Published online 31 May 2006
Published in J Environ Qual 35:969-972 (2006)
DOI: 10.2134/jeq2005.0355
© 2006 American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
Sorption Kinetics of Chlortetracyline and Tylosin on Sandy Loam and Heavy Clay Soils
S. E. Allairea,*,
J. Del Castillob and
V. Juneaua
a Centre de recherche en horticulture, Envirotron, Université Laval, QC, Canada G1K 7P4
b Département de biomédecine vétérinaire, École de médecine vétérinaire, Université de Montréal, QC, Canada G2S 7C6
* Corresponding author (suzanne.allaire{at}sga.ulaval.ca)
Received for publication September 15, 2005.
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ABSTRACT
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Antibiotics may appear in the environment when manure, sewage sludge, and other organic amendments are added to soils. There is concern that the presence of antibiotics in soils may lead to the development of antibiotic-resistant bacteria which may spread to the rest of the environment. This paper aims at evaluating the sorption kinetics of two antibiotics frequently used in pig production. The results indicate that sorption of chlortetracycline (CTC) and tylosin (TYL) in sandy loam and clay occurs very fast. More than 95% of the CTC adsorption is completed within 10 min on both soils and of TYL within 3 h. These results suggest that 24-h soil and antibiotic solution mixtures is enough for sorption studies. Also, there is less likelihood that these antibiotics will leach through soil and appear in the ground water since their sorption on soils is very high unless they are carried by soil particles through preferential flow. There was also no effect of soil sterilization on sorption kinetics of these antibiotics thus suggesting that there is minimal probability of the antibiotics degrading by microorganisms during 24- to 48-h adsorption studies.
Abbreviations: CTC, chlortetracycline • HPLC, high performance liquid chromatography • Kd, linear sorption coefficient • TYL, tylosin
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INTRODUCTION
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IN 1996, about 10.2 million kg of antibiotics were used in Europe, of which 50% were used in veterinary therapeutics and also to promote growth (Kümmerer, 2003). In 2000, more than 22.7 million kg of antibiotics were produced in the United States and more than 40% have been used as feed supplement to enhance animal growth.
Up to 80% of antibiotics used as feed additives or for disease treatment are excreted through urine and feces (Levy, 1992). These urine and feces are then spread on agricultural field thus transferring the antibiotics to the environment. Antibiotics have been detected worldwide in soils, surface water, and ground water (Christian et al., 2003; Hamscher et al., 2002; Kolpin et al., 2002). As an example, 7 mo after liquid manure application, different antibiotics could be found in the soil (15 µg kg–1 of sulfadimidine) and related waters (Christian et al., 2003).
It is believed that antibiotics in soils may favor the development of antibiotic-resistant microorganisms causing different health problems in animals and humans (Halling-Sørensen et al., 1998). Sengeløv et al. (2003) showed that resistance, measured by the presence of resistance genes by polymerase chain reaction (PCR), to tetracycline and streptomycin developed in less than a year in soils treated with pig manure slurry. It is therefore important to understand the behavior of antibiotics in soils.
Good reviews have been published regarding veterinary medications and antibiotics (Boxall et al., 2004). Boxall et al. (2003) listed chlortetracycline (CTC) and tylosin (TYL), among others, as having a high potential of entering the environment. These molecules have already been detected in soils, surface waters, and ground waters (Boxall et al., 2004; Christian et al., 2003) but little is known about their behavior in soils.
These two antibiotics are heavily used in pig production in Québec. Chlortetracycline is used for the treatment or prevention of contagious respiratory infections as well as a growth factor. Chlortetracycline inhibits protein synthesis in prokaryotes and eukaryotes. It also inhibits the binding of aminoacyl-tRNA to ribosomes. Tylosin is used against enteric digestive infections causing diarrhea as well as a growth promoter. Up to 5 and 9 mg L–1 of CTC and TYL, respectively, can be found in liquid manure (Kumar et al., 2004) which is then applied on farm lands.
In spite of their widespread application on farm lands, CTC and TYL behavior in Québec soils has received little attention. Key chemical properties such as water solubility, soil pH, volatility, and sorption influence solute transport in soils. Sorption coefficients (Kd, Koc, Kow) have been used to predict antibiotic behavior in soils. Sorption coefficients reported in literature are usually obtained from experiments conducted over a 24-h period assuming equilibrium and no degradation. In addition, experiments on antibiotics may be conducted with sterilized or unsterilized soils, but the need for sterilization in short-term experiments is not clear. In addition to degradation, kinetics of chemical sorption onto soil particles is very important for solute transport studies and for modeling purposes because antibiotic movement depends on the time lapse between the chemical application (availability of compound in the soil water which depends, in part, on the sorption kinetics) and the first precipitation event. As an initial step for further experimentations and simulations, it is important to better understand sorption kinetics and the importance of sterilization.
The goals of this study were to measure sorption kinetics of CTC and TYL before equilibrium is reached, and the microbial and non-microbial degradation of these compounds during short-term experiments (<48 h).
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MATERIALS AND METHODS
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Chlortetracycline hydrochloride (C22H23ClN2O8·HCl) was obtained from Sigma-Aldrich (St. Louis, MO) at 80% purity. Chlortetracycline has a molecular weight of 515.34 g mol–1, is relatively soluble in water (500 mg L–1), unstable at high pH and under light, but relatively stable under a wide range of temperatures (Budavary et al., 1996). Chlortetracycline is a weak acid. No Kd values for CTC were found in the literature. It has a relatively long half-life in soil of at least 30 d (Gavalchin and Katz, 1994). Chlortetracycline was kept at 4°C and protected against light with aluminium foil. Tylosin (C46H77NO17) was obtained from Sigma-Aldrich as 8000 mg L–1 in 900 mg L–1 NaCl solution. Tylosin has a molecular weight of 916.14 g mol–1 (Budavary et al., 1996), is unstable at room temperature, is a weak base, and has a solubility in pure water slightly above 6000 mg L–1 (Budavary et al., 1996). It has a pKa of 7.1 (Tølls, 2001), a Kd ranging from 8 to 128 L kg–1 in soils (Rabølle and Spliid, 2000), a Koc ranging from 550 to 8000 L kg–1 (Rabølle and Spliid, 2000), and a half-life between 2 and 8 d (Galvalchin and Katz, 1994; Ingeslev and Halling-Sørensen, 2001). Tylosin was kept at –20°C before use.
A sandy loam soil and a clay soil were chosen for their similar pH and low organic carbon content, and because they represent extremes in particle size distribution. Therefore, the difference between the behavior of both chemicals would be mostly related to particle size distribution (Table 1). Difference in organic matter contents should be minimal compared to the difference in clay contents. The aggregates between 0.2 and 0.6 mm were destroyed with a rammer before sieving with a 2-mm sieve. The soil was then oven-dried at 105°C for 24 h before the experiments.
Rain water was captured under trees so that water properties would be as close as possible to rain water falling from canopy. Dust particles were not detectable. Water pH was 4.4 and conductivity was 16.1 µS. Its content (mg L–1) was 0.90 of Ca2+, 0.21 of Mg2+, 1.18 of K+, 0.79 of Na+, 0.25 of NO32–, 0.25 of NH4+, 0.28 of SO42–, and 0.51 of Cl–.
A stock solution was prepared for each treatment, each replicate, and each antibiotic separately. The mixtures were made of 140 g of 200 mg L–1 solution of CTC and 3000 mg L–1 of TYL into 0.025-L polypropylene bottles with (i) soil blanks (no soil but with antibiotics), or with 70 g of (ii) sterilized sandy loam, (iii) non-sterilized sandy loam, (iv) sterilized clay, and (v) non-sterilized clay. Antibiotic blanks (no antibiotics but with soil) were also prepared. Sterilization was performed at 121°C for 20 min using an autoclave and by spreading the soil in fine layer on wide, shallow containers. The sterilized soil was then dried at 105°C for 24 h. The soil was allowed to cool down in a closed container before use.
Optimal soil homogeneity was achieved by destroying the aggregates first, then by sieving the soil, mixing up the soil from bottom to top many times (up to six times), using soil containers smaller than 0.5 L, with at least 15 g of soil and by composite sampling from at least five areas in the containers. A 2:1 solution to soil ratio gave good accuracy (<6% variability) and concentration above detection limits for TYL on both soils but too low solution concentration of CTC sorbing on the clay soil. A 4:1 ratio for CTC sorption on clay was used. In the case of CTC and clay mixtures, 35 g of soil instead of 70 g was used for proper solution to soil ratio. The polypropylene bottles were covered with aluminium tape to prevent light degradation. The mixtures were continuously shaken up to 1 h (depending on the sampling time) and put in darkness. The optimal stroke length and frequency were 0.1 m and 100 stokes per minute below which the solution and soil mixtures did not sufficiently move or above which the bottles' movement would cause friction. The friction results in 2°C temperature increase although the temperature of the environment was controlled. Even with long strokes, soil stratification in the bottles occurred unless they were rotated every 0.25 h. One hour of shaking was sufficient to obtain homogeneous mixtures. The samples were shaken by hand for 1 min right before sampling. A 400-µL of solution were sampled at 0.00, 0.017, 0.08, 0.25, 0.75, 3, 7, 24, and 48 h and put into small vials (Model 410870-840; Alltech, Deerfield, IL). The samples were immediately frozen at –80°C.
The samples were defrosted 1 min before analysis with high performance liquid chromatography (HPLC). The solution was passed through a 0.2-µm filter (nylon filter syringes; Chromatographic Specialties, Brockville, ON, Canada) to remove soil particles. Tests were performed to assure no additional sorption during freezing, storage, and defrosting. Samples were compared with analysis on filtered samples immediate after sampling. Freezing and defrosting both occurred in less than 2 min. The process of freezing, storing, and defrosting may have lead to 3- to 5-min overestimation of sorption kinetics. This is not significant considering that the point of interest of this study was to know if equilibrium was reached within 24 h. Two injections were done for each sample in the HPLC. There were no significant differences between the injections. The error was less than 0.1% except close to the detection limit. Thereafter, the following text refers to the average values of these two injections per sample.
Concentration analysis were performed on HPLC following the method of Loke et al. (2000) and Díaz-Cruz et al. (2003) using a BDS Hypersil C8 100 x 2.1 column (Thermo, Waltham, MA). The HPLC was an autosampler (Model 717; Waters, Milford, MA) with a detector (Model Tm 996; Waters). The flux was 0.25 mL min–1. Two solvents were used, acid formic 0.5% and acetonitrile 100%. Tylosin is reported as the summation of both TYL A and B. Limit of detection was 0.05 mg L–1. Three replications were completed. Statistical analyses were performed using SAS Stat software Version 8.0 (SAS Institute, 1999).
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RESULTS AND DISCUSSION
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There was 6% variability in the concentration of CTC stock solutions between replicates. This variability may be due to the fact that each stock was done on different days with variations in room temperature in addition to small variability that may be randomly induced during storage, handling, freezing, and defrosting before analysis in addition to the HPLC precision. The concentration of TYL soil-blank solution was considerably more variable (up to 20%) than that of CTC (up to 6%). The reason is not clear, but it may be due, in addition to the above mention reasons, to the higher instability of TYL or to isomerization. On four possible isomers, only TYL A and B were measured.
The concentration of soil-blank solutions of both antibiotics did not significantly change over time. There was no significant degradation over a 48-h period at room temperature during storage over many days at –80°C (up to 60 d). Thus, the aluminium foil was sufficient to protect CTC against light degradation, sterilization of solution was not necessary, and the samples were properly stored and handled.
Sorption of TYL and CTC was highly different between soils but not significantly different between replicates neither between sterilized and non-sterilized soils. A period of 48 h was insufficient to measure the impact of microbial degradation for both chemicals in presence of either soil (Fig. 1). Therefore, soil sterilization is not required for short-term experiments (<48 h), which simplifies further experiments.
At equilibrium, CTC sorption on clay was about twice the extent of the sorption on sandy loam. Comparatively, TYL sorption on clay was about 2.4 times that on sandy loam. Thus the impact of soil texture on both chemicals is similar, but TYL is slightly more sensitive than CTC to clay content.
Tylosin sorbed on both soils almost one order of magnitude more than CTC, indicating lower risk of TYL transport in the soil profile. Although detection limits for both chemicals were similar, the variability in TYL sorption was much higher than that of CTC (Fig. 1). Variability of TYL was by far the highest on sandy loam. The difference in variability was not due to handling precision since both experiments were conducted simultaneously by the same people for both antibiotics and the variability of CTC concentration was low (<6%). Besides variability in stock solutions themselves, variability may have been due to small differences in the clay content of the sandy loam. This is because TYL is more sensitive to clay content, it is difficult to obtain perfectly mixed sandy loam soil, and for other unclear reasons. Note that in Fig. 1, each point is an average of two injections on HPLC and three replicates.
Sorption of the two chemicals on both soils occurred very fast (Fig. 1). Chlortetracycline sorption occurred within 10 min on both soils. Tylosin sorption occurred within 5 min on sandy loam and within 3 h on clay. A 24-h equilibrium time is more than enough for studying the sorption of CTC and TYL on both soils. In fact, a 1-h equilibrium time would probably be sufficient in most cases and 4 h for all cases.
The high sorption of the two antibiotics on soils having extreme particle size distribution combined with the very fast sorption kinetics indicates a small probability of leaching through the soil profile toward ground water. These antibiotics may however reach the ground water through preferential flow processes by which soil particles may be carried down in the profile (Kay et al., 2004). The results tend to indicate high probability of reaching surface water through erosion (i.e., attached to the eroded soil particles if erosion would take place). More study is needed on the movement of these chemicals in soil under cold climate.
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CONCLUSIONS
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The soil does not need to be sterilized for TYL and CTC sorption experiments shorter than 48 h. Equilibrium time for CTC sorption occurred within 10 min on both soils. Equilibrium sorption of TYL occurred within 3 h on clay and within 5 min on sandy loam. Only little information is available regarding the behavior of CTC in soils and even less information is available for cold climate conditions such as those found in Canada. More research is needed on CTC and TYL regarding their sorption and potential movement in soils toward surface water and ground water.
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ACKNOWLEDGMENTS
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The authors thank the CORPAQ Quebec and NSERC Canadian funds for their financial support, Francis Beaudry for technical support, and Cindy Dallaire and Pascal Dubé for their hard work in the laboratory.
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ABSTRACT
INTRODUCTION
