Degradation of sulfadiazine by Microbacterium lacus strain SDZm4, isolated from lysimeters previously manured with slurry from sulfadiazine-medicated pigs.

Sulfadiazine (SDZ)-degrading bacterial cultures were enriched from the topsoil layer of lysimeters that were formerly treated with manure from pigs medicated with (14)C-labeled SDZ. The loss of about 35% of the applied radioactivity after an incubation period of 3 years was attributed to CO2 release due to mineralization processes in the lysimeters. Microcosm experiments with moist soil and soil slurries originating from these lysimeters confirmed the presumed mineralization potential, and an SDZ-degrading bacterium was isolated. It was identified as Microbacterium lacus, denoted strain SDZm4. During degradation studies with M. lacus strain SDZm4 using pyrimidine-ring labeled SDZ, SDZ disappeared completely but no (14)CO2 was released during 10 days of incubation. The entire applied radioactivity (AR) remained in solution and could be assigned to 2-aminopyrimidine. In contrast, for parallel incubations but with phenyl ring-labeled SDZ, 56% of the AR was released as (14)CO2, 16% was linked to biomass, and 21% remained as dissolved, not yet identified (14)C. Thus, it was shown that M. lacus extensively mineralized and partly assimilated the phenyl moiety of the SDZ molecule while forming equimolar amounts of 2-aminopyrimidine. This partial degradation might be an important step in the complete mineralization of SDZ by soil microorganisms.

Increased abundance and transferability of resistance genes after field application of manure from sulfadiazine-treated pigs.

Spreading manure containing antibiotics in agriculture is assumed to stimulate the dissemination of antibiotic resistance in soil bacterial populations. Plant roots influencing the soil environment and its microflora by exudation of growth substrates might considerably increase this effect. In this study, the effects of manure from pigs treated with sulfadiazine (SDZ), here called SDZ manure, on the abundance and transferability of sulfonamide resistance genes sul1 and sul2 in the rhizosphere of maize and grass were compared to the effects in bulk soil in a field experiment. In plots that repeatedly received SDZ manure, a significantly higher abundance of both sul genes was detected compared to that in plots where manure from untreated pigs was applied. Significantly lower abundances of sul genes relative to bacterial ribosomal genes were encountered in the rhizosphere than in bulk soil. However, in contrast to results for bulk soil, the sul gene abundance in the SDZ manure-treated rhizosphere constantly deviated from control treatments over a period of 6 weeks after manuring, suggesting ongoing antibiotic selection over this period. Transferability of sulfonamide resistance was analyzed by capturing resistance plasmids from soil communities into Escherichia coli. Increased rates of plasmid capture were observed in samples from SDZ manure-treated bulk soil and the rhizosphere of maize and grass. More than 97% of the captured plasmids belonged to the LowGC type (having low G+C content), giving further evidence for their important contribution to the environmental spread of antibiotic resistance. In conclusion, differences between bulk soil and rhizosphere need to be considered when assessing the risks associated with the spreading of antibiotic resistance.

Long-term sorption and sequestration dynamics of the antibiotic sulfadiazine: a batch study.

Understanding the long-term sequestration of veterinary antibiotics into soil fractions with different bioavailability is important in terms of assessing their eco-toxicological impact. We performed 60-d batch sorption experiments with radiolabeled sulfadiazine (SDZ) using samples from two agricultural soils. Sequential extraction with CaCl/MeOH (easily accessible fraction), microwave (residual fraction, RES), and combustion (nonextractable residues, NER) was used to quantify the sequestration dynamics of the C-derived SDZ-equivalent concentration. Multiple harsh extractions allowed us to mathematically extrapolate to the amount of SDZ equivalents that can be potentially extracted, resulting in halving the NER fraction after 60 d. A modified two-stage model with irreversible sorption combined with global parameter optimization was able to display the sequestration dynamics. We demonstrated this with sterilized samples in which no transformation of the parent compound was observed. This also showed that transformation was primarily biologically driven. These modeling results verified the procedure, which was then applied to nontreated samples from both soils to estimate effective parameter values for SDZ-derived equivalents. Observed initial sorption, to which up to 20% of the kinetic sorption sites attributed, was included in the model. Both the RES and NER fractions reached a sorption plateau, with NER occupying about 30% of the kinetic fraction (RES+NER) for all soils. The sorption and sequestration of SDZ were soil-specific and dominated by kinetics. Sequestration in the RES fraction was much slower (characteristic time: 60 d) than the redistribution in the NER fraction (characteristic time: <6 d). The work presented here contributes to the prediction of the dynamics of (bio-)availability.

Persistence of the fluoroquinolone antibiotic difloxacin in soil and lacking effects on nitrogen turnover.

The environmental risks caused by the use of fluoroquinolone antibiotics in human therapeutics and animal husbandry are associated with their persistence and (bio)accessibility in soil. To assess these aspects, we administered difloxacin to pigs and applied the contaminated manure to soil. We then evaluated the dissipation and sequestration of difloxacin in soil in the absence and presence of plants within a laboratory trial, a mesocosm trial, and a field trial. A sequential extraction yielded antibiotic fractions of differing binding strength. We also assessed the antibiotic's effects on nitrogen turnover in soil (potential nitrification and denitrification). Difloxacin was hardly (bio)accessible and was very persistent under all conditions studied (dissipation half-life in bulk soil, >217 d), rapidly forming nonextractable residues. Although varying environmental conditions did not affect persistence, dissipation was accelerated in soil surrounding plant roots. Effects on nitrogen turnover were limited due to the compound's strong binding and small (bio)accessibility despite its persistence.

Dissipation and sequestration of the veterinary antibiotic sulfadiazine and its metabolites under field conditions.

Veterinary antibiotics introduced into the environment may change the composition and functioning of soil microbial communities and promote the spreading of antibiotic resistance. Actual risks depend on the antibiotic's persistence and (bio)accessibility, which may differ between laboratory and field conditions. We examined the dissipation and sequestration of sulfadiazine (SDZ) and its main metabolites in soil under field conditions and how it was influenced by temperature, soil moisture, plant roots, and soil aggregation compared to controlled laboratory experiments. A sequential extraction accounted for easily extractable (CaCl2-extractable) and sequestered (microwave-extractable, residual) SDZ fractions. Dissipation from both fractions was largely temperature-dependent and could be well predicted from laboratory data recorded at different temperatures. Soil moisture additionally seemed to control sequestration, being accelerated in dry soil. Sequestration, as indicated by increasing apparent distribution coefficients and decreasing rates of kinetic release into CaCl2, governed the antibiotic's long-term fate in soil. Besides, we observed spatial gradients of antibiotic concentrations across soil aggregates and in the vicinity of roots. The former were short-lived and equilibrated due to aggregate reorganization, while dissipation of the easily extractable fraction was accelerated near roots throughout the growth period. There was little if any impact of the plants on residual SDZ concentrations.

Long-term sorption and desorption of sulfadiazine in soil: experiments and modeling.

Antibiotics, such as sulfadiazine (SDZ), may enter arable soil by spreading of manure of medicated husbandry or directly by the excrement of grazing animals. Knowledge of the fate of antibiotics in soils is crucial for assessing the environmental risk of these compounds, including possible transport to ground water. Kinetic sorption of (14)C-labeled SDZ (4-amino-N-pyrimidin-2-yl-benzenesulfonamide) was investigated using the batch technique. The batch sorption-desorption experiments were conducted at various concentration levels (0.044-13 mg L(-1) initial solute concentration) and time scales (0.75-272 d). Sorption of (14)C-SDZ in the investigated silty loam was time dependent and strongly nonlinear in the solution phase concentration. The time to reach an apparent sorption equilibrium was about 20 d. However, desorption was very slow, and 41 d were insufficient to reach the desorption equilibrium. An inverse modeling technique was used to identify relevant sorption processes of (14)C-SDZ during the batch experiments. Among the investigated two- and three-domain sorption models, adsorption and desorption of (14)C-SDZ were best described with a new model defining two sorption domains and four parameters. Whereas sorption in the first sorption domain was nonlinear and instantaneous, solute uptake in the second sorption domain was rate limited following first-order kinetics. Desorption followed the same rate law until an equilibrium distribution was reached. After that, desorption was assumed to be impossible due to partly irreversible sorption. Although the proposed model needs further validation, it contributes to the discussion on complex sorption processes of organic chemicals in soils.

Growth-inhibitory effects of sulfonamides at different pH: dissimilar susceptibility patterns of a soil bacterium and a test bacterium used for antibiotic assays.

The ionic speciation of sulfonamides is pH-driven and this may be crucial for their bioavailability and sorption to soil constituents, as well as for their uptake into bacterial cells. The inhibition behaviour of a bacterial test strain (Pseudomonas aeruginosa; DSM 1117), which was grown in the presence of different concentrations of 8 sulfonamides at pH values from 5 to 8, could be predicted by models that take the speciation of sulfonamides in- and outside of bacterial cells into account. Assuming a pH of 7.5 inside the cells (pH homeostasis), the strongest inhibition was predicted for the lowest external pH and for sulfonamides with the lowest pK(a) values. Growth experiments with Ps. aeruginosa basically reflected this predicted behaviour. However, Pantoea agglomerans -- a bacterial strain isolated from arable soil -- behaved surprisingly different regarding its pH dependency: all sulfonamides showed the strongest effects at pH 7 to 8 instead of being most effective at lowest pH, although the pK(a) dependencies followed the same pattern. Experimental and modeling results could be brought into good agreement for P. agglomerans if the cell-internal pH was admitted to approximate the external pH instead of implying pH homeostasis for modeling calculations. Thus, besides the actual concentration of sulfonamides, the pH dependent mode of reaction of different bacteria to sulfonamides may additionally govern the population dynamics in soils.

Transport of sulfadiazine in soil columns: experiments and modelling approaches.

Antibiotics, such as sulfadiazine, reach agricultural soils directly through manure of grazing livestock or indirectly through the spreading of manure or sewage sludge on the field. Knowledge about the fate of antibiotics in soils is crucial for assessing the environmental risk of these compounds, including possible transport to the groundwater. Transport of (14)C-labelled sulfadiazine was investigated in disturbed soil columns at a constant flow rate of 0.26 cm h(-1) near saturation. Sulfadiazine was applied in different concentrations for either a short or a long pulse duration. Breakthrough curves of sulfadiazine and the non-reactive tracer chloride were measured. At the end of the leaching period the soil concentration profiles were determined. The peak maxima of the breakthrough curves were delayed by a factor of 2 to 5 compared to chloride and the decreasing limbs are characterized by an extended tailing. However, the maximum relative concentrations differed as well as the eluted mass fractions, ranging from 18 to 83% after 500 h of leaching. To identify relevant sorption processes, breakthrough curves of sulfadiazine were fitted with a convective-dispersive transport model, considering different sorption concepts with one, two and three sorption sites. Breakthrough curves can be fitted best with a three-site sorption model, which includes two reversible kinetic and one irreversible sorption site. However, the simulated soil concentration profiles did not match the observations for all of the used models. Despite this incomplete process description, the obtained results have implications for the transport behavior of sulfadiazine in the field. Its leaching may be enhanced if it is frequently applied at higher concentrations.

Fate of the antibiotic sulfadiazine in natural soils: Experimental and numerical investigations.

Based on small-scale laboratory and field-scale lysimeter experiments, the sorption and biodegradation of sulfonamide sulfadiazine (SDZ) were investigated in unsaturated sandy and silty-clay soils. Sorption and biodegradation were low in the laboratory, while the highest leaching rates were observed when SDZ was mixed with manure. The leaching rate decreased when SDZ was mixed with pure water, and was smallest with the highest SDZ concentrations. In the laboratory, three transformation products (TPs) developed after an initial lag phase. However, the amount of TPs was different for different mixing-scenarios. The TP 2-aminopyrimidine was not observed in the laboratory, but was the most prevalent TP at the field scale. Sorption was within the same range at the laboratory and field scales. However, distinctive differences occurred with respect to biodegradation, which was higher in the field lysimeters than at the laboratory scale. While the silty-clay soil favored sorption of SDZ, the sandy, and thus highly permeable, soil was characterized by short half-lives and thus a quick biodegradation of SDZ. For 2-aminopyrimidine, half-lives of only a few days were observed. Increased field-scale biodegradation in the sandy soil resulted from a higher water and air permeability that enhanced oxygen transport and limited oxygen depletion. Furthermore, low pH was more important than the organic matter and clay content for increasing the biodegradation of SDZ. A numerical analysis of breakthrough curves of bromide, SDZ, and its TPs showed that preferential flow pathways strongly affected the solute transport within shallow parts of the soil profile at the field scale. However, this effect was reduced in deeper parts of the soil profile. Due to high field-scale biodegradation in several layers of both soils, neither SDZ nor 2-aminopyrimidine was detected in the discharge of the lysimeter at a depth of 1m. Synthetic 50 year long simulations, which considered the application of manure with SDZ for general agricultural practices in Germany and humid climate conditions, showed that the concentration of SDZ decreased below 0.1 µg/L in both soils below the depth of 50 cm.

A filtration, incubation and staining reactor including a new protocol for FISH.

A newly developed device for performing fluorescence in situ hybridization (FISH) is described. An adapted procedure was compared with two typical FISH protocols. Tests were performed with Pseudomonas cells and the gene probe EUB338. With the novel procedure, we obtained a better recovery of cells and less variability in results.

Enumeration of soil bacteria with the green fluorescent nucleic acid dye Sytox green in the presence of soil particles.

Total counts in soils are usually determined using fluorescent dyes, such as DAPI or Sybr green, due to fluorescence enhancement if they are bound to nucleic acids. Unfortunately, these commonly used dyes stain soil particles as well. Therefore, besides fluorescence enhancement, sufficient spectral differentiation is also required. We present a new procedure that overcomes the problems of visualising bacteria on surfaces in soil and avoids the separation of soil particles to a large extent. Spectral differentiation between bacteria and soil matrix is achieved by using Sytox green and a suboptimal excitation wavelength. Bacteria exhibit a bright green fluorescence, while soil particles fluoresce blue or red. Slight homogenisation and sedimentation of the sand and coarse silt that were too big for microscopic investigations were the only separation steps required. We compared the proposed Sytox green staining with Sybr green staining. The recovery of Sybr green-stained cells amounted to 38%, whereas in samples stained by Sytox green 81% of the spiked cells were counted. Sytox green can also be combined with fluorescence in situ hybridisation (FISH) using deep red dyes such as Cy5.

Structural and functional response of the soil bacterial community to application of manure from difloxacin-treated pigs.

Difloxacin (DIF) belongs to the class of fluoroquinolone antibiotics that have been intensively used for the treatment of bacterial infections in veterinary and human medicine. The aim of this field study was to compare the effect of manure from DIF-treated pigs and untreated pigs on the bacterial community structure and resistance gene abundance in bulk soil and rhizosphere of maize. A significant effect of DIF manure on the bacterial community composition in bulk soil was revealed by denaturing gradient gel electrophoresis (DGGE) of bacterial 16S rRNA gene fragments amplified from total community DNA. In few samples, quinolone resistance genes qnrB and qnrS1/qnrS2 were detected by PCR and subsequent hybridization, while qnrA was not detected. Quantitative PCR revealed an increased abundance of the integrase gene intI1 of class I integrons and sulfonamide resistance genes sul1 and sul2 in DIF manure-treated bulk soil and rhizosphere, relative to 16S rRNA genes, while traN genes specific for LowGC-type plasmids were increased only in bulk soil. Principal component analysis of DGGE profiles suggested a manure effect in soil until day 28, but samples of days 71 and 140 were found close to untreated soil, indicating resilience of soil community compositions from disturbances by manure.

Dynamics of transformation of the veterinary antibiotic sulfadiazine in two soils.

Veterinary antibiotics administered to livestock can be unintentionally released into the environment, for example by the application of manure to soils. The fate of such antibiotics in soils is mostly determined by sorption and degradation processes, including transformation. There is a need to further examine the combined transformation and sorption behavior of these emerging pollutants in soils. Long-term batch sorption experiments with the (14)C-radiolabeled antibiotic sulfadiazine enabled us to simultaneously trace the sorption and transformation dynamics of sulfadiazine. The parent compound and the transformation products were analyzed in the liquid phase and in the extracts from the solid phase after a sequential extraction. We found that of up to six transformation products were formed during degradation and that these products exhibited quite different dynamics in the two soils. Transformation products were formed rapidly and were extractable from the solid phase. We observed identical sets of the transformation products in both phases. The input concentration influenced the course of transformation of the parent substance. We present a detailed analysis including a mathematical description and derive regulatory kinetic endpoints for predicting environmental concentrations.

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.

In a field experiment, the impact of repeated application of the antibiotic sulfadiazine (SDZ)-contaminated pig manure was assessed on functional microbial communities involved in ammonia and nitrite oxidation in the root-rhizosphere complexes (RRCs) of diverse plants composing a pasture. We surveyed the abundance of ammonia-oxidizing archaea (AOA) and bacteria (AOB) as well as Nitrobacter- and Nitrospira-like nitrite-oxidizing bacteria (NOB) by quantitative PCR (qPCR), and the diversity of amoA AOA and Nitrobacter-like nxrA amplicons using a cloning-sequencing approach. Whereas the first SDZ-contaminated manure application caused only slight effects on the investigated microbial communities and did not change the diversity and abundance pattern significantly, the second application of SDZ-contaminated manure induced an up to 15-fold increased ratio of AOA:AOB and a reduction of nrxA genes. The diversity of AOA amoA increased after the second application of SDZ-contaminated manure compared to the control treatment whereas a clear reduction of nrxA OTUs was visible in the same samples. The results indicate that the application of SDZ may principally affect nitrite oxidation by NOB and alternative pathways like nitrite reduction might be favored under these conditions.

Abundance and transferability of antibiotic resistance as related to the fate of sulfadiazine in maize rhizosphere and bulk soil.

Veterinary antibiotics entering agricultural land with manure pose the risk of spreading antibiotic resistance. The fate of sulfadiazine (SDZ) introduced via manure and its effect on resistance gene levels in the rhizosphere were compared with that in bulk soil. Maize plants were grown for 9 weeks in soil fertilized with manure either from SDZ-treated pigs (SDZ treatment) or from untreated pigs (control). CaCl(2) -extractable concentrations of SDZ dissipated faster in the rhizosphere than in bulk soil, but SDZ remained detectable over the whole time. For bulk soil, the abundance of sul1 and sul2 relative to 16S rRNA gene copies was higher in the SDZ treatment than in the control, as revealed by quantitative PCR on days 14 and 63. In the rhizosphere, sampled on day 63, the relative sul gene abundances were also significantly increased in the SDZ treatment. The accumulated SDZ exposure (until day 63) of the bacteria significantly correlated with the log relative abundance of sul1 and sul2, so that these resistance genes were less abundant in the rhizosphere than in bulk soil. Plasmids conferring SDZ resistance, which were exogenously captured in Escherichia coli, mainly belonged to the LowGC group and carried a heterogeneous load of resistances to different classes of antibiotics.

Transformation and sorption of the veterinary antibiotic sulfadiazine in two soils: a short-term batch study.

The worldwide use of veterinary antibiotics poses a continuous threat to the environment. There is, however, a lack of mechanistic studies on sorption and transformation processes for environmental assessment in soils. Two-week batch sorption experiments were performed with the antibiotic sulfadiazine (SDZ) in the plow layer and the subsoil of a loamy sand and a silty loam. The sorption and transformation parameters of SDZ and its main transformation products N1-2-(4-hydroxypyrimidinyl) benzenesulfanilamide (4-OH-SDZ) and 4-(2-iminopyrimidin-1(2H)-yl)aniline (An-SDZ) were estimated using a global optimization algorithm. A two-stage, one-rate sorption model combined with a first-order transformation model adequately described the batch data. Sorption of SDZ was nonlinear, time-dependent, and affected by pH, with a higher sorption capacity for the loamy sand. Transformation of SDZ into 4-OH-SDZ occurred only in the liquid phase, with half-life values of 1 month in the plow layers and 6 months in the subsoils. Under the exclusion of light, An-SDZ was formed in substantial amounts in the silty loam only, with liquid phase half-life values of 2 to 3 weeks. Despite the rather large parameter uncertainties, which may be reduced using additional information obtained from sequential solid phase extraction, the proposed method provides a framework to assess the fate of antibiotics in soils.

Diffuse atrazine pollution in German aquifers.

Until its prohibition in Germany in 1991, atrazine was the most frequently applied herbicide in maize cultivation. Moreover, it was used in orchards and vineyards and as a total herbicide on non-cultivated grounds (railways, factory grounds). Later on, atrazine was substituted mainly by terbutylazine. Terbutylazine and terbutryn are the only s-triazines presently permitted in Germany. Nevertheless, atrazine and its metabolite desethylatrazine are by far the most abundant herbicides detected in near surface groundwater. This might be due to wash-outs from the pools of atrazine and its metabolites from the soil into the groundwater or continuing illegal applications. Samples taken from maize fields in 1994 showed that 6.2% of 471 fields tested were treated with atrazine despite the prohibition of its use. Nevertheless, the overall trend is in fact a slow decrease in atrazine concentrations where it is detected in groundwater and, simultaneously often a slight increase in desethylatrazine concentrations. But this is not the case for all sampling points, and increasing concentrations in several aquifers are observed as well. Factors governing the adsorption, degradation, persistence and the possible transfer into the aquifer and the current situation concerning atrazine occurrence in German aquifers will be discussed.