Metabolism and degradation kinetics of selected veterinary drugs in manure.

Prof Dr. M. Spiteller, Universität Dortmund

Summary

The fate and metabolism of the veterinary drugs sulfadiazine (SDZ) and difloxacin (DIF) was studied after application to fattening pigs by means of newly developed, robust, specific and sensitive analytical LC-MS/MS methods for the quantitation of the applied drugs and their biotransformed metabolites in manure, soil/manure and plants. New transformation products could be identified unambiguously by high resolution mass spectrometry. Knowledge about the metabolite pattern in manure is a prerequisite for risk assessment, as the manure may be spread into the environment. The use of 14C-labelled compounds allowed a complete mass balance of the applied drug and its metabolites after administration to fattening pigs. In the case of SDZ, the analysis of manure revealed two known metabolites, N-acetylsulfadiazine (Ac-SDZ) and 4-hydroxysulfadiazine (4-OH-SDZ), and two hitherto unidentified minor metabolites (N-formylsulfadiazine and N-acetyl-4-hydroxysulfadiazine). While from the excreted radioactivity SDZ accounted for 44%, more than 50% were biotransformation products. The analysis of manure after treating pigs with DIF showed that 90% of DIF was excreted as parent compound. The residual radioactivity contained the bioactive main metabolite Sarafloxacin (10%) and three trace metabolites (<1%). Storage of manure is routine step prior further proceeding in agricultural practice. During the storage (SDZ application) under aerobic and anaerobic conditions, the concentration of SDZ increased due to deacetylation of Ac-SDZ, 4-OH-SDZ concentrations remained constant. Storage of manure after DIF application exhibited no significant transformation or degradation of DIF. Sorption of organic compounds to soil form bound residues or so called non extractable residues (NER). Thus, leaching into groundwater may be avoided and bioavailability is limited. A rapid formation of non-extractable residues could be observed for both antibiotics. Sorption of DIF was fast, very strong and dominated by anionic species. The concentration of extractable SDZ and its metabolites in soil decreased exponentially, Surprisingly Ac-SDZ is directly converted into bound residues via fast process and not converted to SDZ. A significant amount of SDZ was found tightly bound to soil particles and did not desorb. The covalent binding of SDZ to soil humic substances occur via the free amino group as concluded from 15N-NMR experiments. The presence of manure enhanced the hysteresis effect observed and sorption tendency increased significantly. Thus, in fate studies of environmental contaminants the influence of manure should not be underestimated. Photolysis is a major degradation/transformation process in the environment. In aqueous solution and manure SDZ revealed the formation of five known photoproducts and one new compound. The extrusion of the functional SO2 group was found to be the main degradation process. During prolonged photo-degradation of DIF, seven photoproducts were identified, three of which were entirely novel. Residual anti-bacterial activities of DIF, sarafloxacin and also their photoproducts against a group of pathogenic strains were measured with varying degrees of efficacies against the tested bacteria. The plant uptake of SDZ and DIF was studied with maize. Less than 1.5 % (0.2% for DIF) of the applied radioactivity could be detected in the plants. Under the influence of roots a 10 time’s higher mineralization of SDZ and formation of about 3 times less non-extractable residues was observed. Furthermore, the abundance and transferability of bacterial antibiotic resistance genes were most pronounced in the vicinity of roots under conditions of maximum SDZ transformation. Variation of soil moisture (drying, re-wetting) and temperature conditions exhibited no significant influence on the fate of SDZ in soil.

begutachtete Publikationen:

1. Lamshöft, M, Sukul, P, Zühlke, S, Spiteller, M (2007): Metabolism of 14C-labelled and non- labelled Sulfadiazine after administration to pigs. Analytical and Bioanalytical Chemistry 388, 1733¬–1757.
2. Heuer, H, Focks, A, Lamshöft, M, Matthies, M, Spiteller, M (2008): Fate of sulfadiazine administered to pigs and its quantitative effect on the dynamics of bacterial resistance genes in manure and manured soil. Soil Biology & Biochemistry 40, 1892–1900.
3. Sukul, P, Lamshöft, M, Zühlke, S, Spiteller, M (2008): Photolysis of 14C-sulfadiazine in water and manure Chemosphere 71, 717–725.
4. Sukul, P, Lamshöft, M, Zühlke,S, Spiteller, M (2008): Sorption and desorption of sulfadiazine in soil and soil-manure systems. Chemosphere 73, 1344–1350.
5. Sukul, P, Lamshöft, M, Kusari, S, Zühlke, S, Spiteller, M (2009): Metabolism and excretion kinetics of 14C-labeled and non-labeled difloxacin in pigs after oral administration, and antimicrobial activity of manure containing difloxacin and its metabolites. Environmental Research 109, 225–231.
6. Deivasigamani, P, Sukul, P, Lamshöft, M, Mohan, A, Zühlke, S, Spiteller, M (2009): Photolysis of Difloxacin and Sarafloxacin in Aqueous Systems. Chemosphere 77, 739–746.
7. Sukul, P, Lamshöft, M, Kusari, S, Zühlke, S, Spiteller, M (2009): Metabolism and excretion kinetics of 14C-labeled and non-labeled difloxacin in pigs after oral administration, and antimicrobial activity of manure containing difloxacin and its metabolites. Environmental Research 109, 225–231.
8. Kusari, S, Deivasigamani, P, Lamshöft, M, Spiteller, M (2009): In Vitro Residual Anti-bacterial Activity of Difloxacin, Sarafloxacin and their Photoproducts after Photolysis in Water. Environmental Pollution 157, 2722–2730.
9. Lamshöft, M, Sukul, P, Zühlke, S, Spiteller, M (2010): Behavior of 14C-sulfadiazine and 14C-difloxacin during manure storage. Science of the  Total Environment 408, 1563–1568.
10. Kotzerke, A, Hammesfahr, U, Kleineidam, K, Lamshöft, M, Thiele-Bruhn, S, Schloter, M, Wilke, B.-M (2010): Influence of difloxacin-contaminated manure on microbial community structure and function in soils. Biology and Fertility of Soils 47, 177–186.
11. Hammesfahr, U, Kotzerke, A, Lamshöft, M, Wilke, B.-M, Kandeler, E, Thiele-Bruhn S (2011): Effects of sulfadiazine-contaminated fresh and stored manure on a soil microbial community. European Journal of Soil Biology, 47 61–68.
12. Rosendahl. I, Siemens. J, Kindler. R, Groeneweg. J, Zimmermann. J, Czerwinski, S, Lamshöft, M, Laabs. V, Wilke. B.M, Vereecken, H, Amelung. W (2012): Persistence of the fluoroquinolone antibiotic difloxacin in soil and lacking effects on N-turnover. Journal of Environmental Quality, 41 1275–1283.

Dynamics of degradation and residues of veterinary medicines in manure amended soil.

Prof. Dr. A. Schäffer, Dr. B. Schmidt, RWTH Aachen

Summary

Goal of the subproject was the evaluation of the fate of two veterinary pharmaceuticals (sulfadiazine, SDZ, and difloxacine, DIF) in soil using different chemical and analytical methods such as radio-thin layer chromatography (radio-TLC), radio-high performance liquid chromatography (radio-HPLC), gas chromatography mass spectrometry (GC-MS), gas chromatography coupled to a flame ionization detector (GC-FID), and liquid chromatography tandem mass spectrometry (LC-MS/MS). A special focus was to study the influence of maize plants and a potential rhizosphere effect on the fate of the two pharmaceuticals. Only very small quantities of the two pharmaceuticals were taken up by maize plants from the investigated soil. This indicates a poor availability of both antibiotics for uptake by maize. Also the fraction of both 14C- labeled pharmaceuticals that was mineralized to 14CO2 was very small. For both antibiotics the formation of non-extractable residues (NER) dominated their fate in soil. Neither for SDZ nor for DIF significant differences between the extractability from root-free soil and root-near soil were found. Also manure had no effect on the extractability of DIF.

The only metabolite of SDZ was 4-OH-SDZ, which was formed in soil so that its concentrations increased during the course of incubation experiments with soil. No metabolites of DIF were detected in incubations studies, only the parent compound was found in the extracts.

For an in-depth analysis of non-extractable residues, humic substances of the investigated soil were fractionated into fulvic acids, humic acids, and humin. Furthermore, soil that had been extracted using accelerated solvent extraction (ASE) were separated into their sand, silt, and clay particle size fractions in order to assess the distribution of NER in these fractions.

Because large quantities of applied radioactivity were recovered in the fulvic acids, this fraction was investigated in more detail using size exclusion chromatography. The chromatograms showed two radioactivity peaks, a broad one with high molecular weight eluting early and second sharp one eluting later. Based on peak shape and experiments with pure fulvic acids and the two antibiotics we hypothesized that the second low-molecular weight peak represented free molecules of the parent drugs and its metabolites that were not bound to the fulvic acids. A further analysis of the two molecular weight fractions using LC-MS/MS confirmed the existence of the parent compound SDZ in the fulvic acid fraction of NER. During the first days of the incubation experiment, non-extractable SDZ and 4-OH-SDZ occurred almost exclusively in free form in the later-eluting fraction. With increasing incubation time the fraction of free SDZ and 4-OH-SDZ decreased indicating a progressively stronger binding or entrapment of the two compounds into the fulvic acids. An in-depth characterization of DIF-containing fulvic acids with LC-MS/MS was not feasible because only very small amounts of radioactivity could be extracted from soil.

begutachtete Publikationen:

1. Junge, T., Meyer, K.C., Cielinski, K., Adams, A., Schäffer A., Schmidt, B., 2011. Characterization of non-extractable C-14- and C-13-sulfadiazine residues in soil including simultaneous amendment of pig manure. Journal of Environmental Science and Health B 46, 137-149.
2. Junge, T., Classen, N., Schäffer, A., Schmidt, B., (2012): Fate of the veterinary antibiotic C-14-difloxacin in soil including simultaneous amendment of pig manure with the focus on non-extractable residues. Journal of Environmental Science and Health B 47, 858-868.
    

Weitere Publikationen:

1. Junge, T. (2012): Abbau und Rückstandsdynamik von Tierarzneimitteln in Boden-Pflanzen-Systemen. Dissertation, RWTH Aachen, 148 pp.

Sequestration of Veterinary Medicines in Soils.

Prof. Dr. W. Amelung, Dr. V. Laabs, PD Dr. J. Siemens, Universität Bonn

Summary

The environmental effects of veterinary antibiotics that currently provoke increasing public concerns largely depend on the fate of these substances following their application with manure to soil. In this context, sorption and sequestration are key processes that govern the antibiotic’s chemical and biological availability in the short and long run. We therefore investigated these processes using two test compounds (sulfadiazine/SDZ and difloxacin/DIF). Our experiments evolved from laboratory incubations to complex mesocosm and field studies, which increasingly permitted integrating the effects of various environmental variables (soil moisture and soil temperature) and particular soil compartments (rhizosphere, aggregates) on the fate of antibiotics. Following these experiments, we sequentially extracted an easily-extractable fraction (EAS, a proxy for bioaccessibility) and a sequestered residual fraction (RES) from soil to account for antibiotic fractions of different binding strength.

Under controlled laboratory conditions, the dissipation of easily-extractable SDZ was rapid (DT50 < 21 d), while a second more strongly bound residual fraction concomitantly built up in soil and was then very persistent (DT50 > 290 d). Additional laboratory experiments revealed that the sequestration of SDZ is most likely driven by the diffusion of SDZ into soil organic matter (SOM) and – to a smaller extent – into the pores of iron oxides. The sequestration of SDZ was furthermore paralleled by a pronounced formation of non-extractable residues, amounting to approx. 50% of the applied amount after three months. Out of the two major metabolites of SDZ in manure, only 4-OH-SDZ was preserved in soil yet it lacked evidence of toxicity. The other metabolite, N-Ac-SDZ, however, was rapidly reconverted into the target antibiotic. The field experiments showed a similar behavior under field conditions. The presence of plants even accelerated the dissipation of easily-extractable SDZ in the rhizosphere, presumably as a result of an enhanced biological transformation of SDZ. This implies a reduced exposure of the particularly active microbial communities in the rhizosphere to SDZ. Yet, due to remobilization processes, concentrations never reached zero, i.e., the exposure of microorganisms to SDZ was weak but continuous.

The dissipation of both SDZ fractions was largely governed by soil temperature, so that antibiotic concentrations measured in the field could be predicted from a temperature-adjustment of the dissipation rate constants, which was derived from laboratory experiments at different temperatures. The impact of soil moisture, however, was limited to an initially slightly faster dissipation of the EAS-fraction in constantly wet soil relative to a variant with cyclic drying. For SDZ, laboratory results were thus generally transferable to the field situation and this may greatly facilitate the environmental risk assessment of sulfonamides. 

For DIF, we could show that its bioaccessibility constantly amounted to < 2% of the applied amount. Due to the strong sorption of DIF, its fate in soil was not controlled by climatic variables but likely by equilibrium exchanges with bound residues. As a result, the ASE-extractable DIF was highly persistent (DT50 290 d), though dissipation was again accelerated in the rhizosphere. Non-extractable residues of DIF formed at 60–65% of the applied amount. The fate of DIF was identical under all experimental conditions tested, i.e., we conclude that neither soil moisture nor soil temperature affect the behavior of DIF in soil. Instead, the very pronounced sorption of this compound to soil controlled its environmental fate, and likely also its effects.

begutachtete Publikationen:

1. Förster, M, Laabs, V, Lamshöft, M, Pütz, T, Amelung, W (2008): Analysis of aged sulfadiazine residues in soils using microwave extraction and liquid chromatography and tandem mass spectrometry. Analytical and Bioanalytical Chemistry 391, 1029–1038.
2. Förster, M, Laabs, V, Lamshöft, M, Groeneweg, J, Zühlke, S, Spiteller, M, Krauss, M, Kaupenjohann, M, Amelung, W (2009): Sequestration of Manure-Applied Sulfadiazine Residues in Soils. Environmental Science and Technology 43, 1824–1830.
3. Rosendahl, I, Siemens, J, Groeneweg, J, Linzbach, E, Laabs, V, Herrmann, C, Vereecken, H, Amelung, W (2011). Dissipation and Sequestration of the Veterinary Antibiotic Sulfadiazine and Its Metabolites under Field Conditions. Environmental Science and Technology 45, 5216–5222.
4. Rosendahl, I, Siemens, J, Kindler, R, Groeneweg, J, Zimmermann, J, Czerwinski, S, Lamshöft, M, Laabs, V, Wilke, BM, Vereecken, H, Amelung, W (2012): Persistance of the Fluoroquinolone Antibiotic Difloxacin in Soil and Lacking Effects on Nitrogen Turnover. Journal of Environmental Quality 41, 1275–1283.
5. Kopmann, C, Jechalke, S, Rosendahl, I, Groeneweg, J, Krögerrecklenfort, E, Zimmerling, U, Weichelt, V, Siemens, J, Amelung, W, Heuer, H, Smalla, K (2013): Abundance and transferability of antibiotic resistance as related to the fate of sulfadiazine in maize rhizosphere and bulk soil. FEMS Microbiology Ecology 83, 125–134.
6. Müller, T, Rosendahl, I, Focks, A, Siemens, J, Klasmeier, J, Matthies, M (2013): Short-term extractability of sulfadiazine after application to soils. Environmental Pollution 172, 180–185.
7. Jechalke, S, Kopmann, C, Rosendahl, I, Groeneweg, J, Weichelt, V, Krögerrecklenfort, E, Brandes, N, Nordwig, M, Ding, GC, Siemens, J, Heuer, H, Smalla, K (2013): Increased Abundance and Transferability of Resistance Genes after Field Application of Manure from Sulfadiazine-Treated Pigs. Applied and Environmental Microbiology 79, 1704–1711.
8. Reichel, R, Rosendahl, I, Peeters, ETHM, Focks, A, Groeneweg, J, Bierl, R, Leinweber, P, Amelung, W, Thiele-Bruhn, S (2013): Effects of slurry from sulfadiazine (SDZ) and difloxacin (DIF) medicated pigs on the structural diversity of microorganisms in rhizosphere soil. Soil Biology and Biochemistry 62, 92–91.
9. Jechalke, S, Focks, A, Rosendahl, I, Groeneweg, J, Siemens, J, Heuer, H, Smalla, K (2014): Structural and functional response of the soil bacterial community to application of manure from difloxacin-treated pigs. FEMS Microbiology Ecology 87, 78-88.
10. Reichel, R., Patzelt, D., Barleben, C., Rosendahl, I., Ellerbrock, R., Thiele-Bruhn, S. (2014a): Soil microbial community responses to sulfadiazine-contaminated manure in different soil microhabitats. Applied Soil Ecology 80, 15-25.
11. Reichel, R., Radl, V., Rosendahl, I., Albert, A., Amelung, W., Schloter, M., Theile-Bruhn, S. (2014b): Soil microbial community responses to antibiotic-contaminated manure under different soil moisture regimes. Applied Microbiology and Biotechnology 98, 6487-6495.
12. Jechalke, S., Heuer, H., Siemens, J., Amelung, W., Smalla, K. (2014): Fate and effects of veterinary antibiotics in soil. Trends in Microbiology  DOI: http://dx.doi.org/10.1016/j.tim.2014.05.005
     

 Weitere Publikationen:

1. Förster, M. (2011): Sequestration of sulfadiazine in soil, Dissertation, University of Bonn, 97 pp.
2. Rosendahl, I. (2012): Fate of the veterinary antibiotics sulfadiazine and difloxacin in soil, Dissertation, University of Bonn, 133 pp.

Transport of Veterinary Medicines from Soils to Groundwater.

Prof. Dr. H. Vereecken, Dr. T. Pütz, Dr. R. Kasteel, Dr. J. Groeneweg, FZ Jülich

Summary

One objective was to study the sorption and desorption behaviour of sulfadiazine. Short-and long term sorption experiments were performed with the antibiotic sulfadiazine (SDZ) in the plough layer and the subsoil of a loamy sand (Kaldenkirchen, KAL) and a silty loam (Merzenhausen, MRZ). The parameterization of a two-stage, one-rate sorption model combined with a first-order transformation model showed that sorption of SDZ was nonlinear, time-dependent, and affected by pH, with a higher sorption capacity for the loamy sand. Another objective was to study the transport of veterinary medicines using soil columns with special emphasis on preferential flow, the behaviour of 4-hydroxy-sulfadiazine (4-OH-SDZ) and the effect of repeated manuring with sulfadiazine containing piggery waste. No preferential transport or colloid-facilitated transport was observed in any of the column transport studies. An additional (irreversible) sorption site was needed in the model description. Besides chemical interactions, the transport of SDZ was affected by the soils hydraulic behaviour, with less leaching for lower infiltration rates. The sorption and kinetic parameters of 4-OH-SDZ were similar to those of SDZ.  Repeated application of manure from pigs medicated with 14C-labeled SDZ on soil columns with did not show an influence on the shape of the break through curves and on the formation of transformation products. For yet unknown reasons, the transformation product 4-(2-iminopyrimidin-1(2H)-yl) aniline was only formed in considerable amounts in the absence of manure. By upgrading the two-stage one-rate model of Wehrhan (2010), we were able to describe instantaneous sequestration of sulfadiazine in the residual fraction (RES) obtained by harsh extraction of soil and the non extractable fraction (NER). One objective was to provide experimental evidence on the occurrence or non-occurrence of antibiotics in the deep leachate of soils using lysimeters under natural weather conditions and to obtain complete balances on the fate of the veterinary medicine sulfadiazine. In the KAL lysimeter leachate we measured on one occasion 7.6 ng/L sulfadiazine together with the breakthrough of the tracer bromide and on 4 occasion’s 2-amino-pyrimidin up to 36 ng/L. The distribution of radioactivity in the upper 30 cm soil profile of the lysimeter showed a further dislocation in deeper soil layers of the 0.5 m2 lysimeter, which was especially the case in the loamy sand soil (KAL). In the silty loam soil (MRZ) we measured a 30 to 40% loss of radioactivity in three years and most of the leftover radioactivity was found in the first 10 cm. Extraction of the soil samples showed a noticeable difference between the KAL and MRZ soil. At day 218, ~50% of the applied radioactivity was found in the NER-fractions for both soils (a value comparable with the results of Forster et al 2009). At day 1022, we still found all applied radioactivity back in the KAL soil, with an increase in the NER fraction. For MRZ, we measured a strong decrease in the RES-fraction, indicating that SDZ and metabolites were possibly mineralised, but also the NER fraction became smaller, indicating that SDZ and metabolites could have become bioavailable from the NER fraction. A batch experiment with 14C-SDZ added to soil from the MRZ lysimeter confirmed that a mineralisation of up to 10% of the added SDZ after three months is possible, when incubated at 45% of the max. water holding capacity and up to 50-60%, when incubated as slurry. We could isolate and identify a bacterium capable of partly mineralise sulfadiazine and producing the transformation product 2-amino-pyrimidin.Thus, in contrast to previous results, we have strong evidence that mineralisation of SDZ could have been the reason for the loss of radioactivity in the lysimeter experiment and a possible mechanism for a degradation pathway of sulfadiazine in soil.

begutachtete Publikationen:

1. Förster, M, Laabs, V, Lamshöft, M, Pütz, T, Amelung, W (2008). Analysis of aged sulfadiazine residues in soils using microwave extraction and liquid chromatagraphy tandem mass spectrometry.
   Analytical and Bioanalytical Chemistry 391, 1029–1038.
2. Huschek, G, Hollmann, D, Kurowski, N, Kaupenjohann, M, Vereecken, H (2008): Re-evaluation of the conformational structure of sulfadiazine species using NMR and ab initio DFT studies and its implication on sorption and degradation. Chemosphere 72, 1448–1454.
3. Tappe, W, Zarfl, C, Kummer, S, Burauel, P, Vereecken, H, Groeneweg, J (2008): Growth-inhibitory effects of sulfonamides at different pH: Dissimilar susceptibility patterns of a soil bacterium and a test bacterium used for antibiotic assays. Chemosphere 72, 836–843.
4. Unold, M, Kasteel, R, Groeneweg, J, Vereecken, H (2009): Transport and transformation of sulfadiazine in soil columns packed with a silty loam and a loamy sand. Journal of Contaminant Hydrology 103, 38–47
5. Unold, M, Simunek, J, Kasteel, R, Groeneweg, J, Vereecken, H (2009): Transport of Manure-Based Applied Sulfadiazine and Its Main Transformation Products in Soil Columns. Vadose Zone Journal 8, 677–689.
6. Förster, M, Laabs, V, Lamshöft, M, Groeneweg, J, Zühlke, S, Spiteller, M, Krauss, M, Kaupenjohann, M, Amelung, W (2009): Sequestration of manure-applied sulfadiazine in soils. Environmental Science and Technology 43, 1824–1830.
7. Kasteel, R, Mbo, CM, Unold, M, Groeneweg, J, Vanderborght, J, Vereecken, H (2010): Transformation and sorption of the veterinary antibiotic sulfadiazine in two soils: a short-term batch study. Environmental Science and Technology 44, 4651–4657.
8. Wehrhan, A, Streck, T, Groeneweg, J, Vereecken, H, Kasteel, R (2010): Long-term sorption and desorption of sulfadiazine in soil: Experiments and modeling. Journal of Environmental Quality 39, 654–666.
9. Unold, M, Kasteel, R, Groeneweg, J, Vereecken H (2010): Transport of sulfadiazine in undisturbed soil columns: the effect of flow rate and applied mass. Journal of Environmental Quality 39, 2147–2159
10. Rosendahl, I, Siemens, J, Groeneweg, J, Linzbach, E, Laabs, V, Herrmann, C, Vereecken, H, Amelung, W (2011): Dissipation and sequestration of the veterinary antibiotic sulfadiazine and its metabolites under field conditions. Environmental Science and Technology 45, 516–522
11. Rosendahl, I, Siemens, J, Kindler, R, Groeneweg, J, Zimmermann, J, Czerwinsky, S, Lamshöft, M, Laabs, V, Wilke, BM, Vereecken, H, Amelung W (2012): Persistence of the fluoroquinolone antibiotic Difloxacin in soil and lacking effects on N-turnover. Journal of Environmental Quality 41, 1275–1283
12. Sittig, S, Kasteel, R, Groeneweg, J, Vereecken, H (2012): Long-term sorption and sequestration dynamics of the antibiotic sulfadiazine- a batch study. Journal of Environmental Quality 41, 1497–1506
13. Jechalke, S, Kopmann, C, Rosendahl, I, Groeneweg, J, Krögerrecklenfort, E, Zimmerling, U, Weichelt, V, Siemens, J, Amelung, W, Heuer, H, Smalla, K (2013): Abundance and transferability of antibiotic resistance as related to the fate of sulfadiazine in maize rhizosphere and bulk soil. FEMS Microbiology Ecology 83, 125–134.
14. Reichel, R, Rosendahl, I, Peeters, ETHM, Focks, A, Groeneweg, J, Bierl, R, Schlichting, A, Amelung, W, Thiele-Bruhn, S (2013): Effects of slurry from sulfadiazine (SDZ) and Difloxacin (DIF) medicated pigs on the structural diversity of microorganisms in bulk and rhizosphere soil. Soil Biology and Biochemistry 62, 82–91.
15. Jechalke, S, Kopmann, C, Rosendahl, I, Groeneweg, J, Weichelt, V, Krögerrecklenfort, E, Brandes, N, Nordwig, M, Ding, GC, Siemens, S, Heuer, H, Smalla, K (2013): Field application of manure from sulfadiazine treated pigs increased the abundance and transferability of resistance genes. Applied and Environmental Microbiology 79, 1704–1711.
16. Ollivier, J, Schacht, D, Groeneweg, J, Engel, M, Wilke, BM, Kleineidam, K, Schloter, M (2013): Effect of repeated application of sulfadiazine-contamined pig manure on the abundance and diversity of ammonia- and nitrite oxidizers in the root-rhizosphere complex of pasture plants under field conditions. Frontiers in Microbiology 4, Article 22.
17. Tappe, W, Herbst, M, Hofmann, D, Koeppchen, S, Kummer, S, Thiele, B, Groeneweg, J (2013): Degradation of sulfadiazine by Microbacterium lacus strainSDZm4 isolated from lysimeters three years after manuring with slurry frompigsmedicated with 14C-labelled sulfadiazine. Applied and Environmental Microbiology 79, 2572–2577.
18. Sittig, S., Kasteel, R., Groeneweg, J., Hofmann, D., Thiele, B., Köppchen, S., Vereecken, H. (2014): Dynamics of transformation of the veterinary antibiotic sulfadiazine in two soils. Chemosphere 95, 470-477.
     

Weitere Publikationen:

1. Sittig, S. (2014): Sorption, Trabnsformation and transport of sulfadiazine in a loess and a sandy soil. Dissertation, University of Bonn, 103 pp.
2. Unold, M. (2009): Experiments and numerical studies on transport of sulfadiazine in soil columns. Dissertation, University of Bonn, 133 pp.
3. Unold, M.; Kasteel, R., Groeneweg, J.; Vereecken, H. (2008): Der Transport des Antibiotikums Sulfadiazin in Böden: Welchen Effekt hat die Gülle? Mitt. Umweltchem. Ökotox. 14 (4), 98-101.
4. Wehrhan, A. (2006): Fate of veterinary pharmaceuticals in soil: An experimental and numerical study on the mobility, sorption and transformation of sulfadiazine. Dissertation, University of Bonn, 159 pp

Effects of Veterinary Medicines on the Structural Diversity of the Microbial Biomass in Soils.

Prof. Dr. Thiele-Bruhn, Universität Trier

Summary
Veterinary antibiotics reach agricultural soil together with manure as a complex, microbially populated, nutrient-rich substrate. Impacts on the soil microbial community and its structural diversity, functions and resistance level are expected. Hence our central hypothesis in all phases of within FOR566 was that antibiotic pharmaceuticals exert adverse effects on soil microorganisms, reflected in shifts in their structural diversity. This was investigated with samples from microcosm, pot (greenhouse), mesocosm, and field experiments by using PLFA analysis and fingerprints from DGGE separated 16S rRNA genes, in part complemented by subsequent sequencing and FISH analysis. Significant effects of the investigated antibiotics on the microbial community structure were determined in experiments on all spatial scales. Overall, the presence of antibiotics in manure caused shifts from bacteria to fungi and in parts shifts between gram+ and gram‑bacteria within the bacterial community, with Pseudomonas being particularly sensitive. This corresponds to findings of project B2, which also demonstrated shifts from bacteria to archaea after exposure to SDZ-containing manure. Community shifts were often, but not necessarily, accompanied by effects on microbial functions, emphasizing the functional redundancy within soil microbial communities. An increase in the resistance level shown by project B3 clearly documented that community shifts go along with a selection of tolerant and/or antibiotic resistant species. Effects on the community structure evolved a few days after adding antibiotics to soil and further increased and persisted over a long-term of 252 days and possibly beyond. These dynamics did not follow the rapid decline in the bioavailable fraction of the antibiotics, thus leading to an apparent concentration independence of the effects. Furthermore, effects interacted with manure composition and varied with the applied amount of manure. This was because manure acted as substrate and microbial inoculum, whose molecular and microbial composition is already changed within the digestive tract of medicated organisms. Furthermore, parent compounds and metabolites might be transformed during manure storage. Hence, a mixed effect of manure composition, manure-derived microorganisms that survive in soil for several weeks, transformation products of the antibiotic, and the antibiotic itself on soil microorganisms must be expected when soils are fertilized with manure that contains antibiotics. However, storage of manure was not suited to mitigate effects of sulfonamides in soil since SDZ reacted back from the acetyl conjugate to the parent compound. Manure constituents and antibiotics accumulated in outer shells of soil macroaggregates and earthworm burrows. In the latter, significant shifts in the microbial community structure were even significant after 252 days of a field experiment. In contrast, the rhizosphere is a microcompartment of higher resilience, able to mitigate antibiotic effects on the microbial community, which is reflected by a stronger dissipation and transformation of SDZ. Yet, strong effects on plants were identified even at SDZ concentrations that were close to what can be expected in the field. This included hormone-like effects on root geotropism and ramification, and affected plant water and nutrient uptake. Furthermore, antibiotic effects on microbial communities were more pronounced in soils exposed to periodic changes in soil moisture by drying-rewetting dynamics. Drying and rewetting increased the potentially bioaccessible SDZ concentrations. The combined stress from SDZ and strong moisture dynamics lowered the microbial biomass, which trend was also determined under complex field conditions. The studies clearly document the environmental relevance of pharmaceutical antibiotics reaching agricultural soils.

begutachtete Publikationen:

1. Hammesfahr U, Thiele-Bruhn S, Manzke B, Heuer H, Smalla K (2008): Effect of sulfadiazine and pig slurry on the structural diversity of soil microorganisms, Soil Biology and Biochemistry 40, 1583–1591.
2. Hammesfahr U, Bierl R, Thiele-Bruhn S (2011a): Manure and the antibiotic sulfadiazine are interacting on microbial biomass and structure. Journal of Plant Nutrition and Soil Science 174, 614–623.
3. Hammesfahr U, Kotzerke A, Lamshöft M, Wilke BM, Kandeler E, Thiele-Bruhn S (2011b): Sulfadiazine contaminated fresh and stored manure modifies the function and structure of soil microbial community. European Journal of Soil Biology 47, 61–68.
4. Michelini L, Reichel R, Werner W, Ghisi R, Thiele-Bruhn S (2012): Sulfadiazine uptake and effects on Salix fragilis L. and Zea maize L. plants. Water, Air and Soil Pollution 223, 5243–5257.
5. Reichel R, Rosendahl I, Peeters ETHM, Focks A, Groeneweg J, Bierl R, Schlichting A, Amelung W, Thiele-Bruhn S (2013): Effects of slurry from sulfadiazine- (SDZ) and difloxacin- (DIF) medicated pigs on the structural diversity of microorganisms in rhizosphere soil. Soil Biology and Biochemistry 62, 82–91.
6. Reichel, R, Patzelt, D, Barleben, C, Rosendahl, I, Ellerbrock, RH, Thiele-Bruhn, S (2014a): Microbial community structure and function of diverse soil microhabitats respond differently to sulfadiazine antibiotic co-applied with pig manure. Applied Soil Ecology 80, 15-25.
7. Reichel, R., Viviane Radl, Ingrid Rosendahl, Andreas Albert, Wulf Amelung, Michael Schloter, Sören Thiele-Bruhn (2014b): Soil microbial community responses to antibiotic-contaminated manure under different soil moisture regimes. Applied Microbiology and Biotechnology 98, 6487-6495.
    

Weitere Publikationen:

1. Hammesfahr, U. (2011): Alterations in soil microbial structure and function due to different manure and antibiotic loads. PhD thesis, Trierer Bodenkundliche Schriften 16, 98 pp.
2. Thiele-Bruhn, S., Aust, M.-O. (2013) Stoffdatenblatt: Sulfonamide. In: Litz, N., Wilcke, W., Wilke, B.-M. (Hrsg.) Bodengefährdende Stoffe. Kap. VI-2, 1-64. ecomed Verlagsgesellschaft, Landsberg/Lech.
3. Reichel, R. (2014): Soil microbial community responses to antibiotic pharmaceuticals: influence of different soil habitats and moisture regimes. Dissertation. University of Trier, 208 pp.

Effects of veterinary medicines on the functional diversity of the microbial biomass in soils.

Prof. Dr. B.-M. Wilke, TU Berlin, Prof. Dr. M. Schloter, Dr. K. Kleineidam, Helmholz Zentrum München

Summary

Veterinary antibiotics are used in the animal husbandry for therapeutic purposes and for growth promotion. By the application of manure from treated animals as fertilizer in agriculture, excreted antibiotics and their associated metabolites enter the soil environment. In the present project, we investigated the effects of the antibiotics sulfadiazine, amoxicillin and difloxacin, used in veterinary medicine, on the activity and functional diversity of microbial communities. Thereby, an especial emphasis was given to nitrogen cycle, due to the relevance of this nutrient to plant growth, crop production and sustainable use of soils. The focus of the study was to test the hypothesis that the fate and effects of antibiotics is highly determined by the i) type of antibiotic, ii) the mode and number of application to soil as well as iii) the abiotic soil properties and the climatic conditions.
The degree to which the antibiotics affected microbial functions was to a large extent controlled by the exposure of the soil microbial community to the antibiotics, which, in turn, depended on the compound’s fate. Amoxicillin was so readily degraded that its residues hardly affected microbial community functions. Difloxacin in turn was strongly adsorbed by soil particles, so that it was not bioavailable. Both antibiotics had therefore only low potential to affect microbial processes assessed in the experiments. Larger and lasting effects were only reported for sulfadiazine (SDZ). Yet, since SDZ is a bacteriostatic agent, these effects were only observed following the stimulation of microbial activity by adding manure as a substrate.  As expected, the measured effects greatly differed between the investigated soil compartments. While consequences of the application of manure containing antibiotics to microbial communities of bulk soils were relatively low, these were more pronounced in the rhizosphere of maize and clover. Also environmental conditions, like soil moisture, influenced the effect pattern of the applied antibiotics on microbial communities involved in nitrogen transformation.  In general, the bacterial communities that carry on the process of nitrification were more sensitive to the application of manure containing antibiotic than those related to denitrification. This might be explained by the fact that denitrification is a facultative process carried out by very diverse group of microorganisms. Yet, nitrification potential was less affected by the antibiotic application due to the functional redundancy between ammonia oxidizing bacterial (AOB) and ammonia oxidizing archaeal (AOA) communities.
Repeated applications of SDZ-manure led to progressing changes of the microbial community structure and to long-lasting (permanent) decrease of the soil microbial activity. Yet, as shown for AOB, after many applications the community begins to recover due to the selection of either intrinsically better adapted organisms or development of SDZ resistance.
In conclusion, we observed that in order to evaluate the risk of the introduction of antibiotics to soil ecosystems many factors must be considered such as bioavailability of the compound, type of soil, concentration and frequency of application. Besides, the selection of general processes carried out by diverse group of microbes as indicator parameters might mask effects of antibiotics on key soil processes, such as nitrification.

begutachtete Publikationen:

1. Heuer, H, Solehati, Q, Zimmerling, U, Kleineidam, K, Schloter, M, Müller, T, Focks, A, Thiele-Bruhn, S, Smalla, K (2011): Accumulation of sulfonamide resistance genes in arable soils due to repeated application of manure containing sulfadiazine. Applied Environmental Microbiology 77, 2527–2530.
2. Kotzerke, A, Sharma, S, Schauss, K, Heuer, H, Thiele-Bruhn, S, Smalla, K, Wilke, M, Schloter, M (2008): Alterations in soil microbial activity and N-transformation processes due to Sulfadiazine loads in pig-manure. Environmental Pollution 153, 315–322.
3. Kotzerke, A, Kleineidam, K, Horn, M, Drake, H, Wilke, BM, Schloter, M (2010): Manure contaminated with the antibiotic sulfadiazine impairs the abundance of nirK- and nirS-type denitrifiers in the gut of the earthworm Eisenia fetida. Biology Fertility of Soils 46, 415–418.
4. Kotzerke A, Hammesfahr, U, Kleineidam, K, Lamshöft, M, Thiele Bruhn, S, Wilke, BM, Schloter, M (2011a): Influence of difloxacin-contaminated manure on microbial community structure and function in soils. Biology and Fertility of Soils 47, 177–186.
5. Kotzerke, A, Kleineidam, K, Wilke, BM, Schloter, M (2011b): Alterations in total microbial activity and nitrification rates in soil due to amoxicillin spiked pig manure. Plant Nutrition and Soil Science 174, 56–64.
6. Ollivier, J, Kleineidam, K, Reichel, T, Thiele-Bruhn, S, Kotzerke, A, Wilke, BM, Schloter, M (2010): Effect of sulfadiazine-contaminated pig manure on abundance of genes and transcripts involved in nitrogen transformation in the root-rhizosphere complexes of maize and clover. Applied Environmental Microbiology 76, 7903–7909.
7. Ollivier, J, Töwe, S, Bannert, A, Hai, B, Kastl, EM, Meyer, A, Su, MX, Kleineidam, K, Schloter, M (2011): Nitrogen turnover in soil and global change. FEMS Microbiology Ecology 78, 3–16.
8. Ollivier, J, Schacht, D, Kindler, R, Groneweg, J, Engel, M, Wilke, BM, Kleineidam, K, Schloter, M (2013): Effects of repeated application of sulfadiazine-contaminated pig manure on the abundance and diversity of ammonia- and nitrite oxidizers in the root-rhizosphere complex of pasture plants under field conditions. Frontiers in Microbiology 4, 22/1.
9. Rosendahl, I, Siemens, J, Kindler, R, Groeneweg, J, Zimmermann J, Czerwinski, S, Lamshöft, M, Laabs V, Wilke, BM, Vereecken, H, Amelung, W (2012): Persistence of the fluoroquinolone antibiotic difloxacin in soil and lacking effects on nitrogen turnover, Journal of Environmental Quality 41, 1275–1283.
10. Schauss, K, Focks, A, Leininger, S, Kotzerke, A, Heuer, H, Thiele-Bruhn, S, Sharma, S, Wilke, BM, Matthies, M, Smalla, K, Munch, JC, Amelung, W, Kaupenjohann, M, Schleper, C, Schloter, M (2009): Dynamics and functional relevance of ammonia-oxidizing archaea in agricultural soils. Environmental Microbiology 11, 446–456.
     

Weitere Publikationen:

1. Kotzerke, A. (2011): Effects of veterinary antibiotics on the activity and functional diversity of microorganisms in different soils. Dissertation. TU Berlin, 134 pp.
2. Ollivier, J. (2013): Diversity, abundance and activity of microbes involved in nitrogen turnover in the rhizosphere of different plants grown on sites contaminated with the antibiotic sulfadiazine or heavy metals. Dissertation, TU Munich, 147 pp.

Effect of veterinary medicines and manure amendment on the abundance, diversity and transfer of bacterial antibiotic resistance genes in soil bacteria.

Prof. Dr. K. Smalla, Julius Kühn Institut Braunschweig

Summary

Large amounts of antibiotics are applied in veterinary medicine and reach agricultural fields by manure fertilization. In soil, the fate of these substances and their effects on the structure and function of bacterial communities and the development and spread of antibiotic resistance genes and mobile genetic elements were largely unknown. However, the respective knowledge is crucial for an assessment of risks associated with the application of antibiotics with manure to agricultural soils and potential effects on human health.
In this project we aimed at the assessment of effects of the veterinary medicines amoxicillin (AMX), difloxacin (DIF) and sulfadiazine (SDZ) amended via manure to soils on the abundance, diversity, and mobility of bacterial antibiotic resistance genes in soil bacteria, with special focus on the importance of the rhizosphere, the impact of repeated manure applications and the effects of varying soil moisture conditions. Therefore, manure spiked with antibiotics or collected from treated animals was once or repeatedly applied to agricultural soil in microcosm, mesocosm and field experiments and these treatments were compared to soil amended with manure free of antibiotics. The dissipation of the antibiotics in manure, bulk soil and rhizosphere was followed in cooperation with the A3 group and correlated to the abundance and transferability of resistance genes and mobile genetic elements, measured by quantitative real time PCR, Southern blot hybridization and exogenous plasmid isolation. We could demonstrate that manure itself is a reservoir of transferable antibiotic resistance plasmids of different incompatibility groups. In contrast to the diversity of plasmids and gene cassettes, a similar bacterial community structure and consistently high abundance of sulfonamide resistance genes was observed in field scale manures. The application of manure and antibiotics synergistically increased the abundance of antibiotic resistance genes in soil and their transferability. The repeated application of manure containing antibiotics even caused an accumulation of resistance genes, antibiotics and bacterial responders in bulk soil under controlled microcosm conditions. In the field, this accumulation was neither observed in bulk soil nor in the rhizosphere, which might be due to different reasons such as the lower concentrations of sulfadiazine but also organic matter in the manure from treated pigs, and an accelerated dissipation of SDZ in the rhizosphere. Varying soil moisture conditions were also tested as a possible reason for this discrepancy but seemed to have only a minor influence on the fate and effects of veterinary medicines applied with manure to soil. Mobile genetic elements which likely play a major role in horizontal spread of resistance genes between manure and soil bacteria were identified as IncP-1ε plasmids as well as the in this project firstly described LowGC-type plasmids. These plasmids showed a remarkable diversity of antibiotic resistance genes, the ability to efficiently transfer under soil conditions, and a correlation to antibiotic selective pressure which strongly suggest that these plasmids are important vectors for the spread of antibiotic resistance in the agro-ecosystem. Competition experiments in soil microcosms comparing the persistence of A. baylyi BD413 with or without plasmid pHHV216 in soil revealed a fitness advantage of the plasmid carrying strain when the soil was treated with SDZ spiked manure. The results obtained in this project provide significant progress in the understanding of the fate and effects of antibiotics applied with manure to soil and the associated risks for human health.

begutachtete Publikationen:

1. Binh, CTT, Heuer, H, Gomes, NCM, Kotzerke, A, Fulle, M, Wilke, BM, Schloter, M, Smalla, K (2007): Short-term effects of amoxicillin on bacterial communities in manured soil. FEMS Microbiology Ecology 62, 290–302.
2. Binh, CTT, Heuer, H, Kaupenjohann, M, Smalla, K (2008): Piggery manure used for soil fertilization is a reservoir for transferable antibiotic resistance plasmids. FEMS Microbiology Ecology 66, 25–37.
3. Binh, CTT, Heuer, H, Kaupenjohann, M, Smalla, K (2009): Diverse aadA gene cassettes on class 1 integrons introduced into soil via spread manure. Research in Microbiology 160, 427–433.
4. Ding, GC, Radl, V, Hai, B, Jechalke, S, Heuer, H, Smalla, K, Schloter, M (2014): Dynamics of soil bacterial communities in response to repeated application of manure containing sulfadiazine. PLoS ONE 9, e92958.
5. Gaze, WH, Krone, SM, Larsson, DGJ, Li, X, Robinson, JA, Simonet, P, Smalla, K, Timinouni, M, Topp, E, Wellington, EM, Wright, GD, Zhu, YG (2013): Influence of humans on evolution and mobilization of environmental antibiotic resistome. Emerging Infectious Diseases 19, e120871.
6. Heuer, H, Smalla, K (2007): Manure and sulfadiazine synergistically increased bacterial antibiotic resistance in soil over at least two months. Environmental Microbiology 9, 657–666.
7. Heuer, H, Focks, A, Lamshöft, M, Smalla, K, Matthies, M, Spiteller, M (2008): Fate of sulfadiazine administered to pigs and its quantitative effect on the dynamics of bacterial resistance genes in manure and manured soil. Soil Biology and Biochemistry 40, 1892–1900.
8. Heuer, H, Kopmann, C, Binh, CTT, Top, EM, Smalla, K (2009): Spreading antibiotic resistance through spread manure: characteristics of a novel plasmid type with low %G+C content. Environmental Microbiology 11, 937–949
9. Heuer, H, Schmitt, H, Smalla, K (2011): Antibiotic resistance gene spread due to manure application on agricultural fields. Current Opinion in Microbiology 14, 236–243.
10. Heuer H, Solehati Q, Zimmerling U, Kleineidam K, Schloter M, Müller T, Focks A, Thiele-Bruhn S, Smalla K (2011) Accumulation of sulfonamide resistance genes in arable soils due to repeated application of manure containing sulfadiazine. Appl Environ Microbiol 77: 2527-2530.
11. Heuer, H, Binh, CTT, Jechalke, S, Kopmann, C, Zimmerling, U, Krögerrecklenfort, E, Ledger, T, González, B, Top, E, Smalla, K (2012): IncP-1ε plasmids are important vectors of antibiotic resistance genes in agricultural systems: diversification driven by class 1 integron gene cassettes. Frontiers in Microbiology 3, Article 2.
12. Heuer, H, Smalla K (2012): Plasmids foster diversification and adaptation of microbial populations in soil. FEMS Microbiology Reviews 36, 1083–1104.
13. Jechalke, S, Kopmann, C, Rosendahl, I, Groeneweg, J, Weichelt, V, Krögerrecklenfort, E, Brandes, N, Nordwig, M, Ding, GC, Siemens, J, Heuer, H, Smalla, K (2013): Increased abundance and transferability of resistance genes after field application of manure from sulfadiazine-treated pigs. Applied and Environmental Microbiology 79, 1704–1711.
14. Jechalke, S, Dealtry, S, Smalla K, Heuer H (2013). Quantification of IncP-1 plasmid prevalence in environmental samples. Applied and Environmental Microbiology 79, 1410–1413.
15. Jechalke, S, Focks, A, Rosendahl, I, Groeneweg, J, Siemens, J, Heuer, H, Smalla, K (2014): Structural and functional response of the soil bacterial community to application of manure from difloxacin-treated pigs. FEMS Microbiology Ecology 87, 78-88.
16. Jechalke, S, Kopmann, C, Richter, M, Moenickes, S, Heuer, H, Smalla, K (2013): Plasmid-mediated fitness advantage of Acinetobacter baylyi in sulfadiazine-polluted soil. FEMS Microbiology Letters 348, 127–132.
17. Jechalke, S, Focks, A, Rosendahl, I, Groeneweg, J, Siemens, J, Heuer, H, Smalla, K (2014): Structural and functional response of the soil bacterial community to application of manure from difloxacin-treated pigs. FEMS Microbiology Ecology 87, 78-88.
18. Jechalke, S., Heuer, H., Siemens, J., Amelung, W., Smalla, K. (2014): Fate and effects of veterinary antibiotics in soil. Trends in Microbiology  DOI: http://dx.doi.org/10.1016/j.tim.2014.05.005
19. Kopmann, C, Jechalke, S, Rosendahl, I, Groeneweg, J, Krögerrecklenfort, E, Zimmerling, U, Weichelt, V, Siemens, J, Amelung, W, Heuer, H, Smalla, K (2013): Abundance and transferability of antibiotic resistance as related to the fate of sulfadiazine in maize rhizosphere and bulk soil. FEMS Microbiology Ecology 83, 125–134.
     

Weitere Publikationen:

1. Jechalke, S., Heuer, H. Smalla, K (2013): Antibiotikaresistenzgene im Ackerboden. Biospektrum 19, 243-246.

Modeling of interactions between chemical dynamics and effects of veterinary medicines in soil.

Prof. Dr. M. Matthies, PD Dr. J. Klasmeier, Universität Osnabrück

Summary

The overall objective of subproject C was the development of an integrated fate and effect model for veterinary antibiotics (particularly sulfonamides) in soil as basis for sound and comprehensive risk assessment. Based on experimental results within the Research Unit for the model substance sulfadiazine (SDZ), relevant physical, chemical and biological processes that govern fate and effects of sulfonamide antibiotics in soil were identified and integrated into a consistent simulation model. The developed conceptual kinetic fate model well describes concentration dynamics of SDZ and its metabolites 4-hydroxy-SDZ (4-OH-SDZ) and N-acetyl-SDZ (N-Ac-SDZ) observed in laboratory batch and mesocosm experiments and it can also largely explain the dynamics observed in field experiments. The model considers (pseudo-)first-order transformation reactions within the easily extractable fraction (EAS) as well as reversible translocation from EAS into the residual fraction (RES) and irreversible dissipation into the fraction of non-extractable residues (NER). The phenomenon that extractability of SDZ is instantaneously reduced after its application to soil was confirmed in experiments. It was proven that a rapid dissipation process occurs along with the slow first-order kinetics considered in the model. On the field scale, the fate model could preliminary quite well describe EAS concentration dynamics when considering simple temperature and moisture correction functions as well as reasonable adaptions of rate constants for reversible sequestration. Results of targeted experiments with different isotope-labelled compounds on the quantitative effect of temperature on fate processes were not sufficient to deduce consistent correction factors. For de-acetylation and NER formation, temperature sensitivities expressed as Q10 values were much higher than average values known for pesticide degradation in soil, which is hypothesized to be due to overlying effects related to the amendment of the substance with manure. A major finding of the experiments was that a direct pathway from N-Ac-SDZ into NER must exist which had not been considered in the original fate model. For uptake and accumulation of sulfonamides in bacteria, a dynamic model considering intra- and extracellular pH values and the substance specific pKa value was established, which helps to explain the different activities of sulfonamides as inhibitor of bacterial growth. In-vitro microtiter plate tests with E.coli demonstrated that the two major metabolites 4-OH-SDZ and N-Ac-SDZ were not able to exhibit bacteriostatic effects.  For N Ac SDZ, this is in accordance with the inactivation of the aniline moiety by substitution, while for 4-OH-SDZ it is most likely due to keto-enol-tautomerism. Therefore, only the parent compound SDZ has to be considered in risk assessment. Models describing the effect of sulfonamides on bacteria considered growth inhibition of nitrifying bacteria (AOB) and resistance gene selection. It could be demonstrated that ammonia-oxidizing archaea (AOA) maintain the respective soil function in case of deficiency of AOB through the inhibitory effect of antibiotics (functional redundance). The chemical fate model was successfully coupled with the effect model for AOB and AOA. Sensitivity and uncertainty analysis of the integrated fate and effect model clearly showed that the general dynamics of important output variables is robust enough against uncertainties to allow for interpretable predictions of the effect of antibiotics on soil functions such as nitrification.
Due to the extremely strong sorption of the second model compound difloxacin (DIF) to soil, long-term accumulation can be expected, but potential release and effects cannot yet be predicted.

begutachtete Publikationen:

1. Tappe W, Zarfl C, Kummer S, Burauel P, Vereecken H, Groeneweg J (2008): Growth-inhibitory effects of sulfonamides at different pH: Dissimilar susceptibility patterns of a soil bacterium and a test bacterium used for antibiotic assays. Chemosphere 72, 836–843.
2. Heuer H, Focks A, Lamshöft M, Smalla K, Matthies M, Spiteller M (2008): Fate of sulfadiazine administered to pigs and its quantitative effect on the dynamics of bacterial resistance genes in manure and soil. Soil Biology and Biochemistry 40, 1892–1900.
3. Zarfl C, Matthies M, Klasmeier J (2008): A mechanistical model for the uptake of sulfonamides by bacteria. Chemosphere 70, 753–760.
4. Schauss K, Focks A, Heuer H, Kotzerke A, Schmitt H, Thiele-Bruhn S, Smalla K, Wilke BM, Matthies M, Amelung W, Klasmeier J, Schloter M (2009): Analysis, fate and effects of the antibiotic sulfadiazine in soil ecosystems. Trends in Analytical Chemistry 28, 612–618.
5. Schauss K, Focks A, Leininger S, Kotzerke A, Heuer H, Thiele-Bruhn S, Sharma S,  Wilke BM, Matthies M, Smalla K, Munch JC, Amelung W, Kaupenjohann M, Schloter M, Schleper C (2009): Dynamics and functional relevance of ammonia-oxidizing archaea in two agricultural soils. Environmental Microbiology 11, 446–456.
6. Zarfl C, Klasmeier J, Matthies M (2009): A conceptual model describing the fate of sulfadiazine and its metabolites observed in manure-amended soils. Chemosphere77, 720-726.
7. Focks A, Klasmeier J, Matthies M (2010): The mechanistic link between sulfonamide uptake and effect on the growth of bacteria: Model development and application to experimental data from two soil microorganisms. Environmental Toxicology and Chemistry 29, 1445–1452.
8. Heuer H, Solehati Q, Zimmerling U, Kleineidam K, Schloter M, Müller T, Focks A, Thiele-Bruhn S, Smalla K (2011): Accumulation of sulfonamide resistance genes in arable soils due to repeated application of manure containing sulfadiazine. Applied and Environmental Microbiology 77, 2527–2530.
9. Müller T., Rosendahl I, Focks A, Siemens J, Klasmeier J, Matthies M (2013): Short-term extractability of sulfadiazine after application to soils. Environmental Pollution 172, 180–185.
    

Weitere Publikationen:

1. Zarfl C (2008): Chemical Fate of Sulfadiazine in Soil: Mechanisms and Modelling Approaches. PhD Thesis. Shaker Verlag (Aachen). ISBN 978-3-8322-7491-7.
2. Focks A (2009): Effects of Sulfadiazine in Soil: Integrative Modelling Approaches as a Basis for Environmental Risk Assessment. PhD Thesis. Dr.Hut-Verlag (München), ISBN 978‑3‑86853‑041‑4.