Biodegradability of Metronidazole, Olaquindox, and Tylosin and Formation of Tylosin Degradation Products in Aerobic Soil–Manure Slurries

Ecotoxicology and Environmental Safety 48, 311}320 (2001) Environmental Research, Section B doi:10.1006/eesa.2000.2026, available online at http://www.idealibrary.com on

Biodegradability of Metronidazole, Olaquindox, and Tylosin and Formation of Tylosin Degradation Products in Aerobic SoilIManure Slurries Flemming Ingerslev and Bent Halling-S+rensen Institute of Pharmaceutical and Analytical Chemistry, Section of Environmental Chemistry, Royal Danish School of Pharmacy, Universitetsparken 2, Copenhagen DK-2100, Denmark Received November 29, 2000; published online February 5, 2001

The use of veterinary drugs (primarily antibiotics) in animal husbandry harbors the risk that these compounds end up in the farmland when manure is used as fertilizer. The biodegradability of three compounds, olaquindox (OLA), metronidazole (MET), and tylosin (TYL), was simulated in soil+manure slurries with 50 g of soil per liter. Supplemental batch sorption tests revealed that insigni5cant amounts of OLA and MET were located in the soil phase, whereas only 0.1 to 10% of the added amounts of TYL remained in the liquid phase. This may reduce the bioavailability and thus biodegradation rates of TYL. Unidenti5ed metabolites of OLA and TYL and four known TYL metabolites were detected using HPLC. However, none of these substances were seen to persist in the biodegradation experiments, indicating that OLA and TYL most likely were mineralized in the experiments. Neither the use of sandy or clayey soil nor the use of 0, 1, or 10% (V/ V) of manure added to these soils had a signi5cant e4ect on the degradation rates. Degradation half-lives for the primary degradation were 3.3+8.1 days for TYL, 5.8+8.8 days for OLA, and 13.1+26.9 days for MET. Based on comparisons of results obtained with the benchmark chemical aniline and degradation half-lives of this compound in nature, it was assessed that results obtained with the current test method slightly overestimate real-world biodegradation rates.  2001 Academic Press

INTRODUCTION

In intensive farming, substantial amounts of veterinary medicines (i.e., primarily antibiotics) are used as growth promoters or for therapeutic purposes. These substances are often excreted unchanged or as conjugated metabolites in feces or urine (Halling}S+rensen et al., 1998). Compared to industrial chemicals, the exposure routes of veterinary antibiotics to the environment are relatively easy to identify and  To whom correspondence should be addressed. Fax:#45 35 30 60 10. E-mail: "@dfh.dk.

related to speci"c "eld scenarios. Veterinary antibiotics may be spread to the environment either directly when using the drug or more importantly by subsequent excretion from the animals. The dominating pathway of environmental release in the terrestrial compartment is by amendment of arable soil with manure. Regulation dealing with fertilization of arable land with manure is therefore important for prediction of the environmental release of the antibiotics and also to assess the properties of the soil. In EU, a statute with the purpose of preventing nitrate pollution limits the amount of nitrogen applied on the soil in association with manure to 210 kg N/ha (EU, 1991). According to this regulation, the member states may ful"ll this demand by allowing di!erent amounts of manure based on criteria such as climate, soil properties, and animal species. In Denmark the implementation of this law (Danish Environmental Protection Agency, 1998) states that farmers annually are allowed to disperse swine manure from 1.7 animal units/ha and cattle manure from 2.1 animal units/ha. To assess the environmental impact of the use of growth promoters, the fate of these compounds in the farmland must be known. Many factors should be considered, but biodegradation of the chemicals is probably one of the most important. During the biodegradation process, a chemical may be either mineralized or transformed to other degradation products. As several metabolites of pharmaceutical compounds are known to be biologically active (Lanzky and Halling-S+rensen, 1997), the fate of these degradation products is also important. In the scenario of antibiotics in farmland, the biodegradation process is in#uenced by several properties of the soil and the soil biomass. Even though anaerobic conditions often may prevail in subsoils ('0.75 m) and in manurecontaining soils, oxygen is still believed to be present in large parts of the topsoil ((0.25 m). Aerobic biodegradation in the topsoil is therefore believed to be the dominating process. Sorption of the antibiotics to the soil particles may

311 0147-6513/01 $35.00 Copyright  2001 by Academic Press All rights of reproduction in any form reserved.

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as for other chemicals reduce the bioavailability and thus the biodegradability (Hatzinger and Alexander, 1997). Organic materials in the soil can either enhance (Shimp and Pfaender, 1985) or decrease (Zaidi and Mehta, 1995) the biodegradation. The soil biomass consists of a large number of many bacterial species and may therefore have a high capacity for biodegradation of antibiotics. However, this population may be strongly in#uenced by bacteria that are transferred from the manure to the soil as these bacteria previously might have been exposed to antibiotics and therefore may hold a capacity for biodegradation of antibiotics. To simulate biodegradation in soil systems, several biodegradation set-ups are developed. In principle, two basic test types are used: (i) the dry tests, which are methods in which the soil sample is disturbed as little as possible under the exposure to the test chemicals (see e.g., OECD 304A (Organization for Economic Cooperation and Development, 1998)), or (ii) slurry tests in which where the soil sample is diluted in an aqueous medium (see e.g., Bregnard et al. (1996); Ortega-Calvo et al. (1995)). The latter methods are considered as less realistic but are often preferred as the dry test methods require laborious chemical analysis or the availability of the radiolabeled test chemicals. The current study was conducted using three antibiotics used in veterinary treatment: metronidazole (MET), olaquindox (OLA), and tylosin (TYL). The chemical structures of the three compounds are provided in Fig. 1. MET is an antibiotic e!ective against anaerobic bacteria and protozoa (Kapoor, 1976). The compound is applied in this study rather as a model compound for synthetically produced

antibiotics than for the quantity applied. OLA is a synthetic agent (e!ective against gram-negative bacteria) used as a growth promoter mainly for pigs (Bronsch et al., 1976). Tylosin A, which also is used as a growth promoter for pigs, is a microbially synthesized macrolide antibiotic that is active against most gram-positive bacteria (Wilson, 1981). In the current study, a soil slurry technique with two di!erent soil types (sandy and clay soil) was applied with the purpose of providing realistic rate data for the primary transformation of the three model compounds. Furthermore, the purpose was to investigate whether a number of substances known as metabolites of the three model substances but formed either in the human organism (TYL) or by photolysis (OLA) occurred as degradation products in the biodegradation test. For these purposes, the following experiments were performed: (1) Aerobic batch columns were set up with soil slurries (sandy and clayey soil) and increasing quantities of manure (0, 1, or 10%) diluted in a synthetic mineral medium. Results for primary degradation were quanti"ed as half-lives (¹ )  (days). (2) The in#uence of the soil properties on the bioavailability of the test compounds was assessed in simple batch sorption experiments. (3) Viable bacterial counts were performed before and after each experiment in order to investigate the potential active biomass level in the soils. (4) The formation of compounds likely to be formed during biodegradation of the test substances was investigated by analyzing chromatograms of samples obtained during degradation and comparing these with chromato-

FIG. 1. Chemical structures of MET, OLA, and TYL.

313

BIODEGRADABILITY OF THREE ANTIBIOTICS IN SOIL}MANURE SLURRIES

grams of compounds known as metabolites in the human organism (TYL) or formed by photolysis (OLA).

TABLE 2 Characteristics of the Soils Used in the Current Study Lundgaard

MATERIALS AND METHODS

Chemicals

Texture (w/w %)

MET (CAS No. 443-48-1), TYL-tartrate (CAS No. 7461055-2) (purity '89.9% w/v of TYL), and aniline were obtained from Sigma Chemical Co. (St. Louis, MO). OLA (CAS No. 23696-28-8) was purchased from Yick-Vic Chemicals and Pharmaceuticals (Hong Kong) and was of 98.16% purity. Four metabolites of TYL, tylosin B, tylosin C, tylosin D, and demycinosyltylosin, were kindly donated by Professor Hoogmartens (Katholieke Universiteit, Leuven, Belgium). All other chemicals were purchased from Sigma Chemical Co. Chemicals for HPLC analysis were ultrapure (purity '99.9%). All solutions were prepared in Milli-Q water.

Organic C pH Total N Na> K> Ca> Mg> Total CEC Total P

Sampling and Pretreatment of Soil and Manure Manure was obtained from a Danish pig farm using an inorganic method of production. Sampling (5 liters of manure in nalgene bottles) was performed with a bucket during the weekly transfer of manure from the prestorage to the main storage tank. Samples were "ltered within 3}4 h through a nylon cloth (mesh"1 mm) into 1-liter serum bottles while being #ushed with N . To ensure anaerobic  conditions, N #ushing was continued for 15 min before the  bottles were closed with a butyl rubber sceptum. The manure was stored at 43C in these bottles until use (up to 8 weeks). The "ltered manure was analyzed for its major constituents such as dry matter content, ammonia, and total concentrations of nitrogen, phosphorous, potassium, and carbon. These analyses were performed by the Central Laboratory at the Danish Institute of Agricultural Sciences (Danish Institute of Agricultural Sciences, 1999). Results are presented in Table 1. The two soils used in this study are typical Danish agricultural soils according to their physical and chemical characteristics (see Table 2). Both soils are used as reference

TABLE 1 Concentrations of the Major Constituents in the Manure Used in Current Study Constituent Total N (Kjeldahl) Total P Total K Total C Ammonia N Dry matter

Concentration (g/liter) 153.35 1.7 68.9 422.2 98.1 44.3

Clay ((2 lm) Silt (2}20 lm) Fine sand (20}200 lm) Coarse sand (0.2}2 mm) (w/w %) (w/w %) meq/(kg soil) meq/(kg soil) meq/(kg soil) meq/(kg soil) meq/(kg soil) mg/kg

0.052 0.048 0.244 0.632 1.4 6.34 0.1 0.6 2.3 41.9 3.1 67 559

Askov 0.113 0.107 0.379 0.375 1.6 6.78 0.14 1.1 3.7 74.9 4.7 100 703

soils by the Danish Research Center for Sustainable Land Use and Management of Contaminants (Det Strategiske Milj+forskningsprogram, 1999). Topsoils ((0.25 m) from both Askov (clayey soil) (55328N 936E) and Lundgaard (sandy soil) (55328N 9310E) were collected. After sampling, the soils were stored until use in 150-liter stainless steel barrels at 6$13C for up to 6 months. Immediately before being used in the experiments, the soils were "ltered through a nylon "lter (mesh"2 mm). Sorption Sorption of the test compounds to the two soils was investigated in simple batch experiments, which in principle were designed as the biodegradation tests. In 100-ml autoclavable bottles 5 g of a 10% soil}manure mixture and 100 ml of minerals medium (prepared as described for the biodegradation tests) were added. On 2 consecutive days, all bottles were autoclaved (1403C for 1 h) and cooled. As recommended by de Maagd et al. (1998), 10 mmol/liter of NaN was added to prevent microbial growth. The test  compounds were added to #asks in triplicate at six concentrations ranging from 50 to 1000 lg/liter for MET and OLA and from 500 to 25,000 lg/liter for TYL. Also three bottles with no test compounds were included. All #asks were then incubated in darkness at 20$23C and samples for chemical analysis of the liquid phase were taken after 1, 24, and 48 h. Only water phase concentrations (C ) were measured in  the sorption tests. Therefore, soil concentrations (C ) were  calculated on basis of the total added amount of substrate (M ), the mass of soil (m ), and the aqueous volume (< )    using C

M !< ) C  . "   m 

(1)

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INGERSLEV AND HALLING-S"RENSEN

The soil and water concentrations in each experiment were "tted to the Freundlich isoterm C "K ) CL ,   

(2)

where K and n are the Freundlich parameters (see, e.g.,  Schwarzenbach et al. (1993)), using the computer software GraphPad prism version 2.01 (GraphPad, 1998).

Biodegradation Test Degradation experiments were conducted in glass columns (height"50 cm; diameter"5 cm) aerated from the bottom with &1 liter/min of atmospheric air. These columns were equipped with a tap from which slurry samples were withdrawn at regular intervals (i.e., three times during the "rst 2 weeks and weekly thereafter). To each column 1 liter of synthetic minerals medium (prepared as described in OECD 301A-F (Organization for Economic Cooperation and Development, 1998)) was inoculated with 50 g/liter soil containing 0, 1, or 10% (w/v) of manure. The amount of 1% manure in soil is based on the assumption that the applied volume of manure on Danish agricultural soils of 1.7 animal units/ha (see Introduction) corresponds to approximately 30 m/year and is distributed in the top 25-cm layer of the soil. Controls with inhibited biomass were prepared by autoclaving 50 g of soil or soil}manure in 1 liter of minerals medium for 1 h (1403C) on 2 consecutive days. Each sterilized test solution was transferred to the degradation reactors and the test compounds. As the reactors are semiopen systems and therefore susceptible to penetrating bacterial contaminants, a 10 mM concentration of the microbial inhibitor sodium azide was added. The inhibition of the biomass was checked using the biomass analysis as described in the following section. All reactors were covered with aluminum foil to avoid photodegradation. The antibiotics were tested in four experiments, two for each soil type either with MET (approximately 500 lg/liter) and TYL (5000 lg/liter) in a mixture or with OLA (500 lg/liter), which was tested alone. Aniline (150}250 lg/liter) was used as degradation control. Each experiment involved 12 columns and the setup was designed as summarized in Table 3. During the experiments, duplicate 10-ml samples were taken three times during the "rst week and thereafter weekly until degradation was completed. Samples were frozen (!183C) for up to 6 weeks prior to analysis. Biodegradation was assumed to follow simple "rst-order degradation kinetics, i.e., dC "!k ) t  dt U C"C ) eI R, 

(3)

TABLE 3

Reactor No.

Test compounds

Content of manure in soil (% V/W)

Antibiotics Antibiotics Antibiotics

0% 1% 10%

Aniline Aniline Aniline

0% 1% 10%

Antibiotics Antibiotics Antibiotics

0%? 1%? 10%?

1}2 3}4 5}6 7 8 9 10? 11? 12?

Note. The design of the biodegradation experiments involved 12 degradation reactors, which in addition to 1 liter of synthetic test medium were "lled with 50 g of soil}manure and test compounds. ? Test solutions were autoclaved twice and 10 mM NaN was added. 

where C and C are concentrations of substrate at the  beginning or at time, t, and k is the "rst-order rate con stant. Using this model, half-lives, ¹ can be calculated as  ¹ "ln(2)/k .   Identixcation of Degradation Products A qualitative approach was used to investigate the formation of degradation products during biodegradation. Brie#y, chromatograms of samples containing known and unknown metabolites formed by other processes than microbial degradation were compared with chromatograms of samples from the biodegradation tests. The metabolites are described as follows: (i) OLA forms several photodegradation products (metabolites) when exposed to UV light (de Vries et al., 1990). A mixture of these metabolites was produced by exposing 10 mg/liter of OLA to daylight for 10 h. This sample was analyzed with the HPLC method used for OLA. (ii) TYL and its metabolites tylosin B, tylosin C, tylosin D, and demycinosyl-tylosin are described by Knepper et al. (1999). These compounds were identi"ed in the chromatograms by injection of pure samples in the chromatographic system used for TYL. Microbiological Analysis To evaluate changes in active biomass during the study counts of total viable aerobic bacteria were determined at 213C on plate count agar (Danish Standard, 1983). Counts were determined before and after each degradation experiment. Chemical Analysis Prior to analysis all samples were "ltered through a 0.45-lm syringe "lter (Minisart 17598, Sartorius AG, Goet-

BIODEGRADABILITY OF THREE ANTIBIOTICS IN SOIL}MANURE SLURRIES

tingen, Germany). Chemical analysis was made on a Waters HPLC system (Waters 2690, Waters, Milford, MA), equipped with a photodiode detector (Waters, Model 996), a sample cooler, and a column heater. Injection volumes of 100 ll were used and temperatures of the samples and the column were 4 and 403C, respectively. For analysis of MET, OLA, and aniline, a Spherisorp ODS2 (Phase Separation, CT) C-18 chromatographic column (125;4.6 mm, particle size 5 lm) was used. The eluent consisted of solvents A, acetonitrile, and B, 1 mM/liter of EDTA, 16.7 mmol/liter glacial acetic acid adjusted to pH 5 with 4 M NaOH. When analyzing samples for OLA the ratio between solvent A and B was 7.5:92.5, the #ow rate was 1 ml/min, and detection was performed at 260 nm. MET was eluted with 7.5% A and 92.5% B for 4 min and then the composition of the eluent was changed to 75% A and 25% B for 2 min to elute TYL from the column as TYL was not eluted in the previous procedure. Aniline was eluted isocratic with 25% A and 75% B. Detailed information for analysis of TYL is described elsewhere (Loke et al., 2000). RESULTS

Sorption No signi"cant changes in concentrations were measured after 1 and 24 h of incubation in any of the experiments, indicating a rapid equilibrium between solids and the aqueous phase. Therefore, only results from 24 h are presented (Table 4). OLA and MET were seen to be practically unsorbed as Freundlich constants of less than 0.05 were found. Due to the low sorption, di!erences in measured liquid concentrations were smaller than could be precisely analyzed within the analytical uncertainties. Therefore, signi"cant variability of the results for these compounds was obtained and thus no standard deviations are reported for these compounds. TYL, on the other hand, was sorbed to both the sandy and the clay soils with K values of between 2 and 32. 

315

scattered. This was considered as di$culties maintaining a constant aeration #ow rate over time in the di!erent test columns. The air #ow rate in#uences the sedimentation of soil in the column, which, to some extent, can in#uence the homogeneity of the samples taken from the test system. For this reason, the degradation rate data (see Table 5) from the duplicate columns are not presented as average values. The three antibiotics were degraded (primary degradation) without any lag phase, indicating that a capacity for biodegradation of antibiotics were initially available in the biomass. Half-lives were on the order of 3 to 30 days for all three substances, although slightly higher for MET (9.7 to 26.9 days) than for TYL (3.3 to 8.1 days) and OLA (5.8 to 8.7 days). Experiments with aniline demonstrated half-lives ranging from 1 to 5 days. Half-lives obtained with soil from Lundgaard (sandy soil) did not signi"cantly di!er from half-lives obtained with soil from Askov (clayey soil). Similarly, the experiments revealed no e!ects of adding increasing concentrations of manure. Even 10% manure did not a!ect the degradation rate signi"cantly. No degradation of OLA and MET occurred in the inhibited controls but degradation of TYL occurred in these columns after a delay of 5 to 6 days. Biomass analysis of samples from these columns revealed that no bacteria occurred initially in the tests, but after 10 days 2.3;10 to 4.8;10 CFU/ml was formed despite the presence of 10 mM NaN . This "nding is not surprising, as the columns  cannot be considered as a sterile system. Initially measured concentrations of MET and OLA corresponded to the added amounts, whereas concentrations of TYL in the water phase were approximately 2500 lg/liter in experiments with soil from Lundgaard and around 750 lg/ liter when soil from Askov was used. These "ndings re#ect the higher capacity for sorption of TYL in the soil from Askov. This corresponds with the results of the sorption tests. Identixcation of Metabolites

Primary Biodegradation Results of primary biodegradation as presented in Fig. 2 indicate that the data obtained in general were relatively TABLE 4 Freundlich Parameters and Standard Errors in Sorption Experiments Soil Olaquindox Metronidazole Tylosin

Lundgaard Askov Lundgaard Askov Lundgaard Askov

K 

n

0.007 0.007 0.02 0.009 2.03$0.9 32.1$13.2

0.85 0.97 0.55 0.94 0.67$0.07 0.67$0.06

The chromatograms from samples with 10 mg/liter of OLA before and after 10 h of exposure to daylight are presented in Figs. 3A and 3B. As seen in Fig. 3B OLA was completely removed and several metabolites were formed. These metabolites were, however, not detected as degradation products in the biodegradation tests and are therefore either not formed during biological degradation of OLA or rapidly degraded as soon as they are formed. As described in more detail by Loke et al. (2000), the TYL metabolites tylosin B, tylosin C, tylosin D, and desmycinosyltylosin could be separated with the HPLC method used in current study. These substances were also seen as degradation products in the biodegradation tests. Figure 4 presents typical chromatograms obtained when the samples from di!erent sampling times were analyzed for TYL. Due

316

INGERSLEV AND HALLING-S"RENSEN

FIG. 2. Biodegradation curves for the antibiotics in 1-liter slurries with 50 g of either soil (䊐 and 䊏) or soil with 1% (䉭 and 䉱) or 10% (䊊 and 䊉) manure. Experiments with OLA and soil from Lundgaard as well as experiments with TYL and soil from Lundgaard with 1% manure were performed with only one replicate. Experiments with TYL and soil from Lundgaard with 10% manure was not performed.

to impurities in the TYL standard, tylosin B and tylosin D were seen seen immediately after spiking TYL in the test solution. These substances, a third known metabolite (demycinosyltylosin), and several other compounds were formed during the degradation test. As seen, these degradation products disappeared shortly after removal of the parent compound, TYL, from the test solution.

indicated more or less identical viable counts for both soil types. Similarly no signi"cant di!erence in bacterial counts was obtained between the start and the end of the experiments. Table 5 indicates that addition of 10% manure slightly increased the bacterial population of the batch test system. DISCUSSION

Microbial Analysis Table 6 lists the number of aerobic viable counts determined in samples from the biodegradation tests. Data

Chemical analyses of complex matrices like the samples from the biodegradation tests are often di$cult to perform. These problems can be due to impurities in the sample or

BIODEGRADABILITY OF THREE ANTIBIOTICS IN SOIL}MANURE SLURRIES

317

TABLE 5 Half-Lives (Days) during Primary Degradation of Antibiotics in Soil+Manure Slurries Content of manure in soil

Olaquindox

Metronidazol

Tylosin

Anilin

0% 1% 10%

6.3, 5.9 7.1, 8.8 8.7, 7.0

14.1, 14.3 14.5, 14.7 13.7, 9.7

4.1, 4.2 4.1, ND ND

2.9, 4.8 2.6, 3.3 1.4, 3.0

Lundgaard 0% 1% 10%

ND; 5.8 6.3, 6.6 7.5, 6.0

13.1, 16.8 15.3, 14.1 18.0, 26.9

5.2, 6.2 6.7, 5.0 8.1, 3.3

2.1, 4.8 1.1, 3.3 1.6, 2.6

Askov

Note. ND, these experiments were not performed. The two numbers indicate results from each replicate degradation experiment. For aniline, the "rst number indicates results from an experiment conducted in parallel to degradation of olaquindox, whereas the second number indicates results from experiments conducted in parallel with degradation of metronidazol and tylosin.

because the analyte may be in equilibrium with solids or other chemicals in the sample and therefore not detectable if only the liquid phase is analyzed. Furthermore it can be di$cult to state whether the test compound during biodegradation is mineralized or just metabolized to other compounds. To solve these problems a number of experiments were performed in association with the biodegradation tests. One of these experiments was the sorption studies. The results indicate that the sorption of MET and OLA to the soils is negligible and, therefore, the liquid-phase concentrations of these compounds could be considered as total slurry concentrations. TYL, on the other hand, was seen to be sorbed to the soils in considerable amounts (k "32, Askov)  and (k "2.3 Lundgaard). Because nonlinear sorption iso

FIG. 4. Chromatograms of samples from times during the biodegradation experiments with TYL. Of the peaks formed during biodegradation, three were identi"ed as tylosin B, tylosin D, and demycinosyltylosin. The other peaks formed were unknown substances.

terms were obtained, it is di$cult to interpret measured liquid concentrations with respect to the total slurry concentrations of TYL. However, using the Freundlich model, calculations of total slurry concentrations (i.e., the amount of TYL in the soil and liquid phase per slurry volume) indicate that only 0.1 to 10% of the compound added is measured in the liquid phase. A paper by Rab+lle and Spliid (2000) found that TYL desorbed when the liquid phase was replaced by a solution not containing TYL. Although the

FIG. 3. Chromatograms of an aqueous sample with 10 mg/liter of OLA (A) and formation of photometabolites after 10 h of exposure of the sample to daylight (B).

318

INGERSLEV AND HALLING-S"RENSEN

TABLE 6 Determinations of Viable Aerobic Bacteria in the Biodegradation Experiments Content of manure in soil

Start of test (CFUs/ml)

End of test

Ending day

(3.3$1.8);10 (2.5$1.7);10 (7.3$2.7);10 (2.8$1.7);10 (1.7$1.3);10 (3.9$2.0);10

(2.8$1.7);10 (1.2$1.1);10 (3.4$1.8);10 (1.2$1.1);10 (3.2$1.8);10 (6.4$2.5);10

39 44 39 44 39 44

(1.9$1.4);10 (1.4$1.2);10 (2.6$1.6);10 (8.1$2.8);10 (6.4$2.5);10 (1.5$1.2);10

(1.8$1.3);10 (3.0$1.7);10 (7.0$2.6);10 (9.1$3.0);10 (2.9$1.7);10 (2.4$1.5);10

34 48 34 48 34 48

Askov 0% 1% 10% Lundgaard 0% 1% 10%

test conditions were not the same as in the present study, it is believed that even though TYL is sorbed to soil, it is available for the microorganisms when the liquid phase concentration decreases. Another experiment performed to help interpret the biodegradation results was the investigation of the formation of degradation products. In the current study it was found that with the exception of MET, a whole range of known and unknown metabolites were detectable with the analytical techniques used. It was also seen that these metabolites were either rapidly degraded after being detected in the biodegradation tests (TYL) or not detected at all (OLA). The last "nding may indicate either that none of the metabolites were formed at all or that they were degraded immediately after being formed. The complete mineralization of the antibiotics cannot be de"nitively proved, as C-labeled test chemicals were unavailable in the current study. However, there are reasons to believe that both TYL and OLA are mineralized. As for TYL, the unknown metabolites that were formed during degradation have also been observed in abiotic degradation studies by Hoogmartens and coworkers (see Knepper et al., 1999). These authors assume that the observed substances probably are products from the cleaved 16-member lactone ring in TYL; i.e. the compounds converted to unbranched hydrocarbons. Such compounds are easily biodegraded and thus TYL is likely to be mineralized in the current tests. With regard to metabolism of OLA, the HPLC method used here enabled the detection of a range of more lipophilic metabolites. Compounds that are more hydrophilic than OLA might not be detectable, as retention times of these compounds in the chromatographic systems probably are so short that the compounds are eluted simultaneously with the solvent peak. These consid-

erations led to the conclusion that during biodegradation of OLA the compound is mineralized or it is transformed to more polar metabolites that cannot be detected with the chromatographic system used here. An objective of the current study was to investigate di!erences in biodegradability in two soils. As seen, the results from the two soils were uniform and hence the properties of the soils may be considered as unimportant for the biodegradation processes. However, many individual characteristics of the soils with importance to the biodegradation processes, e.g., texture and humidity, might be eliminated by using the slurry technique. This may also explain why no e!ect on the biodegradation is seen when manure is added to the soils. However, other factors are also of importance. The addition of manure contributes bacteria and various organic compounds to the tests. Both factors are well known to enhance biodegradation processes (Alexander, 1985; Schmidt and Alexander, 1985). Apparently, the amount of degraders and organic compounds added with the manure is negliglible compared to what is added to the test solution with the soil. It can be argued that biodegradation studies like the current study have a limited relevance for the environmental situation. The current biodegradation test systems diverged from the environmental situation in several areas. First, the temperature was higher than typical in Denmark, which most likely leads to higher biodegradation rates than would occur in nature. Second, the concentration of the antibiotics was higher than expected in the environment, which, as discussed by several authors (Nyholm and Ingerslev, 1997; Zaidi et al., 1988), may lead to arti"cially large amounts of degraders in the test systems and hence to overestimation of biodegradation rates. The third major di!erence from the natural situation was the large amount of water in the test systems, which was used to enable the concentration of the test compounds to be followed using HPLC analysis. This is believed to a!ect the test results in several ways. Most important is: (i) in natural soils the biomass is more or less immobilized and thus has limited access to the antibiotics as well as inorganic and organic nutrients. This is in contrast to the situation in the soil slurries where increased biodegradation rates therefore could be expected. (ii) It is well known that when chemicals are sorbed to soil they are less biodegradable because their bioavailability is reduced (Verstraete and Devliegher, 1996). Furthermore results have shown that the relative amount of compounds sorbed to soil is higher when the soil/liquid phase ratio is high (Voice and Weber, 1985). This means that in the current test systems the amount of sorbed antibiotics might be lower than would have occurred in the environment. Therefore, a higher bioavailability and thus a higher potential for biodegradation of the antibiotics could be the result. In the current study, this e!ect is of particular importance for TYL, which was absorbed to the soil in the test solution.

BIODEGRADABILITY OF THREE ANTIBIOTICS IN SOIL}MANURE SLURRIES

As discussed above, biodegradation rates determined in the current test systems may for several reasons overestimate biodegradation rates in the environment. However, the magnitude of this error can be assessed by comparing the data with results from other studies. Aniline is an important reference substance that is well known to be readily biodegradable in standard screening tests (Battersby, 1997; Gerike and Fischer, 1979; Painter, 1992). In surface water biodegradation tests, the compound is eliminated after 21 days (Subba-Rao et al., 1982) or with a half-life of 10}20 days (Ingerslev and Nyholm, 2000), or in other environments, a half-life of 5}6 days (seawater) (Nyholm et al., 1992), 0.5}4 days (activated sludge) (Nyholm et al., 1996), and 4.1 days (soil) (Federle et al., 1997). These data indicate that the "nding obtained in the current study might overestimate biodegradation rates in nature. However, it is considered that the present data are still valid for semiquantitative predictions of biodegradation in the environment. CONCLUSIONS

The sorption of OLA and MET to the soils was negligible and is, therefore, unimportant for the biodegradability of these substances. This is in contrast to TYL for which the majority of the compound was sorbed to the soil. Although the sorption appeared to be reversible, the possibility cannot be excluded that the biodegradation of this compound may be in#uenced. A range of metabolites of OLA and TYL were detectable with the analytical methods used in the current study. These were either not detected or rapidly biodegraded after being formed in the test systems. The analytical methods used for detection of the metabolites are believed to cover a range of possible metabolites so wide that no other degradation products are likely to be formed. Thus TYL and OLA are most likely mineralized in the tests. The degradation half-lives of the three compounds (¹ "  4.1}8.1 days for TYL, ¹ "5.9}8.8 days for OLA, and  ¹ "9.7}26.9 days for MET) were not in#uenced by using  di!erent soils or by adding di!erent concentrations of manure in the test systems. The explanation for not observing any di!erences is believed to be linked to the slurry technique used in the tests, as this design may reduce e!ects associated with the charactistics of the soil and manure. Similarly, the slurry technique might overestimate realworld biodegradation rates as bioavailability is believed to be enhanced. Additionally, a comparison of the test results with the antibiotics in parallel experiments conducted with aniline, a ready biodegradable benchmark reference compound, revealed that the antibiotics are much more persistent than this compound (¹ "1.1}4.8 days). Based on  the above "ndings, the overall conclusion with regard to the three test compounds is that they may occur in the soil for considerable periods of time before they are degraded.

319

ACKNOWLEDGMENT The current research project was supported by the Danish Environmental Research Programme (Center for Sustainable Land Use and Management of Contaminents, Carbon, and Nitrogen).

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