Joost Groeneweg

Ammonia oxidation in nitrosomonas at NH3 concentrations near km: Effects of pH and temperature

Abstract Nitrosomonas europaea from continuous pure cultures was incubated with 26.4 μ M NH 3 (= 0.37 mg NH 3 -N l −1 ) at various NH 4 + concentrations, pH values and temperatures. Measured rates of nitrite formation were significantly influenced by pH. Likewise unexpectedly, the maximum ammonia oxidation rate occurred between pH 6.7 and 7.0. Temperature had an even stronger effect on the rate of ammonia oxidation than the availability of NH 3 . It is concluded that the assumption of a strict dependence of the rate of ammonia oxidation on substrate concentration is an unjustified oversimplification. Among the mechanisms which could explain ammonium uptake and oxidation near or below pH 7.0, the formation of NO from HNO 2 is considered.

Diffuse atrazine pollution in German aquifers.

Until its prohibition in Germany in 1991, atrazine was the mostfrequently applied herbicide in maize cultivation. Moreover, it was used in orchards andvineyards and as a total herbicide on non-cultivated grounds (railways, factory grounds).Later on, atrazine was substituted mainly by terbutylazine. Terbutylazine and terbutrynare the only s-triazines presently permitted in Germany. Nevertheless, atrazine and itsmetabolite desethylatrazine are by far the most abundant herbicides detected in near surfacegroundwater. This might be due to wash-outs from the pools of atrazine and its metabolitesfrom the soil into the groundwater or continuing illegal applications. Samples takenfrom maize fields in 1994 showed that 6.2% of 471 fields tested were treated with atrazinedespite the prohibition of its use. Nevertheless, the overall trend is in fact a slow decreasein atrazine concentrations where it is detected in groundwater and, simultaneously often aslight increase in desethylatrazine concentrations. But this is not the case forall sampling points, and increasing concentrations in several aquifers are observedas well. Factors governing the adsorption, degradation, persistence and the possible transferinto the aquifer and the current situation concerning atrazine occurrence in German aquiferswill be discussed.

Increased Abundance and Transferability of Resistance Genes after Field Application of Manure from Sulfadiazine-Treated Pigs

ABSTRACT 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.

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 antibiotics 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 antibiotics 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.

Transport and transformation of sulfadiazine in soil columns packed with a silty loam and a loamy sand

Concerning the transport of the veterinary antibiotic sulfadiazine (SDZ) little is known about its possible degradation during transport. Also its sorption behaviour is not yet completely understood. We investigated the transport of SDZ in soil columns with a special emphasis on the detection of transformation products in the outflow of the soil columns and on modelling of the concentration distribution in the soil columns afterwards. We used disturbed soil columns near saturation, packed with a loamy sand and a silty loam. SDZ was applied as a 0.57 mg L(-1) solution at a constant flow rate of 0.25 cm h(-1) for 68 h. Breakthrough curves (BTC) of SDZ and its transformation products 4-(2-iminopyrimidin-1(2H)-yl)aniline and 4-hydroxy-SDZ were measured for both soils. For the silty loam we additionally measured a BTC for an unknown transformation product which we only detected in the outflow samples of this soil. After the leaching experiments the (14)C-concentration was quantified in different layers of the soil columns. The transformation rates were low with mean SDZ mass fractions in the outflow samples of 95% for the loamy sand compared to 97% for the silty loam. The formation of 4-(2-iminopyrimidin-1(2H)-yl)aniline appears to be light dependent and did probably not occur in the soils, but afterwards. In the soil columns most of the (14)C was found near the soil surface. The BTCs in both soils were described well by a model with one reversible (kinetic) and one irreversible sorption site. Sorption kinetics played a more prominent role than sorption capacity. The prediction of the (14)C -concentration profiles was improved by applying two empirical models other than first order to predict irreversible sorption, but also these models were not able to describe the (14)C concentration profiles correctly. Irreversible sorption of sulfadiazine still is not well understood.

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 antibiotics 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 compounds strong binding and small (bio)accessibility despite its persistence.

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.

Degradation of Sulfadiazine by Microbacterium lacus Strain SDZm4, Isolated from Lysimeters Previously Manured with Slurry from Sulfadiazine-Medicated Pigs

ABSTRACT Sulfadiazine (SDZ)-degrading bacterial cultures were enriched from the topsoil layer of lysimeters that were formerly treated with manure from pigs medicated with 14C-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 14CO2 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 14CO2, 16% was linked to biomass, and 21% remained as dissolved, not yet identified 14C. 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.

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.

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.