J. Plant Nutr. Soil Sci.

2003, 166, 145167

145

Pharmaceutical antibiotic compounds in soils a review

Sren Thiele-Bruhn Institute of Soil Science and Plant Nutrition, University of Rostock, D-18051 Rostock, Germany Accepted 11 February 2003

Antibiotics are highly effective, bioactive substances. As a result of their consumption, excretion, and persistence, they are disseminated mostly via excrements and enter the soils and other environmental compartments. Resulting residual concentrations in soils range from a few lg up to g kg1 and correspond to those found for pesticides. Numerous antibiotic molecules comprise of a non-polar core combined with polar functional moieties. Many antibiotics are amphiphilic or amphoteric and ionize. However, physicochemical properties vary widely among compounds from the various structural classes. Existing analytical methods for environmental samples often combine an extraction with acidic buffered solvents and the use of LC-MS for determination. In soils, adsorption of antibiotics to the organic and mineral exchange sites is mostly due to charge transfer and ion interactions and not to hydrophobic partitioning. Sorption is strongly influenced by the pH of the medium and governs the mobility and transport of the antibiotics. In particular for the strongly adsorbed antibiotics, fast leaching through soils by macropore or preferential transport facilitated by dissolved soil colloids seems to be the major transport process. Antibiotics of numerous classes are photodegraded. However, on soil surfaces this process if of minor influence. Compared to this, biotransformation yields a more effective degradation and inactivation of antibiotics. However, some metabolites still comprise of an antibiotic potency. Degradation of antibiotics is hampered by fixation to the soil matrix; persisting antibiotics were already determined in soils. Effects on soil organisms are very diverse, although all antibiotics are highly bioactive. The absence of effects might in parts be due to a lack of suitable test methods. However, dose and persistence time related effects especially on soil microorganisms are often observed that might cause shifts of the microbial community. Significant effects on soil fauna were only determined for anthelmintics. Due to the antibiotic effect, resistance in soil microorganisms can be provoked by antibiotics. Additionally, the administration of antibiotics mostly causes the formation of resistant microorganisms within the treated body. Hence, resistant microorganisms reach directly the soils with contaminated excrements. When pathogens are resistant or acquire resistance from commensal microorganisms via gene transfer, humans and animals are endangered to suffer from infections that cannot be treated with pharmacotherapy. The uptake into plants even of mobile antibiotics is small. However, effects on plant growth were determined for some species and antibiotics.

Summary Zusammenfassung

Antibiotika sind hochgradig wirksame, bioaktive Substanzen. Infolge ihrer Anwendung, Ausscheidung und Persistenz werden sie meist ber die Exkremente in Bden und andere Umweltkompartimente eingetragen. Die resultierenden Rckstandskonzentrationen in Bden im Bereich von wenigen lg bis zu g kg1 entsprechen in etwa denen von Pflanzenschutzmitteln. Die Moleklstruktur von Antibiotika besteht hufig aus einem unpolaren Kern und polaren Randgruppen. Viele Antibiotika sind amphiphil oder amphoter und bilden Ionen, jedoch weisen die zahlreichen Antibiotika unterschiedlicher Strukturklassen stark divergierende physikochemische Eigenschaften auf. In den vorliegenden Nachweismethoden fr Umweltproben werden hufig sauer gepufferte Lsungsmittel zur Extraktion und eine Bestimmung mittels LC-MS kombiniert. Die Adsorption der Antibiotika an den organischen als auch an den mineralischen Bodenaustauschern erfolgt zumeist durch Ladungs- und Ionenwechselwirkungen und weniger durch hydrophobe Bindungen. Das Verteilungsverhalten hngt dabei entscheidend vom pH-Wert des Mediums ab und beeinflusst die Mobilitt und Verlagerung der Antibiotika. Bei vielen, insbesondere stark adsorbierten Antibiotika sind v. a. schnelle Flievorgnge wie durch prferenziellen und Makroporenfluss sowie der Cotransport mit gelsten Bodenkolloiden von besonderer Bedeutung. Antibiotika vieler Strukturklassen knnen durch Licht abgebaut werden. Dieser Abbaupfad spielt auf Bodenoberflchen jedoch nur eine untergeordnete Rolle. Hingegen kommt es insbesondere durch biologische Transformationsprozesse zu einer intensiven Degradation und Inaktivierung der Antibiotika. Verschiedene Metaboliten weisen jedoch ebenfalls ein antibiotisches Potential auf. Der Abbau der Antibiotika wird durch die Festlegung in Bden gehemmt; dementsprechend wurde eine Persistenz verschiedener Antibiotika nachgewiesen. Trotz der starken bioaktiven Wirkung aller Antibiotika sind die festgestellten Effekte auf Bodenorganismen sehr unterschiedlich. Dies liegt nicht zuletzt an einem Mangel an geeigneten Testmethoden. In der Regel sind jedoch von Dosis und Wirkungsdauer abhngige Effekte insbesondere auf Mikroorganismen festzustellen, die zu Vernderungen der Mikroorganismenpopulation fhren knnen. Lediglich durch Anthelmintika wurden deutliche Wirkungen auf Vertreter der Bodenfauna hervorgerufen. Infolge der antibiotischen Wirkung der Pharmazeutika kann eine Resistenzbildung bei Bodenorganismen ausgelst werden. Zudem hat die Medikation von Antibiotika die Bildung resistenter Mikroorganismen bereits im behandelten Organismus zur Folge. DurchderenanschlieendeAusscheidunggelangenresistenteKeimeauchdirektindieBden.HandeltessichumresistentePathogeneoderkommtes zur bertragung der Resistenzgene zwischen kommensalen und pathogenen Mikroorganismen, so besteht das erhebliche Risiko einer nicht therapierbaren Infektion von Mensch und Tier. Die Aufnahme selbst mobiler Antibiotika in die Pflanzen ist sehr gering. Dennoch wurden bei einigen Pflanzenarten Wirkungen von Antibiotika auf das Wachstum nachgewiesen. PNSS P02/01P

Pharmazeutische Antibiotika in Bden ein berblick

1 Introduction
Antibiosis is a natural chemical regulation mechanism among organisms, especially microorganisms. Consequently, biosynthesis of antibiotics also occurs in soils (Gottlieb, 1976). Today, a wide range of naturally occurring and of synthetic
Correspondence: Dr. S. Thiele-Bruhn; E-mail: soeren.thiele@auf.unirostock.de

antibiotics is frequently used for the therapy of infectious diseases in human and veterinary medicine (Grfe, 1992). For this purpose, antibiotics are designed to act very effectively even at low doses and, in case of intra-corporal administration, to be completely excreted from the body after a short time of residence. Consequently, these substances are released to the environment. Therefore, residual concentrations of pharmaceutical antibiotics are found in 1436-8730/03/0204-145 $17.50+.50/0

2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

146

Thiele-Bruhn

J. Plant Nutr. Soil Sci. 2003, 166, 145167 lg l1 for groundwater. When PEC exceed these trigger values or antibiotics are directly applied to surface water for the treatment of fish, experimental testing in the tiered second phase becomes necessary. Legislation and methods for an environmental risk assessment of pharmaceuticals are dealt with in detail in the book edited by Kmmerer (2001b). There, alternative concepts to the model by Spaepen et al. (1997) are also presented. Therefore, this topic is not addressed in this review. The occurrence and effects of pharmaceuticals in different environmental compartments, especially water, were reviewed by Halling-Srensen et al. (1998), Daughton and Ternes (1999), and Kmmerer (2000). Another review deals with the adsorption of veterinary pharmaceuticals in soils (Tolls, 2001), while Richardson and Bowron (1985) and Hirsch et al. (1999) combined overviews on antibiotics in water with results from their own investigations. However, research and publications on these topics have increased remarkably in the last few years, justifying a review focusing on the input and fate of pharmaceutical antibiotics in soils.

the environment. Contamination of surface, ground and drinking water, of aquatic sediments and soils with pharmaceuticals have been reported (Richardson and Bowron, 1985; Heberer and Stan, 1998; Hirsch et al., 1999; Kmmerer, 2001a; Hamscher et al., 2002a). Soil residues result mostly from the use of contaminated excrements as fertilizer on agricultural land. It has been estimated that loads of up to kilograms per hectare may enter agricultural soils and that a concentration level of antibiotics similar to pesticides is easily reached (van Gool, 1993; Winckler and Grafe, 2000). Due to surface runoff and leaching, soils can even act as a source of antibiotic contaminants for the aqueous environment (Alder et al., 2001). Residues of pharmaceutical antibiotics can provoke resistance in pathogens either directly or indirectly by transfer of plasmids from non-pathogens to pathogenic microorganisms (Wegener et al., 1998). The resulting antibiotic residues and resistant microorganisms can affect the natural soil microbial community and soil functions and may even harm animals and humans via the food chain (Richter et al., 1996; Kennedy et al., 2000). In addition, infections by resistant pathogens lower the efficiency of pharmacotherapies for humans and animals (Richter et al., 1996). However, no regulations exist for concentration limits of antibiotics in soils or soil water. Following the lead of the USA, an environmental risk assessment of veterinary pharmaceuticals was prescribed in the EU in 1998 with the EU directives 81/852/EEC and 92/18/EEC (EMEA, 1997). Thereby, predicted environmental concentrations (PEC) are calculated with the help of a balancing model (Spaepen et al., 1997). The PEC are compared with predicted, biologically noneffective concentrations (PNEC). First, the exposition is evaluated and PEC are compared with trigger values. These trigger values have been set at 10 lg kg1 for the faeces of grazing livestock, at 100 lg kg1 for dung and soils and at 0.1

2 Consumption and physicochemical properties of antibiotics

Antibiotics are defined as chemical compounds that are synthesized through the secondary metabolism of living organisms, with exceptions for semi- or completely synthetic substances. Antibiotics inhibit the activity of microorganisms, viruses, and eucaryotic cells, respectively (Lancini and Parenti, 1982). In human medicine, antibiotics pose the third biggest group among all pharmaceuticals making up more than 6 % of all prescriptions (Schwabe and Paffrath, 2001). In veterinary medicine, more than 70 % of all consumed pharmaceuticals are antibiotic agents (Halling-Srensen et al., 1998). In Europe, two thirds of all pharmaceutical

Table 1: Annual consumption of antibiotics for veterinary medicine, especially livestock, in the European Union, European countries and regions of Germany (without coccidiostatics). Tabelle 1: Jhrlicher Verbrauch an Antibiotika in der Veterinrmedizin, v.a. fr landwirtschaftliche Nutztiere, in der Europischen Union, europischen Staaten und Regionen in Deutschland (ohne Kokzidiostatika). EU 1999a t Therapeutics Tetracyclines Sulfonamidesk Aminoglycosides b-Lactams Macrolides others Ergotropics
a

France 1980b % t 625 117 19 139 22 57 9 6 50 37 n.a. 9 8 6 2 %

Sweden 1996c t 20 2.7 13 2.2 11 1.1 n.a. 1.5 n.a. n.a. 7 5 %

Denmark 1997d t 57 13 13 15 23 23 26 %

Switzerland UK 1997e 2000f t 14 1.0 n.a. n.a. 9.2 64 0.3 36

b

Weser-Ems Brandenburg Mecklenburg1997g 1998/99h Vorpommern 2001i t 88 40 14 3.8 0.2 4.5 18

c j

t 437

t 6.6
j

t 10
j

3902 2575 66 78 1564 351 234 786 468 12

228 52 94 22 12 3 9 3 49 11 41 12 24

57 21 5 0.3 6

4.6 0.9 0.2 0.2 0.01 0.7 0.1

69 14 3 3 0.1 10
d

6.4 41 2.5 16 0.2 0.1 0.1 0.8 n.a. 2 1 1 8

7.7 14 1.7 3 6.5 11 107

7.1 10

226 36

3.8 27

FEDESA (2001), data for percentages of compound classes from 1997; Espinasse (1993); Mudd et al. (1998); Halling-Srensen et al. (2002a); e Swiss Importers of Antibiotics (1998) cited in Alder et al. 2001; f NOAH (2002); g Winckler and Grafe (2000); h Linke and Kratz (2001); i Thiele-Bruhn et al. (2003a); j veterinary prescriptions for feed antibiotics only; k incl. trimethoprim; n.a. = no data available

J. Plant Nutr. Soil Sci. 2003, 166, 145167 antibiotics are used in human medicine and one third for veterinary purposes (FEDESA, 2001). Consequently, tons of antibiotic substances are consumed per year in industrialized countries (Tab. 1). Agricultural livestock, especially poultry and pigs, are treated with the majority of antibiotics while all other domestic animals receive only ca. 1 % of prescriptions within the EU (Ungemach, 2000). To date, the nontherapeutic use of antibiotics as growth promoters is almost completely restricted in the EU and consequently, the consumption of ergotropics is strongly declining. On the other hand, the annual application of therapeutic antibiotics in the EU increased from 1997 to 1999 by 12 %, i.e. to 3900 t (FEDESA, 2001). The antibiotic compound classes primarily administered in veterinary medicine are tetracyclines, sulfonamides, aminoglycosides, b-lactams, and macrolides (Tab. 1). In human medicine b-lactams, tetracyclines and macrolides are mostly prescribed (Schwabe and Paffrath, 2001). In Germany, 250 different antibiotic and antimycotic agents are currently approved for use (Kmmerer, 2001b), a number which might be representative of other countries as well. Antibiotics define a multitude of heterogeneous compounds that are classified with regard to different fields of usage (e.g. antimycotics, antiinfectives, anthelmintics), in different struc-

Antibiotic in soils

147

tural classes (e.g. nucleosides, tetracyclines) and exhibit different molecular structures and diverse chemical and physical properties (Grfe, 1992). Most antibiotics tend to ionize depending on the pH of the medium; pKa values are associated with the different functional groups of the compounds. Ranges of physicochemical properties of important antibiotic compound classes are listed in Tab. 2. In the following section, their physicochemical properties are briefly described with information taken from Booth and McDonald (1988), Grfe (1992), and Schadewinkel-Scherkl and Scherkl (1995), if not indicated otherwise.

Tetracyclines
Tetracyclines (TCs) are polyketides and comprise of a naphthacene ring structure. The TCs are amphoteric compounds as characterized by three pKa values. They are relatively stable in acids, but not in bases, and form salts in both media (Halling-Srensen et al., 2002b). The TCs form chelate complexes with divalent metal ions and b-diketones and strongly bind to proteins and silanolic groups (Oka et al., 2000). Most TCs are sparingly water soluble, while the solubility of the corresponding hydrochlorides is much higher.

Table 2: Representative pharmaceutical antibiotics and typical ranges of physicochemical properties from selected classes of antibiotics (Osol, 1980; Gruber et al., 1990; Asuquo and Piddock, 1993; Nowara et al., 1997; Escribano et al., 1997; National Institutes of Health, 1999; Rablle and Spliid, 2000; Syracuse Research Corporation, 2001). Tabelle 2: Beispiele pharmazeutischer Antibiotika und typische Bereiche physikochemischer Eigenschaften von ausgewhlten Strukturklassen der Antibiotika. Compound class Molar mass g mol1 Water solubility mg l1 log Kow 1.3 0.05

pKa
3.3 / 7.7 / 9.3

Henrys constant Pa l mol1 1.71023 4.81022 1.31012 1.8108 8.51012 4.1108 2.51019 1.21012 7.81036 2.01026 5.21017 3.2108 2.31013 2.71010 negligible 2.81023

Tetracyclines 444.5 527.6 230 52000 chlortetracycline, oxytetracycline, tetracycline

Sulfonamides 172.2 300.3 7.5 1500 0.1 1.7 2 3 / 4.5 10.6 sulfanilamide, sulfadiazine, sulfadimidine, sulfadimethoxine, sulfapyridine, sulfamethoxazole Aminoglycosides 332.4 615.6 kanamycin, neomycin, streptomycin 10 500
a

8.1 0.8

6.9 8.5 2.7 7.7 8.9 8.6 2.4

b-Lactams 334.4 470.3 22 10100 0.9 2.9 penicillins: ampicillin, meropenem, penicillin G; cephalosporins: ceftiofur, cefotiam Macrolides 687.9 916.1 erythromycin, oleandomycin, tylosin 0.45 15 1.6 3.1 1.0 1.6 0.02 3.9 1.0 3.2 5.4 8.5

Fluorquinolones 229.5 417.6 3.2 17790 ciprofloxacin, enrofloxacin, flumequin, sarafloxacin, oxolinic acid Imidazoles 171.5 315.3 fenbendazole, metronidazole, oxfendazole 6.3 407

Polypeptides 499.6 1038 not completely avermectin, bacitracin, ivermectin, virginiamycin Polyethers 670.9 751.0 monensin, salinomycin Glycopeptides vancomycin Quinoxalinederivatives olaquindox
a

2.2106 3.1103 > 1000 1.0106

6.4

2.11018 1.51018 negligible 1.11018

1450.7 263.3

not soluble in octanol 5.0 2.2 10

g l1

148

Thiele-Bruhn

J. Plant Nutr. Soil Sci. 2003, 166, 145167 extractants (SPE) pose serious analytical problems. Thus, for the extraction of most antibiotics, the use of weakly acidic buffers in combination with organic solvents is recommended (Tab. 3). In food analysis, a 0.1 M EDTA-McIlvaine buffer (pH 4.0) is often used (Weimann and Bojesen, 1999; JuhelGaugain et al., 2000). Cooper et al. (1998) and Khne et al. (2000) suggested citric ethylacetate (pH 5.0) for the extraction of TCs. This method was adopted for soil samples by Hamscher et al. (2002a). At least in the case of TCs, this extractant yields better recovery rates from soil samples, although the solubility of numerous antibiotics of different structural classes is considerably smaller in pure ethylacetate as compared to methanol or DMSO (Salvatore and Katz, 1993). Oka et al. (2000) reviewed techniques for the extraction and analysis of TCs and a compilation of methods can be found in Juhel-Gaugain et al. (2000). The use of 0.01 M CaCl2 to extract the mobile, non-adsorbed fraction of xenobiotics as prescribed by OECD (1997), cannot be recommended for TCs. These compounds form sparingly soluble complexes with Ca2+ (Wessels et al., 1998). As an alternative to CaCl2, 0.1 M NH4NO3 was successfully used (Thiele-Bruhn, unpublished data). In general, the sample clean-up is done by SPE or 0.45 lm filtration; extracts from centrifugation and liquid/liquid separation are usually concentrated by evaporation (Tab. 3). For SPE of SAs, reversed phases are very effective. In contrast, the usage of reversed phase materials for SPE of TCs requires pre-treatment of the solid phase with EDTA or silylating agents whereas for conditioning and elution, acidic buffered solvents are recommended (Oka et al., 1991; Zhu et al., 2001; Loke et al., 2002). The TCs bind so strongly to free silanol groups that they cannot be eluted by the usual organic solvents. Lindsey et al. (2001) suggested a macroporous copolymer combined with Na2EDTA as the chelating agent. To overcome the problems resulting from the amphoteric character of many antibiotics, new functionalized SPE materials that separate analytes by their hydrophobic as well as polar properties, e.g. hydrophilic-lipophilic balance cartridges (HLB) and mixed-mode HLB-cation exchange cartridges (MCX), appear to be advantageous (e.g. Kolpin et al., 2002). Golet et al. (2001) extracted FQs from wastewater with a mixed-mode silica based sorbent consisting of a non-polar phase and a strong cation exchanger interacting with the aromatic moiety of the core and the charged amino groups of the substituents. Antibiotics are usually separated by chromatographic techniques and subsequently detected. To avoid dissociation of the compounds or binding to free silanol groups from the widely used silica based chromatographic columns, dilute acids or weakly acidic buffers, e.g. phosphoric, citric, formic, and oxalic acid or EDTA, are commonly used (Oka et al., 2000). Alternatively ion pair-chromatography is applied. Currently, the time and solvent consuming thin layer chromatography has been mostly replaced by high performance liquid chromatography combined with UV and DiodeArray Detection (HPLC-UV, -DAD). Increasingly, liquid chromatography with mass spectrometry (LC-MS) or tandem mass spectrometry (LC-MS/MS) (Thomashow et al., 1997; Oka et al., 2000; Hamscher et al., 2002a) with chemical

The TCs strongly absorb light and thus, are susceptible to photodegradation (Mitscher, 1978).

Sulfonamides
Sulfonamides (SAs) are relatively insoluble in water. They are characterized by two pKa values indicating protonation of the amino group at a pH of 2 to 3 and deprotonation of the R1SO2NHR2 moiety at a pH of 5 to 11 (Ingerslev and HallingSrensen, 2000). In general, the amphoteric SAs behave as weak acids and form salts in strongly acidic or basic solutions. Mostly, SAs, substituted at the amino-N, have greatly reduced antibacterial activity.

Aminoglycosides
Antibiotics from the class of aminoglycosides are basic, strongly polar polycationic compounds. Their molecular structure is characterized by two or more amino sugars that are glycosidically bound to aminocyclitol. They are water soluble, mostly hydrophilic, and susceptible to photodegradation.

b-Lactams
Penicillins and cephalosporins are the two major sub-classes of the b-lactams. The antibiotic effect of penicillins is directly connected to the b-lactam ring. This ring is easily cleaved in acidic and basic media. Cephalosporins are derivatives of 7amino-cephalosporanic acid, condensed with a six-membered heterocycle in contrast to the five-membered heterocycle of penicillins.

Macrolides
Macrolides are defined as lactone structures with cycles of more than 10 C-atoms. Many macrolides are weak bases and are unstable in acids. Their water solubility varies considerably between the different derivatives.

Fluorquinolones
Most fluorquinolones (FQs), also known as quinolones, exhibit large chemical stability. They are insensitive to hydrolysis and increased temperatures, but are degraded by UV light. Their antibiotic potency depends mostly on the aromatic fluorine substituent at the C-6 position (Wetzstein, 2001).

3 Extraction and determination

Numerous antibiotics are comprised of a non-polar core and polar functional groups (Juhel-Gaugain et al., 2000). They are sensitive to bases and strong acids and dissociate or protonate depending on the pH of the medium. Thereby, their distribution behavior changes considerably (Holten Ltzhft et al., 2000). Consequently, incomplete extraction of antibiotics with very polar and non-polar extractants and strong adsorption to polar and non-polar solid phase

Table 3: Selected examples for extraction, separation and detection of antibiotic pharmaceuticals in environmental and food samples. Tabelle 3: Ausgewhlte Beispiele zur Extraktion, Trennung und Bestimmung antibiotischer Pharmazeutika in Umweltproben und Lebensmitteln. Clean-up Reference Hirsch et al., 1998 Campagnolo et al., 2002 Kolpin et al., 2002 C18 1253; 5 lm TCs: C8 1253; 5 lm C8 1502; 3 lm NH4CHO2:CH2O2 buffer pH 3.7/ACN, gradient bioautography ACN:0,01 M H3PO4 (30:70), 1 ml min1 0.05 N H3PO4/ACN (22:78) gradient 2 % acetic acid:ACN (85:15); 0.7 ml min1 UV 290 nm; 5 ng ml1 FLD ex 280 em 444 nm; 0.32 0.87 ng ml1 FLD ex 278 em 445 nm UV 203 nm 50C, 200 ll 25 C, 20 ll UV 290 nm; 5 ng ml1 MS ESI+; < 0.01 lg l1 bromocresol green as indicator dye ion pair chromat., 50 C 10 mM NH4OAc in MS/MS ESI+d; H2O/ACN; TCs: 20 mM < 0.5 lg l1 oxalic acid in H2O/ACN chloramphenicol: ESI-e Column type Eluent Detection & -limits further detailsa

Class/ compound

Sample

Extractant

multiple classesb

manure, water

SPEc: Lichrolute EN + C18, TCs: lyophylisation

multiple classesf var. solvent washings liquid/liquid Hichrom Nucleosil C18 2504.6; 5120 lm Varian MicroPac MCH 10, C18 Inertsil Phenyl agarose gel electro-phoresis at pH 6.0 and 8.0

water

SPE: HLB+MCX with Na2EDTA + C10H14O8-Na2N-H2O

Aminoglycosides

animal feed

MeOHg:Tris-succinate buffer pH 8 (25:75)

Salvatore and Katz, 1993 Long et al., 1989

J. Plant Nutr. Soil Sci. 2003, 166, 145167

Benzimidazoles filtration

milk

ACNh + MeCl2i

plasma, faeces, urine

H2O + conc. NaOH (8:1)

Barker et al., 1986 Holtzapple et al., 1999

Fluorquinolones

animal tissues

on-line immunoaffinity capture column with antibodies

wastewater Alltech RSil C18-HL, 1003.2; 5 lm centrifugation, 0.45 lm filtration SPE: C18 centrifugation, 0.45 lm filtration YMC-Pack ODS-AQ 2504.6; 5 lm Phenomenex ODS Prodigy 2504.6; 5 lm Spherisorb ODS2 C18 1254.6; 5 lm

mixed-phase cation-exchange disks

Discovery C16 2503

25 mM H3PO4/ACN, 0.7 ml min1, gradient ACN:H2O:diethylamine (500:500:0,1), 1 ml min1 2.25 % Na perchlorate (pH 2.5 with HCl):ACN (60:40), 1.0 ml min1 isocratic ACN/1mM EDTA/16.7 mM acetic acid + 4M NaOH (pH 5), 1 ml min1, gradient

Golet et al., 2001 Bens et al., 1982

Macrolides (Tylosin)

ACN:H2O (1:1)

manure

MeOH

UV 290 nm; 5 ng ml1 UV UV 260 nm

35 C, 100 ll

Loke et al., 2000

manure, soil

phosphate buffer + methanol (1:2)

De Liguoro et al., 2002 Ingerslev and Halling-Srensen, 2001

Metronidazole, Olaquindox evaporation, 0.45 lm filtration EtOAck for lipids removal

soil-manure slurry

H2 O

b-Lactams

milk

SPE Carbograph 4

C18 2504.6; 5 lm C2 1505; 10 lm

MeOH/H2O + 10 mM formic acid, 1 ml min1

MS/MS ESI+/ 0.4 3 lg l1 ion pair chromatography DAD phosphate buffer with ion pairing reagent/ACN

50 ll derivatisation: 1,2,4-triazole + HgCl2

Bruno et al., 2001 Marchetti et al., 2001

Antibiotic in soils

(Penicillins) milk

phosphate buffer pH 8, C18 SPE

149

150

Table 3: Continued. Tabelle 3: Fortsetzung. Clean-up Hypersil ODS 2504; 5 lm MeOH:phosphate buffer (pH 4)(9:1), 0.7 ml min1 UV 320 nm; 10 lg kg1 Column type Eluent Detection & -limits further detailsa Reference

Class/ compound

Sample

Extractant

Thiele-Bruhn

Polyethers (Salinomycin, Monensin, Lasalocid) SPE: Silica FLD ex 360 em 420 nm UV 270 nm UV 280 nm 40 C, 50 ll Develosil 5 C18 2504.6; 100 lm C18, 1004.6; 3 lm Phenomenex ODS2 C18 1254.6; 5lm ACN:16.7 mM acetic acid/ (pH 5 with 4M NaOH) (17.5:82.5 ), 1 ml min1 UV 265 nm 0.05 M H3PO4/ACN, gradient MeOH:H2O 97:3, isocratic

animal feed

MeOH/phosphate buffer liquid/liquid MeCl2 (pH 4)(9:1)

100 ll, post column Johannsen, 1991 derivat.: DMA/H2SO4

animal feed

ACN

pre column derivat.: Asukabe et al., 1-(bromoacetyl) 1994 pyrene Samuelsen et al., 1994 Ingerslev and Halling-Srensen, 2000 22 C, 10 ll Thiele, 2000

Sulfonamides centrifugation, 0.45 lm filtration

marine sediment

0.1 N NaOH

centrifugation

sewage sludge

H2O

soil

MeOH

SPE: C18

Nucleosil C18 2504.6; 0.01 M H3PO4 / MeOH, 1 ml min1, 1005 lm gradient Nucleosil C18 1253; 1005 lm Luna C8 1004.6; 3 lm C8 or C18, 1005 lm Waters XTerra C18 1503; 3.5 lm

+Trimethoprim

animal manure

pH 9 with KOH, EtOAc separation of EtOAc, 0.45 lm filtration

H2O:1mM NH4OAc+10 %UV; ACN/ACN, 0.25 ml MS/MS ESI+ min1, gradient NH4formiate/formic acid buffer; 0.6 ml min1, gradient oxalic acid/ACN MeOH/formic acid/ H2O, 0.4 ml min1, gradient MS ESI+; < 1 lg l1 UV 350 375 nm MS/MS ESI+

25 C, 50 ll

Haller et al., 2002

Sulfonam. & Tetracyclines SPE: C18 0.45 lm filtration

water

SPE: macroporous polymer (HLB) + Na2EDTA

Lindsey et al., 2001 Juhel-Gauguin, 2000 22 C Loke et al., 2003

Tetracyclines

animal tissues

McIlvaine EDTA buffer pH4 + MeOH

manure

MeOH

soil

1M citrate buffer (pH 4.7) + EtOAc

evaporation

Puresil C18 1504.6; 5 1mM NH4OAc (pH 2.5)+ 0.5 % formic lm acid/ACN, 1 ml min1

MS/MS ESI+

23 C, 18 ll

Hamscher et al., 2002

J. Plant Nutr. Soil Sci. 2003, 166, 145167

column temperature (C), injection volume (ll); bpenicillins, macrolides, SAs, TCs; csolid phase extraction; delectrospray ionisation positive ion mode; eelectrospray ionisation negative ion mode; FQs, lincosamide, macrolides, polypeptides, SAs, TCs; gmethanol; hacetonitrile; imethylenechloride; jethylacetate

J. Plant Nutr. Soil Sci. 2003, 166, 145167 ionization at atmospheric pressure (APCI) or electrospray ionization (ESI) is used (Oka et al., 1997; Carson et al., 1998). Niessen (1998) reviewed the analysis of antibiotics by LC-MS and stated that this detection method is most sensitive for a multitude of compounds. Numerous applications already exist for LC-MS. Kamel et al. (1999) detected the highest sensitivity for TCs with LC-ESI-MS/MS in the positive ion mode and by addition of 1 % acetic acid in the mobile phase. In comparison, Lindsey et al. (2001) used ammonia formiate/formic acid in combination with a water/ methanol gradient. However, for FQs, fluorescence detection after derivatization and liquid chromatography was superior to LC-MS/MS (Golet et al., 2001). To date, the traditional, semi-quantitative detection of antibiotics by microbial inhibition tests such as agardiffusion and bioautography (Katz and Katz, 1983; Smuth et al., 1987) is carried out only to supplement chemical analysis. However, for a complete assessment of antibiotics, not only their quantity, but also their antibiotic potential must be determined. For this purpose, new techniques that combine chemical extraction, chromatographic separation and determination by microbial assays have been developed (e.g. Sczesny et al., 2003). Hestbjerg Hansen et al. (2001) developed a biosensor for the selective detection of bioavailable and bioactive trace residues of TCs in soil.

Antibiotic in soils

151

4 Input and concentrations in the soil environment

Soils are a habitat and source of indigenous, antibiotics producing microorganisms (Gottlieb, 1976; Thomashow et al., 1997). Among numerous other soil microorganisms, 30 to 50 % of actinomycetes isolated from soil are able to synthesize antibiotics (Topp, 1981). Such antibiotics, biosynthesized in situ, are found especially in the soil rhizosphere with concentrations of up to 5 lg g1 (Soulides, 1965; Lumsden et al., 1992; Shanahan et al., 1992). However, findings of pharmaceutical antibiotics in the environment increase. Like other pharmaceuticals, these compounds are optimized in their pharmacokinetics in such a way that they do not accumulate in the organism. After medication, they are mostly excreted as parent compounds, whereas metabolites might be also bioactive (Bouwman and Reus, 1994; Schadewinkel-Scherkl and Scherkl, 1995; Kmmerer et al., 2000). Excretion rates following the passage through the gastro-intestinal tract are in the range of 40 to 90 % for SAs and TCs (Berger et al., 1986; Winckler and Grafe, 2001). Compilations of excretion rates were published by Zuccato et al. (2001), Halling-Srensen et al. (2001), and Jjemba (2002). Rates vary among the single antibiotic substances, the treated species and depend on the mode of application, as it was shown for SA administered to pigs (Haller et al., 2002).

directly through grazing livestock or indirectly through the use of manure and sewage sludge as fertilizer (Jrgensen and Halling-Srensen, 2000). In addition, wastewater and runoff from agricultural land are mainly responsible for the contamination of aquatic systems (Hirsch et al., 1999; Alder et al., 2001). A further significant source of antibiotics in the environment is their use in aquaculture for fish production. Here they are directly introduced into surface water (Rmbke et al., 1996). Among other compounds, TCs, nitrofurans, and SAs are used mainly for this purpose (Lscher et al., 1994) and result in residual concentrations of several hundred mg kg1 in aquatic sediments (Jacobsen and Berglind, 1988; Samuelsen et al., 1992; Coyne et al., 1994). Flooding of shore soils with contaminated surface water may possibly yield an input of antibiotics. In contrast, environmental contamination due to the production and distribution of pharmaceuticals can mostly be excluded. However, severe ground water contamination following the deposition of pharmaceutical wastes from antibiotic production was reported by Holm et al. (1995). Since the 1950s, antibiotics have been used as pesticides, especially oxytetracycline and streptomycin, which are commonly used in fruit, vegetable, and ornamental plant production. In the USA, 0.5 % of the total antibiotic consumption of approximately 10,000 t is from the application to plants (McManus et al., 2002). In the vicinity of animal houses used for pig and poultry breeding, antibiotics were detected in dust from the exhaust air of the stable ventilation (Hamscher et al., 2002b; Thiele-Bruhn et al., 2003a). Intra-corporal degradation processes usually proceed in the faeces (Langhammer, 1989; Loke et al., 2000). In contrast, antibiotics not metabolized in the organism are often found as recalcitrant after excretion (Bouwman and Reus, 1994; Schadewinkel-Scherkl and Scherkl, 1995). Thus, several antibiotic compounds persist in the environment (Gavalchin and Katz, 1994; Kmmerer et al., 2000; Khne et al., 2000) and are not transformed, e.g. by aeration of manure even at increased ambient temperature (Winckler and Grafe, 2001) or sewage water treatment (Richardson and Bowron, 1985). These substances are likely to reach aquatic sediments through waste water or agricultural soils after fertilization with manure and sewage sludge, respectively.

4.2 Environmental concentrations

The discussed sources of pharmaceuticals result in detectable residual concentrations in diverse environmental compartments and even in drinking water (Heberer and Stan, 1998; Hirsch et al., 1999). In the USA, a nationwide survey of pharmaceutical compounds revealed that among numerous other pharmaceuticals, a number of veterinary and human antibiotics were detected in 27 % of 139 river water samples at concentrations of up to 0.7 lg l1 (Kolpin et al., 2002). In England, representative single substances from the classes of macrolides, SAs, and TCs were determined in river water in concentrations of ca. 1 lg l1 (Watts et al., 1982), a concentration that reduced aqueous microbial activity in biotests (Backhaus and Grimme, 1999). In marine sediment underneath fish farms, residual oxytetracycline concentrations of 500 to 4000 lg kg1 were commonly observed and

4.1 Anthropogenic input

Major portions of antibiotics are excreted after intra-corporal medication or are rinsed from the skin after dermal application. Consequently, antibiotics reach agricultural soils

152

Thiele-Bruhn

J. Plant Nutr. Soil Sci. 2003, 166, 145167 than to the sand size fraction of a soil, the opposite was true for the increase of the partition coefficients at the desorption step (Thiele et al., 2002). As shown for chlortetracycline, the adsorption of epimers and metabolites of an antibiotic may be considerably different from that of the parent compound and in the case of anhydro-chlortetracycline may be even lower (Tjrnelund et al., 2000). This is of special importance when the more mobile metabolite still exhibits antimicrobial activity. Sorption of pharmaceutical antibiotics is especially influenced by soil pH (Holten Ltzhft et al., 2000), SOM (Langhammer, 1989; Gruber et al., 1990), and soil minerals (Batchelder, 1982). Pinck et al. (1961a) and Bewick (1979) reported a much stronger adsorption of aminoglycosides, TCs, and tylosin to expandable three layer clay minerals than to illite and kaolinite. Correspondingly, nearly the opposite sequence was found for the desorption of these antibiotics, while no release of aminoglycosides was determined (Pinck et al., 1961b). Similar results were obtained for the adsorption of these pharmaceuticals and of streptomycin in soils characterized by the investigated different clay minerals (Pinck et al., 1961a; Soulides et al., 1962). It is assumed that besides adsorption, diffusion into porous soil particles also contributed to the fixation. An interlayer adsorption of antibiotics from various classes in the presence of montmorillonite was first described by Pinck et al. (1962). Correspondingly, the strong adsorption of FQs to soils, especially to clay minerals, was accompanied by an expansion of the spacing of montmorillonite (Nowara et al., 1997). The authors proposed coulombic interactions and the adsorption of anionic antibiotics via cation bridging to clay minerals as the main mechanism for FQ adsorption. Thereby, the deprotonated carboxylic group of FQ-carboxylic acids is fixed to the clay minerals while the sorption of the decarboxylated derivative is much smaller. The sorption and fixation of antibiotics is strongly governed by the property of numerous compounds to ionize depending on the pH of the medium (Yeager and Halley, 1990). Octanol/ water coefficients of ionizing compounds change considerably in a pH range around the acid dissociation constant (Holten Ltzhft et al., 2000). Electrostatic forces mostly drive the sorption of these derivatives to charged surfaces of mineral and organic exchange sites (Williams, 1982; Holten Ltzhft et al., 2000). The adsorption coefficients (Kd) of SAs increased from < 1 up to 30 when the soil pH decreased in the range of 8 to 4 (Boxall et al., 2002; Tolls et al., 2002). This was related to the ionization of the amphoteric sulfonamides. Correspondingly, the adsorption of TCs to humic substances and clay minerals was not only influenced by the pH, but also by the ionic strength of the medium (Sithole and Guy, 1987a, b). Oxytetracycline adsorption was 2.5 times greater when Ca2+ was sorbed to clay minerals as compared to Na+ (Sithole and Guy, 1987a), because TCs form reversible complexes with multivalent cations (Wessels et al., 1998). Chelate complexes of TCs preferentially involve the tautomeric C-11-C-12 b-diketone system that is formed under alkalinic conditions from the keto-enol molecule, while with decreasing pH, the dimethylamino group at C-4 position becomes increasingly involved (Loke et al., 2002). Sithole and Guy (1987a, b) proposed three major sorption mechan-

ca. 50 % of red rock crab (Cancer productus) collected after oxytetracycline application exceeded the US FDA limit of 0.1 lg g1 for seafood (Capone et al., 1996). The intra-corporal administration of antibiotics inevitably leads to residual concentrations in excrements. Consequently, antibiotics are frequently found in dung and manure. Manure samples from pigs contained up to 3.5 mg kg1 of SAs and up to 4 mg kg1 of TCs (Hper et al., 2002; Hamscher et al., 2002a; Sengelv et al., 2003). Even larger concentrations of TCs were determined in dung from beef cattle and calves (Patten et al., 1980; Hper et al., 2002). Campagnolo et al. (2002) detected antimicrobial compounds from a multitude of different classes in all manure samples taken from eight pig farms, with the single substances often exceeding 100 lg l1 and the sum of all antibiotics approaching 1000 lg l1. Additionally, surface and groundwater samples nearby the storage lagoons were often contaminated with antibiotics. Residual concentrations of antibiotics were estimated for agricultural soils, ranging for TCs from 450 to 900 lg kg1 (Winckler and Grafe, 2000), for macrolides from 13 to 67 lg kg1 and for FQs from 6 to 52 lg kg1 (Schller, 1998). In soils under conventional landfarming fertilized with manure and monitored for two years, average concentrations of up to 199 lg kg1 tetracycline, 7 lg kg1 chlortetracycline (Hamscher et al., 2002a), and 11 lg kg1 sulfadimidine (Hper et al., 2002) were detected. Contamination of Swiss river water with veterinary antibiotics pointed to a runoff or leaching of antibiotics from soil to surface water (Alder et al., 2001). In contrast, Runsey et al. (1977) detected no antibiotics in weathered manure on pasture and soil and in runoff water. Additionally, b-lactams were rarely found in the environment, most probably due to the fast degradation of the chemically unstable lactam ring (Alder et al., 2001).

5 Fate of antibiotics in soils

5.1 Sorption and fixation
The antibiotics of different structural classes vary considerably in their molecular structures and physicochemical properties (Tab. 2). Some substances are hydrophobic or non-polar, whereas others are completely water soluble or dissociate at pH values typical for soils. Thus, distribution coefficients (Kd) for the adsorption of antibiotics to soil material and aquatic sediments, e.g. as summarized by Tolls (2001), vary for SAs from 0.6 to 4.9, for TCs from 290 to 1620, and for FQs from 310 to 6310 (Tab. 4). From the extractability, Katz and Katz (1983) deduced that the strength of sorption to soil increases in the following sequence: oxytetracycline = chlortetracycline bacitracin < tylosin < erythromycin < streptomycin. For most of the antibiotics, especially the stronger adsorbed TCs and FQs, desorption hysteresis is strong, and partition coefficients multiply in desorption experiments (Nowara et al., 1997; Rablle and Spliid, 2000). Yeager and Halley (1990) found that only 50 % of the adsorbed efrotomycin could be extracted, even with harsh solvents. However, sorption of antibiotics to soil minerals is weaker than to soil organic matter (SOM). Although adsorption of various SAs was stronger to the clay

J. Plant Nutr. Soil Sci. 2003, 166, 145167

Antibiotic in soils

153

Table 4: Sorption coefficients of pharmaceutical antibiotics in soils, sediments and slurry. Tabelle 4: Adsorptionskoeffizienten pharmazeutischer Antibiotika in Bden, Sedimenten und Wirtschaftsdngern. Antibiotic Class Tetracyclines Antibiotic Compound oxytetracycline 33 2000 mg g 2.5 50 2.5 50 2.5 50 2.5 50 285 10.9 Sulfonamides sulfachloropyridazine sulfadimidine 0.05 20 0.05 20 0.2 25 0.2 25 0.2 25 0.2 25 sulfamethazine 0.2 25 0.2 25 0.2 25 0.2 25 sulfapyridine 0.1 500 0.1 500 1.0 10 sulfadiazine sulfadimidine sulfanilamide sulfadimethoxine sulfachloropyridazine sulfadiazine sulfadimidine sulfaisoxazole sulfathiazole Macrolides tylosin 50 mg g
1 1

Concentration lg g1 a

Sample soil: texture / pH / OC% sewage sludge / 6.5 / 37b pig manure 6 h / 24 h sand / 5.6 / 1.4 sandy loam / 5.6 / 1.1 sand / 6.3 / 1.5 organic marine sediment organic marine sediment clay loam / 6.5 / sandy loam / 6.8 / sand / 5.2 / 0.9 loamy sand / 5.6 / 2.3 sandy loam / 6.3 / 1.2 clay silt / 6.9 / 1.1 sand / 5.2 / 0.9 loamy sand / 5.6 / 2.3 sandy loam / 6.3 / 1.2 clay silt / 6.9 / 1.1 silt loam / 7.0 / 1.6 silt loam / 6.9 / 2.4 silt loam / 7.0 / 1.6 silt loam / 7.0 / 1.6 silt loam / 7.0 / 1.6 silt loam / 7.0 / 1.6 silt loam / 7.0 / 1.6 clay-loam / 6.2 / 3.1 clay-loam / 6.2 / 3.1 clay-loam / 6.2 / 3.1 clay-loam / 6.2 / 3.1 clay-loam / 6.2 / 3.1 clay-loam / 6.2 / 3.1 kaolinite illite montmorillonite bentonite
1 d

Kd l kg1
3020 83.2 / 77.6 680 670 1026 417 2590 663 1.8 0.9 1.3e 3.5
e

Koc l kg1
8160a 195 42500 47880 93320 27790

Kclay Reference l kg1

Holten-Ltzhoftc Loke et al., 2002 Rablle and Spliid, 2000

loamy sand / 6.1 / 1.6

Smith and Samuelsen, 1996 Boxall et al., 2002 139 151 170 80 174 125 208 82 101 308 217 124 149 106 143 323 129
e

Langhammer and Bning-Pfaue, 1989 Langhammer, 1989

2.0e 0.9e 1.2 3.1 2.0 1.0 1.6 7.4 3.5 2.0 2.4 1.7 2.3 10e 4
e

Thiele, 2000 Thiele et al., 2002

1.0 10 1.0 10 1.0 10 1.0 10

Tolls et al., 2002

2.5 3e 1.5 3e

81 97 48 97 0.7 3.9 0.7 3.1 Bewick, 1979

50 mg g1 500 mg g1 500 mg g1 100 2000 mg g Macrolides tylosin 1.25 25 1.25 25 1.25 25 1.25 25 Fluorquinolones ciprofloxacin 250 lg l 2 200 enrofloxacin 2 200 2 200
1

pig manure 6 h / 24 h sand / 5.6 / 1.4

45.7 / 240 128 10.8 62.3 8.3

b

110 7990 771 5660 553 1127b 61000 186340 768740 2240

Loke et al., 2002 Rablle and Spliid, 2000

loamy sand / 6.1 / 1.6 sandy loam / 5.6 / 1.1 sand / 6.3 / 1.5 sewage sludge / 6.5 / 37 loamy sand / 5.3 / 0.70 clay / 4.9 / 1.63 loam / 5.3 / 0.73

417 427 3037 5612

HallingSrensen, 2000 Nowara et al., 330 1997

154

Thiele-Bruhn

J. Plant Nutr. Soil Sci. 2003, 166, 145167

Table 4: Continued. Tabelle 4: Fortsetzung. Antibiotic Class Antibiotic Compound Concentration lg g1 a 2 200 2 200 2 200 decarboxylated enrofloxacin ofloxacin Imidazoles metronidazole 2 200 2 200 100 2000 mg g 1.25 25 1.25 25 1.25 25 1.25 25 fenbendazole Polypeptides avermectin 0.5 100 0.5 100 0.006 2.17 0.006 2.17 0.006 2.17 Quinoxalinederivatives olaquindox 1.25 25 1.25 25 1.25 25 1.25 25 Lipoglycosides efrotomycin 1.0 135 1.0 135 Diaminopyrimidines trimethoprim 500 lg l1
1

Sample soil: texture / pH / OC% loamy sand / 6.0 / 1.23 loam / 7.5 / 1.58 loamy sand / 5.3 / 0.70 loamy sand / 5.3 / 0.70 loamy sand / 5.3 / 0.70 pig manure 6 h / 24 h sand / 5.6 / 1.4 sandy loam / 5.6 / 1.1 sand / 6.3 / 1.5 silt loam / 7.0 / 1.6 silt loam / 6.9 / 2.4 clay loam / 6.6 / 4.8 sand / 7.5 / 0.1 silt loam / 7.5 / 2.1 loamy sand / 6.1 / 1.6 sand / 5.6 / 1.4 sandy loam / 5.6 / 1.1 sand / 6.3 / 1.5 silt loam / 7.5 / 2.1 loam / 6.7 / 2.5 sandy loam / 7.5 / 1.1 clay loam / 5.0 / 4.6 sewage sludge / 6.5 / 37b
d

Kd l kg1
1230 260 496 7.7 309 n.d. 0.67 0.54 0.62 0.57 0.91 0.84 147 17.4 80.2 20.4 / 9.77 1.67 1.21 1.27 0.69 18 8.3 51 290 76

Koc l kg1
99980 16510 70910 1100 44140 n.d. 42 39 56 38 57 35 5300 30000 6600 50 104 86 116 46 1460 580 8000 11000 205b

Kclay Reference l kg1

2276 140 2480

Loke et al., 2002 Rablle and Spliid, 2000

loamy sand / 6.1 / 1.6

Thiele and Leinweber, 2000 Gruber et al., 1990 Loke et al., 2002 Rablle and Spliid, 2000

100 2000 mg g1 pig manure 6 h / 24 hd

Yeager and Halley, 1990

Halling-Srensen et al., 2002b

a if not indicated otherwise b Koc estimates for OC in dry matter from own data; c Holten-Ltzhft and Halling-Srensen cited in Stuer-Lauridsen et al. (2000); d adsorption time; e data derived from figures; n.d. = not detectable

isms for TCs: complexation by divalent cations, ion exchange, and hydrogen bridging from acidic groups of humic acids to polar groups of the TCs. Adsorption of antibiotics to SOM is strong and depends on the quantity and the composition of SOM as well, as it was shown for sulfapyridine (Thiele, 2000). When a Chernozem was physically dispersed and separated into particle size fractions, the adsorption of SAs to the clay size fraction with stable organo-mineral complexes was about two times greater than to the sand size fraction, which was mostly characterized by particulate organic matter of plant origin (Thiele et al., 2002). The adsorption of sulfapyridine was significantly correlated with the concentration of lipids and lignin dimers in the SOM of the particle fractions. Correspondingly, the adsorption of oxytetracycline increased with increasing aromaticity of organic soil components (Suan and Dmitrenko, 1994a). As for soil minerals, the adsorption of oxytetracycline to humic acid varies significantly with pH

(Sithole and Guy, 1987b). The TCs bind to humic acids and proteins especially via anionic functional groups (Loke et al., 2002). In mildly alkalinic manure (pH 7.8), tylosin A is partly positively charged thus, the resulting sorption is likely due to ion binding to negative groups of the manure particles, but not to complexation with metal ions (Loke et al., 2002). Since Koc was sufficiently estimated from Kow, a major contribution of hydrophobic partitioning was concluded. However, the Koc concept is not valid for the majority of polar antibiotics (Thiele et al., 2002). The much stronger sorption of TCs to dissolved organic matter (DOM) than expected from Kow clearly stresses that sorption is not attributable to hydrophobic partitioning, but ionic interactions and hydrogen bonds (Tolls, 2001). In general, the adsorption of antibiotics like FQs and SAs to faeces that are rich in organic matter is strong (Marengo et al., 1997; Thiele-Bruhn and Aust, 2003). However, distribution coefficients for oxytetracycline and tylosin are smaller in manure than in soils (Loke et al., 2002). Accordingly, Boxall et al. (2002) determined decreas-

J. Plant Nutr. Soil Sci. 2003, 166, 145167 ing Kd values for sulfachloropyridazine with an increasing proportion of manure in soil. This was not related to the mobilizing effect of DOM, but to the pH effect of the alkalinic manure. Similar effects of manure additions to soil were found for other SAs (Thiele-Bruhn and Aust, 2003). However, the slurry used in the latter investigation was acidic and above a manure to soil ratio of 1:10, Kd values strongly increased. Adsorption of most antibiotics to soils is fast. Efrotomycin, a lipoglycoside, and SAs reach sorption equilibrium in soil after several hours (Langhammer and Bning-Pfaue, 1989; Yeager and Halley, 1990; Thiele, 2000). The adsorption of TCs to various exchange sites is characterized by two processes of different kinetics (Sithole and Guy, 1987b; Suan and Dmitrenko, 1994b) that can be interpreted as a fast initial adsorption to outer surfaces, followed by a penetration into interlayers of clay minerals and micropores. In contrast, for salinomycin surface adsorption to SOM is energetically preferred to incorporation into the humic substance as was revealed from molecular modelling by computational chemistry (Schulten, 2002). Adsorption mostly reduces the antibiotic potency of the compounds (Ingerslev and Halling-Srensen, 2000). It is assumed that this is especially the case when the bioactive functionality associates with the exchange sites (Wessels et al., 1998; Thiele, 2000). Correspondingly, desorption yields a reactivation of the antimicrobial potency (Samuelsen et al., 1994; Halling-Srensen et al., 2002b). However, sorption or fixation does not necessarily result in a complete elimination of the antimicrobial activity (Halling-Srensen et al., 2003).

Antibiotic in soils

155

for oxytetracycline. Soil sorptive properties also governed the extent of sulfadimidine translocation in 30 cm soil columns filled with three soil materials of different properties (Langhammer and Bning-Pfaue, 1989). In contrast, sulfachloropyridazine was not leached through a sandy soil, while for a structured clay soil, rapid preferential transport of the SA into drainage water was observed within seven days (Boxall et al., 2002).

5.3 Degradation and inactivation

Numerous antibiotic compounds such as FQs, SAs, and TCs are susceptible to photodegradation (e.g. Burhenne et al., 1997; Halling-Srensen et al., 2003). Accordingly, chlortetracycline was degraded on the surface of a Chernozem at a DT50 of 5.8 days (Thiele-Bruhn et al., 2003b). However, under similar conditions no significant abiotic degradation of fenbendazole and sulfapyridine was determined. It is well known that photodecomposition in water already declines with increasing water depth and turbidity (Lunestad et al., 1995). Thus, it was assumed that photodegradation has no significant effect on the concentration of antibiotics in soils, especially when they are spread onto soils as contaminants in sludge or slurry. As competing processes, fixation to and penetration into voids of the soil solids protect antibiotics from photodecomposition. Hydrolysis, another major abiotic process, also yields transformation of antibiotics (HallingSrensen, 2000). The concentration of chlortetracycline aged in sterile soil possibly declined due to this process, while the extractable concentrations of fenbendazole and sulfapyridine did not change after an initial strong fixation (Thiele-Bruhn et al., 2003b). The degradation of xenobiotics in soils is mainly driven by microbial processes and numerous antibiotics are susceptible to enzymatic transformation reactions like oxidative decarboxylation and hydroxylation (Chen et al., 1997; McGrath et al., 1998; Al-Ahmad et al., 1999). Biodegradation was shown for ceftiofur-Na from the class of cephalosporins (Gilbertson et al., 1990). The antibiotic was quickly degraded in fortified cattle faeces, whereas no degradation was determined in sterile excrements. Thereby, intra-corporal metabolism proceeds in the environment. In mammals, antibiotics are mostly metabolized by a biphasic mechanism. First, functional groups are coupled to the molecule by monooxygenases, reductases, and hydrolases, followed by a covalent conjugation in the second phase, rendering the molecule more hydrophilic, excretable (Daughton and Ternes, 1999) and mostly antibiotic inactive (Halling-Srensen et al., 1998). The conjugation reactions are reversible though, and reactions back to the parent compound have been observed in the environment (Hussar et al., 1968; Langhammer, 1989; Halling-Srensen et al., 2002b). However, antibiotics usually further degrade in dung, manure, and soil (Halling-Srensen, 2000; Ingerslev and Halling-Srensen, 2000; Wetzstein et al., 2002). Additions of manure or sludge, containing high numbers of microorganisms, mostly result in increased biodegradation of antibiotics in soil (Ingerslev and Halling-Srensen, 2001; Ingerslev et al., 2001). Examples for degradation rates of antibiotics in manure and soils are listed in Tab. 5.

5.2 Mobility and transport

Field investigations revealed that point sources caused ground water contamination resulting from transport of antibiotics through soil as determined in the vicinity of manure lagoons (Campagnolo et al., 2002) and at a disposal site of a pharmaceutical plant (Holm et al., 1995). A diffuse contamination of surface water by antibiotic leaching from agricultural soils has also been reported (Alder et al., 2001). In contrast, antibiotics were only detected in a small number of ground water samples from regions with intensive livestock production (Hirsch et al., 1999). However, to date, only a few systematic investigations related to the mobility and transport of antibiotics in soil exist. Numerous antibiotics have a low water solubility, they are relatively polar, and strongly retarded in soils (Tab. 2, Tab. 4). It is assumed that significant transport of such antibiotics like TCs is restricted to fast preferential and macropore flow or is facilitated by co-transport with mobile colloids like DOM. In submerged marine sediment, oxytetracycline transport within 220 days was restricted to 2 to 4 cm (Samuelsen et al., 1992). Also, leaching of avermectin in soil columns was small but increased significantly with preferential flow within the cracks of a structured silt loam (Gruber et al., 1990). In accordance with results from sorption experiments, the weakly adsorbing olaquindox completely leached through soil columns, while the stronger adsorbing tylosin was retained in different depths depending on the soil properties (Rablle and Spliid, 2000). No transport was found

156

Thiele-Bruhn

J. Plant Nutr. Soil Sci. 2003, 166, 145167 for SAs declined dramatically after respiking the soil with the same or a similar antibiotic.

Degradation of antibiotics is governed by their molecular composition. Macrolides and penicillins are targets for fast degradation in soil (Gavalchin and Katz, 1994; Midtvedt, 2001). The intense transformation of sarafloxacin (Marengo et al., 1997) and virginiamycin (Weerasinghe and Towner, 1997) yielded numerous metabolites of minor concentration (< 10 %) and followed first order kinetics. In the presence of wood rotting fungi, FQs are inactivated through metabolism at the amine-position of the molecule while the heterocyclic ring is very persistent (Wetzstein, 2001). Thus, the xenobiotic fluorine moiety of FQs is quickly eliminated and the resulting metabolites exhibit no or strongly reduced antibiotic potential (Wetzstein et al., 1997). From the group of SAs, sulfadimethoxine is possibly demethylated in sediments (Samuelsen et al., 1994). However, fixation of antibiotic compounds to surfaces or in pores of the soil matrix may effectively protect them from biodegradation (Samuelsen et al., 1992; Gavalchin and Katz, 1994) without alterations to their molecular structure (HallingSrensen et al., 2002b). Consequently, bioavailable concentrations possibly decline below a threshold concentration for the initiation of biological degradation (Alexander, 1999). Thus, even compounds like TCs, which are reactive in standard solutions, persist in the soil for several months (van Gool, 1993; Hper et al., 2002). In aquatic sediments, no or only minor degradation of FQs, TCs, and SAs was determined over a period of 180 days (Samuelsen et al., 1994; Hektoen et al., 1995). Besides of the physicochemical properties of the antibiotics, the extent and kinetics of degradation are also considerably affected by temperature (Gavalchin and Katz, 1994) and adsorption to soil (Weerasinghe and Towner, 1997) (Tab. 4). For the FQ enrofloxacin, it was shown that unspecific adsorption to SOM reduced degradation, although it was not completely stopped (Martens et al., 1996; Wetzstein et al., 1997). The products of biodegradation may still exhibit antimicrobial potential, as it was found for several metabolites of FQs (Weerasinghe and Towner, 1997; Marengo et al., 1997; Wetzstein et al., 2000). Furthermore, the decline in concentration of TCs and various antibiotics of other structural classes was not always mirrored by a decline in microbial toxicity (Halling-Srensen et al., 2002b, 2003). For example, tylosin A was converted to the antibiotic tylosin B in acidic medium, whereas in neutral and alkaline medium, tylosin A aldol was detected along with a number of polar decomposition products which were less bioactive (HallingSrensen et al., 2003). Generally, the degradation of most xenobiotics is faster and more complete under aerobic as compared to anaerobic conditions. Correspondingly, degradation of oxytetracycline, tylosin, sulfadiazine, streptomycin, metronidazole, and olaquindox in activated sludge, soil and surface water was similar or slightly lower under anaerobic as compared to aerobic conditions, while ciprofloxacin was not degraded under anaerobic conditions (Ingerslev et al., 2001; HallingSrensen et al., 2003; Tab. 5). Additionally, Ingerslev and Halling-Srensen (2000) demonstrated that acclimation of degrading organisms occurs in soil, when degradation times

5.4 Effects on soil organisms and plants

Pharmaceutical antibiotics are designed to affect mainly microorganisms. Hence, the toxic dose for microorganisms is often several magnitudes smaller than for higher organisms (Wollenberger et al., 2000). Accordingly, dose related effects on soil microorganisms were determined (Herron et al., 1998; Pfeiffer et al., 1998). Selected results are listed in Tab. 6. Effects and effective doses vary with time (Thiele and Beck, 2001). When a Gleyic Podzol was incubated with tetracycline, the metabolic quotient was significantly affected by a concentration of 10 lg kg1 after 8 weeks (Hper et al., 2002). After 16 weeks, effects were significant only at an initial concentration of 10 mg kg1. Some antibiotics inhibit microorganisms (Colinas et al., 1994; Thiele and Beck, 2001), while others promote their growth and activity (Patten et al., 1980; Hossain and Alexander, 1984). Antibiotics like streptomycin and cycloheximide are generally used to selectively inhibit growth of bacteria and fungi in soil experiments. Consequently, other pharmaceutical antibiotics cause changes in the composition of the indigenous soil microbial population as well (Ingham and Coleman, 1984; Mc Cracken and Foster, 1993). In this manner, even small extractable concentrations of oxytetracycline and sulfapyridine produced longer lasting significant effects (Thiele and Beck, 2001). In contrast, species of soil fauna were not affected by even excessive doses of antibiotics (Gomez et al., 1996; Herron et al., 1998; Baguer et al., 2000). While no effects of selected antibiotics on enchytreids and springtails were observed at environmentally relevant concentrations, the anthelmintic ivermectin significantly increased the mortality of springtails (Jensen et al., 2001b). Also, anthelmintics can change the fauna in and underneath cow-pats and hamper the dung decomposition (Madsen et al., 1988; McCracken and Foster, 1993) and inhibit nematodes (Tomlinson et al., 1985) and earthworms (Gunn and Sadd, 1994; Tab. 6) The effects of antibiotics on organisms are essentially influenced by their bioavailability that depends on the soil properties, the availability of nutrients, and the presence of root exudates (da Gloria Britto de Oliveira et al., 1995; Herron et al., 1998). Multivalent cations inhibit the antibiotic potential of TCs and FQs (Froehner et al., 2000). First results confirming the reduction of antibiotic potency due to sorption and degradation in soils were published by Jefferys (1952). Degradation products of tylosin, sulfadiazine, streptomycin, ciprofloxacin, and olaquindox showed no significant potency in a soil bacterial assay (Halling-Srensen et al., 2003). However, a transformation of pharmaceuticals does not necessarily yield a decline in their antibiotic potential. Various metabolites of TCs still exhibited bacterial toxicity in sewage sludge and soil (Halling-Srensen et al., 2002b). The period over which antibiotics are effective depends on their persistence (Samuelsen et al., 1994). The antibiotic potential can increase with time (Dojmi di Delupis et al., 1992)

J. Plant Nutr. Soil Sci. 2003, 166, 145167

Table 5: Degradation of pharmaceutical antibiotics in soils and manure. Tabelle 5: Abbau pharmazeutischer Antibiotika in Bden und Wirtschaftsdngern. Class Tetracyclines Compound chlortetracycline 5.6 4.7 lg kg1 tetracycline 50300 lg kg 10 10
1

Antibiotic in soils

157

Concentration lg g1 a

Sample soil: texture / pH / OC% cattle manure sandy loam / 6.1+ manure soil soil poultry manure manure + soil pig manure, aerated pig manure, non aerated

Degradation % 24 88 0 0 65b 100 50 50 50 0 50 0 0 / 50c 0 / 50

c b

Time d 84 30 ca. 180 ca. 180 84 14 4.5 9 55105 180 43.8 14 28 / 0.4c 28 / 1.6 28 / 0.4 28 / 3.8c 64 64 22 41 30 30 22.2 49.0 41.4
c

Reference Runsey et al., 1977 Gavalchin and Katz, 1994 Hamscher et al., 2001 Jagnow, 1977 Khne et al., 2000 Winckler and Grafe, 2001 van Gool, 1993 Ingerslev et al., 2001 Frankenberger and Tabatabai, 1982 Ingerslev and Halling-Srensen, 2000 Langhammer et al., 1990 Halling-Srensen, 2000 Gavalchin and Katz, 1994 Gilbertson et al., 1990

20 100 lg l1 oxytetracycline

pig manure soil + contam. manure sediment slurry, aerobic

Sulfonamides

sulfanilamide sulfabenzamide sulfadiazine sulfameter sulfanilamide sulfadimidine trimethoprim 2501000 lg l1 1.0 1.0 500 lg l1 5.6 5.6

various soils manure / sludge manure / sludge manure / sludge manure / sludge loamy sand / 5.6 / 2.3 clay silt / 6.9 / 1.1 sewage sludge sandy loam / 6.1+ manure sandy loam / 6.1+ manure clay loam sand silty clay loam

0 / 50c 0 / 50c 0.2 / 0.3d,e 0.3 / 0.7 50 0 0 50e 50e 50e 50 25 70b 0 50 50 50 0.58e 0.57e 0.49e 30.3e 0.1-0.7 / 0.712.8e
d,e

Aminoglycosides streptomycin b-Lactams penicillin ceftiotur

mecillinam Macrolides erythromecin spiramycin tylosin

500 lg l1 5.6

sewage sludge sandy loam / 6.1+ manure poultry manure

0.5 0.7 Halling-Srensen, 2000 30 28 30 4.2 5.7 > 2.5 80 80 80 56 56 Martens et al., 1996 Wetzstein et al., 1997 Gavalchin and Katz, 1994 Jagnow, 1977 Gavalchin and Katz, 1994 Ingerslev and Halling-Srensen, 2001 Loke et al., 2000 Marengo et al., 1997

5.6 100 100 25 mg l

1

sandy loam / 6.1+ manure slurry + sand / 6.3 / 1.4 slurry+sandy loam /6.8/1.6 pig manure, anaerobic loam / 7.9 sandy loam / 7.6 silt loam / 7.6 sandy loam / 5.4 / 1.3 cattle manure + basidiomycetes

Fluorquinolones

sarafloxacin

3.4 3.4 3.4

enrofloxacin

10 10

158

Thiele-Bruhn

J. Plant Nutr. Soil Sci. 2003, 166, 145167

Table 5: Continued. Tabelle 5: Fortsetzung. Class Compound ciprofloxacin Concentration lg g1 a 10 250 lg l1 Imidazoles metronidazole 10 10 Sample soil: texture / pH / OC% mineral. media + wood rotting fungi sewage sludge slurry + sand / 6.3 / 1.4 slurry+sandy loam /6.8/1.6 sediment slurry, aerobic sediment slurry, anaerobic Polypeptides bacitracin Zn-bacitracin virginiamycin 5.6 25 25 1.0 1.0 1.0 1.0 1.0 1.0 Polyethers Phospholipoglycosides monensin flavomycin 5.6 sandy loam / 6.1+ manure poultry manure manure + soil sandy silt / 8.2 silty sand / 6.3 silty sand / 5.7 silty clay loam / 6.1 silty clay loam / 5.6 clay loam / 5.4 manure, anaerobic sandy loam / 6.1+ manure poultry manure, aerobic poultry manure, anaerobic slurry + sand / 6.3 / 1.4 slurry+sandy loam /6.8/1.6 sediment slurry, aerobic sediment slurry, anaerobic Siderophores
a d

Degradation % 2.2 35.3e 50 50 50 50 50 33 90b 100 50 50 50 21 12 18 30 40 0 100b 0

b b

Time d 56 1.6 2.5 14.2 15.0 14 75 74.5 30 2 7 87 116 173 64 64 64 70 30 119 119 6.1 5.8 4.0 7.0 21.5 60

Reference Wetzstein et al., 1999 Halling-Srensen, 2000 Ingerslev and Halling-Srensen, 2001 Ingerslev et al., 2001 Gavalchin and Katz, 1994 Jagnow, 1977 Weerasinghe and Towner, 1997

Donoho, 1984 Gavalchin and Katz, 1994 Jagnow, 1977 Ingerslev and Halling-Srensen, 2001 Ingerslev et al., 2001 Hbener et al., 1992

flavophospholipol 10 10 Quinoxalinederivatives olaquindox 10 10

50 50 50 50 50

cyclosporin A

200 250

compost soil

if not indicated otherwise; b concentration determined from antibiotic activity; c second values after respiking the antibiotic to the sample; determined at 10 and 20 C, respectively; e 14CO2 determined from radio-labelled antibiotics

from what may be due to bioaccumulation (Lo and Hayton, 1981; Migliore et al., 1993). Bioaccumulation of 15 FQs was determined in E. coli, S. aureus and P. aeruginosa. However, the resulting intra-corporal concentrations were not correlated with the antibiotic effects of the pharmaceuticals (Asuquo and Piddock, 1993). In total, the knowledge about the ecotoxicity of antibiotics is still scarce, while their effects on humans and other mammals are well documented. There is a lack of special test methods and the adoption of unsuited methods produces erroneous results. No toxicity of antibiotics was observed with the bioluminescence test following the usual procedure, while after an extension of the duration of the assay, clear effects were determined (Backhaus and Grimme, 1999). Also, an

elongation of the substrate induced respiration test from 24 to 48 hours was necessary to obtain dose response relationships from antibiotics in selected soil samples (Thiele and Beck, 2001). The nitrification inhibition test proved not to be suitable for antibiotic testing in wastewater (Alexy et al., 2001). This might also explain for the nitrification rate in sewage sludge, manure, and soil, which was not affected by numerous antibiotics in various concentrations (Warman, 1980; Patten et al., 1980; Gomez et al., 1996).

5.4.1 Antibiotic resistance

Antibiotics released into the environment can provoke the formation of resistance, and even cross- and multiple resistance, in organisms (Nygaard et al., 1992; Wegener et

J. Plant Nutr. Soil Sci. 2003, 166, 145167 al., 1998; Al-Ahmad et al., 1999). Pathogens as well as commensal bacteria are affected, the latter constituting a potential reservoir of resistance genes for pathogenic bacteria. The transfer of such pathogens through the food chain is possible and consequently lowers the success of pharmacotherapies for curing humans and animals (Richter et al., 1996). The initiation of resistance is promoted by continuing a sublethal dosage of antibiotics (Gavalchin and Katz, 1994). This is put into effect by the repeated spreading of contaminated faeces onto agricultural soils (van Gool,

Antibiotic in soils

159

1993). The application of tetracycline contaminated manure induced antibiotic resistance in soil microorganisms that lasted for weeks (Frnd et al., 2000). Accordingly, tetracycline and oleandomycin resistant clostridia were significantly accumulated in manure, manure fertilized soils, and the ground water below (Huysman et al., 1993). However, survival of microorganisms in the presence of antibiotics is not necessarily due to acquired resistance. Many investigations revealed that numerous soil microorganisms have a natural tolerance towards antibiotics (Esiobu et al., 2002).

Table 6: Effects of pharmaceutical antibiotics on soil organisms and plants. Tabelle 6: Wirkungen pharmazeutischer Antibiotika auf Bodenorganismen und Pflanzen. Class Tetracyclines Compound chlortetracycline oxytetracycline chlortetracycline oxytetracycline oxytetra. + penicillin oxytetracycline chlortetracycline oxytetracycline Sulfonamides Tetracyclines sulfapyridine tetracycline Habitat / Organism Phaseolus vulgaris biomass biomass in sandy loam biomass in sandy loam hyphae of fungi: length and activity bacteria in sand soil methane production in manure silty sand / loamy sand 13 microorg. strains 8 soil microorg. strains oxytetracycline springtails F. fimetaria earthworms A. caliginosa enchytreids E. crypticus Macrolides tylosin springtails F. fimetaria earthworms A. caliginosa enchytreids E. crypticus Fluorquinolones Sulfonamides Aminoglycosides Imidazoles Macrolides Pleuromutilin Fluorquinolones Quinoxalines -Lactam Sulfonamides Tetracyclines ciprofloxacin trimethoprim mecillinam streptomycin metronidazole tylosin tiamulin oxolinic acid olaquindox penicillin G sulfadiazine chlortetracycline oxytetracycline tetracycline sewage sludge bacteria Effect / Inhibition Concentration lg g1 a 70 % 85 % 51 % 56 % 48 % 71 % 100 % ED10 SIR ED10 SIR ED10 SIR ED10 SIR MIC MIC LC10/EC10 LC10/EC10 LC10/EC10 LC10/EC10 LC10/EC10 LC10/EC10 EC50 EC50 EC50 EC50 NOEC EC50 EC50 EC50 EC50 EC50 NOEC EC50 EC50 EC50 10 mg l1 10 mg l1 160 160 10 10 18 mg l1 0.81 / 0.93 19.1 / 31.2 1.17 / 11.5 0.05 / 6.20 < 1 1000 lg l1 van Dijck and van de Voorde, 1976 10 lg l1 >5000/>5000 >5000 / 1954 >5000 / 3000 >5000 / 149 >5000 / 3306 2501 / 632 0.61 mg l1 17.8 mg l
1

Reference Batchelder, 1981

Colinas et al., 1994 Fedler and Day, 2002 Thiele and Beck, 2001

van Gool, 1993 Baguer et al., 2000

Halling-Srensen, 2000 Halling-Srensen, 2001

62.1 mg l1 0.47 mg l1 100 mg l1 54.7 mg l

1

14.3 mg l1 0.10 mg l1 95.7 mg l1 84.6 mg l1 60 mg l1 0.4 mg l1 1.2 mg l1 2.2 mg l1

160

Thiele-Bruhn

J. Plant Nutr. Soil Sci. 2003, 166, 145167

Table 6: Continued. Tabelle 6: Fortsetzung. Class Aminoglycosides Fluorquindones Macrolides Sulfonamides Tetracyclines Aminoglycosides Compound streptomycin ciprofloxacin tylosin sulfadiazine oxytetracyclines streptomycin Habitat / Organism sewage sludge bacteria soil fungi/bacteria/protozoa cycloheximid Anthelmintics ivermectin soil fungi/bacteria/protozoa springtails springtails reproduction soil respiration substrate induced respiration DMSO-reduction Polyether monensin soil respiration substrate induced respiration DMSO-reduction various enchytreids springtails springtails reproduction Sulfonamindes sulfadimethoxine Panicum miliaceum roots Panicum miliaceum stems Pisum sativum roots Pisum sativum stems Zea mays roots Zea mays stems carrot root / stem / leaf corn root / stem / leaf millet root / stem / leaf pea root / stem / leaf
a

Effect / Inhibition EC50 0/10 h EC50 0/10 h EC50 0/10 h EC50 0/10 h EC50 0/10 h /0/+ +//
b b

Concentration lg g1 a 0.42 / 0.61 mg l1 0.025 / 0.008 mg l 17.5 / 24.9 mg l1 15.9 / 16.8 mg l1 0.12 / 0.27 mg l1 3 30 1 1 10 0.5 11 11 11 176 176 176
1

Reference Halling-Srensen et al., 2003

Ingham et al., 1986 Ingham and Coleman, 1984 Jensen et al., 2000 Pfeiffer et al., 1998

no effect no effect LD50 EC10 +b 0

b

0b EC50 +
b

+b LD10 LD10 EC10 plant concentrations from bioaccumulation

oxytetracycline, tylosin, tiamulin, metronidazole, olaquindox > 1000 > 1000 100 2071 110 178 60.2 269 12.5 / / b // //
b

Jensen et al., 2001a

Migliore et al., 1995

1 mM 1 mM 1 mM 1 mM

Migliore et al., 1996

0 / 0 / 0b
b

if not indicated otherwise;

= inhibiton, 0 = no effect, + = promotion

Out of 36 strains of microorganisms from uncontaminated soil and water only seven were susceptible to 21 diverse antibiotics (van Dijck and van de Voorde, 1976). In particular, pseudomonads are often intrinsically resistant to antibiotics (Halling-Srensen et al., 2003).

5.4.2 Input of resistant microorganisms into soils

Resistance of soil microorganisms is not only provoked by the input of antibiotics into the environment. It appears to be even more important that resistant microorganisms are directly introduced with faeces into soils (Ali-Shtayeh et al., 1998). By dairy farm manure, resistance against ampicillin, penicillin,

tetracycline, vancomycin, and streptomycin was significantly increased in a garden soil to a frequency of 70 % (Esiobu et al., 2002). The medication of pigs with chlortetracycline was followed by the excretion of microorganisms resistant towards chlortetracycline and a multitude of other antibiotics (Langlois et al., 1978). A high prevalence of resistance against various antibiotics was determined in numerous faeces samples from pigs in the Netherlands and Sweden (van den Bogaard et al., 2000). Differences in the extent of resistance were related to the different intensity of antibiotic application in the two countries. Of special concern is the possible induction of antibiotic resistance in pathogens either directly or indirectly after transfer of genes encoding antibiotic resistance from non-pathogenic to pathogenic microorgan-

J. Plant Nutr. Soil Sci. 2003, 166, 145167 isms (Wegener et al., 1998). Transfer of resistance genes in soil was reviewed by Seveno et al. (2002). Mobile genetic elements conferring antibiotic resistance were readily obtained from microbial communities of environmental habitats (Smalla and Sobecky, 2002). As the major disseminating antibiotic resistance genes, bacterial IncQ plasmids were identified in pig manure (Smalla et al., 2000). Additionally, genetic transfer in soil was shown to increase by fertilization with pig manure, since it consisted of plasmids with high mobility (Gtz and Smalla, 1997). Accordingly, the extent of the increase of TC resistance in soil following manure fertilization was related to the amount of applied manure (Sengelv et al., 2003). However, the resulting increased resistance level in soils and water was lower than in faeces (Langlois et al., 1978) and vanished within one month (Sengelv et al., 2003).

Antibiotic in soils

161

5.4.3 Uptake in and effects on plants

The uptake and effects on plants varies considerably between reports and depends on the antibiotic substance and plant species (Patten et al., 1980; Langhammer, 1989; Migliore et al., 1995; Tab. 6). Yields and nutrient uptake by radish, wheat, and corn were increased in the presence of 160 mg kg1 of TCs (Batchelder, 1982). In contrast, performance of pinto beans (Phaseolus vulgaris) was significantly reduced in a sandy loam, but not clay loam that is most likely due to the inhibition of root nodulation by rhizobia (Batchelder, 1982). The effects of antibiotics on plants were reviewed by Jjemba (2002). The author stated that negative impacts of therapeutic compounds on plants were mostly determined by in vitro experiments at concentrations that are unlikely to occur in field soils. Negative impacts of contaminated manure on field soils were most likely related to excessive nitrogen or heavy metals, but not antibiotics. Accordingly, the plant growth inhibiting effect of sulfadimethoxine was smaller in vivo than in vitro (Migliore et al., 1996). This was related to a slow bioaccumulation of the antibiotic from the nutrient solution into the plant (Migliore et al., 1998) that may have been suppressed in soil by the aging of antibiotics. The translocation of 14C-sulfadimidine from soil into maize plants declined from 15 to 3 % after 32 days of aging in soil (Langhammer et al., 1990). Additionally, the transfer from roots to shoots was less than 0.04 % of the total radiolabel. No uptake of TCs into pinto beans and coconut trees was observed even after direct application (McCoy, 1976; Batchelder, 1981), although growth of beans in liquid cultures was significantly reduced at a concentration of 10 mg l1 chlortetracycline and oxytetracycline (Batchelder, 1981). However, endangerment of humans via the food chain is possible (Kennedy et al., 2000).

known about the numerous antibiotics from classes less often administered to humans and animals. Only a few investigations exist on the mobility and resulting transport and bioavailability of antibiotics. Degradation pathways and kinetics and on the other hand the degree and causes for persistence of antibiotics in soils need further elucidation. Ecotoxicity resulting from long-term exposure to low doses and mixtures of compounds are a special problem associated with antibiotics in the environment. Additionally, the introduction of resistance as opposed to existing intrinsic resistance of soil microorganisms is not completely understood. Hence, a comprehensive evaluation of residual concentrations in the soil environment is not possible. Existing arbitrary trigger values are not scientifically based, while limit values are missing. Effective and harmonized analytical methods exist for food, but not for soils. Also, methods to evaluate ecotoxicity need to be adapted or developed. The actual used methods according to OECD guidelines were developed for the testing of pesticides and are often not suitable for the chemically different antibiotics. To reduce the risk of environmental contamination and to save the indispensable antibiotics as effective drugs against infectious diseases, a responsible use and reduction of the consumption is needed. Especially in agriculture, antibiotics must not be abused to compensate for insufficient hygiene in stables and for not species appropriate animal husbandry.

Acknowledgments
The help of S. L. Foran for language editing and of G. Jandl and M.-O. Aust for proof reading of the manuscript is gratefully acknowledged.

Abbreviations
Antibiotics: FQs fluoroquinolones; SAs sulfonamides; TCs tetracyclines Extraction: ACN acetonitrile; EtOAc ethylacetate; MeCl2 dichloromethane; MeOH methanol; NH4OAc ammonia oxalate; SPE solid phase extraction; C8/C18 reversed octyl/ octadecyl silica phases; HLB hydrophilic-lipophilic balance cartridges; MCX mixed-mode HLB-cation exchange cartridges Detection: HPLC high performance liquid chromatography; DAD diode array detector; FLD fluorescence light detector; UV ultraviolet light absorption detector; LC-MS liquid chromatography-mass spectrometry; MS/MS tandem mass spectrometer; APCI chemical ionisation at atmospheric pressure ; ESI electrospray ionization Soil: DOM dissolved organic matter; OC organic carbon; SOM soil organic matter Effects on organisms: EC/ED effective concentration/dose; EC10/EC50 10 %/50 % difference from control; LC/LD lethal concentration/dose; MIC minimal inhibiting concentration; NOEC no observable effect concentration

6 Conclusions
Residues of pharmaceutical antibiotics occur quiet often in soils. Hence, environmental risk assessment of pharmaceutical antibiotics is necessary and legally prescribed. However, there is still a considerable lack of knowledge, although the number of investigations on the input and fate of antibiotics in soils strongly increased in the last years. Especially little is

162

Thiele-Bruhn

J. Plant Nutr. Soil Sci. 2003, 166, 145167

K. M., and McGeehin, M. (2002): Antimicrobial residues in animal waste and water resources proximal to large-scale swine and poultry feeding operations. Sci. Total Environ. 299, 8995. Capone, D. G., Weston, D. P., Miller, V., and Shoemaker, C. (1996): Antibacterial residues in marine sediments and invertebrates following chemotherapy in aquaculture. Aquaculture 145, 5575. Carson, M. C., Ngoh, M. A., and Hadley, S. W. (1998): Confirmation of multiple tetracycline residues in milk and oxytetracycline in shrimp by liquid chromatography-particle beam mass spectrometry. J. Chromatogr. B 712, 113128. Chen, Y., Rosazza, J. P., Reese, C. P., Chang, H. Y., Nowakowski, M. A., and Kiplinger, J. P. (1997): Microbial models of soil metabolism: biotransformations of danofloxacin. J. Ind. Microbiol. Biotechnol. 19, 378384. Colinas, C., Ingham, E., and Molina, R. (1994): Population responses of target and non-target forest soil-organisms to selected biocides. Soil Biol. Biochem. 26, 4147. Cooper, A. D., Stubbings, G. W., Kelly, M., Tarbin, J. A., Farrington, W. H., and Shearer, G. (1998): Improved method for the on-line metal chelate affinity chromatography-high-performance liquid chromatographic determination of tetracycline antibiotics in animal products. J. Chromatogr. A 812, 321326. Coyne, R., Hiney, M., OConnor, B., Kerry, J., Cazabon, D., and Smith, P. (1994): Concentration and persistence of oxytetracycline in sediments under a marine salmon farm. Aquaculture 123, 3142. da Gloria Britto de Oliveira, R., Wolters, A. C., and van Elsas, J. D. (1995): Effects of antibiotics in soil on the population dynamics of transposon Tn5 carrying Pseudomonas fluorescens. Plant Soil 175, 323334. Daughton, C. G., and Ternes, T. A. (1999): Pharmaceuticals and personal care products in the environment: agents of subtle change? Environ. Health Perspect. 107, 907938. Dojmi di Delupis, G., Macri, A., Civitareale, C., and Migliore, L. (1992): Antibiotics of zootechnical use: Effects of high and low dose contamination on Daphnia magna. Aquatic. Toxicol. 22, 5360. Donoho, A. L. (1984): Biochemical studies on the fate of monensin in animals and in the environment. J. Anim. Sci. 58, 15281539. EMEA (1997): Note for guidance: Environmental risk assessment for veterinary medicinal products other than GMO-containing and immunological products. Committee for Veterinary and Medicinal Products, London, p. 42. Escribano, E., Calpena, A. C., Garrigues, T. M., Freixas, J., Domenech, J., and Moreno, J. (1997): Structure-absorption relationships of a series of 6-fluoroquinolones. Antimicrob. Agents Chemother. 41, 19962000. Esiobu, N., Armenta, L., and Ike, J. (2002): Antibiotic resistance in soil and water environments. Int. J. Environ. Health Res. 12, 133144. Espinasse, J. (1993): Responsible use of antimicrobials in veterinary medicine: perspective in France. Vet. Microbiol. 35, 289301. FEDESA (2001): Antibiotic use in farm animals does not threaten human health. Press release, Visby, Sweden. 13.06.2001. Fedler, C. B., and Day, D. L. (2002): Anaerobic digestion of swine manure containing an antibiotic inhibitor. Agric. Waste Utiliz. Manag. 523530. Frankenberger, W. T., and Tabatabai, M. A. (1982): Transformations of amide nitrogen in soils. Soil Sci. Soc. Am. J. 46, 280284. Froehner, K., Backhaus, T., and Grimme, L. H. (2000): Bioassays with Vibrio fischeri for the assessment of delayed toxicity. Chemosphere 40, 821828.

References
Al-Ahmad, A., Daschner, F. D., and Kmmerer, K. (1999): Biodegradability of cefotiam, ciprofloxacin, meropenem, penicillin G, and sulfamethoxazole and inhibition of waste water bacteria. Arch. Environ. Contam. Toxicol. 37, 158163. Alder, A. C., McArdell, C. S., Golet, E. M., Ibric, S., Molnar, E., Nipales, N. S., and Giger, W. (2001): Occurrence and fate of fluoroquinolone, macrolide, and sulfonamide antibiotics during wastewater treatment and in ambient waters in Switzerland. In C. G. Daughton and T. Jones-Lepp: Pharmaceuticals and Personal Care Products in the Environment: Scientific and Regulatory Issues, pp. 5669. American Chemical Society, Washington, D.C. Alexander, M. (1999): Biodegradation and Bioremediation. Academic Press, San Diego, CA, p. 453. Alexy, R., Kmpel, T., Drner, M., and Kmmerer, K. (2001): Effects of antibiotics against environmental bacteria studied with simple tests. SETAC Europe 11th Annual Meeting, 610 May, Madrid. Ali-Shtayeh, M. S., Jamous, R. M., and Abu-Ghdeib, S. I. (1998): Ecology of cycloheximide-resistant fungi in field soils receiving raw city wastewater or normal irrigation water. Mycopathologia 144, 3954. Asukabe, H., Murata, H., Harada, K., Suzuki, M., Oka, H., and Ikai, Y. (1994): Improvement of chemical-analysis of antibiotics. 21. Simultaneous determination of three polyether antibiotics in feeds using high-performance liquid-chromatography with fluorescence detection. J. Agric. Food Chem. 42, 112117. Asuquo, A. E., and Piddock, L. J. (1993): Accumulation and killing kinetics of fifteen quinolones for Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa. J. Antimicrob. Chemother. 31, 865880. Backhaus, T., and Grimme, L. H. (1999): The toxicity of antibiotic agents to the luminescent bacterium Vibrio fischeri. Chemosphere 38, 32913301. Baguer, A. J., Jensen, J., and Krogh, P. H. (2000): Effects of the antibiotics oxytetracycline and tylosin on soil fauna. Chemosphere 40, 751757. Batchelder, A. R. (1981): Chlortetracycline and oxytetracycline effects on plant growth and development in liquid cultures. J. Environ. Qual. 10, 515518. Batchelder, A. R. (1982): Chlortetracycline and oxytetracycline effects on plant growth and development in soil systems. J. Environ. Qual. 11, 675678. Berger, K., Petersen, B., and Bning-Pfaue, H. (1986): Persistenz von Glle-Arzneistoffen in der Nahrungskette. Arch. Lebensmittelhyg. 37, 99102. Bewick, M. W. M. (1979): The adsorption and release of tylosin by clays and soils. Plant Soil 51, 363372. Booth, N. H., and McDonald, L. E. (1988): Veterinary Pharmacology and Therapeutics. Iowa State University Press, Ames, p. 1227. Bouwman, G. M., and Reus, J. A. W. A. (1994): Persistence of Medicines in Manure. Centre for Agriculture & Environment, CLM, 163, p. 26. Boxall, A. B. A., Blackwell, P., Cavallo, R., Kay, P., and Tolls, J. (2002): The sorption and transport of a sulphonamide antibiotic in soil systems. Toxicol. Lett. 131, 1928. Burhenne, J., Ludwig, M., Nikoloudis, P., and Spiteller, M. (1997): Photolytic degradation of fluoroquinolone carboxylic acids in aqueous solution. 1. Primary photoproducts and half-lives. Environ. Sci. Pollut. Res. 4, 1015. Campagnolo, E. R., Johnson, K. R., Karpati, A., Rubin, C. S., Kolpin, D. W., Meyer, M. T., Esteban, J. E., Currier, R. W., Smith, K., Thug,

J. Plant Nutr. Soil Sci. 2003, 166, 145167

Frnd, H.-C., Schlsser, A., and Westendarp, H. (2000): Effects of tetracycline on the soil microflora determined with microtiter plates and respiration measurement. Mitteilgn. Dtsch. Bodenkundl. Gesellsch. 93, 244247. Gavalchin, J., and Katz, S. E. (1994): The persistence of fecal-borne antibiotics in soil. J. Assoc. Off. Anal. Chem. Int. 77, 481485. Gilbertson, T. J., Hornish, R. E., Jaglan, P. S., Koshy, K. T., Nappier, J. L., Stahl, G. L., Cazers, A. R., Nappier, J. M., Kubicek, M. F., Hoffman, G. A., and Hamlow, P. J. (1990): Environmental fate of ceftiofur sodium, a cephalosporin antibiotic role of animal excreta in its decomposition. J. Agric. Food Chem. 38, 890894. Golet, E. M., Alder, A. C., Hartmann, A., Ternes, T. A., and Giger, W. (2001): Trace determination of fluoroquinolone antibacterial agents in urban wastewater by solid-phase extraction and liquid chromatography with fluorescence detection. Anal. Chem. 73, 36323638. Gomez, J., Mendez, R., and Lema, J. M. (1996): The effect of antibiotics on nitrification processes. Batch assays. Appl. Biochem. Biotechnol. 5758, 869876. Gottlieb, D. (1976): The production and role of antibiotics in soil. J. Antibiot. 29, 9871000. Gtz, A., and Smalla, K. (1997): Manure enhances plasmid mobilization and survival of Pseudomonas putida introduced into field soil. Appl. Environ. Microbiol. 63, 19801986. Grfe, U. (1992): Biochemie der Antibiotika: Struktur Biosynthese Wirkmechanismus. Spektrum Akademischer Verlag, Heidelberg, p. 389. Gruber, V. F., Halley, B. A., Hwang, S.-C., and Ku, C. C. (1990): Mobility of avermectin B1a in soil. J. Agric. Food Chem. 38, 886890. Gunn, A., and Sadd, J. W. (1994): The effect of ivermectin on the survival, behavior and cocoon production of the earthworm Eisenia fetida. Pedobiologia 38, 327333. Haller, M. Y., Muller, S. R., McArdell, C. S., Alder, A. C., and Suter, M. J. F. (2002): Quantification of veterinary antibiotics (sulfonamides and trimethoprim) in animal manure by liquid chromatographymass spectrometry. J. Chromatogr. A 952, 111120. Halling-Srensen, B. (2000): Algal toxicity of antibacterial agents used in intensive farming. Chemosphere 40, 731739. Halling-Srensen, B. (2001): Inhibition of aerobic growth and nitrification of bacteria in sewage sludge by antibacterial agents. Arch. Environ. Contam. Toxicol. 40, 451460. Halling-Srensen, B., Nors Nielsen, S., Lanzky, P. F., Ingerslev, F., Holten Ltzhft, H. C., and Jrgensen, S. E. (1998): Occurrence, fate and effects of pharmaceutical substances in the environment a review. Chemosphere 36, 357393. Halling-Srensen, B., Jensen, J., Tjrnelund, J., and Montforts, M. H. M. M. (2001): Worst-case estimations of predicted environmental soil concentrations (PEC) of selected veterinary antibiotics and residues used in Danish agriculture. In K. Kmmerer: Pharmaceuticals in the Environment: Sources, Fate, Effects and Risks. Springer, Berlin, pp. 143157. Halling-Srensen, B., Nors Nielsen, S., and Jensen, J. (2002a): Environmental Assessment of Veterinary Medicinal Products in Denmark. Environmental Project No. 659 2002. Danish Environmental Protection Agency, Copenhagen, p. 97. Halling-Srensen, B., Sengelv, G., and Tjrnelund, J. (2002b): Toxicity of tetracyclines and tetracycline degradation products to environmentally relevant bacteria, including selected tetracyclineresistant bacteria. Arch. Environ. Contam. Toxicol. 42, 263271. Halling-Srensen, B., Sengelv, G., Ingerslev, F., and Jensen, L. B. (2003): Reduced antimicrobial potencies of oxytetracycline, tylosin,

Antibiotic in soils

163

sulfadiazin, streptomycin, ciprofloxacin, and olaquindox due to environmental processes. Arch. Environ. Contam. Toxicol. 44, 716. Hamscher, G., Sczesny, S., Hper, H., and Nau, H. (2001): Tierarzneimittel als persistente organische Kontaminanten in Bden. In Niederschsisches Landesamt fr Bodenforschung: 10 Jahre Boden-Dauerbeobachtung in Niedersachsen. Hannover. Hamscher, G., Sczesny, S., Hper, H., and Nau, H. (2002a): Determination of persistent tetracycline residues in soil fertilized with liquid manure by high-performance liquid chromatography with electrospray ionization tandem mass spectrometry. Anal. Chem. 74, 15091518. Hamscher, G., Pawelzick, H. T., Nau, H., and Hartung, J. (2002b): Detection of antibiotics in dust originating from a pig fattening farm. SETAC Europe 12th annual meeting. 12 16 May 2002, Vienna. Heberer, T., and Stan, H.-J. (1998): Arzneimittelrckstnde im aquatischen System. Wasser Boden 50, 2025. Hektoen, H., Berge, J. A., Hormazabal, V., and Ynestad, M. (1995): Persistence of antibacterial agents in marine sediments. Aquaculture 133, 175184. Herron, P. R., Toth, I. K., Heilig, G. H. J., Akkermans, A. D. L., Karagouni, A., and Wellington, E. M. H. (1998): Selective effect of antibiotics on survival and gene transfer of streptomycetes in soil. Soil Biol. Biochem. 30, 673677. Hestbjerg Hansen, L. H., Ferrari, B., Srensen, A. H., Veal, D., and Srensen, S. J. (2001): Detection of oxytetracycline production by Streptomyces rimosus in soil microcosms by combining whole-cell biosensors and flow cytometry. Appl. Environ. Microbiol. 67, 239244. Hirsch, R., Ternes, T., Haberer, K., and Kratz, K. L. (1999): Occurrence of antibiotics in the aquatic environment. Sci. Total Environ. 225, 109118. Holm, J. V., Rugge, K., Bjerg, P. L., and Christensen, T. H. (1995): Occurrence and distribution of pharmaceutical organic-compounds in the groundwater downgradient of a landfill (Grindsted, Denmark). Environ. Sci. Technol. 29, 14151420. Holten Ltzhft, H. C., Vaes, W. H., Freidig, A. P., Halling-Srensen, B., and Hermens, J. L. (2000): 1-Octanol/water distribution coefficient of oxolinic acid: influence of pH and its relation to the interaction with dissolved organic carbon. Chemosphere 40, 711714. Hper, H., Kues, J., Nau, H., and Hamscher, G. (2002): Eintrag und Verbleib von Tierarzneimittelwirkstoffen in Bden. Bodenschutz 4, 141148. Hossain, A. K. M., and Alexander, M. (1984): Enhanzing soybean rhizosphere colonization by Rhizobium japonicum. Appl. Envir. Microbiol. 48, 468472. Hbener, R., Dornberger, K., Zielke, R., and Grfe, U. (1992): Abbau von Cyclosporin A durch Bodenmikroorganismen. UWSF Z. Umweltchem. kotox. 4, 227230. Hussar, D. A., Niebergall, P. J., Sugita, E. T., and Doluisio, J. T. (1968): Aspects of the epimerization of certain tetracycline derivatives. J. Pharm. Pharmacol. 20, 539546. Huysman, E., van Renterghem, B., and Verstraete, W. (1993): Antibiotic resistant sulphite-reducing Clostridia in soil and groundwater as indicator of manuring practices. Water Air Soil Pollut. 69, 243255. Ingerslev, F., and Halling-Srensen, B. (2000): Biodegradability properties of sulfonamides in activated sludge. Environ. Toxicol. Chem. 19, 24672473. Ingerslev, F., and Halling-Srensen, B. (2001): Biodegradability of metronidazole, olaquindox, and tylosin and formation of tylosin

164

Thiele-Bruhn

J. Plant Nutr. Soil Sci. 2003, 166, 145167

Kmmerer, K. (2000): Drugs, diagnostic agents and disinfectants in wastewater and water a review. Schriftenr. Ver. Wasser Boden Lufthyg. 105, 5971. Kmmerer, K. (2001a): Drugs in the environment: emission of drugs, diagnostic aids and disinfectants into wastewater by hospitals in relation to other sources a review. Chemosphere 45, 957969. Kmmerer, K. (2001b): Pharmaceuticals in the environment sources, fate, effects and risks. Springer, Berlin, p. 265. Kmmerer, K., Al Ahmad, A., and Mersch-Sundermann, V. (2000): Biodegradability of some antibiotics, elimination of the genotoxicity and affection of wastewater bacteria in a simple test. Chemosphere 40, 701710. Lancini, G., and Parenti, F. (1982): Antibiotics: An integrated View. Springer, New York, Heidelberg, p. 253. Langhammer, J.-P. (1989): Untersuchungen zum Verbleib antimikrobiell wirksamer Arzneistoffe als Rckstnde in Glle und im landwirtschaftlichen Umfeld. PhD thesis, Univ. Bonn, p. 138. Langhammer, J.-P., and Bning-Pfaue, H. (1989): Bewertung von Arzneistoff-Rckstnden aus der Glle im Boden. Lebensmittelchem. Gerichtl. Chem. 43, 103113. Langhammer, J.-P., Fhr, F., and Bning-Pfaue, H. (1990): Verbleib von Sulfonamid-Rckstnden aus der Glle in Boden und Nutzpflanze. Lebensmittelchemie 44, 93. Langlois, B. E., Cromwell, G. L., and Hays, V. W. (1978): Influence of chlortetracycline in swine feed on reproductive-performance and on incidence and persistence of antibiotic resistant enteric bacteria. J. Animal Sci. 46, 13691382. Lindsey, M. E., Meyer, T. M., and Thurman, E. M. (2001): Analysis of trace levels of sulfonamide and tetracycline antimicrobials in groundwater and surface water using solid-phase extraction and liquid chromatography/mass spectrometry. Anal. Chem. 73, 46404646. Linke, I., and Kratz, W. (2001): Tierarzneimittel in der Umwelt Erhebung von Tierarzneimittelmengen im Land Brandenburg fr den Zeitraum von Juli 1998 bis Juni 1999. LUA Brandenburg, Potsdam, p. 34. Lo, I. H., and Hayton, W. L. (1981): Effects of pH on the accumulation of sulfonamides by fish. J. Pharmacokin. Biopharm. 9, 443459. Loke, M. L., Ingerslev, F., Halling-Srensen, B., and Tjrnelund, J. (2000): Stability of tylosin A in manure containing test systems determined by high performance liquid chromatography. Chemosphere 40, 759765. Loke, M. L., Tjrnelund, J., and Halling-Srensen, B. (2002): Determination of the distribution coefficient (log Kd) of oxytetracycline, tylosin A, olaquindox and metronidazole in manure. Chemosphere 48, 351361. Loke, M.-L., Jespersen, S., Vreeken, R., Halling-Srensen, B., and Tjrnelund, J. (2003): Determination of oxytetracycline and its degradation products by high-performance liquid chromatographytandem mass spectrometry in manure containing anaerobic test systems. J. Chromatogr. B 783, 1123. Lscher, W., Ungemach, F. R., and Kroker, R. (1994): Grundlagen der Pharmakotherapie bei Haus- und Nutztieren. P. Parey, Berlin, p. 437. Lumsden, R. D., Locke, J. C., Adkins, S. T., Walter, J. F., and Rideout, C. J. (1992): Isolation and localization of the antibiotic gliotoxin produced by Gliocladium virens from alginate prill in soil and soilless media. Phytopathol. 82, 230235. Lunestad, B. T., Tore, B., Samuelsen, O. B., Fjelde, S., and Ervik, A. (1995): Photostability of eight antibacterial agents in seawater. Aquaculture 134, 217225.

degradation products in aerobic soil-manure slurries. Ecotoxicol. Environ. Saf 48, 311320. Ingerslev, F., Torang, L., Loke, M. L., Halling-Srensen, B., and Nyholm, N. (2001): Primary biodegradation of veterinary antibiotics in aerobic and anaerobic surface water simulation systems. Chemosphere 44, 865872. Ingham, E. R., and Coleman, D. C. (1984): Effects of streptomycine, cycloheximide, fungizone, captan, carbofuran, cygon, and PCNB on soil microorganisms. Microb. Ecol. 10, 345358. Ingham, E. R., Cambardella, C., and Coleman, D. C. (1986): Manipulation of bacteria, fungi and protozoa by biocides in lodgepole pine forest soil microcosms effects on organism interactions and nitrogen mineralization. Can. J. Soil Sci. 66, 261272. Jacobsen, P., and Berglind, L. (1988): Persistence of oxytetracycline in sediment from fish farms. Aquaculture 70, 365370. Jagnow, G. (1977): Mikrobieller Abbau der Futtermittelantibiotika Zink-Bacitracin, Flavophospholipol, Spiramycin und von Tetracyclin in feucht gelagertem und in mit Boden vermischtem Hhnerkot. Landwirtsch. Forsch.1, 227234. Jefferys, E. G. (1952): The stability of antibiotics in soils. J. Gen. Microbiol. 7, 295312. Jensen, J., Krogh, P. H., and Sverdrup, L. (2001a): Environmental risk of veterinary medicines to soil fauna. SETAC Europe 11th Annual Meeting, 610 May, Madrid. Jensen, L. B., Baloda, S., Boye, M., and Aarestrup, F. M. (2001b): Antimicrobial resistance among Pseudomonas spp. and the Bacillus cereus group isolated from Danish agricultural soil. Environ. Int. 26, 581587. Jjemba, P. K. (2002): The potential impact of veterinary and human therapeutic agents in manure and biosolids on plants grown on arable land: a review. Agric. Ecosyst. Environ. 93, 267278. Johannsen, F. H. (1991): Zur Analytik von Wirkstoffen. 2. Bestimmung von Ionophor-Antibiotika in Futtermitteln und Lebensmitteln mittels Hochleistungsflssigchromatographie und Nachsulenderivatisierung. Agribiol. Res. 44, 7989. Jrgensen, S. E., and Halling-Srensen, B. (2000): Drugs in the environment. Chemosphere 40, 691699. Juhel-Gaugain, M., McEvoy, J. D., and van Ginkel, L. A. (2000): Measurements for certification of chlortetracycline reference materials within the European Union: Standards, measurements and testing programme. Fres. J. Anal. Chem. 368, 656663. Kamel, A. M., Brown, P. R., and Munson, B. (1999): Electrospray ionization mass spectrometry of tetracycline, oxytetracycline, chlorotetracycline, minocycline, and methacycline. Anal. Chem. 71, 968977. Katz, J. M., and Katz, S. E. (1983): Microbial assay systems for determining antibiotic residues in soils. J. Assoc. Anal. Chem. 66, 640645. Kennedy, D. G., Cannavan, A., and McCracken, R. J. (2000): Regulatory problems caused by contamination, a frequently overlooked cause of veterinary drug residues. J. Chromatogr. A 882, 3752. Kolpin, D. W., Furlong, E. T., Meyer, M. T., Thurman, E. M., Zaugg, S. D., Barber, L. B., and Buxton, H. T. (2002): Pharmaceuticals, hormones, and other organic wastewater contaminants in US streams, 19992000: A national reconnaissance. Environ. Sci. Technol. 36, 12021211. Khne, M., Ihnen, D., Mller, G., and Agthe, O. (2000): Stability of tetracycline in water and liquid manure. J. Vet. Med. A 47, 379384.

J. Plant Nutr. Soil Sci. 2003, 166, 145167

Madsen, E. L., Francis, A. J., and Bollag, J. M. (1988): Environmental factors affecting indole metabolism under anaerobic conditions. Appl. Environ. Microbiol. 54, 7478. Marengo, J. R., Kok, R. A., OBrien, K., Velagaleti, R. R., and Stamm, J. M. (1997): Aerobic biodegradation of (14 C)-sarafloxacin hydrochloride in soil. Environ. Toxicol. Chem. 16, 462471. Martens, R., Wetzstein, H. G., Zadrazil, F., Capelari, M., Hoffmann, P., and Schmeer, N. (1996): Degradation of the fluoroquinolone enrofloxacin by wood-rotting fungi. Appl. Environ. Microbiol. 62, 42064209. McCoy, R. E. (1976): Uptake, translocation, and persistence of oxytetracycline in coconut palm. Phytopathol. 66, 10381042. McCracken, D. I., and Foster, G. N. (1993): The effect of ivermectin on the invertebrate fauna associated with cow dung. Environ. Toxicol. Chem. 12, 7384. McGrath, J. W., Hammerschmidt, F., and Quinn, J. P. (1998): Biodegradation of phosphonomycin by Rhizobium huakuii PMY1. Appl. Environ. Microbiol. 64, 356358. McManus, P. S., Stockwell, V. O., Sundin, G. W., and Jones, A. L. (2002): Antibiotic use in plant agriculture. Annu. Rev. Phytopathol. 40, 443465. Midtvedt, T. (2001): Antibiotics in the Environment: Zinc bacitracin environmental toxicity and breakdown. In K. Kmmerer: Pharmaceuticals in the Environment: Sources, Fate, Effects and Risks. Springer, Berlin, pp. 7779. Migliore, L., Brambilla, G., Grassitellis, A., and Dojmi di Delupis, G. (1993): Toxicity and bioaccumulation of sulphadimethoxine in Artemia (Crustacea, Anostraca). Int. J. Salt Lake Res. 2, 141152. Migliore, L., Brambilla, G., Cozzolino, S., and Gaudio, L. (1995): Effect on plants of sulphadimethoxine used in intensive farming (Panicum miliaceum, Pisum sativum and Zea mays). Agric. Ecosyst. Environ. 52, 103110. Migliore, L., Brambilla, G., Casoria, P., Civitareale, C., Cozzolino, S., and Gaudio, L. (1996): Effects of antimicrobials for agriculture as environmental pollutants. Fres. Envir. Bull. 5, 735739. Migliore, L., Civitareale, C., Cozzolino, S., Casoria, P., Brambilla, G., and Gaudio, L. (1998): Laboratory models to evaluate phytotoxicity of sulphadimethoxine on terrestrial plants. Chemosphere 37, 29572961. Mitscher, L. A. (1978): The Chemistry of the Tetracycline Antibiotics. Marcel Dekker, Basel, p. 330. Mudd, A. J., Lawrence, K., and Walton, J. (1998): Study of Swedens model on antimicrobial use shows usage has increased since 1986 ban. Feedstuffs 10. National Institutes of Health (1999): U.S. National Library of Medicine. Toxnet Toxicology Data Network. http://www.toxnet.nlm.nih.gov. Niessen, W. M. A. (1998): Analysis of antibiotics by liquid chromatography mass spectrometry. J. Chromatogr. A 812, 5375. NOAH (2002): Sales of antimicrobial Products used as veterinary Medicines, Growth Promoters and Coccidiostats in the UK in 2000. National Office of Animal Health, UK. Nowara, A., Burhenne, J., and Spiteller, M. (1997): Binding of fluoroquinolone carboxylic acid derivatives to clay minerals. J. Agric. Food Chem. 45, 14591463. Nygaard, K., Lunestad, B. T., Hektoen, H., Berge, J. A., and Hormazabal, V. (1992): Resistance to oxytetracycline, oxolinic acid and furazolidone in bacteria from marine sediments. Aquaculture 104, 3136. OECD (1997): Adsorption/desorption using a batch equilibrium method. OECD guidelines for testing of chemicals, test guideline

Antibiotic in soils

165

106, revised Draft Document. Organization for Economic Cooperation and Development, Paris. Oka, H., Ikai, Y., Kawamura, N., and Hayakawa, J. (1991): Limited survey of residual tetracyclines in tissues collected from diseased animals in Aichi prefecture, Japan. J. Assoc. Off. Anal. Chem. Int. 74, 894896. Oka, H., Ikai, Y., Ito, Y., Hayakawa, J., Harada, K., Suzuki, M., Odani, H., and Maeda, K. (1997): Improvement of chemical analysis of antibiotics. XXIII. Identification of residual tetracyclines in bovine tissues by electrospray high-performance liquid chromatographytandem mass spectrometry. J. Chromatogr. B 693, 337344. Oka, H., Ito, Y., and Matsumoto, H. (2000): Chromatographic analysis of tetracycline antibiotics in foods. J. Chromatogr. A 882, 109133. Osol, A. (1980): Remingtons Pharmaceutical Sciences. Mack Publishing Co., Easton, PA, p. 1928. Patten, D. K., Wolf, D. C., Kunkle, W. E., and Douglass, L. W. (1980): Effect of antibiotics in beef cattle feces on nitrogen and carbon mineralization in soil and on plant growth and composition. J. Environ. Qual. 9, 167172. Pfeiffer, C., Emmerling, C., Schrder, D., and Niemeyer, J. (1998): Antibiotika (Ivermectin, Monensin) und endokrine Umweltchemikalien (Nonylphenol, Ethinylstradiol) im Boden: Mgliche Auswirkungen von synthetischen Umweltchemikalien auf mikrobielle Eigenschaften eines landwirtschaftlich genutzten Bodens. Umweltwiss. Schadst. Forsch. 10, 147153. Pinck, L. A., Holton, W. F., and Allison, F. E. (1961a): Antibiotics in soils: 1. Physico-chemical studies of antibiotic-clay complexes. Soil Sci. 91, 2228. Pinck, L. A., Soulides, D. A., and Allison, F. E. (1961b): Antibiotics in soils: II. Extent and mechanisms of release. Soil Sci. 91, 9499. Pinck, L. A., Soulides, D. A., and Allison, F. E. (1962): Antibiotics in soils: 4. Polypeptides and macrolides. Soil Sci. 94, 129131. Rablle, M,. and Spliid, N. H. (2000): Sorption and mobility of metronidazole, olaquindox, oxytetracycline and tylosin in soil. Chemosphere 40, 715722. Richardson, M. L., and Bowron, J. M. (1985): The fate of pharmaceutical chemicals in the aquatic environment. J. Pharm. Pharmacol. 37, 112. Richter, A., Lscher, W., and Witte, W. (1996): Leistungsfrderer mit antibakterieller Wirkung: Probleme aus pharmakologisch-toxikologischer und mikrobiologischer Sicht. Prakt.Tierarzt 7, 603624. Rmbke, J., Knacker, T., and Stahlschmidt-Allner, P. (1996): Umweltprobleme durch Arzneimittel Literaturstudie. UBA Texte, Berlin, p. 341. Runsey, T. S., Miller, R. W., and Dinius, D. A. (1977): Residue content of beef feedlot manure after feeding diethylstilbestrol, chlortetracycline and Ronnel and the use of stirofos to reduce population of fly larvae in feedlot manure. Arch. Environ. Contam. Toxicol. 6, 203212. Salvatore, M. J., and Katz, S. E. (1993): Solubility of antibiotics used in animal feeds in selected solvents. J. Assoc. Off. Anal. Chem. Int. 76, 952956. Samuelsen, O. B. (1994): Simultaneous determination of ormethoprim and sulphadimethoxine in plasma and muscle of Atlantic salmon (Salmo salar). J. Chromatogr. B 660, 412417. Samuelsen, O. B., Torsvik, V, and Ervik, A. (1992): Long-range changes in oxytetracycline concentration and bacterial resistance toward oxytetracycline in a fish farm sediment after medication. Sci. Total Environ. 114, 2536. Samuelsen, O. B., Lunestad, B. T., Ervik, A., and Fjelde, S. (1994): Stability of antibacterial agents in an artificial marine aquaculture

166

Thiele-Bruhn

J. Plant Nutr. Soil Sci. 2003, 166, 145167

human pharmaceuticals in Denmark after normal therapeutic use. Chemosphere 40, 783793. Suan, D. T., and Dmitrenko, L. V. (1994a): Effect of the structure of sulfocationites on the sorption of the antibiotic tetracycline. Appl. Biochem. Microbiol. 30, 629633. Suan, D. T., and Dmitrenko, L. V. (1994b): Sorption kinetics of the antibiotic oxytetracycline on ion exchange materials. Appl. Envir. Microbiol. 30, 634636. Smuth, R., Eberspcher, J., Haag, R., and Springer, W. (1987): Biochemisch-mikrobiologisches Praktikum. Georg Thieme Verlag, Stuttgart, p. 409. Syracuse Research Corporation (2001): Environmental Fate Data Base. http://esc.syrres.com/efdb.htm.. Thiele, S. (2000): Adsorption of the antibiotic pharmaceutical compound sulfapyridine by a long-term differently fertilized loess Chernozem. J. Plant Nutr. Soil Sci. 163, 589594. Thiele, S., and Beck, I.-C. (2001): Wirkungen pharmazeutischer Antibiotika auf die Bodenmikroflora Bestimmung mittels ausgewhlter bodenbiologischer Testverfahren. Mitteilgn. Dtsch. Bodenkundl. Gesellsch. 96, 383384. Thiele, S., and Leinweber, P. (2000): Bedeutung der Huminstoffe fr Bindung und Umsatz organischer Fremdstoffe am Beispiel ausgewhlter Veterinrantibiotika. Rostocker Agr. Umweltwiss. Beitr. 8, 265273. Thiele, S., Seibicke, T., and Leinweber, P. (2002): Sorption of sulfonamide antibiotic pharmaceuticals in soil particle size fractions. SETAC Europe 12th Annual Meeting, 12 16 May 2002, Vienna. Thiele-Bruhn, S., and Aust, M. O. (2003): Effects of slurry amendment on soil sorption of sulfonamide antibiotics, in preparation. Thiele-Bruhn, S., Mogk, A., and Freitag, D. (2003a): Einsatz von Tierarzneimitteln zur Anwendung bei landwirtschaftlichen Nutztieren in Mecklenburg-Vorpommern. Ber. Landwirtsch., submitted. Thiele-Bruhn, S., Peters, D., Halling-Srensen, B., and Leinweber, P. (2003b): Photodegradation and ageing of antibiotic pharmaceuticals on soil surfaces. ENVIRPHARMA, European Conference on Human and Veterinary Pharmaceuticlas in the Environment, 14 16 April 2003. Thomashow, L. S., Bonsall, R. F., and Weller, D. M. (1997): Antibiotic production by soil and rhizosphere microbes in situ. In C. J. Hurst, G. R. Knudson, M. J. McInerney, L. D. Stetzenbach and M. V. Walter: Manual of environmental microbiology. ASM Press, Washington, D.C., pp. 493499. Tjrnelund, J., Jensen, R. B., Ma, H. P., and Halling-Srensen, B. (2000): Sorption of chlortetracycline and its decomposition products on soil. Dansk Kemi 81, 1214. Tolls, J. (2001): Sorption of veterinary pharmaceuticals in soils: a review. Environ. Sci. Technol. 35, 33973406. Tolls, J., Gebbink, W., and Cavallo, R. (2002): pH-dependence of sulfonamide antibiotic sorption: data and model evaluation. SETAC Europe 12th Annual Meeting 12 16 May 2002, Vienna. Tomlinson, G., Albuquerque, C. A., and Woods, R. A. (1985): The effects of amidantel (Bay D-8815) and its deacylated derivative (Bay D-9216) on Caenorhabditis-Elegans. Europ. J. Pharmacol. 113, 255262. Topp, W. (1981): Biologie der Bodenorganismen. Quelle & Meier UTB, Heidelberg, p. 224. Ungemach, F. R. (2000): Figures on quantities of antibacterials used for different purposes in the EU countries and interpretation. Acta Vet. Scand. Suppl 93, 8997.

sediment studied under laboratory conditions. Aquaculture 126, 283290. Schadewinkel-Scherkl, A.-M., and Scherkl, R. (1995): Antibiotika und Chemotherapeutika in der tierrztlichen Praxis. Gustav Fischer, Jena, Stuttgart, p. 153. Schller, S. (1998): Anwendung antibiotisch wirksamer Substanzen beim Tier und Beurteilung der Umweltsicherheit entsprechender Produkte. 3. Statuskolloquium kotoxikologischer Forschungen in der Euregio Bodensee, 3 4 December 1998. Schulten, H. R. (2002): New approaches to the molecular structure and properties of soil organic matter: humic-, xenobiotic-, biological-, and mineral-bonds. In Violante, A., Huang, P. M., Bollag, J.-M., and Gianfreda, L.: Developments in Soil Science. Soil Mineral-Organic Matter-Microorganisms Interactions and Ecosystem Health 28A, Elsevier, Amsterdam, pp. 351381. Schwabe, U., and Paffrath, D. (2001): Arzneiverordnungs-Report 2001. Springer Verlag, Berlin, Heidelberg, p. 982. Sczesny, S., Nau, H., and Hamscher, G. (2003): Residue analysis of tetracyclines and their metabolites in eggs and in the environment by HPLC coupled with a microbiological assay and tandem mass spectrometry. J. Agric. Food Chem. 51, 697703. Sengelv, G., Agerso, Y., Halling-Srensen, B., Baloda, S. B., Andersen, J. S., and Jensen, L. B. (2003): Bacterial antibiotic resistance levels in Danish farmland as a result of treatment with pig manure slurry. Environ. Int. 28, 587595. Seveno, N. A., Kallifidas, D., Smalla, K., van Elsas, J. D., Collard, J. M., Karagouni, A. D., and Wellington, E. M. H. (2002): Occurrence and reservoirs of antibiotic resistance genes in the environment. Rev. Med. Microbiol. 13, 1527. Shanahan, P., Borro, A., OGara, F., and Glennon, J. D. (1992): Isolation, trace enrichment and liquid chromatographic analysis of diacetylphloroglucinol in culture and soil samples using UV and amperometric detection. J. Chromatogr. A 606, 171177. Sithole, B. B., and Guy, R. D. (1987a): Models for oxytetracycline in aquatic environments. I. Interaction with bentonite clay systems. Water Air Soil Pollut. 32, 303314. Sithole, B. B., and Guy, R. D. (1987b): Models for oxytetracycline in aquatic environments. II. Interactions with humic substances. Water Air Soil Pollut. 32, 315321. Smalla, K., and Sobecky, P. A. (2002): The prevalence and diversity of mobile genetic elements in bacterial communities of different environmental habitats: insights gained from different methodological approaches. FEMS Microbiol. Ecol. 42, 165175. Smalla, K., Heuer, H., Gotz, A., Niemeyer, D., Krogerrecklenfort, E., and Tietze, E. (2000): Exogenous isolation of antibiotic resistance plasmids from piggery manure slurries reveals a high prevalence and diversity of IncQ-like plasmids. Appl. Environ. Microbiol. 66, 48544862. Smith, P., and Samuelsen, O. B. (1996): Estimates of the significance of out-washing of oxytetracycline from sediments under Atlantic salmon sea-cages. Aquaculture 144, 1726. Soulides, D. A. (1965): Antibiotics in soils. VII. Production of streptomycin and tetracyclines in soils. Soil Sci. 100, 200206. Soulides, D. A., Pinck, L. A., and Allison, F. E. (1962): Antibiotics in soils: V. Stability and release of soil-adsorbed antibiotics. Soil Sci. 4, 239244. Spaepen, K. R. I., van Leemput, L. J. J., Wislocki, P. G., and Verschueren, C. (1997): A uniform procedure to estimate the predicted environmental concentration of the residues of veterinary medicines in soil. Environ. Toxicol. Chem. 16, 19771982. Stuer-Lauridsen, F., Birkved, M., Hansen, L. P., Ltzhft, H. C., and Halling-Srensen, B. (2000): Environmental risk assessment of

J. Plant Nutr. Soil Sci. 2003, 166, 145167

van den Bogaard, A. E., London, N., and Stobberingh, E. E. (2000): Antimicrobial resistance in pig faecal samples from the Netherlands (five abattoirs) and Sweden. J. Antimicrob. Chemother. 45, 663671. van Dijck, P., and van de Voorde, H. (1976): Sensitivity of environmental microorganisms to antimicrobial agents. Appl. Environ. Microbiol. 31, 332336. van Gool, S. (1993): Possible effects on the environment of antibiotic residues in animal manure. Tijdschr. Diergeneeskd. 118, 810. Warman, P. R. (1980): The effect of amprolium and aureomycin [antibiotic] on the nitrification of poultry manure-amended soil. Soil. Sci. Soc. Am. J. 44, 13331334. Watts, C. D., Crathorne, B., Fielding, M., and Killops, S. D. (1982): Non-volatile organic-compounds in treated waters. Environ. Health Persp. 46, 8799. Weerasinghe, C. A., and Towner, D. (1997): Aerobic biodegradation of virginiamycin in soil. Environ. Toxicol. Chem. 16, 18731876. Wegener, H. C., Aarestrup, F. M., Jensen, L. B., Hammerum, A. M., and Bager, F. (1998): The association between the use of antimicrobial growth promoters and development of resistance in pathogenic bacteria towards growth promoting and therapeutic antimicrobials. J. Animal Feed Sci. 7, 714. Weimann, A., and Bojesen, G. (1999): Analysis of tetracyclines in raw urine by column-switching high-performance liquid chromatography and tandem mass spectrometry. J. Chromatogr. B 721, 4754. Wessels, J. M., Ford, W. E., Szymczak, W., and Schneider, S. (1998): The complexation of tetracycline and anhydrotetracycline with Mg2+ and Ca2+: a spectroscopic study. J. Phys. Chem. B 102, 93239331. Wetzstein, H. G. (2001): Chinolone in der Umwelt: Biologische Abbaubarkeit der Gyrasehemmer. Pharmazie in unserer Zeit 30, 450457. Wetzstein, H. G., Schmeer, N., and Karl, W. (1997): Degradation of the fluoroquinolone enrofloxacin by the brown rot fungus Gloeophyllum striatum: identification of metabolites. Appl. Environ. Microbiol. 63, 42724281.

Antibiotic in soils

167

Wetzstein, H. G., Stadler, M., Tichy, H. V., Dalhoff, A., and Karl, W. (1999): Degradation of ciprofloxacin by basidiomycetes and identification of metabolites generated by the brown rot fungus Gloeophyllum striatum. Appl. Environ. Microbiol. 65, 15561563. Wetzstein, H. G., Karl, W., Hallenbach, W., Himmler, T., and Petersen, U. (2000): Residual antibacterial activity of metabolites derived from the veterinary fluroquinolone enrofloxacin. 100th General Meeting of the American Society for Microbiology, Los Angeles, CA. Wetzstein, H. G., Schneider, S., and Karl, W. (2002): Kinetics of the biotransformation of enrofloxacin in aging cattle dung. 102nd General Meeting of the American Society for Microbiology, Salt Lake City, UT. Williams, S. T. (1982): Are antibiotics produced in soil? Pedobiologia 23, 427435. Winckler, C., and Grafe, A. (2000): Stoffeintrag durch Tierarzneimittel und pharmakologisch wirksame Futterzusatzstoffe unter besonderer Bercksichtigung von Tetrazyklinen. UBA-Texte 44/00, Berlin, p. 145. Winckler, C., and Grafe, A. (2001): Use of veterinary drugs in intensive animal production: evidence for persistence of tetracycline in pig slurry. J. Soils Sed. 1, 6670. Wollenberger, L., Halling-Srensen, B., and Kusk, K. O. (2000): Acute and chronic toxicity of veterinary antibiotics to Daphnia magna. Chemosphere 40, 723730. Yeager, R. L., and Halley, B. A. (1990): Sorption/desorption of [14C]efrotomycin with soils. J. Agric. Food Chem. 38, 883886. Zhu, J., Snow, D. D., Cassada, D. A., Monson, S. J., and Spalding, R. F. (2001): Analysis of oxytetracycline, tetracycline, and chlortetracycline in water using solid-phase extraction and liquid chromatography-tandem mass spectrometry. J. Chromatogr. A 928, 177186. Zuccato, E., Bagnati, R., Fioretti, F., Natangelo, M., Calamari, D., and Fanelli, R. (2001): Environmental loads and detection of pharmaceuticals in Italy, in K. Kmmerer: Pharmaceuticals in the Environment Sources, Fate, Effects and Risks. Springer, Berlin, pp. 1927.