Chemosphere 60 (2005) 497507 www.elsevier.

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Column studies to investigate the fate of veterinary antibiotics in clay soils following slurry application to agricultural land
Paul Kay *, Paul A. Blackwell 1, Alistair B.A. Boxall
Craneld Centre for EcoChemistry, Craneld University, Shardlow Hall, Shardlow, Derby DE72 2GN, UK Received 20 May 2004; received in revised form 14 January 2005; accepted 18 January 2005 Available online 23 February 2005

Abstract The environment may be exposed to veterinary medicines administered to livestock due to the application of organic fertilisers to land. For other groups of substances that are applied to agricultural land (e.g. pesticides), preferential ow in underdrained clay soils has been identied as an extremely important pathway by which pollution of surface waters can occur. Three soil column leaching experiments have therefore been carried out using a clay soil to investigate the fate of a range of antibiotics from the sulphonamide, tetracycline and macrolide groups. These column studies complemented a range of other experiments at the eld and semi-eld scales, as well as modelling studies which are being reported in separate papers. Each column study had a dierent objective. The rst examined the eect of pig slurry on the mobility of antibiotics in clay loam soil. The second experiment investigated changes in soil water pH due to the application of slurry. The nal experiment quantied the extent to which soil tillage prior to slurry application can reduce the leaching of antibiotic residues found in slurry. It was found that slurry had no impact on the leaching of oxytetracycline although soil water pH was aected signicantly by slurry application. It was also shown that pretillage can substantially reduce the leaching of antibiotic residues through macroporous clay soils. 2005 Elsevier Ltd. All rights reserved.
Keywords: Veterinary antibiotics; Column studies; Clay soil; Tillage; Slurry

1. Introduction Veterinary medicines are used in agriculture to prevent disease in livestock and treat illness. Following
* Corresponding author. Present address: ADAS Gleadthorpe, Meden Vale, Manseld, Nottingham NG20 9PF, UK. Tel.: +44 0 1623 844331; fax: +44 0 1623 844472. E-mail address: paul.kay@adas.co.uk (P. Kay). 1 Present address: Environment Agency, National Laboratory Service, Nottingham Laboratory, Meadow Lane, Nottingham NG2 3HN, UK.

administration, quantities of these drugs may be excreted as the parent compound and/or metabolites and enter the environment due to the spreading of manure and slurry on agricultural land, or due to direct deposition by grazing livestock (Halling-Srenson et al., 1998). The presence of veterinary medicines in the environment may be a cause for concern as they are designed to have a biological eect (Halling-Srenson et al., 1998). These chemicals may aect terrestrial and aquatic organisms (Wollenberger et al., 2000) as well as play a role in the development of antimicrobial resistance (Chee-Sanford et al., 2001). Data exist which show that

0045-6535/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2005.01.028

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residues may be present in manure and slurry applied to land (Haller et al., 2002). Some studies have subsequently measured concentrations of veterinary antibiotics in soil, surface water and groundwater (Hirsch et al., 1999; Hamscher et al., 2002). A considerable amount of work has been carried out on the environmental fate of pesticides in clay soils (e.g. Kladivko et al., 1991; Heppell et al., 1999, 2002). These studies have shown that the application of chemicals to underdrained clay soils poses a signicant risk to surface water quality. This is due to by-pass ow via macropores which allows substances to reach sub-surface drains without interacting with the surrounding soil to the extent which they otherwise would. It therefore seems possible that the application of manure and slurry to such soils will create the greatest risk of surface water contamination by antibiotic residues in the manure/slurry. A recent eld study has indeed shown that veterinary antibiotics applied to macroporous clay soils can be rapidly transported to surface waters in drainow (Kay et al., 2004). There remain gaps in the information available however. No data have been produced to describe how slurry aects the environmental fate of veterinary drugs. The fact that veterinary antibiotics will be present in the environment in combination with manure and slurry makes them dierent to pesticides and this is one of the reasons why further investigations into their fate are important. Previous work on pesticides (Edwards and Daniel, 1993; Worrall et al., 1995; de Jonge et al., 1998; Hesketh et al., 2001) as well as polynuclear aromatic hydrocarbons (Magee et al., 1991) has found that the mobility of highly sorptive substances will increase in the presence of colloidal matter and dissolved organic carbon (DOC). As manure and slurry are particularly rich in these materials (Hsu and Lo, 1999; Lopez Periago et al., 2000) it may be expected that their presence would signicantly increase the mobility of antibiotics with high sorption coecients. A further implication of the presence of manure/ slurry may be that the speciation of a veterinary medicine is aected by changes in soil water pH, caused by the high levels of ammonia present in the manure (Tolls, 2001). Studies that have quantied pH changes resulting from manure application are also lacking from the literature and so a further column study has been carried out to investigate this phenomenon. Practical measures to reduce the risk of surface water pollution by veterinary antibiotic residues are obviously highly desirable. The aforementioned eld study (Kay et al., 2004) indicated that soil tillage prior to slurry application may oer a means of reducing the transport of veterinary antibiotic residues to tile drains and surface water. It was therefore deemed worthwhile to further investigate this phenomenon in a more replicated fashion using laboratory soil columns.

Laboratory soil columns oer the potential for more replication than eld and semi-eld studies as it is easier to collect and manage multiple small cores. It is also easier to investigate particular processes as small soil cores can be manipulated more easily. Soil column leaching tests are a common laboratory scale tool for assessing the fate of chemicals in the environment (Lynch, 1995; Sadeghi et al., 2000). It is envisaged that these studies will provide information that can be used by scientists and policy makers to further research and to assess the eectiveness of present environmental management strategies which aim to reduce the environmental impact of veterinary medicines. These include environmental risk assessment procedures and agricultural codes of practise.

2. Materials and methods 2.1. Study compounds The test substances investigated in the study were chosen as they are commonly used in veterinary medicine (Veterinary Medicines Directorate, 2002), have a range of sorption and degradation characteristics (e.g. Rablle and Spliid, 2000; Ingerslev et al., 2001) and are largely excreted as the parent molecule (Partt, 1999). They comprised three antibiotics (Table 1), which are used in greater quantities than other classes of veterinary medicine (Boxall et al., 2002; Koschorreck et al., 2002). These were oxytetracycline (OTC), sulphachloropyridazine (SCP) and tylosin (TYL). 2.2. Test system A clay loam soil collected from an agricultural eld in Leicestershire, UK, was used for all three column experiments. The properties of the soil have already been reported in Kay et al. (2004). The test system varied slightly in each experiment. The soil chromatography test detailed in Lynch (1995) was used in the rst experiment to investigate the eect of slurry on the mobility of the most sorptive study compound, OTC. Only OTC was investigated in this experiment as the presence of DOC and colloidal matter is likely to have a much greater eect on the mobility of substances with high sorption coecients. This test system is commonly used as a screening test for observing the leaching potential of different pesticides relative to each other. The method employs soil columns of 30 cm length and 5 cm diameter which contain repacked air dried and sieved (<2 mm) soil. Many other studies have employed a similar test system using repacked dried and sieved soil to study the mobility of pesticides and slurry constituents (Nunez-Delgado et al., 1997; de Jonge and de Jonge, 1999; Novak, 1999; Lopez Periago et al., 2000; Worrall

P. Kay et al. / Chemosphere 60 (2005) 497507 Table 1 The study compounds used in the soil column experiments Test substance Oxytetracycline Structure Water sol. (g l1)a 0.3 Koc (l kg1)b 27 79293 317d DT50 slurry (d)c 79e

499

DT50 soil (d)c 18.2e

Sulphachloropyridazine

8.2

4182f

127e

3.5e

Tylosin
O HO O O O O
a b c d e f g h

O O N HO O O OH OH O O OH

5537988d

<2g

3.38.1h

Boxall, A.B.A., personal communication. Organic carbon sorption coecient. Degradation half-life. Rablle and Spliid (2000). Blackwell, P., personal communication. Boxall et al. (2002). Loke et al. (2000). Ingerslev and Halling-Srensen (2001).

et al., 2001; Haberhauer et al., 2002). One of the advantages of this kind of test is that the eects of soil structure, which in macroporous clay soils is highly variable, are eliminated and, thus, dierent observations can be attributed more easily to contrasting soil treatments or chemical properties than when using undisturbed soils (Lynch, 1995). Whilst this sort of test system is useful for comparing the mobility of dierent chemicals or the same chemical under dierent treatments, as was the aim of the rst study, the system may not directly mimic the processes occurring in the environment. Undisturbed soil cores were therefore used for the following studies where pH values and antibiotic concentrations likely to be present in the environment were required. Undisturbed soil cores were collected using a metal Coremaster barrel (5 cm internal diameter) (Hampshire Glass Limited, Hampshire, UK) with an acetate sleeve inserted. The barrel was attached to a metal coring

handle and manually hammered into the soil to the top of the barrel and then rotated through 180 to break the base of the core from the surrounding soil before extraction. The acetate sleeve was removed from the barrel and the soil core returned to the laboratory in an upright position. In the pH experiment the cores were 10 cm in length although in the tillage experiment the cores were 20 cm in length due to the presence of the tilled layer. For the tillage experiment, an articial macropore was created from the top to the bottom of each of the cores to mimic conditions observed when slurry is usually applied to arable clay soils in autumn (Kladivko et al., 1991; Johnson et al., 1996). This was done by inserting and withdrawing a metal rod of 6 mm diameter through the soil. In half of the cores, the top 10 cm of soil was disturbed to mimic the eect of pre-tillage (discing). This was done using a handheld trowel to create aggregates with a long axis length of 23 cm (Fig. 1).

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(a)
Soil surface

(b)
Soil surface

Top 10 cm of soil tilled

Acetate sleeve

Acetate sleeve

Soil

Soil

Artificial macropore Nylon mesh

Artificial macropore Nylon mesh

HDPE funnel 500 ml amber glass bottle

HDPE funnel 500 ml amber glass bottle

Fig. 1. Experimental setup showing a soil core with the articial macropore in contact with the soil surface (a) and the soil tilled to 10 cm depth (b).

2.3. Slurry and antibiotic application Slurry from a working pig farm in Leicestershire, UK, was used in the experiments. The pigs were treated continuously with tylosin at 100 g/tonne of feed and the slurry had been stored for between 0 and 3 months in concrete lined pits under the houses. Where OTC and SCP were applied to the soil columns, the slurry was spiked with the compounds at concentrations predicted using the model developed by Spaepen et al. (1997). These were 18.85 mg l1 for OTC and 25.58 mg l1 for SCP (nominal concentrations). These concentrations are within the same order of magnitude as peak concentrations measured in slurry in recent studies for the sulphonamides (Campagnolo et al., 2002; Haller et al., 2002) and tetracyclines (Hamscher et al., 2002). The slurry was applied at a rate of 45 000 l ha1, in-line with normal agricultural practise (Kay et al., 2004), using a glass volumetric ask. In the rst experiment (eect of slurry on OTC leaching) the columns received three dierent treatments. Three columns were treated with an OTC/slurry mixture, three columns received the OTC (in MeOH) and slurry separately and a further three columns received

the OTC solution but no slurry. A further soil column did not receive the OTC or slurry and acted as a control. As these dierent treatments meant that varying quantities of liquid were applied to the dierent cores, 0.01 M CaCl2 solution was applied to some columns so that the moisture content of each remained the same. In the second experiment (eect of slurry on soil water pH) half (7) of the soil cores were amended with slurry whilst the other half were not. In the tillage experiment, slurry containing OTC, SCP and TYL was applied to three undisturbed cores and three tilled cores. One undisturbed core and one which had been tilled remained untreated as controls. 2.4. Irrigation The soil columns were irrigated with 0.01 M CaCl2 solution. In the rst experiment the equivalent of 200 mm was applied over a 48-hour period (Lynch, 1995). This was applied using a glass beaker in four separate quantities spaced equidistantly in time. In the remaining two experiments the undisturbed soil was taken to eld capacity before the slurry was applied. They were then irrigated following precipitation patterns

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501

recorded in the eld (Kay et al., 2004) using a plastic syringe. 2.5. Sample collection In each experiment, leachate samples were collected in 500 ml amber glass bottles. Where samples were to be analysed for antibiotics the leachate was transferred to high density polyethylene (HDPE) bottles and frozen at 24 C prior to analysis. Leachate pH measurements were made immediately after sample collection using a DOLMEN 19 pH probe and digital pH meter (ETI Ltd., Sussex, UK). At the end of experiments 1 and 3, the soil columns were sectioned and analysed for antibiotic residues. 2.6. Chemical analysis Soil and water samples were analysed for antibiotic parent compounds using high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection. The methods used have been described in detail in previous papers (Blackwell et al., 2004a,b) and so they are only described briey here. Prior to extraction, soil cores were sectioned whilst they were still frozen using a Draper chop saw tted with a diamond tipped blade and, when defrosted, the sections were placed in aluminium foil trays and homogenised by hand. Sub-samples (4 g) of the soil were placed in plastic centrifuge tubes and 2.5 ml of buer (100 ml 0.1 M Na2EDTA (AnalaR grade, BDH, Poole, Dorset, UK) (to chelate with metals), 60 ml 0.2 M citric acid, 40 ml 0.4 M Na2HPO4 and 2 ml H3PO4 (to adjust pH to 7)) and methanol (all HiPerSolv for HPLCTM grade, BDH, Poole, Dorset, UK) were added. Each sample was then vortex mixed at 2500 rpm for 30 s using a Yellowline vortex mixer and, following this, sonicated in an Ultrawave sonic bath for 10 min. The samples were then centrifuged for 15 min at 3500 rpm to separate the solid and liquid phases. The liquid phase (5 ml) was poured into a duran bottle and the extraction procedure repeated a further two times to give a total of 15 ml of extract. The extracts were then made up to 400 ml with distilled water and 200 ll of H3PO4 added to adjust the pH to 2.8. The three antibiotics were extracted from the solution by solid phase extraction (SPE), using an

Isolute SAX (strong anion exchange) (International Sorbent Technology, Mid-Glamorgan, UK) cartridge to remove interfering humic materials in tandem with an Oasis HLB (hydrophiliclipophilic-balanced) cartridge (Waters, Watford, UK) to extract the compounds. The SPE cartridges were preconditioned using MeOH and buer (comprising 400 ml distilled water, 3.75 ml EDTA, 1.55 ml 0.4 M Na2HPO4, 0.3 ml 0.2 M citric acid and 200 ll H3PO4) and eluted after washing (5 ml buer, 5 ml 0.1 M NaOAc (AnalaR grade, BDH, Poole, Dorset, UK), 5 ml distilled water, 2 ml 20% MeOH) with 2 1 ml of methanol. A ow rate of 10 ml/min was used for the extractions. After defrosting, leachate samples were initially centrifuged at 3500 rpm for 15 min. The water was then ltered through a 0.8 lm membrane lter (Whatman International Ltd., Maidstone, UK). Concentrations of the antibiotics in the soil and water extracts were determined by HPLC with UV detection using a Dionex (Sunnyvale, CA, USA) Summit system, comprising a GINA 50 autosampler and a P580 quaternary gradient pump with a UVD 170S UV/visible spectrophotometric detector. Separations were performed using a GENESIS 4 lm C18 150 cm 4.6 mm internal diameter column with 4 m packing (Jones Chromatography, Mid-Glamorgan, UK). Analysis was performed using gradient elution over 25 min with a tetrahydrofuran (THF), acetonitrile (ACN) and 0.05% triuoroacetic acid in water (TFA) mobile phase (all HiPerSolv for HPLCTM grade, BDH, Poole, Dorset, UK). This contained 5% THF throughout the analysis. At the start of the analysis, the mobile phase contained 2.5% ACN and 92.5% TFA which was maintained for 4 min, the composition then changed linearly between 4 and 18 min to give a composition of 75% ACN and 20% TFA. Between 18 and 20 min the composition changed linearly to 2.5% ACN and 92.5% TFA and these conditions were then maintained for the remainder of the run. 20 ll of each extract was injected into the HPLC and the compounds were detected at 285 nm (SCP and TYL) and 355 nm (OTC). The method was validated by spiking river water to give a concentration of 1 or 10 lg l1. Recoveries and detection limits are shown in Table 2. The soil concentrations reported are corrected for recoveries.

Table 2 Recoveries and limits of quantication for the study compounds in soil and water samples Compound Recovery (%) Soil Oxytetracycline (OTC) Sulphachloropyridazine (SCP) Tylosin (TYL) 35 5 65 10 50 10 Water 99.6 4.6 99.9 2.2 94.9 2.4 Limit of quantication (lg kg1 or lg l1) Soil 10 10 10 Water 0.35 0.25 0.35

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8.6 8.4 8.2 8 Slurry applied Slurry amended Untreated

2.7. Statistical treatment of data Signicance tests were carried out using ANOVA. The data were rst tested for normality using the Shapiro-Wilk W test and homogeneity of variance using Levenes test and the statistical analyses carried out in GenStat (NAG Ltd., Oxford, UK).

pH

7.8 7.6 7.4

3. Results In the rst soil column experiment, which looked at the eects of slurry on the mobility of highly sorptive compounds, OTC was not detected in any leachate sample. Residues were, however, present throughout the soil columns at the end of the experiment (Fig. 2). Concentrations of OTC were generally highest in the top 5 cm of soil and then declined with depth. The average peak concentration detected in the 05 cm layer was around 1 mg kg1. The presence of slurry and how the OTC was applied in combination with the slurry (mixed together or separate) had no signicant impact on the distribution of OTC residues throughout the soil column ( p = 0.984). In the next experiment, where the eect of slurry application on soil water pH was investigated, very similar amounts of leachate were produced by all of the soil cores and the amount of eective irrigation for all of the cores was 43%. Leachate samples obtained prior to slurry application showed little dierence in pH, with values ranging between 7.6 and 7.88 (Fig. 3). Slurry application signicantly aected leachate pH however (p = 0.002). The pH of leachate from the slurry amended cores after the slurry has been applied ranged from 8.14 to 8.44

7.2 7 -13 -8 -3 2 7 12 17 22 27 32 37 42 47 52

Days after slurry application

Fig. 3. Mean pH (1 S.D.) of leachate from slurry amended and untreated soil cores.

whereas the range was 7.527.75 from the untreated soil cores. This dierence was still evident 50 days after slurry application. In the following experiment, investigating the eect of soil tillage prior to slurry application on the leaching of antibiotic residues, close to 100 ml of leachate was collected from each core at every sampling timepoint. This equated to an eectiveness of 7183%. SCP and OTC were only detected in leachate in the rst samples collected following slurry application to the soil cores (Fig. 4). Concentrations of SCP from the untilled columns were signicantly higher than concentrations from the tilled columns (p = 0.013). All three cores which had not been tilled produced leachate with high concentrations of SCP (mean 1264 lg l1) whilst none was detected in the leachate from the tilled soil. Thus, the mass loss of SCP from the non-tilled soil was

antibiotics and slurry mixed 0-5cm antibiotics and slurry separate antibiotics only

antibiotics and slurry mixed 5-10cm antibiotics and slurry separate antibiotics only

antibiotics and slurry mixed 10-20cm antibiotics and slurry separate antibiotics only

antibiotics and slurry mixed 20-30cm antibiotics and slurry separate antibiotics only 0.1 1.0 10.0 100.0
-1

1000.0

10000.0

OTC concentration (g kg )

Fig. 2. Distribution of OTC residues in soil columns used to investigate the eects of the presence of slurry in soil on the mobility of highly sorptive antibiotics.

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1600 SCP No tillage

503

Antibiotic concentration (g l-1)

1400 1200 1000 800 600 400 200 0

SCP Tilled OTC No tillage OTC Tilled

19

32

43

64

Days after slurry application

Fig. 4. Concentrations of SCP and OTC in leachate from soil columns used to investigate the eect of pre-tillage on the leaching of antibiotics in slurry.

54.6% compared to 0% from the tilled cores. SCP was detected throughout the soil cores at the end of the experiment (64 days after the slurry had been applied). Concentrations in the non-tilled soil were between 200 and 600 lg kg1 (Fig. 5a) whereas in the tilled soil concentrations were signicantly lower (p = 0.016) and were no greater than 300 lg kg1 (Fig. 5b). In contrast, OTC residues (0.2 lg kg1) were only detected in one single sample collected from the 05 cm layer of one soil core. As for leachate, no TYL was detected in the soil.

4. Discussion A range of laboratory soil column experiments has been carried out in order to investigate the fate of veterinary antibiotics in agricultural clay soils. A soil chromatography experiment indicated that the presence of slurry in combination with highly sorptive antibiotics, such as oytetracycline, will not increase their mobility due to facilitated transport, associated with colloidal matter and DOC from the slurry. This is surprising given that previous work has found that the mobility of pesticides (e.g. Edwards and Daniel, 1993; Worrall et al., 1995; Hardy, 1997; de Jonge et al., 1998; Hesketh et al., 2001) and polynuclear aromatic hydrocarbons (Magee et al., 1991) will be increased in the presence of dissolved organic matter (DOM). Additionally, 77% of the OTC detected in drainow in a eld study where the substance was investigated (Kay et al., 2004) was in the aqueous phase (<0.8 lm), and it seemed possible that the reason for this was facilitated transport by dissolved organic matter. Furthermore, this nding does not agree with previous observations which have found colloidal transport of pesticides to be more important than sediment associated transport (Worrall et al., 1999).

If DOM was not responsible for OTC transport in drainow then we must assume that this transport was in the true aqueous phase. In fact, some studies have indeed found that the presence of DOM in soil water has little or no eect on the transport of some pesticides (Spark and Swift, 2002) and polycyclic aromatic hydrocarbons (Conte et al., 2001). Some studies have suggested that it is not the presence of dissolved humic substances which increases or decreases transport, but the composition of these substances. It has previously been found that the presence of humic acid increased the mobility of the pesticide MCPA whereas fulvic acids retarded movement (Haberhauer et al., 2002). It is possible that the lack of observed dierences was due to the nature of the test system used in this study (Lynch, 1995). This test system was used as a screening method as the aim was only to observe whether a dierence existed in OTC mobility between soil treatments and not to determine probable environmental concentrations. The behaviour of OTC may have been dierent in the soil chromatography test, however, due to higher rates of leaching than in the environment due to articially high irrigation rates and higher ow rates through the disturbed soil (Brown et al., 1995). If this is the case, however, this has serious implications for the use of data from previous studies which have employed this test system. The sorption behaviour of veterinary medicines may be inuenced by changes in the pH of soil interstitial water brought about by levels of ammonia in manure and slurry applied to soils (Tolls, 2001; Boxall et al., 2002). A laboratory soil column leaching experiment has therefore been carried out to measure changes in soil water pH following slurry amendment. Based on available information in the literature, an assessment of the eects of pH changes on the mobility of the study antibiotics has been made. As pH increases, both acidic and basic compounds will become more mobile as sorption decreases. Acidic compounds have a negative charge at higher pH and so will sorb less to soil which also has a negative charge. Basic compounds exist as positive ions at lower pH, as sorption of the positive ion is greater then mobility also increases with rising pH. Slurry amendment has been found to signicantly increase the pH of soil water for a period of weeks after application in this particular experiment. The increase induced by the slurry was found to be approximately one half of a pH unit. This is slightly more than has been found in sorption experiments where the soil was amended with pig slurry at the eld application rate and an increase of approximately 0.1 of a pH unit was reported (Boxall et al., 2002). The pH measurements made in leachate from soil cores comprising of the top 10 cm of the eld soil, indicated that the pH of soil water can be aected to at least this depth. This may be important for the fate of surface applied chemicals which will

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0-5

Depth (cm)

5-10

10-15

15-20

100

200

300

400

500
-1

600

700

(a)

Antibiotic concentration (g kg )

0-5

Depth (cm)

5-10

10-15

15-20

100

200

300

400

500
-1

600

700

(b)

Antibiotic concentration (g kg )

Fig. 5. Mean SCP concentrations (1 S.D.) in soil from cores which had not been tilled (a) or tilled to 10 cm depth (b) prior to slurry amendment, 64 days after application.

obviously interact most with the surface layer of the soil (Hamscher et al., 2002). The consequences of this may be that they increase in mobility and therefore pose a potentially greater risk to ground and surface waters and plants through the uptake of soil water. Increased bioavailability may however lead to faster degradation by microbes. In the associated eld experiment (Kay et al., 2004) no eect on drainow pH was measured however so it may be that this phenomenon is limited to the topsoil only. This may be due to the interaction of a range of factors in the eld such as the application of inorganic fertiliser which tends to keep soil pH in the range of 67.5 (Scheer and Schachtschabel, 1989).

Drainow also comprised partly of new water, which would have diluted the old soil interstitial water. Previous work has shown that SCP sorbs less strongly with increasing pH as the molecule is deprotonated (Boxall et al., 2002). The study indicated that changes in mobility are likely to be small however as Kd values varied very little (between 0.6 and 2) in the pH range likely to be experienced. This may be explained by the fact that SCP has a pKa outside the range of those normally found in agricultural soils (5.1) (Cavallo and Tolls, 2001). As concluded by these authors, the mobility of SCP is therefore unlikely to be aected by pH changes caused by manure/slurry.

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There is a lack of data in the literature quantifying the mobility of OTC in non-amended and slurry treated soils. The compound does however have a pKa of 7.7, associated with the C11C12 ketoenol system (Loke et al., 2003) (as well as further pKa values of 3.3 and 9.1), indicating that its speciation will change over the range of pH values found in agricultural soils. Whilst TYL was not detected throughout the study, it also has a pKa within the range of normal environmental pH and could thus be aected by the presence of manure/slurry. In the presence of manure it will be partially positively charged and will not form complexes with metal ions in the manure and soil. Strong ionic bonds may however be formed with negatively charged components. Van der Waals induced dipole bindings with components of manure displaying some degree of positive character may also result in sorption (Loke et al., 2003). Many other antimicrobials have been identied with dissociation constants in the range of environmental pH including the 4-quinolones and elfamycins (Holten-Lutzhft, 2000; Tolls, 2001), which could therefore be aected by changes in soil water pH. Even though it is possible that slurry application to agricultural land may increase the pH of the soil and interstitial water, available information indicates that, although changes in sorption characteristics of some veterinary medicines may occur, these are likely to be small as pH will only increase by around half of one pH unit at the most. Moreover, other processes will be operating at the same time which will inuence the behaviour of a compound. For instance, the hydrological pathways present in a system have been shown to have a large inuence on the potential for sorption and degradation (Kay et al., 2004, 2005). A laboratory soil column leaching study was carried out to further investigate the phenomenon that soil tillage prior to slurry application can signicantly reduce losses of antibiotics to tile drainage systems (Kay et al., 2004). This is due to the breakage of soil macropores which leads to greater interaction between solutes and the soil. The results of this laboratory experiment agree with the ndings of the eld study and clearly support the theory that soil tillage prior to slurry application can signicantly reduce antibiotic concentrations in leachate. When the connectivity of the macropores with the soil surface was broken in the soil columns by tillage, losses of SCP fell from over 50% of that applied to zero. Losses of OTC in leachate were not reduced so dramatically although they were, nevertheless, 60% lower from the tilled soil columns. Whilst we might expect the reduction in transport to be greater for OTC due to the substances higher sorption potential, the greater interaction between water and soil could have encouraged the water to move into the soil matrix and, thus, transport the hydrophilic

SCP with it. In contrast, a greater proportion of the OTC may have remained on the ped surfaces or in voids, sorbed to slurry solids. When irrigation was applied, a greater fraction of the OTC may therefore have been available for transport (Hesketh et al., 2001). In contrast, if this was the case, the irrigation water would not have interacted with the SCP in the relatively immobile matrix water to this extent (Thomas and Phillips, 1979). At the end of the experiment, SCP concentrations were considerably higher in the soil which had not been tilled, despite mass losses in leachate being much greater from these cores. This indicates that the SCP degraded much more quickly in the soil that had been tilled. This phenomenon was also observed in the eld where concentrations of the two compounds were lower when the soil had been tilled prior to application. This may have been due to the compound interacting with the soil to a greater extent and therefore undergoing greater exposure to degrading microorganisms. OTC appears to have been almost entirely degraded in the soil, more rapidly than SCP. This contradicts the ndings of the eld experiment and most other work on the degradation of tetracyclines (Ingerslev et al., 2001; Halling-Srensen et al., 2003). As there was no difference in degradation rates between the tilled and nontilled cores, soil treatment does not seem to oer an explanation. Thus, there appears no obvious explanation for the degradation of OTC, although OTC metabolites were not analysed. TYL was again probably not detected due to degradation in slurry during storage prior to application.

5. Conclusion Three soil column leaching studies have been carried out in order to investigate the fate of veterinary antibiotics in clay soils. Chemical application to these soils has previously been shown to represent a signicant risk to surface water quality (e.g. Kladivko et al., 1991; Brown et al., 1995). It has been indicated that pig slurry does not have a signicant impact on the mobility of oxytetracycline. This may be due to the make up of the dissolved organic matter in the slurry or the nature of the test system used. It has also been shown that the application of pig slurry will cause a signicant increase in soil water pH in the top 10 cm of the soil. The rise of only half of one pH unit and the inuence of other processes may mean however that any eect on antibiotic fate will not be signicant. The nal soil column experiment helped to conrm that soil tillage prior to slurry application is a method which farmers can practically employ to reduce the environmental risk posed by veterinary antibiotics.

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P. Kay et al. / Chemosphere 60 (2005) 497507 Haller, M.Y., Muller, S.R., McArdell, C.S., Alder, A.C., Suter, M.J.-F., 2002. Quantication of veterinary antibiotics (sulfonamides and trimethoprim) in animal manure by liquid chromatographymass spectrometry. J. Chromatogr. A 952, 111120. Halling-Srensen, B., Lykkeberg, A., Ingerslev, F., Blackwell, P., Tjrnelund, J., 2003. Characterisation of the abiotic degradation pathways of oxytetracyclines in soil interstitial water using LCMSMS. Chemosphere 50 (10), 13311342. Halling-Srenson, B., Nors Nielsen, S., Lanzky, P.F., Ingerslev, F., Holten Lutzhft, H.C., Jrgensen, S.E., 1998. Occur rence, fate and eect of pharmaceutical substances in the environmenta review. Chemosphere 36 (2), 357393. Hamscher, G., Sczesny, S., Hoper, H., Nau, H., 2002. 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. Hardy, I.A.J., 1997. Water quality from contrasting drained soils: the relative importance of sorbed and aqueous phase transport mechanisms. Unpublished PhD thesis, Craneld University, Silsoe. Heppell, C.M., Burt, T.P., Williams, R.J., Haria, A.H., 1999. The inuence of hydrological pathways on the transport of the herbicide, isoproturon, through an underdrained clay soil. Water Sci. Technol. 39 (12), 7784. Heppell, C.M., Worrall, F., Burt, T.P., Williams, R.J., 2002. A classication of drainage and macropore ow in an agricultural catchment. Hydrol. Process. 16, 2746. Hesketh, N., Brookes, P.C., Addiscott, T.M., 2001. Eect of suspended soil material and pig slurry on the facilitated transport of pesticides, phosphate and bromide in sandy soil. Eur. J. Soil Sci. 52, 287296. Hirsch, R., Ternes, T., Haberer, K., Kratz, K.L., 1999. Occurrence of antibiotics in the aquatic environment. Sci. Total Environ. 225, 109118. Holten-Lutzhft, H.C., 2000. Environmental risk assessment of antimicrobials. Unpublished PhD thesis, The Royal Danish School of Pharmacy, Copenhagen. Hsu, J.-H., Lo, S.-L., 1999. Chemical and spectroscopic analysis of organic matter transformations during composting of pig manure. Environ. Pollut. 104, 189196. Ingerslev, F., Halling-Srensen, B., 2001. Biodegradability of metronidazole, olaquindox and tylosin and formation of tylosin degradation products in aerobic soil/manure slurries. Chemosphere 48, 311320. Ingerslev, F., Torang, L., Loke, M.-L., Halling-Srensen, B., Nyholm, N., 2001. Primary biodegradation of veterinary antibiotics in aerobic and anaerobic surface water simulation systems. Chemosphere 44, 865872. Johnson, A.C., Haria, A.H., Bhardwaj, C.L., Williams, R.J., Walker, A., 1996. Preferential ow pathways and their capacity to transport isoproturon in a structured clay soil. Pest. Sci. 48, 225237. Kay, P., Blackwell, P.A., Boxall, A.B.A., 2004. Fate of veterinary antibiotics in a macroporous tile drained clay soil. Environ. Toxicol. Chem. 23 (5), 11361144. Kay, P., Blackwell, P.A., Boxall, A.B.A., 2005. A lysimeter experiment to investigate the leaching of veterinary antibiotics through a clay soil and comparison with eld data. Environ. Pollut. 134 (2), 333341.

Acknowledgment This work was funded by EU FP5, project EVK1CT-1999-00003 (ERAVMIS).

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