a range of concentrations of the tested substance over several generations. The test solutions are incubated for a period of 16 hours, after which the cell density is measured in each solution. The growth inhibition in relation to the control culture is determined. The purpose of this test is to determine the effects of a substance on the growth of a bacterial species (CNI, 1995b). Pseudomonas putida growth inhibition test was performed according to the European Standard EN ISO 10712:1995 and the Czech Standard (CNI, 1995b). The test was performed in the miniaturised version as described in the article of Schmitz at al. (Schmitz et al., 1998). The test was performed in 96-well microplates. Cryopreserved cultures of Pseudomonas putida, stored ◦ ◦ at -80 C in glycerol, were used. The microplates were incubated at 23±1 C for 16 hours on a shaker in the dark. The growth inhibition was evaluated as the change of the absorbance of the bacterial culture measured at 590 nm (using Tecan GENios or BioTek Absorbance Microplate Reader) before and after exposure and expressed as the percentage of control.

Vibrio fischeri test on bioluminescence inhibition Revitalised cultures of luminescent marine bacteria Vibrio fischeri are exposed to various concentrations of the test substance for 30 minutes. The purpose of this test is to determine the effects of a substance on the luminescence ability of a bacterial species. The inhibition of luminescence in relation to the control culture is determined (ISO, 1998). Vibrio fischeri test on bioluminescence inhibition was performed according to the Czech standard (CNI, 2000) based on the European Standard (ISO, 1998). 2% solution of natrium chloride was used as a test medium. For testing, freeze-dried cultures of Vibrio fischeri purchased from the In- stitute of Microbiology, Academy of Sciences of the Czech Republic were used. The cell suspension was mixed with the test medium in polystyrene tubes. The solutions of the studied compounds were added in five different ◦ concentrations. The incubation was run at 15 C for 30 minutes. The bioluminescence was measured in the cuvette luminometer Lumino M90a before the tested compound was added. Subsequently, the bioluminescence was measured after 15 and 30 min of exposure. The inhibition of bioluminescence of bacterial culture was evaluated as a percentage of luminescence decrease in the tested samples in comparison with the control at the end of

58 3. MATERIALS AND METHODS the test.

Genotoxicity – SOS-chromotest Exponentially-growing cultures of genetically modified bacteria Escherichia coli are exposed to various concentrations of the test substance. The purpose of this test is to determine the induction of the SOS-response in conse- quence of the alteration of genetic information in a bacterial strain. The test solutions are incubated with the bacterial culture for 2 hours. Afterwards, the viability of bacterial cells and the activity of the DNA repair system are measured, as described below. The genotoxicity expressed as the induction factor in relation to the negative controls is determined. The SOS-chromotest was performed using genetically modified bacterial tester strain Escherichia coli PQ 37 (Quillardet et al., 1982). The test was performed in the miniaturised version, using the 96-well microtitre plate format, as described previously (White et al., 1996; Xu et al., 1989; Bar- tos et al., 2005) with and without metabolic activation using S9 rat liver homogenate (Institute of Public Health Ostrava, Czech Republic). After 2 hours of incubation with compounds of interest, the activity of β-galactosidase, the reporter enzyme for genotoxicity induced along with DNA repair system, was measured using a chromogenic substrate ortho-nitrophenyl-β- D-galactopyranoside. At the same time, activity of alkaline phosphatase, the marker of viability, was assessed using p-nitrophenyl phosphate chromogenic substrate. Both genotoxicity and viability were measured spec- trophotometrically as the change of colour of the bacterial suspension induced by the reaction of the appropriate enzyme with the chromogenic substrate. Cytotoxic effects were quantified as the percentage of inhibition of the alkaline phosphatase in comparison with the negative control. The concentrations causing more than 50% inhibition were excluded from genotoxicity evaluations. The SOS induction factor (IF) was then calculated for each tested concentration as the ratio of activities of β-galactosidase and alkaline phosphatase related to the negative control. The minimal genotoxic concentration (MGC), at which the IF reaches the critical value 1.5, was determined.

Genotoxicity – umu-test The principle of the umu-test is similar to the principle of the SOS-chromotest, except that another bacterial tester strain is used. The sensitivity of the test might hence be different. Further, the cytotoxicity of the tested compound is determined by determining growth inhibition, rather than the metabolic viability of the culture.

59 3. MATERIALS AND METHODS

The umu-test was performed according to the international standard ISO/FDIS 13829 (DIN 38415-3) (ISO, 1999). The genetically modified bacterial tester strain Salmonella choleraesius subsp. chol. was used. The genotoxin- dependent induction of the umuC-gene was compared with the spontaneous activation of the untreated control culture. The test was performed in 96-well microplates with and without metabolic activation using S9 rat liver homogenate (Trinova Biochem GmbH, Giessen, Germany). After the 2 hours of initial incubation of bacteria with the tested compounds (with or without S9 homogenate), reaction mixtures were diluted with exposure media ten fold and incubated for another 2 hours. Optical density was then measured to assess the bacterial growth (cytotoxicity) ) using Labsystems iEMS Reader MF. Subsequently, the β-galactosidase activity was measured using a chromogenic substrate ortho-nitrophenyl-β-D-galactopyranoside. Cy- totoxic effects were quantified as the percentage of inhibition of growth in comparison to the negative control. The concentrations causing more than 50% inhibition were excluded from genotoxicity evaluations. The induction factor was calculated for each tested concentration and the experimental minimal genotoxic concentration was determined as the first tested concentration, at which the IF reached over the critical value of 1.5.

Genotoxicity - GreenScreen assay GreenScreen assay (GSA) is a rapid genotoxicity assay using eukaryotic yeast cells Saccharomyces cerevisiae based on assessment of the green fluorescent protein (GFP) fused to the DNA-damage inducible promoter of the RAD54 gene (Afanassiev et al., 2000). This assay responds to genotoxic agents that activate the DNA repair pathway (Cahill et al., 2004), and the amount of accumulated GFP reflects the genotoxic effect of the tested substance. The GreenScreen assay was performed using two modified Saccharomy- ces cerevisiae yeast strains (GenT01 testing strain and GenC01 as the control for cytotoxicity) as described previously (Bartos et al., 2006; Cahill et al., 2004). The assay was carried out in 96-well microplates (black, clear bot- ◦ tom) using the overnight exposure of 16 hours at 30 C, without shaking. The cytotoxicity, i.e. growth inhibition was evaluated as the change of the absorbance of the yeast culture measured at 620 nm before and after exposure. The genotoxicity potential was determined as the rate of synthesis of the green fluorescent protein, which was measured as fluorescence at 520 nm in the POLARstar fluorimeter with fluorescence polarisation (BMG LABTECH). The induction factor was calculated for each tested concentration, and the minimal genotoxic concentration was determined as the

60 3. MATERIALS AND METHODS concentration, at which the IF reaches the critical value of 1.3. Only noncytotoxic concentrations were included in the evaluation.

Chemical analyses Chemical analyses were performed to check the stability of cytostatic agents during acute and reproduction tests with Daphnia magna. Samples were collected, ﬁltered and diluted with acetonitrile (1:1) and the concentrations measured using HPLC/DAD or HPLC/MS-MS as described in detail previously (Kovalova et al., 2009). Analyses revealed good stability of the studied compounds during the tests with maximum decline less than 10% (data not presented).

3.3 Data analysis

For ecotoxicological assays, the homogeneity of variance was controlled by Levene‘s test. Values of LOEC (the lowest tested concentration causing statistically significant effect) were evaluated using Analysis of Variance (ANOVA) followed by the Dunnet’s test or non-parametric Mann-Whitney U test. The highest tested concentrations with no observable effects (NOEC values) were determined experimentally as the nearest lower tested concentration to LOEC. Concentrations causing 50% effects (EC50) were estimated using the probit analysis in the U.S. Environmental Protection Agency Pro- bit software (U.S. EPA,Washington, D.C.) in the first study, and using sigmoidal non-linear dose-response regression in GraphPad Prism 4.0 (Graph- Pad Software, Inc.), in the subsequent studies. Minimum genotoxic concentrations (MGC) were derived as the mean concentrations that reached the induction factors value of 1.5 (SOS-chromotest) or 1.3 (GSA assay), in the first study. In the following studies, MGCs were derived experimentally, as the first tested concentration that exceeded the induction factors value of 1.5 in both SOS-chromotest and the umu-test. All calculations were performed with Microsoft Excel(r) (Microsoft, Redmond, WA, USA) and Statistica(r) for Windows 6.0 and 7.1 (StatSoft, Tulsa, OK, USA).

3.4 Risk characterisation

Risk quotients (RQ) for the aquatic environment were calculated using the MEC (measured environmental concentration) or PEC (predicted environmental concentration) reported in the literature and the PNECs (predicted no-effect concentrations) based on the current study. For PNEC, the lowest observable effect concentration (LOEC) values from our studies were used.

61 3. MATERIALS AND METHODS

An assessment factor (AF) of 1000 was used for extrapolation of the values from acute ecotoxicological tests, following the equation, Eq. 3.1.

PNEC = LOEC/AF (3.1)

The ﬁnal risk quotient was calculated by dividing the worst case (the highest found) MEC or PEC by the PNEC (Eq. 3.2.).

RQ = MEC/P NEC (3.2)

Ecological risk is not expected if the risk quotient is calculated to be less than 1 (EMEA, 2008).

62 Chapter 4 Results and discussion

4.1 Cytostatics

There are only limited studies of the ecotoxicity of cytostatic pharmaceuticals available in the literature, although cytostatics and their metabolites have the deﬁnite potential to affect most organisms due to their mode of action, which often targets the structure or function of DNA. Potentially, any growing eukaryotic organism could be harmed by them (Johnson et al., 2008). We have performed two studies of the ecotoxicity of cytostatics. The initial study evaluated the ecotoxicity and genotoxicity of ﬁve cytostatic compounds used in high quantities. The particular compounds were selected in cooperation with the hospital pharmacy of Masaryk Memorial Cancer Institute, Brno, Czech Republic, based on their relatively common use in the Czech Republic. The second study addressing cytostatic ecotoxicity was performed mainly at Institute of Hygiene and Environmental Health of RWTH Aachen in Germany, within the scope of a Marie-Curie Early Stage Research Train- ing programme. This study focused on the ecotoxicity and genotoxicity of three cytostatic pharmaceuticals and their main human metabolites, including chronic effects.

4.1.1 Ecotoxicity and genotoxicity assessment of cytostatic pharmaceuticals (Paper I)

The ecotoxicity and genotoxicity of cyclophosphamide, 5-ﬂuorouracil, cisplatin, doxorubicin and etoposide were investigated. The compounds studied are used in large volumes both in the Czech Republic and worldwide. 5-ﬂuorouracil is, very likely, the most common cytostatic drug in the UK, by quantity, followed by cyclophosphamide and platinum compounds. Ap- proximately 1 ton of 5-FU is used in the UK per year, together with 1.7 tons of capecitabine, which is the prodrug of 5-FU giving the combined value of

63 4. RESULTS AND DISCUSSION

2.7 tons/pa (Johnson et al., 2008). For more information about the studied compounds see Chapter 3.1.1. In evaluating ecotoxicity, representatives of all three trophic levels were used. Producers - green algae - were represented in the growth inhibition test with Pseudokirchneriella subcapitata. Consumers - fresh water crustaceans - were represented in the acute immobilization test with Daphnia magna Strauss. Decomposers - bacteria - were involved in the growth inhibition test with Pseudomonas putida. Genotoxicity was evaluated in both prokaryotic and eukaryotic bioassays - both SOS-chromotest with Escherichia coli PQ 37 genetically modified tester strain and GreenScreen assay using yeast cells Saccharomyces cerevisiae. The main objective of this study was to obtain comprehensive comparative data on the potential ecotoxicological impact of cytostatics, which may aid further evaluation of the related environmental risks. All tested cytostatics except for cyclophosphamide exhibited significant effects in all bioassays. The effects of tested cytostatic agents, i.e. values for no observable effect concentrations (NOEC), the lowest observable effect concentrations (LOEC), concentrations causing 50% effect (EC50), and minimal genotoxic concentrations (MGC) are listed in Tables 4.1 and 4.2. Full dose-response curves in ecotoxicological tests are illustrated in Figure 4.1. Finally, results of genotoxicity test are presented in Figure 4.2. In general, 5-FU was the most toxic compound with effect concentrations ranging from 10 µg/L. In contrast, the effective concentrations of CP, the least toxic agent, were four to five orders of magnitude higher. With the exception of cyclophosphamide, our results suggest that ecotoxic potencies of cytostatics might be higher than expected from what was previously reported for other compounds (see Table 2.3, Chapter 2.2.2). CP was nontoxic to D. magna in this study with EC50 higher than 1000 mg/L. This value may perhaps be comparable to the EC50 of 1795 mg/L reported in the literature (Sanderson et al., 2003). Differences in bioassay sensitivities were observed. 5-FU was the most toxic compound for P.putida and P.subcapitata with LOEC values of 10 µg/L. Doxorubicin and cisplatin were most toxic for D. magna with LOEC values one order of magnitude higher. Generally, the values of effect concentrations for D. magna (Tab. 4.1) are relatively high. The fact that D. magna is less sensitive to cytostatics than other testing organisms might be some- how connected with their mode of action and the experiment design. In the acute immobilization test, only non-specific effects such as non-polar narcosis are observed, whereas mechanisms affecting nucleic acid and protein synthesis (and thus cell division) might be manifested in growth inhibition

64 4. RESULTS AND DISCUSSION tests. Also, bacterial toxicity data for 5-FU are one order of magnitude lower then the value reported in the literature (V. ﬁscheri luminescence inhibition, 30-min EC50 = 0.122 mg/L (Backhaus & Grimme, 1999)). The explana- tion might be similar - no protein or nucleic acid synthesis is necessary for measuring the endpoint in luminescence inhibition test. Moreover, the exposure time might be too short for compounds with such a speciﬁc mode of action to express. The issue of the differences between short-term and prolonged bioassays for testing of pharmaceutical substances is mentioned also in the antibiotics section of this work and is discussed in the article of Backhaus (Backhaus et al., 1997) at some length.

Table 4.1: Ecotoxicity of the tested cytostatic compounds. Values in the parentheses indicate 95% conﬁdence interval. NOEC = no observable effect concentration (experimental); LOEC = lowest observable effect concentration (experimental); EC50 = concentration causing 50% effect.

Assay/compound NOEC (mg/L) LOEC (mg/L) EC50 (mg/L)

Pseudomonas putida 5-ﬂuorouracil 0.003 0.01 0.027 (0.015–0.045) Cisplatin 0.03 0.1 1.2 (1.0–1.40) Cyclophosphamide 1000 >1000 >1000 Doxorubicin 1 10 >1000 Etoposide 200 250 630 (580–830)

Pseudokirchneriella subcapitata 5-ﬂuorouracil 0.001 0.01 0.11 (0.03–0.3) Cisplatin 0.1 1 2.3 (1.7–2.9) Cyclophosphamide 250 500 930 (700–1100) Doxorubicin 1 10 13 (12–17) Etoposide <10 10 250 (120–460)

Daphnia magna 5-ﬂuorouracil 1 10 36 (12–70) Cisplatin 0.2 0.5 0.64 (0.4–0.85) Cyclophosphamide ≥ 1000 >1000 >1000 Doxorubicin 0.01 0.1 2.0 (0.52–4.8) Etoposide 10 30 30 (16–40)

There are only limited data on the toxicity of cytostatics to producers such as algae in the literature. The toxicity of cyclophosphamide in our study (EC50 of 930 mg/L; Table 4.1) is much lower than the reported QSAR-

65 4. RESULTS AND DISCUSSION

Table 4.2: Genotoxicity (minimum genotoxic concentrations, MGC) of the tested cytostatic compounds in the bacterial SOS-chromotest and eukaryotic yeast GSA test. Values in parentheses indicate 95% conﬁdence interval. NS = no signiﬁcant toxicity.

MGC (mg/L) SOS chromotest GSA Compound without metabolic activation with metabolic activation 5-ﬂuorouracil 1.4 (1.2–29) NS 0.02 (0.018–0.021) Cisplatin 0.17 (0.1–0.37) 0.09 (0.03–0.37) 0.44 (0.32–1.6) Cyclophosphamide NS NS 470 (270–750) Doxorubicin 0.074 (0.02–0.12) 0.098 (0.05–0.5) 2.8 (2.0–3.6) Etoposide 2.4 (1.3–7.7) 6.4 (4.8–20) 150 (140–168)

predicted value (EC50 of 11 mg/L (Sanderson et al., 2003), Table 2.3 in Chapter 2.2.2). Cyclophosphamide generally had very low toxicity in all bioassays performed (Table 4.1). This corresponds to the fact that cyclophosphamide, as many other cytotoxic drugs, is a prodrug. It is biologically ineffective until transformed into its active metabolites by intracellular or hepatic enzymes (Klener, 2002). The important question for environmental risk assessment is whether the same activating enzymes and hence toxic actions might occur in aquatic organisms (Johnson et al., 2008). The possible environmental effects of active metabolites of cyclophosphamide (phosphoramide mustard, acrolein, and 4-hydroxycyclophosphamide, al- dophosphamide) and metabolites of cytostatic drugs in general have not been studied in any detail yet. All tested compounds exhibited significant genotoxicity in at least one test. Doxorubicin was the most genotoxic compound in the bacterial assay (MGC below 0.1 mg/L in both versions without and with metabolic activation, Tab. 9). The most genotoxic compound overall was 5-fluorouracil with MGC in the yeast assay of 0.02 mg/L (Tab. 9). The differences between the two bioassays and between both versions of the SOS-chromotest were observed. Cyclophosphamide, for instance, significantly induced reporter green fluorescent protein in eukaryotic yeast GSA test, although it was non-genotoxic in bacterial SOS-chromotest (Fig. 4.2). Similar results were obtained in the study of Anderson et al. (Anderson et al., 1995). CP has been reported as mutagenic to Salomonella typhimurium, but not to Es- cherichia coli. The genotoxic concentration of CP in GSA is, indeed, environmentally irrelevant.

66 4. RESULTS AND DISCUSSION

Figure 4.1: Effects of tested cytostatics (FU 5-ﬂuorouracil, CP - cyclophosphamide, CS - cisplatin, DX - doxorubicin, EP - etoposide) on the growth growth of green alga Pseudokirchneriella subcapitata, immobilization of Daphnia magna and of bacteria Pseudomonas putida.

67 4. RESULTS AND DISCUSSION

Figure 4.2: Genotoxicity (expressed as Induction Factors, IF) of ﬁve studied cytostatics in the yeast GSA test (white bars) and bacterial SOS-chromotest (without metabolic activation – dashed bars and with metabolic activation – black bars). Data represent mean ± standard deviation of at least three independent experiments. Values of IF>1.3 and IF>1.5 indicate signiﬁcant genotoxicity in GSA and SOS assays, respectively.

68 4. RESULTS AND DISCUSSION

For doxorubicin and etoposide, the GSA was less sensitive than the SOS- chromotest. The results differ from each other by one or two orders of magnitude. For etoposide, we have observed fairly good agreement between the results reported in the literature (MGC of 125 mg/L (Cahill et al., 2004)) and our experiments. For cisplatin, our results correspond to a previous study (Quillardet & Hofnung, 1993), which demonstrated mutagenicity in the Ames test. The GSA displayed significant genotoxic potential of cisplatin, which was approximately 40 times higher than previously reported value (MGC of 18.75 mg/L with similar test system (Cahill et al., 2004)). No major differences have been observed between the SOS-chromotest results of cisplatin, doxorubicin, and etoposide with or without metabolic activation (Fig. 4.2). On the other hand, 5-fluorouracil showed no genotoxicity in the SOS-chromotest with metabolic activation, although the effects in the variant without metabolic activation, as well as in the GSA (in principle without metabolic activation, but eukaryotic testing system) were significant in relatively low concentrations. These findings may be explained by the possible inactivation during metabolization (Klener, 2002). Overall, we have determined the eco- and genotoxic potential of some compounds with LOEC and MGC values below 0.1 mg/L (5-fluorouracil in P. putida and P. subcapitata growth inhibition test and GSA, cisplatin in P. putida growth inhibition test and doxorubicin in SOS-chromotest), with substantial differences in bioassay responses. Special matrices, such as hospital effluents, may contain cytostatics concentrations within the range of observed acute toxicity (see Table 2.2, Chapter 2.2.3), and possible chronic effects may increase possible risks further. More discussion about the risk assessment is provided in the Chapter 4.3.

4.1.2 Ecotoxicity and genotoxicity assessment of cytotoxic antineoplastic drugs and their metabolites (Paper II)

Many pharmaceuticals used in human medicine are not completely metabolized. Such pharmaceuticals, including cytostatics, are excreted either slightly transformed or unchanged, often conjugated to polar molecules. These conjugates can easily be cleaved during sewage treatment and the original drugs might then be released into the aquatic environment (Heberer, 2002). Other pharmaceuticals, albeit still including cytostatics, are metabolized. The metabolites might, however, still be active and toxic. In some cases, the metabolites may be more toxic than the parent compound (Halling- Sorensen et al., 1998). If the elimination of drugs and their metabolies during waste water treatment is limited, they may persist in the environment

69 4. RESULTS AND DISCUSSION

(Heberer, 2002). Although some experimental studies investigated the toxicity of cytostatic compounds in acute ecotoxicological tests and the genotoxicity of hospital effluents as an indirect marker of cytostatics contamination (Giu- liani et al., 1996; Jolibois et al., 2003; Jolibois & Guerbet, 2006), to our knowledge, chronic effects of these compounds and ecotoxicity of their metabolites have not been reported yet. The present study aims to evaluate ecotoxicity and genotoxicity of three cytostatic drugs and their major human metabolites. All compounds studied - 5-fluorouracil, cytarabine and gemcitabine - belong to the family of pyrimidine antimetabolites, which rank among the most widely used cytostatics. All of these compounds are metabolized and excreted in urine, mostly as inactive metabolites. Only approximately 10 % of the administered drug is excreted in the unchanged form (Heggie et al., 1987; Hamada et al., 2002; Abbruzzese et al., 1991). 5-FU is not biodegraded in waste water, while gemcitabine and cytarabine are biodegraded only partially, posing a risk to the environment (Kummerer & Al-Ahmad, 1997). For more information about the tested compounds, see Chapter 3.1.1. The ecotoxicity and genotoxicity of the compounds were investigated with three acute ecotoxicity bioassays on all trophic levels of ecosystem. First, acute immobilization test with Daphnia magna was performed, second, growth inhibition test with green alga Desmodesmus subspicatus and third, growth inhibition test with bacteria Pseudomonas putida. Finally, one chronic bioassay - reproduction test with Daphnia magna – and a bacterial genotoxicity assay – umu-test – were performed. All tested parent cytostatic agents exhibited significant toxicity. The effects of tested drugs and their metabolites, i.e. full dose-response curves in ecotoxicological bioassays, are summarized in Figures 4.2 and 4.4. Values of the no observable effect concentrations (NOEC), lowest observable effect concentrations (LOEC) and 50% effective concentrations (EC50) are listed in Table 4.3. The toxicity of the tested cytostatic pharmaceuticals was variable, with acute EC50 values ranging from 44 µg/L (5-fluorouracil in the Pseu- domonas putida growth inhibition test) to 200 mg/L (cytarabine in Daphnia magna acute immobilization test). Most EC50 values were between 10 and 100 mg/L or even higher. 5-fluorouracil was the most toxic compound in this study. It showed high toxicity in two assays (reproduction toxicity to D. magna and P. putida growth inhibition, EC50 = 0.04–0.05 mg/L), while less pronounced effects were observed in the algal growth inhibition test (EC50 = 75 mg/L). 5-fluorouracil was found to be highly toxic in majority of the conducted bioassays also in the previous study.

70 4. RESULTS AND DISCUSSION 50 10 0.1 > 10 > 10 -fluoro- α reproduction 3.7 > 10 > 10 > 1.0 > 1.0 > 1.0 > 1.0 ∗ ∗ ∗ ∗ 1.0 10 10 0.01 0.05 1.0 1.0 NOEC LOEC EC Daphnia magna 50 > 100 > 100 > 100 EC – concentration causing 50% effect. acute 50 50 110 (45–520) 5.0 15 (5.2–45) 100 200 (63–810) 100 > 100 > 100 Daphnia magna ∗ ∗ 50 60 5.0 1.0 100 100 NOEC LOEC 50 > 200 > 200 40 53 (29–95) 10 45 (12–170) 40 48 (44–51) 25 80 (28–230) 200 > 200 D. subspicatus ∗ 0 0 20 10 20 200 NOEC LOEC EC -D-arabinofuranoside (AraU), 2’,2’-difluorodeoxyuridine (dFdU) in ecotoxicological beta " indicate the highest concentrations used in the respective assay. 50 ∗ > 500 > 500 EC 17 (4.7–64) 100 (34–300) 140 (110–180) 10 50 100 > 500 > 500 Pseudomonas putida ∗ ∗ 0 0 50 500 500 -alanine (FBAL), uracil-1- Summary of the effects of cytostatic drugs 5-fluorouracil, cytarabine and gemcitabine, and their metabolites biotests. All toxicity values areable in effect mg/L (values concentration, in LOEC parentheses – show lowest 95% confidence observable intervals); effect NOEC concentration, – EC no observ- β Numbers with the sign " Cytarabine Gemcitabine FBAL Test substance NOEC5-fluorouracil LOEC 0.02 0.03 0.044 (0.025–0.077) AraU dFdU Table 4.3:

71 4. RESULTS AND DISCUSSION

Toxicity values derived in our study are also well comparable with effective concentrations of other compounds from the family of pharmaceuticals and personal care products (Sanderson et al., 2003; Cleuvers, 2003; Halling- Sorensen et al., 1998; Wollenberger et al., 2000) (see Table 2.1, Chapter 2.3.1). Given the limited availability of standards, only limited data seem to exist on metabolites and derivatives of parent pharmaceuticals. The metabolites of cytotoxic drugs tested in the present study, which are considered inactive in cancer therapies, were expected to be less cytotoxic than the parent compounds. The lower toxicity of metabolites was confirmed in our study. Only FBAL (metabolite of 5-fluorouracil) showed pronounced toxicity and EC50 values were derived for two assays with bacteria and algae (Table 4.3). In spite of lower toxicity, FBAL might still be classified as harmful to aquatic organisms, according to the EU (Cleuvers, 2003). Interest- ingly, FBAL elicited comparable toxicity as its parent compound, 5-FU, in the growth inhibition test with D. subspicatus. Concerning sensitivity of acute assays, the growth inhibition test with Pseudomonas putida was the most sensitive in the present study. For 5-fluorouracil, for example, it had the same sensitivity as the long-term reproduction test with D. magna (Table 4.3). It was only in the case of gemcitabine, that the growth inhibition test with D. subspicatus was more sensitive than P. putida test. A considerable difference becomes apparent when comparing our results with P. putida growth inhibition test with those published in the literature. Kummerer and Al Ahmad (Kummerer & Al-Ahmad, 1997) tested biodegradability of 5-FU, CYT and GemC and performed the growth inhibition test with P. putida as a control. For all antitumour agents, concentrations exhibiting no inhibitory effects were above 128 mg/L. The difference between our measurements and the published ones is about four to five orders of magnitude. (Kummerer & Al-Ahmad, 1997) expected only little toxicity of 5-FU against Gram-negative bacteria but the present work demonstrates significant toxicity. Gram-negative bacteria form an important part of the activated sludge in the sewage treatment plants (Kummerer & Al-Ahmad, 1997), and consequently, possibility of the STP bacterial communities being influenced by the cytostatic compounds present in hospital effluents cannot be excluded. Large differences among toxicities of studied drugs were observed in the acute vs. 21-day reproduction tests with D. magna. For 5-fluorouracil and cytarabine, EC50 in the reproduction test was nearly three orders of magnitude lower than acute immobilization. In contrast, no pronounced effects in the reproduction test were recorded for gemcitabine up to the

72 4. RESULTS AND DISCUSSION

Figure 4.3: Ecotoxicity (concentration-response curves) of the studied cytostatic drugs and their metabolites. A – Daphnia magna acute immobilization test. B – Growth inhibition test with Desmodesmus subspicatus. C – Growth inhibition test with Pseudomonas putida. 5-FU: 5- fluorouracil, CytR: cytarabine, GemC: gemcitabine, FBAL: α-fluoro- β-alanine, dFdU: 2’,2’-difluorodeoxyuridine. Compounds, which did not induce significat toxicity are not presented in respective plots.

73 4. RESULTS AND DISCUSSION

Figure 4.4: Effects of 5-ﬂuorouracil (5-FU) and cytarabine (CytR) on the reproduction of Daphnia magna (numbers of offsprings) in the 21-d chronic test.

74 4. RESULTS AND DISCUSSION highest concentration tested (1 mg/L). In this respect, other studies with drug sulfamethazine (De Liguoro et al., 2009) or derivatives of polycyclic aromatic hydrocarbons (Feldmannova et al., 2006) reported differences in EC50 between acute and chronic assays with D. magna of 1 to 2 orders of magnitude. Higher differences recorded in the present study might be attributed to the mechanism of toxicity of the studied drugs, acting via DNA damage. Similar results have been discussed in other studies already. For example, dramatically stronger effects on the luminescence of Vibrio fischeri were observed after prolonged 24 hours incubations in comparison with traditional 30 min exposures (Backhaus et al., 1997). Also Kummerer et al. (2004) concluded that standardized short term respiration test with bacteria may underestimate risks of pharmaceuticals. These conclusions correspond well to large differences in toxicity of two studied drugs between the acute and reproduction assays with D. magna in the present study. Never- theless, it should be emphasized that the standardized chronic assays, such as the 21-day reproduction toxicity with D. magna, in fact represent only one generation assay. Prolonged multigeneration exposures might result in a number of unexpected effects, especially for compounds acting on DNA (Vandegehuchte et al., 2009a, 2009b). Our genotoxicity results are summarized in Figure 4.5 and Table 4.4. Cytarabine and gemcitabine showed genotoxicity in both variants, with and without metabolic activation, but the effects were observed only at relatively high concentrations > 100mg/L (Table 4.4). For gemcitabine, slightly stronger genotoxicity after metabolic activation was observed at concentrations around 100 mg/L. This seems to correspond to the fact that gemcitabine itself is a pro-drug, and its activity depends on the intracellular conversion to two active metabolites (Abbruzzese et al., 1991). Also 5-fluorouracil was genotoxic without S9 activation (induction factors > 4) at concentrations around 100 mg/L with MGC ≈ 40 mg/L. However, its high cytotoxicity (over 50% growth inhibition) complicates the interpretation, and the results should not be considered valid, according to the standard ISO method. On the other hand, other studies clearly demonstrated that 5-fluorouracil belongs among strong genotoxic compounds in bacterial assays (Oda, 1987; Barclay et al., 2001). In our previous study, genotoxicity of 5-FU was also demonstrated in two tests. No genotoxicity was observed up to the highest tested concentrations in the metabolites of cytarabine and gemcitabine. The only genotoxic response was recorded for FBAL, a metabolite of 5-fluorouracil, at the highest concentration (667 mg/L) tested without metabolic activation. In general, umu-test for genotoxicity seems to be of lower sensitivity to the studied drugs. Other models based on eukary-

75 4. RESULTS AND DISCUSSION otic organisms such as yeast Saccharomyces cerevisiae could be recommended for future studies, in addition to bacterial genotoxicity tests, as their higher sensitivity to cytotoxic drugs such as 5-ﬂuorouracil was demonstrated in the previous study. The effective toxic concentrations of the studied cytotoxic drugs and their metabolites appear to be mostly higher than the expected environmental levels. Environmental risk assessment was carried out subsequently (see Chapter 4.3). Lower toxicities were observed in the metabolites. The metabolites seem to be the dominant contaminants, as parent cytotoxic drugs in the unchanged form represent only 10 % of the excreted volume (Heg- gie et al., 1987; Hamada et al., 2002; Abbruzzese et al., 1991), approximately. This fact further increases relative importance of the drug metabolites in the complex mixture, but only limited data are still available on their concentrations in waste waters.

Table 4.4: Genotoxicity in the umu-test of cytostatic drugs 5-fluorouracil, cytarabine and gemcitabine, and their metabolites α-fluoro-β-alanine (FBAL), uracil-1-β-D-arabinofuranoside (AraU), 2’,2’-difluorodeoxyuridine (dFdU); MGC – minimal genotoxic concentration.

umu test MGC (mg/L) Test substance without metabolic activation with metabolic activation

5-ﬂuorouracil - - Cytarabine 167 333 Gemcitabine 167 42

FBAL 667 > 667 AraU > 667 > 667 dFdU > 667 > 667

76 4. RESULTS AND DISCUSSION Results of genotoxicity (induction factorstions (triangles on with the dashed left lines; Y-axes;(GemC), right diamonds and Y-axes) of with a cytostatics metabolite continuous 5-fluorouracil of lines)(empty (5-FU), 5-fluorouracil and symbols) cytarabine (FBAL) growth and in (CytR), without inhibi- the gemcitabine metabolic umu-test.induction activation Results (filled for of symbols) genotoxicity the are assays (induction presented. with factorinhibition). Horizontal metabolic = dashed activation line 1.5); – dash-dotted critical line – critical value of the growth factor (0.5; 50% Figure 4.5:

77 4. RESULTS AND DISCUSSION

4.2 Complex evaluation of ecotoxicity and genotoxicity of antimicrobials oxytetracycline and ﬂumequine widely used in aquaculture

Oxytetracycline (OTC) and flumequine (FLU) are the most frequently used antimicrobials in fish farming (Lalumera et al., 2004). In the Czech Repub- lic, there are only two antibiotics preparations registered for application to fish at the time of writing, namely Flumiquil 50 % plv. ad us. vet. with flumequine as the active ingredient, and Rupin Special gran. ad us. vet. with oxytetracycline as the active ingredient (Svobodova et al., 2006). The consumption of veterinary antimicrobials for therapeutic purposes and as growth promoters is high and is growing. In the Czech Republic, 49 tons of veterinary antibiotics were used in 2003. By 2008, this amount had increased to 80 tons, from which 45 tons were tetracyclines1. It has been estimated that 70–80 % of administered antimicrobials end up in the environment (Hektoen et al., 1995). Drug concentrations of antibacterial activity were found in sediment underneath fish farms (Samuelsen et al., 1992). The main objective of this study was to derive comparative data on the potential ecotoxicological impact of these two veterinary antibiotics. We have investigated ecotoxicity and genotoxicity of flumequine and oxytetracycline hydrochloride on the representatives of all three major trophic levels, i.e. producers, consumers and decomposers. Duckweed (Lemna minor) growth inhibition test and algal growth inhibition test with Pseu- dokirchneriella subcapitata were performed as the tests on producers. Acute immobilization test with Daphnia magna Strauss and reproduction test with D. magna were performed as the tests on consumers. Finally, growth inhibition test with Pseudomonas putida was used as the test on decomposers. Genotoxicity was evaluated in prokaryotic bioassay SOS-chromotest with Escherichia coli PQ 37 bacterial tester strain. Both antimicrobials tested exhibited significant effects in the majority of the bioassays with effect concentrations ranging from 0.04 mg/L (LOEC of OTC in the growth inhibition test with P. putida) to 500 mg/L (MGC of OTC in the SOS-chromotest). OTC was the more toxic compound, on the whole, with the lowest EC50 value of this study in the P. putida growth inhibition test, but FLU elicited major toxicity in most of the other tests, including the genotoxicity test. Effects of both tested compounds in the ecotoxicological and genotoxicity tests, i.e. values of the 50% effective concentrations

1Available on-line on: http://www.uskvbl.cz/attachments/339_spot%C5%99eba%20ATB%202003-2008.doc

78 4. RESULTS AND DISCUSSION

(EC50), the lowest observable effect concentrations (LOEC), no observable effect concentration (NOEC) and minimal genotoxic concentrations (MGC) are summarized in Table 4.5. Full dose-response curves are presented in Figures 4.6, 4.7, and 4.8. Table 2.5 in Chapter 2.3.6 surveys results on OTC and FLU ecotoxicity and genotoxicity available in the literature.

Table 4.5: Summary of the effects of oxytetracycline hydrochloride and ﬂume- quine in ecotoxicological biotests. All toxicity values are in mg/L (values in parentheses show 95% conﬁdence intervals); EC50 – concentration causing 50% effect; LOEC – lowest observable effect concentration; NOEC – the highest concentration causing no observable effect; MGC – minimal genotoxic concentration.

Oxytetracycline hydrochlorid Flumequine

Bioassay NOEC LOEC EC50 NOEC LOEC EC50 [mg/L] [mg/L] [mg/L] [mg/L] [mg/L] [mg/L] Pseudomonas putida < 0.04 0,04 0.22 (0.14–0.25) < 0.2 0,2 0.82 (0.81–1.1)

Vibrio ﬁsheri (Microtox) 2,5 5.0 21.0 (13–35) 0.31 0.63 11 (4.4–28)

Pseudokirchneriella subcapitata 0,78 1.6 3.1 (1.5–6.3) < 1.6 1.6 2.6 (0.45–15.0)

Lemna minor < 1.0 1.0 2.1 (1.7–3.0) < 1.0 1.0 3.0 (0.5–7.0)

Daphnia magna acute test 400 - - 12,5 25 59 (16–227)

Daphnia magna reproduction test < 20.0 20.0 86 (48–155) < 0.75 0,75 1.2 (0.44–3.1)

MGC [mg/L] MGC [mg/L] SOS-chromotest 500 0,25

For both antimicrobials tested, P. putida was the most sensitive organism. Effect concentrations in this test were at least one order of magnitude lower than in other tests for both compounds tested. LOEC derived in the present study was nearly two orders of magnitude lower than the minimum growth inhibitory concentration previously reported for Pseudomonas sp. (Halling-Sorensen et al., 2002) (Tables 2.5 and 4.5). Similar result might have been expected, considering that antibiotics are primarily designed to kill bacteria.

79 4. RESULTS AND DISCUSSION Ecotoxicity (concentration-response curves) of thequine (FLU). studied antimicrobial drugs oxytetracycline (OTC) and ﬂume- Figure 4.6:

80 4. RESULTS AND DISCUSSION

Figure 4.7: Comparison of toxicity of the studied antimicrobial drugs in the acute and reproduction test with Daphnia magna. OTC - oxytetracycline hydrochloride, FLU - ﬂumequine.

The test with marine bacteria V. fischeri was not as sensitive as one may expect, taking into account the statement above. OTC was actually two orders of magnitude less toxic for V. fischeri than for P. putida. The low sensitivity of this test to antibiotics was reported also by other authors (Christensen, A.M. et al., 2006). Lower sensitivity of V. fischeri to the antimicrobials, namely OTC, can be explained by its mode of action on protein synthesis, which is not of importance during the short term bioluminescence testing. However, a drastic increase in the sensitivity of V. fischeri was

81 4. RESULTS AND DISCUSSION

Figure 4.8: Comparison of genotoxicity of the studied antimicrobial drugs in the SOS-chromotest. OTC - oxytetracycline, FLU - ﬂumequine.

82 4. RESULTS AND DISCUSSION shown during the prolonged 24 h incubation (Backhaus et al., 1997). Isidori et al. (2005) studied toxicity and genotoxicity of six antibiotics including OTC. For V. fischeri EC50 of 65 mg/L was reported, which is comparable with the result of the present study. On the other hand, the 30-min EC50 of OTC for V. fischeri reported by Lalumera et al. (2004) was ten times higher. Interestingly, the results of the same authors for FLU were in accordance with the present study. Both representatives of producers – unicellular green alga P. subcapitata and aquatic vascular plant L. minor demonstrated comparable sensitivity to both tested compounds. Both compounds could be classified as medium- toxic with effective concentrations in the low mg/L range. Three different values for algal growth inhibition test with OTC were found in the literature (Table 2.5, Chapter 2.3.6) and they were generally comparable with the results of the present study (Lutzhoft et al., 1999; Pro et al., 2003; Robinson et al., 2005). The least sensitive organism was D. magna for both tested antimicrobials in the acute test. In particular, OTC demonstrated no toxicity up to 400 mg/L, the highest concentration tested. Only low toxicity was observed for OTC in the chronic reproduction test. Similar differences in sensitivity of crustaceans vs. algae were previously reported by Isidori et al. (2005). Low toxicity of OTC was also reported by Wollenberger et al. (2000). Toxicity of FLU to D. magna in the reproduction test was much higher. The LOEC value was two orders of magnitude lower than LOEC of OTC. Robinson et al. (2005) reported minor acute toxicity of FLU for D. magna with NOEC of 10 mg/L, which is similar to our results, but we are unaware of any study that would have addressed the chronic toxicity of FLU in the reproduction test with D. magna. Our study seems to indicate parallels in responses of D. magna in the reproduction test and E. coli in the SOS-chromotest. Both D. magna and E. coli were markedly more sensitive to FLU than to OTC, which might be related to the mode of action of both antimicrobials. While OTC affects the protein synthesis in bacteria, FLU acts directly on the DNA by inhibiting the topoisomerase II enzyme (Rigos et al., 2003; Samuelsen, 2006). Low genotoxicity of OTC was observed in the present study. The MGC value of 500 mg/L is very high and environmentally non-relevant. This corresponds to the study of Isidori et al. (2005), who reported no mutagenicity of OTC, neither in the Ames nor SOS chromotest. Genotoxicity of FLU was markedly higher. Interestingly, genotoxicity of FLU was not previously reported, although studies with other quinolone antibiotics are available (Itoh et al., 2006). The significance of the quinolone compounds

83 4. RESULTS AND DISCUSSION was suggested by Hartmann et al. (1998), who concluded that genotoxicity detected in hospital wastewaters is caused mainly by fluoroquinolone antibiotics. Moreover, Lancieri et al. (2004) demonstrated teratogenic effects of FLU during acute exposures of the early stages of fish Danio rerio. This study provides complete data on ecotoxicity and genotoxicity of two widely used veterinary antimicrobials which do occur in the environment. OTC is the more toxic agent from those two and also is occurring in higher concentrations. FLU elicited significant genotoxicity. Environmen- tal risk assessment has been carried out (Chapter 4.3). The results indicate potential risk of veterinary antimicrobials for aquatic environment.

4.3 Environmental relevance of data obtained in experimental laboratory studies

4.3.1 Cytostatic agents

The evaluation of risks connected with the presence of cytostatic drugs in the environment is limited by rare monitoring data on cytostatics. Cy- clophosphamide and ifosfamide were detected in the hospital wastewater treatment effluents in concentrations of up to 4.5 µg/L. In another study, concentrations up to 140 ng/L were detected in the effluents of a sewage treatment plant (Steger-Hartmann et al., 1996). Maximum concentrations of 5-fluorouracil, gemcitabine and its metabolite 2’,2’-difluorodeoxyuridine reported from a Swiss cantonal hospital were 27, 38 and 840 ng/L, respectively (Kovalova et al., 2009). In the study of Mullot et al. (2009), concentrations up to 4.0 µg/L of unchanged 5-fluorouracil were reported in sewage from a hospital located in Paris. Another study (Mahnik et al., 2004) reported three orders of magnitude higher levels of 5-fluorouracil in sewage from an oncology in-patient-treatment ward, i.e. concentrations 20–122 µg/L. Ecotoxicity and genotoxicity of 5-fluorouracil observed in our studies (Tables 4.1, 4.2, 4.3, and 4.4) were fairly close to some of these reported environmental concentrations. Nearly all mentioned concentrations come from hospital effluents and further dilution in sewer system, STPs, and even surface water should be taken into account. Consequently, final concentrations in surface water are expected to be by several orders of magnitude lower. A predicted environmental concentration (PEC) of 0.8 ng/L was calculated for ifosfamide in surface water in Germany (Kummerer et al., 1997). The study of Johnson et al. (2008) predicts 5-50 ng/L concentration of 5- fluorouracil in rivers during low flow conditions in a small catchment area

84 4. RESULTS AND DISCUSSION in the UK. Assuming that cyclophosphamide occurs in similar concentration as ifosfamide (being used in similar amounts, having similar structure and thus undergoing similar processes in STPs, sewer system and environment), final risk quotient for cyclophosphamide would lie within the range − of 10 6. However, the risk quotient would be different for other cytostatics such as 5-fluorouracil. As the effects of 5-fluorouracil occurred at much lower concentrations, the risk quotient would be between 0.5 and 5.0. Although these values represent the worst case scenario, they seem to indicate a considerable risk. Lower toxicities were observed in the metabolites of cytostatics. The metabolites seem to be the dominant contaminants, as parent cytotoxic drugs in the unchanged form represent only about 10 % of the excreted volume (Heggie et al., 1987; Hamada et al., 2002; Abbruzzese et al., 1991). This fact may increase the relative importance of the drug metabolites in the complex mixture but only limited data are still available on their concentrations in the environment. Concerning the risk for humans, the additional intake risk calculated for CP and IFA is low, considering emissions into surface water used further as drinking water (no treatment of water considered). However, this approach may have some shortcomings. It documents current lacks in methodol- ogy for the risk assessment of carcinogenic pharmaceuticals. IFA and CP, as well as other antineoplastics, directly interact with the DNA and therefore, a safe threshold concentration for these compounds cannot be defined (Kummerer & Al-Ahmad, 2010). A question remains, whether a similar approach should be applied also for environmental risk assessment. This issue will however require further research.

4.3.2 Aquaculture antimicrobials

Observed ecotoxicity values were compared with reported environmental concentrations (see Chapter 3.1). Investigated veterinary antimicrobials OTC and FLU have been found in many environmental matrices in different concentrations (see Table 2.4, Chapter 2.3.6). Tetracyclines are not expected to be found dissolved in the aquatic environment in high concentrations, due to their precipitation, sorption to the organic matter and possible accumulation in sediments. Nevertheless, OTC was detected in surface water samples in the U.S. at the concentration of 0.34 µg/L (Kolpin et al., 2002). This concentration was taken as MEC for our calculations of risk. FLU was detected in concentrations up to 32.0 ng/L in the Seine River

85 4. RESULTS AND DISCUSSION inner estuary (Tamtam et al., 2008). For OTC, some risk assessments were previously reported but lead to contradictory conclusions. Specifically, Park and Choi (2008) reported the risk quotient of 2 using MEC of 0.34 µg/L for the calculation. Their PNEC was derived from the EC50 value of 0.17 mg/L in the P. subcapitata growth inhibition test. On the other hand, Isidori et al. (2005) reported the risk quotient lower than 1 using MEC of 0.05 µg/L and the same value of PNEC as in the study of Park and Choi (2008). Our results are comparable to the findings of Park and Choi (2008) – using MEC of 0.34 µg/L, we have calculated the risk quotient of 8.5 indicating thus considerable risk. For FLU, low or acceptable risk can be predicted (calculated risk quotient of 0.16) but its significant genotoxicity should not be overlooked. Values used for the risk assessment of both veterinary antimicrobials and cytostatic agents including final risk quotients are summarized in Table 4.6, allowing comparison of particular values.

Table 4.6: Summary of risk assessment of veterinary antimicrobials and cytostatic agents. MEC (measured environmental concentration) or PEC (predicted environmental concentration) values derived from literature, PNEC (predicted no-effect concentration) calculated on the basis of our results. The worst-case scenario is presented – the highest MEC (PEC) available was used for calculation and PNEC was derived from the lowest available LOEC.

Pharmaceutical MEC or PEC the lowest LOEC (mg/L) PNEC Risk quotient (µg/L) (bioassay) (µg/L) − Cyclophosphamide 0, 0008a 500 (P. subcapitata) 500 1.6 × 10 6 5-ﬂuorouracil 0.005 − 0.05b 0.01 (P. putida) 0,01 0.5–5.0 Oxytetracycline 0, 34c 0.04 (P. putida) 0,04 8,5 Flumequine 0, 032c 0.2 (P. putida) 0,2 0,16 a: (Kummerer et al., 1997) b: (Johnson et al., 2008) c: (Kolpin et al., 2002) d: (Tamtam et al., 2008)

86 Chapter 5 General discussion and conclusions

The dissertation studied ecotoxicological properties of two important groups of pharmaceuticals: cytostatic (antineoplastic) agents and veterinary antimicrobials as important environmental pollutants. Until recently, studies of toxicity of pharmaceuticals had been restricted to the potential toxicity for target organisms, such as humans in human medicine or animals in veterinary medicine. As the consumption of pharmaceuticals and other personal care products increases, it seems evident, that the issue of their release in the environment needs to be studied. Up to now, studies of the effects of individual pharmaceuticals focused on acute effects of the original compounds, mostly those used in high quantities. Only limited studies of chronic effects of pharmaceuticals or effects of mixtures are available in the literature (Wollenberger et al., 2000; Christensen, A.M. et al., 2006; Cleuvers, 2004; Pomati et al., 2006), and to our knowledge no studies considered the effects of metabolites. The present work studied both acute and chronic effects of selected pharmaceutical compounds, along with their genotoxicity as well as effects of their metabolites. To our knowledge, our study represents one of the first investigations of reproduction toxicity of cytostatics and their metabolites in ecotoxicological bioassays. The results obtained with cytostatics indicate generally lower risks of acute ecotoxic effects for concentrations expected in the environment. Sig- nificant effects of studied compounds in the acute assays were observed at relatively high concentrations, mostly in the range of tens mg/L. The LOEC and EC50 values in acute tests are quite comparable with other tradi- tionally studied pharmaceuticals (Table 2.1, Chapter 2.1.3). However, risks should be evaluated with care considering specificity of cytostatic mechanism of action. The changes in the structure of DNA, an effect inherent to anticancer chemotherapies, might become manifested after prolonged exposures with unexpected consequences. As discussed in Chapter 4.3, a threshold concentration cannot be given for compounds directly reacting with DNA (Kummerer & Al-Ahmad, 2010).

87 5. GENERALDISCUSSIONANDCONCLUSIONS

5-fluorouracil was, generally, the most toxic compound with low effective concentrations. Besides the acute ecotoxicity, significant genotoxicity of several tested compounds both in prokaryotic and eukaryotic test were observed. The metabolites studied were mostly non-toxic. The metabolite of 5-fluorouracil, however, elicited effects comparable with toxicity of the less toxic parent compounds in some tests. Besides the acute toxicity, chronic effects may occur at much lower concentrations and they may also contribute to environmental risks of this class of pharmaceuticals. Significance is supported by the chronic EC50 values obtained in the reproduction test with D. magna (up to three orders of magnitude lower EC50 in comparison with the acute test). Waste water and hospital effluents, in particular, may contain relatively high concentrations of cytostatics. Some studies reported concentrations within the range close to the experimentally derived LOECs or EC50s (Mah- nik et al., 2004). Considering dilutions in the sewer system, pharmaceutical pollution with individual chemicals seems to be rather low but specific sources of human pharmaceuticals such as hospital effluents should be carefully monitored. In spite of many discussions, chronic ecotoxicity of pharmaceuticals was not investigated in detail and there are also other issues, which will require further research attention. Studies should investigate degradation pathways of the drug metabolites, their continuous release and pseudopersistence. These problems are of special interest as some studies indicated that chronic effects may remain hidden for many years (Jones et al., 2001), and there might be unexpected toxicity interactions among drugs within low concentration (Pomati et al., 2008). The next problem investigated in this dissertation is the ecotoxicity assessment of veterinary antimicrobials. The Paper III provides comprehensive data on aquatic ecotoxicity and genotoxicity of two veterinary antimicrobials frequently used in aquaculture. EC50 values lower than 1 mg/L were derived for both antimicrobials tested in P. putida growth inhibition test. Algae and vascular plants were affected by studied drugs with EC50 in low mg/L range. Minor effects were observed at D. magna but FLU had significant effects during the chronic reproduction study. The results thus indicate potential risk for non-target organisms resulting from exposure to low levels of veterinary antimicrobials. Comparing the results from all the studies conducted within the frame of this dissertation, it is complicated to derive and generalize conclusions about pharmaceutical risks. Although it may seem that cytostatics are "more toxic and more dangerous", the differences between ecotoxicities of the two

88 5. GENERALDISCUSSIONANDCONCLUSIONS groups of pharmaceuticals were not particularly significant but there were some specificities. For example, in the P. putida growth inhibition test, 5- fluorouracil was more toxic than both antimicrobials, but other tested cytostatics elicited much lower toxicity. Analogous results were obtained in algal growth inhibition test, where 5-fluorouracil and cisplatin were more toxic than the antimicrobials (which were one or two orders of magnitude more toxic than other tested cytostatics). Genotoxicity of studied compounds can be compared only bearing in mind that tests with different species were carried out for particular pharmaceuticals. The results from the prokaryotic test suggest that the genotoxicity of antimicrobials such as flumequine is comparable with genotoxicity of several cytostatic compounds. In the eukaryotic testing system, cytostatics such as 5-fluorouracil exhibited higher genotoxicity. This corresponds to the fact that antimicrobials are active against prokaryots, such as bacteria, whereas cytostatics were developed to affect eukaryotic cancer cells. Nev- ertheless, antimicrobials targeted other structures or processes than those related to DNA, seem to be less genotoxic. Also, sensitivity of testing organisms can be compared for the two groups of pharmaceuticals tested. Pseudomonas putida growth inhibition test showed the highest sensitivity from all acute assays used in this work. For antimicrobials, Pseudomonas putida was more sensitive than D. magna reproduction test, and the most sensitive test in general. For cytostatics, D. magna reproduction test appeared to be the most sensitive assay. One may also consider that P. putida growth inhibition test is – in fact – also "chronic". Comparing duration of the test (16 h) with doubling time of the bacteria (approximately 40–120 min) (Molina et al., 2000; Sauer & Camper, 2001; Espinosa-Urgel et al., 2000), one could consider this assay as "multi-generation". On the other hand, the reproduction test with D. magna still follows only one generation of organisms. Monitoring of risks connected with the use of particular compounds is of high importance. While a number of environmental risk assessments for antimicrobial substances were conducted leading to variable results, environmental risks of cytostatics were not evaluated in detail yet. Calcula- tions based on the results of the present dissertation seem to indicate significant risks for both veterinary antimicrobials and highly toxic cytostatic compounds. As has been shown, antimicrobials and cytostatic drugs share a number of features. Both groups were developed to control the growth of cells. Many cytostatics and antimicrobials target the DNA, and also sensitivity of testing model ecotoxicological organisms to both groups of pharmaceuti-

89 5. GENERALDISCUSSIONANDCONCLUSIONS cals is similar. Therefore, it seems that diverse pharmaceuticals may cause fairly comparable risks. For pharmaceuticals and other compounds with specific mode of action, which are present in low concentrations in the environment, the results from chronic assays are needed as they represent more relevant data for risk assessment (Jones et al., 2001; Kummerer et al., 2004; Seiler, 2002). Further research should thus address chronic effects of pharmaceuticals in more detail. In summary, this work contributes to the environmental research of pharmaceuticals as an example of micropollutants with a specific mode of action. Compounds used and consumed in high quantities may represent a significant risk for the environment especially when considering their potential long-term effects.

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I. Zounková, R., Odráška, P., Doležalová, L., Hilscherová, K., Maršálek, B., and Bláha, L. (2007): Ecotoxicity and Genotoxicity Assessment of Cytostatic Pharmaceuticals. Environmental Toxi- cology and Chemistry 26(10): 2208-2214

II. Zounková, R., Kovalová, L., Bláha, L., Dott, W.: Ecotoxicity and Genotoxicity Assessment of Cytotoxic Antineoplastic Drugs and their Metabolites. Chemosphere (accepted with moderate revision)

III. Zounková, R., Klimešová, Z., Nepejchalová, L., Hilscherová, K., Bláha, L.: Complex Evaluation of Ecotoxicity and Genotoxicity of Antimicrobials Oxytetracycline and Flumequine used in Aqua- culture (submitted to Environmental Toxicology and Chemistry)

IV. Curriculum vitae

Paper I

Environmental Toxicology and Chemistry, Vol. 26, No. 10, pp. 2208–2214, 2007 ᭧ 2007 SETAC Printed in the USA 0730-7268/07 $12.00 ϩ .00

ECOTOXICITY AND GENOTOXICITY ASSESSMENT OF CYTOSTATIC PHARMACEUTICALS

RADKA ZOUNKOVA´ ,† PAVEL ODRA´ SˇKA,†‡ LENKA DOLEZˇ ALOVA´ ,‡ KLA´ RA HILSCHEROVA´ ,†§ BLAHOSLAV MARSˇA´ LEK,†§ and LUDEˇ K BLA´ HA*†§ †Research Centre for Environmental Chemistry and Ecotoxicology (RECETOX), Faculty of Science, Masaryk University, Kamenice 126/3, 625 00 Brno, Czech Republic ‡Masaryk Memorial Cancer Institute, Zˇ luty´ kopec 7, 656 53 Brno, Czech Republic §Academy of Sciences of the Czech Republic, Institute of Botany, Department of Experimental Phycology and Ecotoxicology, Kveˇtna´8, 603 65 Brno, Czech Republic

(Received 15 February 2007; Accepted 17 May 2007)

Abstract—The fate and effects of cytostatic (anticancer or antineoplastic) pharmaceuticals in the environment are largely unknown, but they can contaminate wastewater treatment effluents and consequently aquatic ecosystems. In this paper, we have focused on five cytostatic compounds used in high amounts (cyclophosphamide, cisplatin, 5-fluorouracil, doxorubicin, and etoposide), and we have investigated their ecotoxicity in bacterial Pseudomonas putida growth-inhibition test, algal Pseudokirchneriella subcapitata growth-inhibition test, and Dapnia magna acute immobilization test. Genotoxicity also was assessed with Escherichia coli SOS- chromotest (with and without metabolic activation) and the GreenScreen Assay using yeast S. cerevisiae. All tested compounds showed significant effects in most of the assays with lowest-observed-effect concentrations and concentrations causing 50% effects (EC50s) values ranging within ␮g/L to mg/L. The most toxic compound was 5-fluorouracil in the assays with P. putida (EC50 ϭ 0.027 mg/L) and P. subcapitata (EC50 ϭ 0.11 mg/L), although cisplatin and doxorubicin were the most toxic to D. magna (EC50 ϭ 0.64 and 2.0 mg/L, respectively). These two chemicals were also the most genotoxic in the SOS-chromotest (minimum genotoxic concentrations [MGC] ϭ 0.07–0.2 mg/L), and 5-fluorouracil was the most genotoxic in the eukaryotic yeast assay (MGC ϭ 0.02 mg/L). Our investigation seems to indicate generally lower risks of acute effects at concentrations expected in the environment. However, some effective concentrations were relatively low and chronic toxicity of cytostatics (and/or their transformation products), as well as specific sources of human pharmaceuticals such as hospital effluents, require research attention.

Keywords—Cytostatic agents Antineoplastics Ecotoxicity Genotoxicity Pharmaceutical

INTRODUCTION Because information on cytostatics is lacking, we have investigated the ecotoxicity and genotoxicity of five compounds During recent years, great consideration has been given to (cyclophosphamide, 5-fluorouracil, cisplatin, etoposide, and the issue of environmental contamination by veterinary and doxorubicin), which are used in large volumes in the Czech human pharmaceuticals. Human pharmaceuticals and their me- Republic and worldwide (Table 1). For ecotoxicity evaluations tabolites may be excreted into the hospital or municipal waste- we have used representatives of three major trophic levels, i.e., water treatment effluents by patients in considerable amounts, producers (green algae), consumers (crustaceans), and des- and they may induce effects in nontarget organisms [1]. These truents (bacteria). Genotoxicity was evaluated in prokaryotic compounds have been detected in the effluents of sewage treat- (bacterial) and eukaryotic (yeast) bioassays. The main objec- ment plants and in surface and even drinking waters but little tive of our study was to obtain complex comparative data is still known about their fate and effects [2]. Several groups concerning the potential ecotoxicological impact of cytostatics, of pharmaceuticals, such as antibiotics and hormones, already which may aid in further evaluation of environmental risks. have been studied but less attention has been paid to other toxic compounds such as cytostatic agents. MATERIALS AND METHODS Cytostatics are a group of pharmaceuticals used for cancer Chemicals therapy. They are considered genotoxic, mutagenic, carcino- The cytostatic compounds cisplatin, cyclophosphamide, genic, teratogenic, and embryotoxic [2–4]. Total used amounts doxorubicin, etoposide, and 5-fluorouracil were obtained from of cytostatic compounds are below those of antibiotics or an- a hospital pharmacy of Masaryk Memorial Cancer Institute, algesics but they may pose a risk to the environment, and only Brno, Czech Republic (cytostatics in original packaging that limited data is available on their ecotoxicity [1,5,6]. remained from on-demand prepared therapeutic infusions). Because cytostatics interfere with the function of DNA, Names of the preparatives, producers, chemical structures, which is common to all taxa, these compounds have the po- Chemical Abstract Services numbers, anatomical therapeutic tential to affect nonhuman target organisms. Some experi- chemical classification codes, and mechanisms of cytostatic mental studies [7,8] reported genotoxicity of hospital waste- action are listed in Table 1. All operations carried out with water effluents samples collected both above and below the cytostatics were in compliance with safety standards for deal- sewage treatment plants but genotoxicity of only few pure ing with cytostatics used in the hospital pharmacy. All oper- compounds have been investigated in ecotoxicological bio- ations were conducted in a safety hood or laminar flow box. assays with bacteria and yeast [7,9]. All instruments and materials that were in contact with tested compounds were disposable and treated as a hazardous waste. * To whom correspondence may be addressed With the exception of etoposide (an ethanol used as carrier, ([email protected]) up to 1% v/v) all compounds initially were dissolved in the

2208 Ecotoxicity and genotoxicity of cytostatic pharmaceuticals Environ. Toxicol. Chem. 26, 2007 2209

Table 1. Studied cytostatic agents (commercial names and producers in parentheses) plus Chemical Abstract Services numbers (CAS no.), anatomical therapeutic chemical classiﬁcation (ATC) codes, chemical structures, and major mechanisms of action

Compound (producer) CAS no./ATC code Structure Mechanism of action

5-Fluorouracil (LA-FU, Pliva-Lachema Antimetabolite; inhibition of DNA synthesis a.s., Zagreb, Croatia) 51-21-8/L01BC02

Cisplatin (Platidiam, Pliva-Lachema a.s.) Alkylating agent; covalent binding to DNA 15663-27-1/L01XA01

Cyclophosphamide (Endoxan, Baxter Oncology, Alkylating agent; covalent binding to DNA Round Lake, IL, USA) 50-18-0/L01AA01

Doxorubicin (Doxorubicin-Teva 0.2%, Teva Intercalator; noncovalent binding to DNA Pharmaceuticals, Israel) 23214-92-8/L01DB01

Etoposide (Etoposide-Teva, Teva Topoisomerase II inhibitor; inhibition of Pharmaceuticals, Israel) 33419-42-0/L01CB01 DNA replication

buffered saline solution (human infusions) and further diluted [14]; 50% medium prepared by mixing Zehnder Z-medium, with corresponding test media. Appropriate solvent controls Bristol modiﬁed Bold (BB) medium, and distilled water in the were always tested. ratio 1:1:2 were used for cell cultures and testing (http:// www.butbn.cas.cz/ccala/index.php). Cells in the exponential Bioassays growth phase were used, and light absorbance (680 nm; cor- Pseudomonas putida growth-inhibition test was performed responding to the amount of chlorophyll) was determined in according to the European norm based on International Stan- the beginning of the exposure and then every 24 h. Total du- dardization Organization Standard EN ISO 10712:1995 and ration of the bioassay was 96 h when the growth inhibition the Czech Standard [10] in the miniaturized version [11]. We was evaluated. have used cryopreserved cultures of P. putida (stored at Ϫ80ЊC Acute immobilization test with D. magna was based on in glycerol), and the growth inhibition was evaluated as the Czech standard [15], which is identical with European Stan- change in the absorption of bacterial culture (590 nm) after a dard EN ISO 6341:1996. Newly hatched neonates (less than 16-h exposure. 24 h old) obtained from the continuous laboratory culture were Algal growth-inhibition test was performed according to used (20 animals for each tested concentration and control). the European standard EN ISO 8692:1989 [12] and the Czech Total duration of the exposure was 48 h. Immobilized organ- standard [13] using Pseudokirchneriella subcapitata. This test isms were counted and the results were expressed as the per- assay was modiﬁed and miniaturized for 96-well microplates centage of control. 2210 Environ. Toxicol. Chem. 26, 2007 R. Zounkova´ et al.

Bacterial genotoxicity test SOS-chromotest [16] used genetically modified bacterial tester strain Escherichia coli PQ 37 [17]. The test was performed in the 96-well microtitre plate format [18,19] with and without metabolic activation using S9 rat liver homogenate. After a 2-h incubation with compounds of interest, ␤-galactosidase activity was measured (reporter enzyme for genotoxicity induced along with DNA repair system) using a chromogenic substrate ortho-nitrophenyl-␤-D- galactopyranoside. At the same time, activity of alkaline phosphatase (marker of viability/cytotoxicity) was assessed using p-nitrophenyl phosphate chromogenic substrate. Cytotoxic effect was quantified as a percentage of inhibition of the alkaline phosphatase in comparison with the negative control. The concentrations causing more than 50 % inhibition were excluded from genotoxicity evaluations. The SOS induction factor then was calculated for each tested concentration, and the minimal genotoxic concentration ([MGC], the concentration at which the induction factor reaches the value 1.5) was determined. GreenScreen assay (GSA) is a rapid genotoxicity assay using eukaryotic yeast cells Saccharomyces cerevisiae based on assessment of the green fluorescent protein fused to the DNA damage–inducible promoter of the RAD54 gene [20]. This assay responds to genotoxic agents that activate the DNA repair pathway [21], and the amount of accumulated green fluorescent protein reflects the genotoxic effect of the tested substance. Two yeast strains have been used for the assay (GenT01 testing strain and GenC01 as the control for cytotoxicity). The assay was carried out in 96-well microplates (black, clear bottom) using overnight exposure (16 h) at 30ЊC, without shaking [22], and MGC values (induction factor Ͼ 1.3) were calculated using noncytotoxic concentrations.

Statistics For all bioassays, experiments included initial testing of a wide range of concentrations (1:10 dilutions), followed by detailed assessment around 50% effective concentration (EC50) values. Full dose-response curves for all assays (3–4 replicates per treatment) were repeated independently at least three times. Results were pooled and analyzed. For ecotoxicological assays, the highest tested concentration with no observable effects and the lowest tested concentration causing statistically significant effect were evaluated using analysis of variance followed by Dunnet’s test. Concentrations eliciting Fig. 1. Effects of tested cytostatics (5-fluorouracil [FU], cyclophos- 50% effects were estimated using the U.S. Environmental Pro- phamide [CP], cisplatin [CS], doxorubicin [DX], etoposide [EP]) on tection Agency Probit software (U.S. EPA, Washington, D.C.). the growth of bacteria Pseudomonas putida (A), growth of the green alga Pseudokirchneriella subcapitata (B), and immobilization of Minimum genotoxic concentration was derived (according to Daphnia magna (C). Data points represent mean of at least three standard procedures of both assays) as the mean concentration independent experiments; coefficient of variance was below 30% (er- that exceeded the induction factors value of 1.5 (SOS-chrom- ror bars not presented). otest) or 1.3 (GSA assay). Calculations were performed with Microsoft Excel௡ (Microsoft, Redmond, WA, USA) and Sta- tistica௡ for Windows 6.0 (StatSoft, Tulsa, OK, USA) sensitivities were observed. For example, 5-fluorouracil was the most toxic for P. putida and P. subcapitata, and cisplatin RESULTS AND DISCUSSION and doxorubicin were the most toxic for D. magna (Table 2). Previously published ecotoxicological data for cytostatics Ecotoxicity results mostly were obtained with daphnids (Table 4). With the ex- Effects of cytostatic agents, i.e. full dose-response curves ception of cyclophosphamide (nontoxic to D. magna in our in ecotoxicological and genotoxicity bioassays, are summa- study with EC50 Ͼ1,000 mg/L, a value comparable to the rized in Figures 1 and 2, and values of no-observable-effects literature EC50 ϭ1,795 mg/L [5]), our results suggest that concentration, lowest-observable-effect concentration, EC50, ecotoxic potencies of cytostatics may be higher than expected and MGC are in Tables 2 and 3. In general, 5-fluorouracil was from previous reports for other compounds (compare D. mag- among the most toxic compound in contrast to the least toxic na results in Tables 2 and 4). Similarly, results for the 5- cyclophosphamide with effective concentrations about four or- fluorouracil derived from the bacterial assay with P. putida ders of magnitude higher. Apparent differences in bioassay (EC50 ϭ 0.027 mg/L; Table 2, Fig. 1) indicate higher toxicity Ecotoxicity and genotoxicity of cytostatic pharmaceuticals Environ. Toxicol. Chem. 26, 2007 2211

Fig. 2. Genotoxicity (expressed as induction factors [IF]) of ﬁve studied cytostatics in the yeast GreenScreen assay ([GSA]; white bars) and bacterial SOS-chromotest (without metabolic activation, dashed bars; with metabolic activation, black bars). Data represent mean Ϯ standard deviation of at least three independent experiments. Values of IF Ͼ1.3 and IF Ͼ1.5 indicate signiﬁcant genotoxicity in GSA and SOS-chromotest, respectively.

Table 2. Ecotoxicity of the tested cytostatic compounds. Values in the parentheses indicate 95% conﬁdence interval. NOEC ϭ no-observed- effect-concentration (experimental); LOEC ϭ lowest-observed-effect concentration (experimental); and EC50 ϭ concentration causing 50% effect

Assay/compound NOEC (mg/L) LOEC (mg/L) EC50 (mg/L)

Pseudomonas putida 5-Fluorouracil 0.003 0.01 0.027 (0.015–0.045) Cisplatin 0.03 0.1 1.2 (1.0–1.40) Cyclophosphamide 1,000 Ͼ1,000 Ͼ1,000 Doxorubicin 1 10 Ͼ1,000 Etoposide 200 250 630 (580–830) Pseudokirchneriella subcapitata 5-Fluorouracil 0.001 0.01 0.11 (0.03–0.3) Cisplatin 0.1 1 2.3 (1.7–2.9) Cyclophosphamide 250 500 930 (700–1,100) Doxorubicin 1 10 13 (12–17) Etoposide Ͻ10 10 250 (120–460) Daphnia magna 5-Fluorouracil 1 10 36 (12–70) Cisplatin 0.2 0.5 0.64 (0.4–0.85) Cyclophosphamide Ն1,000 Ͼ1,000 Ͼ1,000 Doxorubicin 0.01 0.1 2.0 (0.52–4.8) Etoposide 10 30 30 (16–40) 2212 Environ. Toxicol. Chem. 26, 2007 R. Zounkova´ et al.

Table 3. Genotoxicity (minimum genotoxic concentrations [MGC]) of tested cytostatic compounds in the bacterial SOS-chromotest and eukaryotic yeast GreenScreen Assay (GSA). Values in parentheses indicate 95% conﬁdence interval. NS ϭ no signiﬁcant genotoxicity

MGC (mg/L)

SOS chromotest

Without metabolic With metabolic Compound activation activation GSA

5-Fluorouracil 1.4 (1.2–29) NS 0.02 (0.018–0.021) Cisplatin 0.17 (0.1–0.37) 0.09 (0.03–0.37) 0.44 (0.32–1.6) Cyclophosphamide NS NS 470 (270–750) Doxorubicin 0.074 (0.02–0.12) 0.098 (0.05–0.5) 2.8 (2.0–3.6) Etoposide 2.4 (1.3–7.7) 6.4 (4.8–20) 150 (140–168) than the V. fisheri literature values (EC50 ϭ 0.122 mg/L [23]; cated further by the results of Quillardet and Hofnung [25] Table 4). However, in this case, differences in EC50 values who demonstrated cyclophosphamide genotoxicity in the stan- may be explained by different exposures (16-h growth inhi- dard Ames test with bacteria Salmonella typhimurium. bition vs. 30-min luminescence inhibition) as well as by pos- Obviously, genotoxicity of cytostatic agents depends on the sible species-specific sensitivity. mode of action of individual pharmaceutical and/or its metab- Only limited information is available on the toxicity of olites. For cisplatin (MGC ϭ 0.1–0.2 mg/L in SOS-chromotest, cytostatics to producers such as algae (Table 4). The toxicity Table 3) our results correspond to the previous study that dem- of cyclophosphamide in our study (EC50 ϭ 930 mg/L; Table onstrated mutagenicity in the Ames test [25]. The GSA also 2, Fig. 1) was much lower than the literature quantitative- showed significant genotoxic potential of cisplatin (MGC ϭ structure activity relationship–predicted value (EC50 ϭ 11 0.44 mg/L) being about 40 times higher than previously re- mg/L [5]; Table 4). Nevertheless, we consider our results more ported value MGC ϭ 18.75 mg/L with similar yeast system reliable because cyclophosphamide generally had low toxicity [21]. Interestingly, for another tested compound (etoposide), in all of our bioassays (Table 2), and it also is known to be we have observed fairly good agreement between the same biologically ineffective until activated by metabolism [24]. On literature source (MGC ϭ 125 mg/L; [21]) and our experiments the other hand, active metabolites of cyclophosphamide (phos- (MGC ϭ 150 mg/L in GSA). phoramide mustard, acrolein, and 4-hydroxycyclophospha- Our study also demonstrated differences in genotoxicity re- mide [24]) may be excreted into the environment (rather than sulting from the bioactivation. The SOS-chromotest results of parent cyclophospamide) but ecotoxicity of pharmaceutical cisplatin, doxorubicin, and etoposide were independent of the metabolites in general has not been studied in detail yet. bioactivation (Fig. 2, Table 3). On the other hand, 5-fluorouracil was highly genotoxic in the yeast GSA (no bioactivation; Table Genotoxicity results 3) as well as in the SOS-chromotest without bioactivation. Sup- Doxorubicin was the most genotoxic compound in both pressions in 5-fluorouracil genotoxicity in variants with S9 frac- bacterial and yeast assays (MGC below 1 mg/L for bacterial tion were observed (Fig. 2), and this finding may be explained SOS-test; Table 3, Fig. 2) but differences between biotests were by possible inactivation during metabolization [24,26]. Taken to- observed for other compounds. For example, cyclophospha- gether, we have determined relatively high genotoxic potential of mide significantly induced reporter green fluorescent protein some compounds with MGC below 0.1 mg/L (5-fluorouracil in in eukaryotic yeast GSA test, although it was nongenotoxic GSA and doxorubicin in SOS-chromotest) with substantial dif- in bacterial SOS-chromotest (Fig. 2). This finding is compli- ferences in bioassay responses.

Table 4. Summary of the literature ecotoxicity data of cytostatics

Ecotoxicity Compound Bioassays (EC50 [mg/L]) Reference

Cyclophosphamide Scenedesmus subspicatus (alga, 72-h growth) 11 (QSAR)a [5] Daphnia magna (crustacean, 48-h immobilization) 1,795 (QSAR) [5] Brachydanio rerio (fish, 96-h mortality) 70 (QSAR) [5] Fluorouracil Vibrio fisheri (bacterial luminescence) 0.122 (24 h) [23] Pimephales promelas (fish, 120-h growth) 400 (LOEC ϭ 20)b [32] Fluorouracil V. fisheri (bacterial luminescence) 0.122 (24 h) [24] P. promelas (fish, 120-h growth) 400 (LOEC ϭ 20) [32] Methotrexate S. subspicatus (alga) 72-h growth 260 [6] D. magna (crustacean, 48-h immobilization) Ͼ1,000 [6] B. rerio (fish, 96-h mortality) 85 [6] V. fisheri (bacterial luminescence) 1,220 (30 min) [6] Tetrahymena pyriformis (protozoan, 48-h growth) 45 [6] Xenopus laevis (frog, 96-h embryo malformations) 0.015 [33] Cladribine D. magna (crustacean, 48-h immobilization) 233 [31] Paclitaxel D. magna (crustacean, 48-h immobilization) Ͼ0.74 [31] Thiotepa D. magna (crustacean, 48-h immobilization) 546 [31] a QSAR ϭ values estimated by quantitative structure activity relationship. b LOEC ϭ lowest-observable-effect concentration. Ecotoxicity and genotoxicity of cytostatic pharmaceuticals Environ. Toxicol. Chem. 26, 2007 2213

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Paper II

Zounková, R., Kovalová, L., Bláha, L., Dott, W.: Ecotoxicity and Genotoxicity Assessment of Cytotoxic Antineoplastic Drugs and their Metabolites. Chemosphere (accepted with moderate revision)

Ecotoxicity and genotoxicity assessment of cytotoxic antineoplastic drugs and their metabolites

Radka Zounkováa,b,*, Lubomíra Kovalováa,c, Luděk Bláhab,d, Wolfgang Dotta a RWTH Aachen University, Institute of Hygiene and Environmental Health, Pauwelsstraβe 30, 52072 Aachen, Germany b RECETOX – Research Centre for Environmental Chemistry and Ecotoxicology, Faculty of Science, Masaryk University, Kamenice 126/3, 625 00 Brno, Czech Republic c Eawag, Swiss Federal Institute of Aquatic Science and Technology, Environmental Chemistry, Überlandstrasse 133, CH-8600 Dübendorf, Switzerland d Academy of Sciences of the Czech Republic, Institute of Botany, Department of Experimental Phycology and Ecotoxicology, Květná 8, 603 65 Brno, Czech Republic

Abstract In spite of growing scientific concern about pharmaceuticals in the environment, there is still a lack of information especially with regard to their metabolites. The present study investigated ecotoxicity and genotoxicity of three widely used cytostatic agents 5-fluorouracil (5-FU), cytarabine (CYT) and gemcitabine (GemC) and their major human metabolites, i.e. α-fluoro-β- alanine (FBAL), uracil-1-β-D-arabinofuranoside (AraU) and 2’,2’-difluorodeoxyuridine (dFdU), respectively. Effects were studied in acute immobilization and reproduction assays with crustacean Daphnia magna and growth inhibition tests with alga Desmodesmus subspicatus and bacteria Pseudomonas putida. Genotoxicity was tested with umu-test employing Salmonella choleraesius subsp. chol. Toxicity was relatively high at parent compounds with EC50 values ranging from 44 microgram/L (5-fluorouracil in the P. putida test) to 200 milligram/L (cytarabine in D. magna acute test). In general, the most toxic compound was 5-FU. Studied metabolites showed low or no toxicity; only FBAL (metabolite of 5-FU) showed low toxicity to D. subspicatus and P. putida with EC50 values 80 and 140 milligram/L, respectively. All parent cytostatics showed genotoxicity with minimum genotoxic concentrations (MGC) ranging from 40 to 330 milligram/L. From metabolites, only FBAL was genotoxic in high concentrations. To our knowledge, the present study provides some of the first ecotoxicity data for both cytostatics and their metabolites, which might further serve for serious evaluation of ecological risks. The observed EC50 values within the microgram/L range were fairly close to concentrations reported in hospital sewage water, which indicates further research needs, especially studies of chronic toxicity.

Keywords: cytostatics, cytotoxic drugs, metabolites, ecotoxicity, genotoxicity, chronic effects

1. Introduction Public and scientific concern about the occurrence of pharmaceuticals in the environment increased during recent years as adverse effects of these compounds and their metabolites in the environment were demonstrated (Halling-Sorensen et al., 1998; Heberer, 2002; Kummerer, 2001). Pharmaceuticals are continuously released into the environment in waste water or directly via human and animal excretion (Halling-Sorensen et al., 1998). It is known that many pharmaceuticals used in the human medicine are not completely metabolized, and they are excreted either slightly transformed or unchanged mostly conjugated to polar molecules. These conjugates can easily be cleaved during sewage treatment and the original drugs might then be released into the aquatic environment (Heberer, 2002). Elimination of some drugs during the waste water treatment and their biodegradation might be limited, so they can persist in the environment (Heberer, 2002). Pharmaceuticals and especially their metabolites are often hydrophilic and therefore highly mobile in aquatic environment (Kummerer, 2001). Cytostatic (antineoplastic) drugs are a class of pharmaceuticals used in cancer therapy. They often act by interfering with the structure and function of DNA, and they were shown to have cytotoxic, genotoxic, mutagenic, carcinogenic or teratogenic effects in non target organisms (Kummerer et al., 2000). Due to their molecular mode of action, all organisms in general might be susceptible to their toxicity (Johnson et al., 2008). Although some research consideration has been given to the issue of ecotoxicity of cytostatics (Zounkova et al., 2007), there is still a lack of information about particular compounds including their fate and effects in the environment (Kummerer et al., 1996; Mahnik et al., 2007; Steger-Hartmann et al., 1997). Residues of cytostatic drugs were detected in hospital sewage at concentrations up to the low μg/L level (Kovalova et al., 2009; Mahnik et al., 2007; Mullot et al., 2009; Steger-Hartmann et al., 1997), and some studies reported antineoplastic agents also in treated hospital waste water effluents (Aherne et al., 1990; Steger-Hartmann et al., 1996). According to Scherrer and Daschner (1997), cytostatics - with the exception of gemcitabine - are not readily biodegraded during water treatment, and they may become persistent in the sewage sludge (Kummerer et al., 1996). To our knowledge, only limited data exist on ecotoxicity of cytostatics, and acute effects were mostly covered (Al-Ahmad and Kummerer, 2001; AlAhmad et al., 1997; Hirose et al., 2005; Zounkova et al., 2007). Some experimental studies also investigated genotoxicity of hospital effluents as an indirect marker of cytostatics contamination (Giuliani et al., 1996; Jolibois and Guerbet, 2006; Jolibois et al., 2003). The present study aims to evaluate ecotoxicity and genotoxicity of three cytostatic drugs and their major human metabolites. Studied compounds - 5-fluorouracil, cytarabine and gemcitabine - belong to the family of pyrimidine antimetabolites, which are among the most widely used cytostatics. They inhibit DNA polymerase, stop the cell cycle and induce apoptosis (Klener, 2002). All of these compounds are metabolized and excreted in urine mostly as inactive metabolites, while approximately 10% is excreted in the unchanged form (Heggie et al., 1987; Hamada et al., 2002; Abbruzzese et al., 1991). According to some studies, 5-fluorouracil is not biodegraded in waste water, while gemcitabine and cytarabine are partially biodegraded, and they may pose risks to the environment (Kummerer and Al-Ahmad, 1997). As mentioned, some previous studies focused on ecotoxicity of the parent cytotoxic drugs but to our knowledge, chronic effects of these compounds and toxicity of their metabolites were not reported. In this study we report our investigations of three cytostatics and their metabolites testing genotoxicity with the umu-test as well as acute and chronic ecotoxicity in the assays with crustacean Daphnia magna, alga Desmodesmus subspicatus, bacteria Pseudomonas putida.

2. Materials and methods

2.1. Chemicals (Tested compounds) 5-fluorouracil (5-FU), cytarabine (CytR) and their metabolites α-fluoro-β-alanine (FBAL) and uracil-1-β-D-arabinofuranoside (AraU) were purchased as standards for chemical analysis from Sigma-Aldrich (Seelze, Germany). Gemcitabine hydrochloride (GemC) and its metabolite 2’,2’- difluorodeoxyuridine (dFdU) were kindly provided by the Eli-Lilly Research Laboratories (Indianapolis, IN, USA). Concentrated stock solutions 1000 mg/L in water were prepared for all compounds. All operations and handling with tested compounds were carried out in a safety hood or laminar flow box.

Table 1: Description and structures of the studied antineoplastic drugs and their metabolites. CAS no. - Chemical Abstract Services number, ATC code – the code in accordance with the Anatomical Therapeutic Chemical Classification System.

Compound CAS no./ Structure Description (abbreviation) ATC code 5-fluorouracil (5-FU) 51-21-8/ O cytostatic L01BC02 F N

O N

Cytarabine 147-94-4/ N cytostatic (CytR) L01BC01 N

O N

O O

Gemcitabine (GemC) 95058-81-4/ N cytostatic L01BC05 N

O N F O F

O O

α-fluoro-β-alanine 3821-81-6 O 5-FU (FBAL) F metabolite O

uracil-1-β-D- 3083-77-0 O CytR arabinofuranoside N metabolite (araU) O N

O O

O O 2’,2’- 114248-23-6 O GemC difluorodeoxyuridine metabolite (dFdU) N

O N F O F

O O

2.2. Bioassays The biotests involved all three trophic levels of ecosystems in the battery of bioassays including producers (green algae), consumers (crustaceans) and decomposers (bacteria). Algal growth-inhibition test was conducted according to the European standard EN ISO 8692:1989 (ISO, 1989b) and DIN 38412 Part 9 (DIN, 1993) using green alga Desmodesmus subspicatus. The assay was performed in 24-well microplates. Cells in the exponential growth phase were used with the initial cell density adjusted to 105 cells ml-1. The cell suspension was mixed in the test wells with medium and the solution of the studied compound in five different concentrations. The plate was incubated at 23°C under continuous light and discontinuous shaking (30 min 400 rpm, 15 min break). Fluorescence of chlorophyll was measured in the beginning of the exposure and then every 24 hours, absorbance at 650 nm was measured at the end of the test. Total duration of the bioassay was 72 hours when the average growth rates were calculated and the growth inhibition evaluated. Acute immobilization test with Daphnia magna was based on the European Standard EN ISO 6341:1996 (ISO, 1989a) with some modifications. For the breed (as well for the test) we have used the ADaM medium (Aachener Daphnien Medium) imitating natural fresh water (Klüttgen et al., 1994). Juveniles of D. magna (less than 24 hours old) obtained from the continuous laboratory culture were used. Twenty animals for each tested concentration and for the control were used and subdivided into four replicates (each containing five daphnids) in the glass beakers. Total duration of the exposure (in the dark at 20°C) was 48 h, immobilized organisms were counted after 24 and 48 h. Number of immobilized animals after 48 hours was taken as endpoint. Results were expressed as the percentage of control. The reproduction test with Daphnia magna was conducted in accordance with the OECD TG 211 standard (OECD, 1998) with modifications. As in the acute test, the ADaM medium (Klüttgen et al., 1994) was used. Newly hatched daphnids were transferred into the glass beakers (one animal per beaker) with 50 ml of the medium containing specific concentration of the tested compound or control. During 21 days of exposure, the survival and the reproduction were monitored. Medium with the tested compounds was changed three times a week and daphnids were fed with the suspension of green alga Desmodesmus subspicatus. Experiments were run at temperatures 21 ± 1°C and photoperiod 16:8 light/dark. The growth inhibition test with Pseudomonas putida was conducted according to the European norm based on International Standardization Organization Standard EN ISO 10712:1995 and the Czech Standard (CSN, 1995) in the miniaturized version (Schmitz et al., 1998). Cryopreserved cultures of P. putida stored at -80 °C in glycerol were used. The test was performed in 96-well microplates, incubated in the dark for 16 hours at 23 ± 1 °C on a shaker. The growth inhibition was evaluated as the change of the absorbance of the bacterial culture measured at 590 nm before and after exposure. Bacterial genotoxicity test (umu-test) was performed in accordance with the international standard ISO/FDIS 13829 (DIN 38415-3) (ISO, 1999). The genetically modified bacterial tester strain Salmonella choleraesius subsp. chol. was used. The genotoxin-dependent induction of the umuC-gene was compared with the spontaneous activation of the untreated - control - culture. The test was performed in 96-well microplates with and without metabolic activation using S9 rat liver homogenate (Trinova Biochem GmbH, Giessen, Germany). After 2 hours of initial incubation (exposure) of bacteria and compounds of interest (with or without S9 homogenate), reaction mixtures were ten fold diluted with exposure media and incubated for another 2 hours. Optical density was then measured to assess the bacterial growth (cytotoxicity) at 600 nm and then the β-galactosidase activity was measured at 420 nm after a 30 min incubation with a chromogenic substrate ortho-nitrophenyl-β-D-galactopyranoside. Cytotoxic effect was quantified as a percentage of inhibition of growth in comparison to the negative control. The concentrations causing more than 50 % inhibition were excluded from genotoxicity evaluations. The induction factor was then calculated for each tested concentration. Chemical analyses were performed to check the concentrations during acute and reproduction tests with Daphnia magna. Samples were collected, filtered and diluted with acetonitrile (1:1) and the concentrations measured using HPLC/DAD or HPLC/MS-MS as described in detail previously (Kovalova et al., 2009). Analyses with samples from other tests have not been performed due to extremely small amounts of medium in testing microwell plates.

2.3. Statistical evaluations For all bioassays, experiments included initial testing of a wide range of concentrations (1:10 dilutions). If necessary, more detailed assessment around the 50% effective concentration (EC50) followed. For ecotoxicological assays, the homogenity of variance was controlled using Levene`s test and the lowest tested concentrations causing statistically significant effect (LOEC) were evaluated using Analysis of Variance (ANOVA) followed by the Dunnet’s test or non- parametric Mann-Whitney U test. Concentrations causing 50% effects (EC50) were estimated using the probit analysis in the U.S. Environmental Protection Agency Probit software (U.S. EPA,Washington, D.C.). For the umu-test, minimum genotoxic concentration was derived as the first tested concentration that exceeded the induction factors value of 1.5. Calculations were performed with Microsoft Excel® (Microsoft, Redmond, WA, USA) and Statistica® for Windows 6.0 (StatSoft, Tulsa, OK, USA).

3. Results and discussion Three short-term ecotoxicological tests (growth inhibition tests with bacteria and green algae, acute immobilization test with fresh water crustaceans), one chronic test for reproduction and a genotoxicity test were carried out to assess the effects of three cytostatic pharmaceuticals and their major metabolites. Analyses of studied compounds by HPLC showed only minor declines in the concentrations of all tested compounds within the period of media exchanges. Maximum decline by 15% was observed at gemcitabine. In general, good stability of tested compounds during toxicological experiments was confirmed.

3.1. Ecotoxicity results Effects of cytostatic agents and their metabolites in nominal concentrations, i.e. full dose- response curves in ecotoxicological bioassays, are summarized in Figures 1 and 2. Values of the lowest observable effect concentrations (LOEC) and 50% effective concentrations (EC50) are listed in Table 2. The toxicity of the tested cytostatic pharmaceuticals was variable, with acute EC50 concentrations ranging from 44 μg/L (5-fluorouracil in the Pseudomonas putida growth inhibition test) to 200 mg/L (cytarabine in Daphnia magna acute immobilization test). Most EC50 values were between 10 and 100 mg/L or even higher. The most toxic compound in our study was 5-fluorouracil. It showed high toxicity in two assays (reproduction toxicity to D.magna and P.putida growth inhibition, EC50 = 0.04-0.05 mg/L), while less pronounced effects were observed in the algal growth inhibition test (EC50 = 75 mg/L). These values are well comparable with effective concentrations of other compounds from the family of pharmaceuticals and personal care products (PPCP`s) (Sanderson et al., 2003; Cleuvers, 2003; Halling-Sorensen et al., 1998; Wollenberger et al., 2000). Also in our previous study (Zounkova et al., 2007), 5-fluorouracil was highly toxic in majority of the bioassays.

Table 2. Summary of the effects of cytostatic drugs 5-fluorouracil, cytarabine and gemcitabine, and their metabolites α-fluoro-β-alanine (FBAL), uracil- 1-β-Darabinofuranoside (AraU), 2’,2’-difluorodeoxyuridine (dFdU) in ecotoxicological biotests. All toxicity values are in mg/L (values in parentheses show 95% confidence intervals); NOEC – no observable effect concentration, LOEC – lowest observable effect concentration, EC50 – concentration causing 50% effect. Numbers with the sign “>” indicate the highest concentrations used in the respective assay.

D. magna Test substance P. putida D. subspicatus D. magna acute reproduction

LOEC EC50 LOEC EC50 LOEC EC50 LOEC EC50

5-fluorouracil 0.03 0.044 (0.025 - 0.077) 40 48 (44-51) 5.0 15 (5.2-45) 0.05 0.1 Cytarabine 10 17(4.7-64) 40 53 (29-95) 100 200(63-810) 3.7 10 Gemcitabine 50 100 (34-300) 10 45 (12-170) 50 110(45-520) > 1.0 > 1.0

FBAL 100 140 (110-180) 25 80 (28-230) > 100 > 100 > 10 > 10 AraU > 500 > 500 > 200 > 200 > 100 > 100 > 10 > 10 dFdU > 500 > 500 200 > 200 100 > 100 > 1.0 > 1.0

Table 3. Genotoxicity in the umu-test of cytostatic drugs 5-fluorouracil, cytarabine and gemcitabine, and their metabolites α-fluoro-β-alanine (FBAL), uracil-1-β-Darabinofuranoside (AraU), 2’,2’-difluorodeoxyuridine (dFdU); MGC – minimal genotoxic concentration.

umu test MGC (mg/L) without metabolic with metabolic Test substance activation activation

5-fluorouracil - - Cytarabine 167 333 Gemcitabine 167 42

FBAL 667 > 667 AraU > 667 > 667 dFdU > 667 > 667

The EU-Directive 93/67/ EEC (EC, 1996) classifies substances into different classes according -1 -1 to their EC50 values: EC50 < 1 mg L are very toxic to aquatic organisms; 1-10 mg L are toxic to aquatic organisms and 10-100 mg L-1 are classified as harmful to aquatic organisms. -1 Substances with an EC50 above 100 mg L would not be classified (Cleuvers, 2003). In our study, EC50 below 1 mg/L were observed for 5-fluorouracil in two tests (P. putida growth inhibition test and D. magna reproduction test) and for cytarabine below 10 mg/L in D. magna reproduction test. Correspondingly, 5-fluorouracil may be classified as very toxic, cytarabine as toxic, and gemcitabine harmful to aquatic organisms. Jones (2002) proposed yet another category of extremely toxic compounds with EC50 < 0.1 mg/L. According to their study, cytostatic drugs should be considered very toxic to microorganisms (EC50 = 0.1-1 mg/L) and harmful to crustaceans and fish (EC50 = 10-100 mg/L). Comparing with our results, 5- fluorouracil would be categorized as extremely toxic to both microorganisms and crustaceans and harmful to green algae. With respect to limited accessibility to standards, only limited data still exist on metabolites and derivatives of parent pharmaceuticals and other PPCPs. The metabolites of cytotoxic drugs, which are considered inactive in the cancer therapy, are expected to be less cytotoxic than the parent compounds, and lower toxicity of metabolites was revealed also in our study. Only FBAL (metabolite of 5-fluorouracil) showed pronounced toxicity and EC50 values could be derived for two assays with bacteria and algae (Table 2). In spite of lower toxicity, FBAL might still be classified as harmful to aquatic organisms according to the EU (Cleuvers, 2003). With respect to sensitivity of the acute assays, the growth inhibition test with Pseudomonas putida was the most sensitive in the present study. For example, for 5-fluorouracil it had the same sensitivity as the long-term reproduction test with D. magna (Table 2). In our previous report focused on five different parent cytotoxic drugs (Zounkova et al., 2007), sensitivity of the P. putida was comparable with the algal growth inhibition test. Similar pattern was observed in the present study for gemcitabine, where the growth inhibition test with D. subspicatus was even more sensitive than P. putida test. Large differences among studied drugs were observed in the acute vs. 21-day reproduction tests with D. magna. For 5-fluorouracil and cytarabine, EC50 for reproduction toxicity was nearly three orders of magnitude lower than acute immobilization. In contrast, no pronounced effects in the reproduction test were recorded for the third drug gemcitabine up to the highest tested concentration (1 mg/L). With this respect, other studies with drug sulfamethazine (De Liguoro et al., 2009) or derivatives of polycyclic aromatic hydrocarbons (Feldmannova et al., 2006) reported 1 to 2 orders of magnitude large differences in EC50 between acute and chronic assays with D. magna. Even higher differences recorded in the present study might be attributed to the mechanism of toxicity of the studied drugs acting via DNA damage but this issue will require further research. For pharmaceuticals and similar compounds, which are present in low concentrations in the environment, the results from chronic assays are more relevant than those from acute tests. This was shown for example with Vibrio fisheri, where dramatically stronger effects on the luminescence were observed after prolonged 24h incubations in comparison with traditional 30 min exposures (Backhaus et al., 1997). Also (Kummerer et al., 2004) concluded that standardized short term respiration test with bacteria may underestimate risks of pharmaceuticals. These conclusions well correspond to large differences in toxicity of two studied drugs between the acute and reproduction assays with D. magna in the present study. However, it should be emphasized that the standardized chronic assays, such as the 21-day reproduction toxicity with D. magna, in fact represent only one generation assay. Prolonged multigeneration exposures might result in number of unexpected effects, especially for compounds acting on DNA (Vandegehuchte et al., 2009a; 2009b).

A 120 5-FU

100 CytR GemC 80 dFdU 60

40 immobilization [%] 20

0 1 10 100 1000 concentration [mg/L]

B 100 5-FU FBAL 75 CytR

GemC 50

[%] inhibition growth 25

0 10 100 1000 concentration [mg/L]

C 5-FU 100 FBAL

CytR 75 GemC

[%] inhibition growth 25

0 0.01 0.1 10 100 1000 concentration [mg/L]

Fig. 1: Ecotoxicity (concentration-response curves) of the studied cytostatic drugs and their metabolites. A - Daphnia magna acute immobilization test. B - Growth inhibition test with Desmodesmus subspicatus. C - Growth inhibition test with Pseudomonas putida. 5-FU: 5-fluorouracil, CytR: cytarabine, GemC: gemcitabine, FBAL: α-fluoro-β-alanine, dFdU: 2’,2’-difluorodeoxyuridine. Compounds, which did not induce significat toxicity are not presented in respective plots.

5-FU control 60 0.001 mg/L

50 0.01 mg/L

0.1 mg/L 40 1.0 mg/L 30 10 mg/L

offspring of No normalized 10

0 0 3 6 9 12 15 18 21 day

CytR control 80 0.001 mg/L 70 0.01 mg/L 60 0.1 mg/L

50 1.0 mg/L 40 10 mg/L

20 normalized No of offspring of No normalized 10

0 0 3 6 9 12 15 18 21 day

Fig. 2: Effects of 5-fluorouracil (5-FU) and cytarabine (CytR) on the reproduction of Daphnia magna (numbers of offsprings) in the 21-d chronic test.

3.2. Genotoxicity results Besides the ecotoxicity, genotoxic effects of the cytotoxic drugs and their metabolites were tested, and the results are summarized Figure 3 and Table 3. Two of the tested parent cytostatics (cytarabine and gemcitabine) showed genotoxicity in the umu-test in both variants with and without metabolic activation, but the effects were observed at relatively high concentrations > 100mg/L (Table 3). For gemcitabine, slightly stronger genotoxicity after metabolic activation was observed at concentrations around 100 mg/L. This seems to correspond to the fact, that gemcitabine itself is a pro-drug, and its activity depends on the intracellular conversion to two active metabolites (Abbruzzese et al., 1991). Also 5-fluorouracil was genotoxic without S9 activation (induction factors > 4) at concentrations around 100 mg/L with MGC ~ 40 mg/L. However, its high toxicity (over 50% growth inhibitions in the umu-test) complicates the interpretation, and the results should not be considered valid according to the standard ISO method. On the other hand, other studies clearly demonstrated that 5-fluorouracil belongs among strong genotoxic compounds in bacterial assays (Oda, 1987; Zounkova et al., 2007; Barclay et al., 2001).

Fig. 3: Results of the genotoxicity (induction factors on the left Y-axes; diamonds with continuous lines) and growth inhibitions (triangles with dashed lines; right Yaxes) of cytostatics and their metabolites in the umu-test. Results of the assays with metabolic activation (empty symbols) and without metabolic activation (filled symbols) are presented. 5-FU: 5-fluorouracil, CytR: cytarabine, GemC: gemcitabine, FBAL: α-fluoro-β- alanine, dFdU: 2’,2’-difluorodeoxyuridine, AraU: uracil-1-β-D-arabinofuranoside. Horizontal dashed line - critical induction for genotoxicity (induction factor = 1.5); dash-dotted line - critical value of the growth factor (0.5; 50% inhibition).

Regarding the metabolites of cytarabine and gemcitabine, no genotoxicity was observed up to the highest tested concentrations. The only genotoxic effects were recorded at FBAL, a metabolite of 5-fluorouracil, at the highest dose (667 mg/L) tested without metabolic activation. In general, umu-test for genotoxicity seemed to be less sensitive to the studied drugs than other acute and chronic ecotoxicity assays. Other models based on eukaryotic organisms such as yeast Saccharomyces cerevisiae could be recommended for future studies in addition to bacterial genotoxicity tests, as the higher sensitivity to cytotoxic drugs such as 5-fluorouracil was demonstrated (Zounkova et al., 2007).

3.3. Environmental relevance Effective toxic concentrations of the studied cytotoxic drugs and their metabolites observed in the present study appear to be higher than expected environmental levels. Maximum concentrations reported from the Swiss cantonal hospital for 5-fluorouracil, gemcitabine and its metabolite 2',2'-difluorodeoxyuridine were 27, 38 and 840 ng/L respectively (Kovalova et al., 2009). In the study of Mullot et al. (2009) higher concentrations of unchanged 5-fluorouracil (0.09 to 4.0 μg/L) were reported in sewage from a hospital located in Paris. Another study (Mahnik et al., 2004) reported three orders of magnitude higher levels of 5-fluorouracil in waste water from an oncologic in-patient-treatment ward (concentrations of 20-122 μg/L), and they were close to LOEC and even EC50 values derived in this study. Although lower toxicities were observed at the metabolites, these seem to be the dominant contaminants as parent cytotoxic drugs are excreted only in about 10% in the unchanged form (Heggie et al., 1987; Hamada et al., 2002; Abbruzzese et al., 1991). This fact further increase relative importance of the drug metabolites in the complex mixture but only limited data are still available on their concentrations in waste waters.

4. Conclusions Our study brings some of the first experimental data on ecotoxicity and genotoxicity of relatively poorly studied group of antineoplastic pharmaceuticals and especially their metabolites. We have found that cytotoxic agents 5-fluorouracil, cytarabine and gemcitabine were toxic in various assays but significant effects were observed at relatively high concentrations (mg/L). Studied metabolites were mostly non-toxic including minor effects in the chronic reproduction assay with D. magna. Considering dilutions in the sewer system, pharmaceutical pollution by individual chemicals seems to be rather low but some studies reported environmental concentrations within the range close to the experimentally derived LOECs or EC50 in the biotests. In spite of many discussions, limited experimental data on chronic ecotoxicity of pharmaceuticals still exist. There are also other issues, which will require further research attention such as degradation pathways of the drug metabolites, their continuous release and pseudopersistence as well as chronic effects that may be hidden for many years (Jones et al., 2001). Unexpected toxicity interactions among drugs within low concentration ranges are also of concern (Pomati et al., 2008).

Acknowledgements The study was conducted at RWTH University Aachen and at Masaryk University. Research was supported by the Marie Curie Early Stage Research Fellowship of the European Community`s Sixth Framework Programme (contract number MEST-CT-2004-505169) and partially by the Czech Ministry of Education grant no. 0021622412 "INCHEMBIOL". Authors would like to acknowledge Lilly Research Laboratoires (Indianapolis, USA) for providing some compounds. We would like to thank Juliane Hollender from EAWAG, Switzerland for valuable consultation and Rita Hochstrat who coordinated the AQUA-base project.

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Paper III

Complex evaluation of ecotoxicity and genotoxicity of antimicrobials oxytetracycline and flumequine used in aquaculture

Radka Zounková†*, Zdeňka Klimešová†, Leona Nepejchalová‡, Klára Hilscherová†, Luděk Bláha†

†RECETOX – Research Centre for Environmental chemistry and Ecotoxicology, Faculty of Science, Masaryk University, Kamenice 126/3, 62500 Brno, Czech Republic

‡Institute for State Control of Veterinary Biologicals and Medicaments, Hudcova 56, 62100 Brno, Czech Republic

* To whom correspondence may be addressed: Radka Zounkova; Masaryk University, RECETOX, Kamenice 126/3, CZ62500 Brno, Czech Republic; Tel: +420776037798; E-mail: [email protected]

ABSTRACT Ecotoxicity and genotoxicity of widely used veterinary antimicrobials oxytetracycline and flumequine was studied with 6 model organisms (V. fischeri, P. putida, P. subcapitata, L. minor, D. magna, E. coli). Overall EC50 values ranged from 0.22 mg/L to 86 mg/L. Pseudomonas putida was the most sensitive organism (EC50 values for 16-h growth inhibition were 0.22 mg/L and 0.82 mg/L for oxytetracycline and flumequine, respectively), followed by duckweed Lemna minor (7-d growth inhibition, EC50 2.1 mg/L and 3.0 mg/L) and green alga Pseudokirchneriella subcapitata (4-d growth inhibition, EC50 3.1 mg/L and 2.6 mg/L). The least sensitive organism was Daphnia magna (48-h immobilization, LOEC of oxytetracycline of 400 mg/L). Oxytetracycline showed limited genotoxicity (SOS-chromotest with Escherichia coli, minimal genotoxic concentration of 500 mg/L), flumequine was genotoxic at 0.25 mg/L. Based on the reported measured concentrations (MECs) and predicted no effect concentrations (PNECs) derived from our data, we conclude that oxytetracycline is of ecotoxicological concern (calculated risk quotient = 8), while flumequine seems to represent lower risk.

KEYWORDS oxytetracyline, flumequine, ecotoxicity, genotoxicity, veterinary antibiotics

INTRODUCTION During recent years significant attention has been given to the occurrence of drugs in the environment. It has become clear, that the use and disposal of pharmaceutical substances may have adverse effects in the environment. Taking into account that drugs are specially designed to affect biological systems, this is not surprising. Antibiotics are an important group of pharmaceuticals in the present-day medicine. In addition to the treatment of human infections, they are also used in veterinary medicine [1]. Veterinary antimicrobials are widely used both therapeutically and also as growth promoters in intensive farming. About 200 tons of antimicrobial agents are administered annually in Denmark, from that approximately 10 tons in fish farming [2, 3]. Estimated antibiotic consumption worldwide is between 100000 and 200000 tons per year. In 1996, about 10200 tons of antibiotics were used in EU, of which approximately 50 % was applied in veterinary medicine [4]. In the Czech Republic, 49 tons of veterinary antibiotics were used in 2003 and this amount increased to 80 tons in 2008,

from which 45 tons were tetracyclines (available on-line on http://www.uskvbl.cz/attachments/339_spot%C5%99eba%20ATB%202003-2008.doc). To treat bacterial infection in intensive fish farming, antibacterial agents are administered directly to the water as feed additives, mostly as medicated feed pellets [5] [2], or by simple addition to water [1]. Thus, most of these pharmaceuticals end up directly in the environment. As mostly hydrophilic compounds, antibiotics and their metabolites are highly mobile in the aquatic environment [4]. Moreover, they often have a low biodegradability [2]. Although antibiotics are designed to kill bacteria, they may have effects on organisms from all trophic levels in the ecosystem. Antibiotic residues in the environment are also suspected to induce resistances in pathogenic bacterial strains causing thus a threat for public health [1]. Two veterinary antimicrobials are commonly used in fish farming. Those are oxytetracycline (OTC) and flumequine (FLU) [6]. In the Czech Republic, there are only two antibiotics preparations registered for the application to aquaculture, namely Flumiquil with flumequine as an active ingredient, and Rupin Special with oxytetracycline as an active ingredient [7]. Oxytetracycline hydrochloride (OTC, CAS number 2058-46-0) is a tetracycline broad-spectrum antibiotic with bacteriostatic action against various gram-positive and gram-negative bacteria. It is produced by Streptomyces spp. fungi. It inhibits protein synthesis by reversibly binding to 30S ribosomal subunit of the microbial ribosome. Oxytetracycline has become the most widely used antimicrobial agent for the treatment of bacterial fish diseases [8]. Flumequine (FLU, CAS number 42835-25-6) is a quinolone broad-spectrum antibacterial agent with bactericidal action especially against gram-negative bacteria, which is widely used in intensive aquaculture. The mode of action of quinolones is inhibition of bacterial growth by interfering with the enzyme DNA-gyrase (topoisomerase II), which is essential for coiling and uncoiling of DNA, and thus terminating the normal DNA synthesis [9]. Both oxytetracycline and flumequine are persistent in surface sediments with half-life of approximately 150 days [10]. The main objective of this study was to derive comparative data on the potential ecotoxicological impact of veterinary antibiotics. We investigated ecotoxicity and genotoxicity of flumequine and oxytetracycline hydrochloride. For ecotoxicity evaluation representatives of all three trophic levels, i.e. producers (green algae, aquatic plant), consumers (fresh water crustacean) and decomposers (bacteria) were used. Genotoxicity was evaluated in prokaryotic (bacterial) bioassay.

MATERIALS AND METHODS Chemicals Veterinary antimicrobials, both flumequine and oxytetracycline hydrochloride, were obtained from Institute for State Control of Veterinary Biologicals and Medicines, Brno, Czech Republic. Bioassays Pseudomonas putida growth inhibition test was performed according to the Czech standard based on European standard EN ISO 10712:1995 [11] in the miniaturized version [12]. Cryopreserved cultures of Pseudomonas putida (stored at -80°C in glycerol) were used, the growth inhibition was evaluated as the change in the absorption of bacterial culture (590 nm) after 16 hours of exposure in the dark at 23 ± 1 °C on a shaker. Vibrio fischeri test on bioluminescence inhibition was performed according to the Czech standard based on ISO norm [13]. We used freeze-dried cultures of Vibrio fischeri purchased from the Institute of Microbiology, Academy of Sciences of the Czech Republic. The decrease of bioluminescence of bacterial culture was evaluated after 15 and 30 min exposure. Algal growth inhibition test was performed according to the Czech norm based on the European standard EN ISO 8692:1989 [14] using unicellular alga Pseudokirchneriella subcapitata. This assay was performed in modified and miniaturized version using 96-well microplates, as described in the article of Rojickova et al. [15], 50% ZBB medium was used as culture and test medium (prepared by mixing of Zehnder Z-medium and Bristol modified Bold (BB) medium (available on-line on http://www.butbn.cas.cz/ccala/index.php) and distilled water in the ratio 1:1:2). Cells in the

exponential growth phase were used, growth was evaluated as the change of light absorbance (680nm; corresponding to the amount of chlorophyll) at the beginning of the exposure and then every 24 hours. Total duration of the test was 96 hours when the growth inhibition was evaluated. Growth inhibition test with Lemna minor was performed according to the European standard [16]. The growth inhibition was evaluated as the change of total frond number in comparison to the control. The test was performed in 50 ml polystyrene vessels in cultivating room at 24 ± 2 °C under continual light. Total duration of the exposure was 7 days. Acute immobilization test with Daphnia magna was conducted according to the Czech standard [17] which is identical with European Standard EN ISO 6341:1996. Newly hatched neonates (less than 24 hours old) obtained from the continuous laboratory culture were used (twenty animals for each tested concentration and control). Total duration of the exposure was 48 hours. Immobilized organisms were counted and the results were expressed as the percentage of control. Daphnia magna reproduction test was performed according to the OECD Standard 221 and the Czech Standard [18]. Newly hatched neonates (less than 24 hours old, only females) obtained from the continuous laboratory culture were used (ten animals for each concentration and control, each animal separately in 50 ml of M4 medium in test vessel). Total duration of the exposure was 3 weeks. Organisms were fed with mixture of green algae three times a week, simultaneously the medium was changed and offspring produced by parent animals was counted and removed. Survival of parent animals and number of live offspring were evaluated and expressed as the percentage of control. Bacterial genotoxicity test (SOS-chromotest) was performed using genetically modified bacterial tester strain Escherichia coli PQ 37. The test was performed without metabolic activation in the 96- well microtitre plate as described previously [19]. After 2 hours of incubation with compounds of interest, β-galactosidase (reporter enzyme for genotoxicity induced along with DNA repair system) activity was measured using a chromogenic substrate ortho-nitrophenyl-β-D-galactopyranoside. At the same time, activity of alkaline phosphatase (marker of viability/cytotoxicity) was assessed using p-nitrophenyl phosphate chromogenic substrate. Cytotoxic effect was quantified as a percentage of inhibition of the alkaline phosphatase in comparison with the negative control and the concentrations causing more than 50 % inhibition were excluded from genotoxicity evaluations. The SOS induction factor (IF) was then calculated for each tested concentration, and the minimal genotoxic concentration (MGC – the concentration, at which the IF reaches the critical value 1.5) was determined. Data analysis For all bioassays, experiments included initial testing of a wide range of concentration (1:10 dilutions) followed by a detailed assessment around EC50 values. Full dose-response curves for all bioassays were repeated independently at least three times. Results were pooled and analyzed. For ecotoxicological assays, homogeneity of variance was controlled by Levene`s test. Values of NOEC (the highest tested concentration with no observable effects) and LOEC (the lowest tested concentration causing statistically significant effect) were evaluated using Analysis of Variance (ANOVA) followed by the Dunnet’s test or non-parametric Mann-Whitney U test. Concentrations eliciting 50 % effects (EC50) were estimated using sigmoidal non-linear dose-response regression in the GraphPad Prism 4.0 software (GraphPad Software, Inc.). For the genotoxicity test, minimum genotoxic concentration (MGC) was derived according to standard procedure of the assay as the mean concentration that exceeded the induction factor value of 1.5 (SOS-chromotest). Calculations were performed with MS-Excel and Statistica for Windows 7.1 (StatSoft, Tulsa, OK, USA). Risk characterization Risk quotients for the aquatic environment were calculated using the MECs (measured environmental concentrations) reported in the literature and the PNECs based on the current study. For PNEC values the lowest LOEC (lowest observable effect concentration) values from this study were used. An assessment factor (AF) of 1000 was used following the equation, Eq. 1. PNEC = LOEC / AF (1)

The final risk quotient was calculated by dividing the worst case (the highest found) MEC by the PNEC (Eq. 2.). RQ = MEC / PNEC (2) If a risk quotient is calculated to be less than 1, ecological risk is not expected [20].

RESULTS Effects of both tested compounds in all ecotoxicological and genotoxicity tests, i.e. values of the 50% effective concentrations (EC50), the lowest observable effect concentrations (LOEC), no observable effect concentration (NOEC) and minimal genotoxic concentrations (MGC) are summarized in Table 1. Both antimicrobials showed significant effects in majority of the bioassays with effect concentrations in a wide range from 0.04 mg/L (LOEC of OTC in the growth inhibition test with P. putida) to 500 mg/L (MGC of OTC in the SOS-chromotest). The more toxic compound was OTC with the lowest EC50 value of this study in the P. putida growth inhibition test, but FLU elicited major toxicity in most of the other tests including the genotoxicity test. In the tests on producers, the toxicities were similar for both tested compounds. In the tests with D. magna and SOS-chromotest, the differences between the two antimicrobials were more obvious. Table 1 also presents calculation of risk quotients. Risk quotient of OTC reached the value of 8.5 which exceeds the critical value of 1 (due to a very low LOEC of 0.04 mg/L). Full dose-response curves are presented in Figures 1 - 3. Further, Table 2 shows available literature results on OTC and FLU ecotoxicity and genotoxicity.

DISCUSSION For both tested antimicrobials P. putida was the most sensitive organism, which was expected as antibiotics are primarily designed to kill bacteria. LOEC derived in the present study was nearly two orders of magnitude lower than the minimum growth inhibitory concentration previously reported for Pseudomonas sp. [21] (Table 2). The test with marine bacteria V. fischeri was much less sensitive but also other authors reported low sensitivity of this test to antibiotics [22]. Isidori et al. [23] studied toxicity and genotoxicity of six antibiotics including OTC and reported EC50 value of 65 mg/L for V. fischeri, which is comparable with the result of the present study. On the other hand, the 30-min EC50 of OTC for V. fischeri reported by Lalumera et al. [6] was ten times higher. Interestingly, the results of the same authors for FLU [6] were in accordance with the present study. Lower sensitivity of V. fischeri to the antimicrobials, namely OTC, can be explained by its mode of action on protein synthesis, which is not of importance during the short term bioluminescence testing. However, drastic increase in the V. fischeri sensitivity was shown during the prolonged 24 h incubation [24]. Both representatives of producers – green alga P. subcapitata and aquatic vascular plant L. minor demonstrated more or less the same sensitivity for both tested compounds. Three different values for algal growth inhibition test with OTC were found in the literature (Table 2) and they were in general comparable with the results of the present study [5, 25, 26]. The least sensitive organism was D. magna, for which OTC in acute test demonstrated no toxicity up to 400 mg/L and only low toxicity was observed in the chronic reproduction test. Similar differences in sensitivity of rotifers and crustaceans vs. algae were previously reported by Isidori [23]. On the other hand, toxicity of FLU to D. magna was much higher in the reproduction test (two orders of magnitude lower LOEC). Low toxicity of OTC for D. magna was also previously reported by Wollenberger et al. [2] but other authors observed higher toxicity with the EC50 value of 22.6 mg/L [23]. Robinson et al. [26] reported minor toxicity of FLU for D. magna with NOEC of 10 mg/L, which is similar to our result but to our knowledge, no study previously addressed chronic toxicity of FLU in the reproduction test with D. magna. Our study seems to indicate parallels in responses of the D. magna (during the reproduction test) and E. coli in the SOS-chromotest. Both Daphnia and E. coli were markedly more sensitive to FLU than to OTC, which might be related to the mode of action of both antimicrobials. While OTC has

effect on the protein synthesis in bacteria, FLU acts directly on the DNA by inhibiting the topoisomerase II enzyme [8] [9]. Low genotoxicity of OTC was observed in the present study (very high and environmentally non- relevant MGC of 500 mg/L), which corresponds to study of Isidori et al. [23], who reported no mutagenicity of OTC, neither in the Ames nor SOS-chromotest. On the other hand, genotoxicity of FLU was markedly higher. Interestingly, genotoxicity of FLU was not previously reported although studies with other quinolone antibiotics are available. For example, Itoh et al. [27] showed DNA alterations induced by various quinolone antimicrobials in the comet assay and in vitro micronucleus test. Significance of the quinolone compounds was suggested by Hartmann et al. [29] who concluded that genotoxicity detected in hospital wastewaters is caused mainly by fluoroquinolone antibiotics. Moreover, Lancieri at al. [28] demonstrated teratogenic effects of FLU during acute exposures of the early stages of fish Danio rerio. Many antibiotics have been found in sewage influent and effluent samples, surface waters and even ground and drinking water. In Italy, flumequine and oxytetracycline were indicated as priority chemicals with possible side effects in aquaculture [6]. Due to their precipitation, tetracyclines are not expected to be present dissolved in the aquatic environment but they sorb to the organic matter and may accumulate in sediments. Nevertheless, OTC was detected in the U.S. surface water samples at concentration 0.34 μg/L [30]. Higher concentrations (55.7 μg/L) were reported in the overland flow water [31]. In the Czech Republic, concentrations of OTC in the fish farm bottom sediments reached up to 1516 μg/kg two months after application [32]. FLU was detected in concentrations up to 32 ng/L at 5 sampling locations in the Seine River inner estuary [33]. Reported concentrations were compared with the observed ecotoxicity LOECs and risk quotients were calculated (see Table 1). For OTC, some risk assessments were previously reported but lead to contradictory conclusions. In part, Park and Choi [34] reported the risk quotient of 2 using MEC of 0.34 μg/L for the calculation. Their PNEC was derived from the EC50 value of 0.17 mg/L in the P. subcapitata growth inhibition test. On the other hand, Isidori et al. [23] reported the risk quotient lower than 1 using MEC of 0.05 μg/L and the same value of PNEC as in the study of Park and Choi [34]. Our results are comparable to the findings of Park and Choi [34]: using MEC of 0.34 μg/L, we have calculated the risk quotient to be 8.5. No previously reported risk assessment for FLU has been found in the literature but according to the results of present study, low or acceptable risk can be predicted (the risk quotient of 0.16) but significant genotoxicity of FLU should not be overlooked.

CONCLUSIONS This study provides comprehensive data on aquatic ecotoxicity and genotoxicity of two veterinary antimicrobials frequently used in aquaculture. Pseudomonas putida was found to be the most sensitive species with EC50 values lower than 1 mg/L. Algae and vascular plants were also affected by studied drugs with EC50 in low mg/L range. Minor effects were observed at D. magna but FLU had significant effects during the chronic reproduction study. Risk quotient for OTC highly exceeded 1 indicating thus possible environmental risk, while FLU seems to have only minor impact. The results indicate potential risk for non-target organisms resulting from exposure to low levels of veterinary antimicrobials.

ACKNOWLEDGEMENTS The research was supported by the Czech Ministry of Education CR (project INCHEMBIOL No. 0021622412).

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21. Halling-Sorensen, B, Sengelov, G,Tjornelund, J. 2002. Toxicity of tetracyclines and tetracycline degradation products to environmentally relevant bacteria, including selected tetracycline-resistant bacteria. Arch Environ Contam Toxicol 42: 263-271. 22. Christensen, AM, Ingerslev, F,Baun, A. 2006. Ecotoxicity of mixtures of antibiotics used in aquacultures. Environ Toxicol Chem 25: 2208-2215. 23. Isidori, M, Lavorgna, M, et al. 2005. Toxic and genotoxic evaluation of six antibiotics on non- target organisms. Sci Total Environ 346: 87-98. 24. Backhaus, T, Froehner, K, et al. 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. 25. Pro, J, Ortiz, JA, et al. 2003. Effect assessment of antimicrobial pharmaceuticals on the aquatic plant Lemna minor. Bull Environ Contam Toxicol 70: 290-295. 26. Robinson, AA, Belden, JB,Lydy, MJ. 2005. Toxicity of fluoroquinolone antibiotics to aquatic organisms. Environ Toxicol Chem 24: 423-430. 27. Itoh, T, Mitsumori, K, et al. 2006. Genotoxic potential of quinolone antimicrobials in the in vitro comet assay and micronucleus test. Mutat Res, Genet Toxicol Environ Mutagen 603: 135- 144. 28. Hartmann, A, Alder, AC, et al. 1998. Identification of fluoroquinolone antibiotics as the main source of umuC genotoxicity in native hospital wastewater. Environ Toxicol Chem 17: 377- 382. 29. Lancieri, M, Cozzolino, S, et al. 2002. Toxicity and teratogenic effects of flumequine on Danio rerio embryos. Fresenius Environ Bull 11: 642-646. 30. Kolpin, DW, Furlong, ET, et al. 2002. Pharmaceuticals, hormones, and other organic wastewater contaminants in US streams, 1999-2000: A national reconnaissance. Environ Sci Technol 36: 1202-1211. 31. Kay, P, Blackwell, PA,Boxall, ABA. 2005. Transport of veterinary antibiotics in overland flow following the application of slurry to arable land. Chemosphere 59: 951-959. 32. Nepejchalova, L, Svobodova, Z, et al. 2008. Oxytetracycline Assay in Pond Sediment. Acta Vet Brno 77: 461-466. 33. Tamtam, F, Mercier, F, et al. 2008. Occurrence and fate of antibiotics in the Seine River in various hydrological conditions. Sci Total Environ 393: 84-95. 34. Park, S,Choi, K. 2008. Hazard assessment of commonly used agricultural antibiotics on aquatic ecosystems. Ecotoxicology 17: 526-538. 35. Eguchi, K, Nagase, H, et al. 2004. Evaluation of antimicrobial agents for veterinary use in the ecotoxicity test using microalgae. Chemosphere 57: 1733-1738. 36. Migliore, L, Civitareale, C, et al. 1997. Toxicity of several important agricultural antibiotics to Artemia. Water Res 31: 1801-1806.

TABLES AND FIGURES

Table 1. Summary of the effects of tested veterinary antimicrobials oxytetracycline hydrochloride and flumequine in ecotoxicological biotests. All toxicity values are in mg/L (values in parentheses show 95% confidence intervals); EC50 - concentration causing 50% effect; LOEC - lowest observable effect concentration; NOEC – the highest concentration causing no observable effect; MGC – minimal genotoxic concentration. Oxytetracycline Flumequine

LOEC NOEC LOEC NOEC ECOTOXICITY EC50 [mg/L] [mg/L] [mg/L] EC50 [mg/L] [mg/L] [mg/L]

Pseudomonas putida 0.22 (0.14 - 0.25) 0.04 < 0.04 0.82 (0.81 - 1.1) 0.2 < 0.2

Vibrio fischeri 21.0 (13 - 35) 5.0 2.5 11 (4.4 - 28) 0.63 0.31

Pseudokirchneriella subcapitata 3.1 (1.5 - 6.3) 1.6 0.78 2.6 (0.45 - 15.0) 1.6 < 1.6

Lemna minor 2.1 (1.7 - 3.0) 1.0 < 1.0 3.0 (0.5 - 7.0) 1.0 < 1.0

Daphnia magna acute test - - 400 59 (16 - 227) 25 12.5

Daphnia magna reproduction test 86 (48 - 155) 20.0 < 20.0 1.2 (0.44 - 3.1) 0.75 < 0.75

GENOTOXICITY MGC [mg/L] MGC [mg/L] SOS-chromotest 500 0.25

RISK EVALUATION PNEC [μg/L]* 0.04 0.2 MEC [μg/L] ** 0.34 0.032 MEC / PNEC 8.5 0.16 * PNEC (predicted no effect concentration) was calculated by applying factor of 1000 to the lowest LOEC value ** MEC (measured environmental concentration) is the highest value reported in the literature (for oxytetracycline [30], for flumequine [33])

Table 2. Aquatic ecotoxicity data of tested antimicrobials reported in literature. All toxicity values in mg/L.

Bioassay Endpoint Toxicity Reference Oxytetracycline

Activated sludge bacteria growth inhibition 48-EC50 0.08 (0.06 – 0.1) mg/L [21] Pseudomonas sp. 24-h MIC 1.0 ± 0.0 mg/L [21]

V. fisheri luminescence inhibition 30-min EC50 64.50 (47.2–88.3) mg/L [23] 120 (112 - 130) mg/L [6]

15-min IC50 87.0 (50.8–148.9) mg/L [34]

Microcystis aeruginosa growth inhibition 7-d EC50 0.21 (0.18 – 0.25) mg/L [5]

Chlorella vulgaris growth inhibition 48-EC50 6.4 (4.9 – 8.4) mg/L [25]

P. subcapitata growth inhibition 72-h EC50 4.5 (2.3 – 86) mg/L [5] 0.342 (0.321 - 0.364) mg/L [35] 0.17 (0.11– 0.25) mg/L [23]

Rhodomonas salina growth inhibition 72-h EC50 1.6 (0.4 – 6.1) mg/L [5]

Lemna minor growth inhibition 168-h EC50 4.92 (3.6 – 6.8) mg/L [25]

D. magna immobilization 48-EC50 22.64 (17.19–29.81) mg/L [23] 621.2 (437.71–804.8) mg/L [34] 48-h LOEC 100 mg/L [2]

C. dubia immobilization 48-EC50 18.65 (15.96–21.79) mg/L [23]

D. magna reproduction 21-d EC50 46.2 mg/L [2] Brachionus calyciflorus population growth inhibition 48-EC50 1.87 (1.19–2.96) mg/L [23]

C. dubia population growth inhibition 7-d EC50 0.18 (0.11–0.26) mg/L [23] Flumequine

V. fisheri luminescence inhibition 30-min EC50 12.7 (12.0 - 13.5) [6]

Microcystis aeruginosa growth inhibition 7-d EC50 0.16 (0.066 – 0.38) mg/L [5]

5-d EC50 1.96 (1.76 - 2.16) mg/L [26]

P. subcapitata growth inhibition 72-h EC50 5.0 (1.6 – 16) mg/L [5] 5.0 (4.8 - 5.2) mg/L [26]

Rhodomonas salina growth inhibition 72-h EC50 18 (10- 31) mg/L [5]

L. minor growth inhibition 7-d EC50 2.47 (1.65 - 3.3) mg/L [26]

Atemia salina mortality (ArToxKit) 72-h EC50 96.0 (39 – 240) mg/L [36]

P. putida growth inhibition V. fisheri luminescence inhibition

100 OTC 100 OTC FLU FLU 80 75

growth inhibition[%] 25 20 luminescence inhibition [%]

0 0 0.1 1 0.1 1 10 100 concentration [mg/L] concentration [mg/L]

P. subcapitata growth inhibition L. minor growth inhibition 100 100 OTC OTC

FLU FLU 75 75

50 50

growth inhibition [%] 25 growth inhibition [%] 25

0 0 1 10 1 10 concentration [mg/L] concentration [mg/L] Fig. 1. Ecotoxicity (concentration-response curves) of the studied antimicrobial drugs. A – Pseudomonas putida growth inhibition test. B – Inhibition of luminescence of Vibrio fischeri. C - Growth inhibition test with Pseudokirchneriella subcapitata. D – Growth inhibition test with Lemna minor. OTC: oxytetracycline hydrochloride, FLU - flumequine

D. magna immobilization

100 OTC FLU 75

immobilization [%] 25

0 1 10 100 1000 concentration [mg/L]

D. magna reproduction

OTC

100 FLU

inhibition [%] 40

0 1 10 100 concentration [mg/L]

Fig. 2. Comparison of toxicity of the studied antimicrobial drugs in the acute and reproduction test with Daphnia magna. A – Acute immobilization test with D. magna. B – Reproduction test with D. magna. OTC: oxytetracycline hydrochloride, FLU – flumequine

OTC 3.5

3.0 2.5

2.0

1.5 1.0 factor Induction 0.5 0.0 0.05 0.50 5.00 50.00 500.00 concentration [mg/L]

FLU 6

factor Induction 1

0 0.0625 0.1250 0.2500 0.5000 1.0000

concentration [mg/L]

Fig. 3. Comparison of genotoxicity of the studied antimicrobial drugs in the SOS-chromotest. OTC: oxytetracycline hydrochloride, FLU: flumequine

Curriculum vitae Radka Zounková

Curriculum vitae

Mgr. Radka Zounková

Personal data

Date of birth 23th of November 1976 Place of birth Brno, Czech Republic Citizenship Czech Republic Permanent adress Riegrova 8, 612 00, Brno, Czech Republic E-mail [email protected]

Education

2003 – present Ph.D. candidate in Environmental Chemistry RECETOX – Research Centre for Environmental Chemistry and Ecotoxicology, Faculty of Science, Masaryk University, Brno, Czech Republic (www.recetox.muni.cz)

2001 – 2003 Ecotoxicology – Master’s studies (without degree) RECETOX – Research Centre for Environmental Chemistry and Ecotoxicology, Faculty of Science, Masaryk University, Brno, Czech Republic

1995 – 2000 M.Sc. in Pharmacy University of Veterinary and Pharmaceutical Sciences Brno, Faculty of Pharmacy Master thesis in Pharmaceutical Chemistry: Synthesis of new potential antioxidants

Professional

2009 – present pharmacist – assistant Pharmacy at the Hospital, Vyškov, Czech Republic

2008 – 2009 research and pedagogic assistant, part-time RECETOX – Research Centre for Environmental Chemistry and Ecotoxicology, Faculty of Science, Masaryk University, Brno, Czech Republic

2007 – 2008 research scientist, full-time – research fellowship within the scope of the Marie-Curie Early Stage Research Training programme, project AQUAbase (www.aquabase.rwth-aachen.de) Institute of Hygiene and Environmental Health, RWTH Aachen, Germany

2006 – 2007 research assistant, part-time RECETOX – Research Centre for Environmental Chemistry and Ecotoxicology, Faculty of Science, Masaryk University, Brno, Czech Republic

2001 – 2002 research and pedagogic assistant, part-time Department of chemical drugs, University of Veterinary and Pharmaceutical Sciences Brno, Faculty of Pharmacy

Research interests ecotoxicological biotests, environmental toxicology, risk assessment, effects of drugs in the environment, speciﬁc effects of organic pollutants

Languages Czech (native), English (ﬂuent), German (passive)

Research projects, fellowships, trainings

2007 – 2008 AQUAbase – Organic Micropollutants in Aquatic Environment – Interdisciplinary Concepts for Assessment and Removal; Marie-Curie Early Stage Research Training programme (www.aquabase.rwth-aachen.de) AQB-SP1: Ecotoxicological biotests for pharmaceuticals – principal investigator 2006 – 2007 EU Waste Ringtest 2006/2007 – Evaluation of a biotest battery for the ecotoxicological characterisation of waste and waste eluates by an interlaboratory test – participation

2005 – 2007 Project EC "ECODIS" (6th FWP contract No. 518043-1); Dynamic Sensing of Chemical Pollution Disasters and Predictive Modelling of their Spread and Ecological Impact – participation

2005 Cytostatics and their effects in the environment – ecotoxicologial study; Project of Ministry of Education, Youth and Sports of the Czech Republic FRVS G4 2546/2005 – principal investigator

2005 Summer school – Pharmaceuticals and hormones in the environment; RECETO – Research School of Environmental Chemistry and Ecotoxicology – participation

Publications

• Zounková, R., Odráška, P., Doležalová, L., Hilscherová, K., Maršálek, B., and Bláha, L. (2007): Ecotoxicity and Genotoxicity Assessment of Cytostatic Pharmaceuticals. Environmental Toxicology and Chem- istry 26(10): 2208-2214

• Zounková, R., Kovalová, L., Bláha, L., Dott, W.: Ecotoxicity and Geno- toxicity Assessment of Cytotoxic Antineoplastic Drugs and their Meta- bolites. Chemosphere (accepted with moderate revision)

• Zounková, R., Klimešová, Z., Nepejchalová, L., Hilscherová, K., Bláha, L.: Complex Evaluation of Ecotoxicity and Genotoxicity of Antimicro- bials Oxytetracycline and Flumequine used in Aquaculture (submitted to Environmental Toxicology and Chemistry) Contribution at international conferences

Oral presentations

• Zounková, R., Odráška, P., Hilscherová, K., Bláha, L. (2006): Cyto- statics and their effects in the environment. Students science conference, Bratislava, Slovak republic, April 26, 2006, Book of Reviewed Abstracts: 257, ISBN: 80-88870-58-5 (Czech language)

• Zounková, R., Odráška, P., Hilshceotvá, K., Bláha, L. (2006): Ecotox- icological investigation of cytostatic pharmaceuticals contaminating surface waters. AQUAbase Workshop - Risk Assessment of Organic Micropollutants in the Aquatic Environment, Aachen, Germany, No- vember 28-29, 2006

Poster presentations

• Zounková, R., Maršálek, B., Hilscherová, K., Bláha, L. (2004): Pharma- ceuticals as potential toxic compounds in the environment. First Bien- nial Central & Eastern European Environmental Health Conference, International Health Sciences Solving Common Problems, Prague, Oc- tober 24-27, 2004, Book of Abstracts: 37

• Zounková, R., Hilscherová, K., Odráška, P., Bartoš, T., Bláha, L., Maršá- lek, B., Holoubek, I. (2005): Ecotoxicity and genotoxicity assessment of cytostatic pharmaceuticals. SETAC Europe 15th Annual Meeting, Lille, France, May 22-26, 2005, Book of Abstracts: 126-127

• Zounková, R., Odráška, P., Hilscherová, K., Bláha, L. (2005): The use of classical ecotoxicological biotests for testing of special (hospital) waste waters. ECOTOX 2005 – Advances and Trends in Ecotoxicol- ogy, Brno, Czech Republic, September 5-7, 2005, Book of Abstracts: 242, ISBN 80-210-3799-7

• Zounková, R., Odráška, P., Hilscherová, K., Bláha, L. (2006): Evalua- tion of ecotoxicity and genotoxicity of special (hospital) waste waters. SETAC Europe – 16th Annual Meeting, The Hague, The Netherlands, May 7-11, 2006

• Zounková, R., Bláha, L., Dott, W. (2008): Ecotoxicity and genotoxicity assessment of cytostatics and their metabolites. SETAC Europe – 18th Annual Meeting, Warsaw, Poland, May 25-29, 2008, Book of Abstracts: 83 Contribution at national conferences and seminars

• Zounková, R. (2005): Ecotoxicological bioassays and effects studies of pharmaceuticals in the environment; lecture in "Modern ecotoxicology: ecological and health consequences of chronic effects of chemical compounds in the environment" - seminar of Czechoslovak Biological Society, Brno, April 13, 2005 (Czech language)