Björn Thiele

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.

Direct Analysis of Underivatized Amino Acids in Plant Extracts by LC-MS-MS

In this chapter, we describe a method for quantification of 20 proteinogenic amino acids as well as 13 (15)N-labeled amino acids by liquid chromatography-mass spectrometry without the need for derivatization and use of organic solvents. Analysis of the underivatized amino acids is performed by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS-MS) in the positive ESI mode. Separation is achieved on a strong cation exchange (SCX) column (Luna 5 μm SCX 100 Å) with 30 mM ammonium acetate in water (A) and 5% acetic acid in water (B). Quantification is accomplished by use of d(5)-phenylalanine as internal standard achieving limits of detection of 0.1-3.0 μM. The method was successfully applied for the determination of proteinogenic and (15)N-labeled amino acids in plant extracts.

DRY–WET CYCLES INCREASE PESTICIDE RESIDUE RELEASE FROM SOIL

Soil drying and rewetting may alter the release and availability of aged pesticide residues in soils. A laboratory experiment was conducted to evaluate the influence of soil drying and wetting on the release of pesticide residues. Soil containing environmentally long-term aged (9–17 years) 14C-labeled residues of the herbicides ethidimuron (ETD) and methabenzthiazuron (MBT) and the fungicide anilazine (ANI) showed a significantly higher release of 14C activity in water extracts of previously dried soil compared to constantly moistened soil throughout all samples (ETD: p < 0.1, MBT and ANI: p < 0.01). The extracted 14C activity accounted for 44% (ETD), 15% (MBT), and 20% (ANI) of total residual 14C activity in the samples after 20 successive dry–wet cycles, in contrast to 15% (ETD), 5% (MBT), and 6% (ANI) in extracts of constantly moistened soils. In the dry–wet soils, the dissolved organic carbon (DOC) content correlated with the measured 14C activity in the aqueous liquids and indicated a potential association of DOC with the pesticide molecules. Liquid chromatography MS/MS analyses of the water extracts of dry–wet soils revealed ETD and MBT in detectable amounts, accounting for 1.83 and 0.01%, respectively, of total applied water-extractable parent compound per soil layer. These findings demonstrate a potential remobilization of environmentally aged pesticide residue fractions from soils due to abiotic stresses such as wet–dry cycles. Environ. Toxicol. Chem. 2012; 31: 1941–1947.

An efficient laboratory workflow for environmental risk assessment of organic chemicals

In this study, we demonstrate a fast and efficient workflow to investigate the transformation mechanism of organic chemicals and evaluate the toxicity of their transformation products (TPs) in laboratory scale. The transformation process of organic chemicals was first simulated by electrochemistry coupled online to mass spectrometry (EC-MS). The simulated reactions were scaled up in a batch EC reactor to receive larger amounts of a reaction mixture. The mixture sample was purified and concentrated by solid phase extraction (SPE) for the further ecotoxicological testing. The combined toxicity of the reaction mixture was evaluated in fish egg test (FET) (Danio rerio) compared to the parent compound. The workflow was verified with carbamazepine (CBZ). By using EC-MS seven primary TPs of CBZ were identified; the degradation mechanism was elucidated and confirmed by comparison to literature. The reaction mixture and one primary product (acridine) showed higher ecotoxicity in fish egg assay with 96 h EC50 values of 1.6 and 1.0 mg L(-1) than CBZ with the value of 60.8 mg L(-1). The results highlight the importance of transformation mechanism study and toxicological effect evaluation for organic chemicals brought into the environment since transformation of them may increase the toxicity. The developed process contributes a fast and efficient laboratory method for the risk assessment of organic chemicals and their TPs.

Long-term persistence of various 14C-labeled pesticides in soils

The fate of the 14C-labeled herbicides ethidimuron (ETD), methabenzthiazuron (MBT), and the fungicide anilazine (ANI) in soils was evaluated after long-term aging (9-17 years) in field based lysimeters subject to crop rotation. Analysis of residual 14C activity in the soils revealed 19% (ETD soil; 0-10 cm depth), 35% (MBT soil; 0-30), and 43% (ANI soil; 0-30) of the total initially applied. Accelerated solvent extraction yielded 90% (ETD soil), 26% (MBT soil), and 41% (ANI soil) of residual pesticide 14C activity in the samples. LC-MS/MS analysis revealed the parent compounds ETD and MBT, accounting for 3% and 2% of applied active ingredient in the soil layer, as well as dihydroxy-anilazine as the primary ANI metabolite. The results for ETD and MBT were matching with values obtained from samples of a 12 year old field plot experiment. The data demonstrate the long-term persistence of these pesticides in soils based on outdoor trials.

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.

Leaf wounding or simulated herbivory in young N. attenuata plants reduces carbon delivery to roots and root tips.

AbstractMain conclusionInNicotiana attenuataseedlings, simulated herbivory by the specialistManduca sextadecreases root growth and partitioning of recent photoassimilates to roots in contrast to increased partitioning reported for older plants. Root elongation rate in Nicotiana attenuata has been shown to decrease after leaf herbivory, despite reports of an increased proportion of recently mobilized photoassimilate being delivered towards the root system in many species after similar treatments. To study this apparent contradiction, we measured the distribution of recent photoassimilate within root tissues after wounding or simulated herbivory of N. attenuata leaves. We found no contradiction: herbivory reduced carbon delivery to root tips. However, the speed of phloem transport in both shoot and root, and the delivery of recently assimilated carbon to the entire root system, declined after wounding or simulated herbivory, in contrast with the often-reported increase in root partitioning. We conclude that the herbivory response in N. attenuata seedlings is to favor the shoot and not bunker carbon in the root system.

Copper amine oxidase 8 regulates arginine-dependent nitric oxide production in Arabidopsis thaliana

Highlight Copper amine oxidase in polyamine metabolism modulates arginine-dependent nitric oxide production by affecting arginase activity in Arabidopsis thaliana.

OrganoCat pretreatment of perennial plants: Synergies between a biogenic fractionation and valuable feedstocks

A successful biorefinery needs to align suitable pretreatment with sustainable production of biomasses. Herein, four perennial plants, (Sida, Silphium, Miscanthus and Szarvasi) regarded as promising feedstocks for biorefineries were subjected to the OrganoCat pretreatment. The technology was successfully applied to the different perennial plants revealing that pretreatment of grasses was more efficient than of non-grasses. Thorough analyses of the lignocellulose - before and after fractionation - enabled a detailed description of the fate of cellulosic, non-cellulosic polysaccharides and lignin during the pretreatment. Especially Szarvasi pulp displayed outstanding results in terms of fractionation efficiency and enzymatic digestibility, though in all cases successful lignocellulose fractionation was observed. These insights into the structural composition of different perennial plant species and the impact of the OrganoCat pretreatment on the plant material leads to useful information to strategically adapt such processes to the individual lignocellulosic material aiming for a full valorisation.

Analysis of Alkaloids in Single Plant Cells by Capillary Electrophoresis

In this chapter, capillary electrophoresis (CE) is demonstrated to be a useful technique for the determination of alkaloids in microsamples of single plant cells. A single cell sampling technique with microcapillaries that includes extraction of sample volumes in the pl range from single cells, division into aliquots, addition of internal standard, and injection into the CE capillary is described. The danger of contamination and evaporation of such low sample volumes has been avoided by handling them under an inert protective layer of silicone oil. For the determination of alkaloids in cell samples, CE with direct ultraviolet detection using a high concentration of citric acid as background electrolyte provides sufficient sensitivity.