Project

Sulfonamides; Nonextractable Residues; High Resolution Mass Spectrometry; Soil Organic Matter; Sorption; soil; antibiotics

Lead
Lay summary
The input of veterinary antibiotics such as sulfonamides into the environment is of concern because of their potential to invoke bacterial resistance or to exert adverse effects on microbial communities. After input into soils, a major part cannot be recovered by conventional extraction methods. The added sulfonamides thus form "nonextractable residues" (NER). The mechanisms postulated for NER formation are either physical entrapment in organic matter or covalent bond formation. Our own and other studies suggest that the rapid initial NER formation of sulfonamides is probably caused by covalent bonding. Based on long- and short-term batch studies with humic acids, quinone and other carbonyl-containing monomers, we could show that the main pathway of covalent bond formation of sulfonamides occurs by nucleophilic addition rather than by radical reactions, and the formation of reactive quinones by oxidative enzymes or Mn oxides is the rate-limiting step. Currently, we are developing a method to detect NERs of sulfonamides based on high pressure size-exclusion chromatography coupled to soft ionization high resolution mass spectrometry (HPSEC-MS/MS) to detect sulfonamide NERs in organic matter isolates. The objectives of the project are (i) combining this HPSEC-MS/MS method with a sequential extraction procedure to detect sulfonamide NER in soils, and (iv) to study the relevant mechanisms of sulfonamide covalent bond formation in soil by selective sample manipulation. For these studies, 14C-labeled sulfamethazine will be used. The sequential extraction procedure starts with a pressurized liquid extraction (PLE) of sulfonamides. To release organic matter and sulfonamide NER from the PLE-extracted soil, we will evaluate two different extraction procedures and develop appropriate purification steps to make these extracts SEC-MS amendable. The relevant mechanisms in soils will be assessed by applying this method to soil samples, in which either Mn oxides are selectively removed, oxidative enzymes are inhibited or Mn oxides and free enzymes are added prior to incubation. Furthermore, the nucleophilic addition reaction itself will be targeted by addition of a competing nucleophile and addition of selected model hydroquinones to increase the pools of compounds available to form quinones. From a direct comparison of the different treatments we will get insights in the limiting factors and mechanisms driving the NER formation of sulfonamides in soils from both, macroscopic distribution among different fractions and application of the HPSEC-MS/MS method.

Direct link to Lay Summary

Last update: 21.02.2013

Active participation

Number	Title	Start	Funding scheme

The input of veterinary antibiotics such as sulfonamides into the environment is of concern because of their potential to invoke bacterial resistance or to exert adverse effects on microbial communities. After input into soils, a major part cannot be recovered by conventional extraction methods. The added sulfonamides thus form “nonextractable residues” (NER). The mechanisms postulated for NER formation are either physical entrapment in organic matter or covalent bond formation. Our own and other studies suggest that the rapid initial NER formation of sulfonamides is probably caused by covalent bonding. Based on long- and short-term batch studies with humic acids, quinone and other carbonyl-containing monomers, we could show that the main pathway of covalent bond formation of sulfonamides occurs by nucleophilic addition rather than by radical reactions, and the formation of reactive quinones by oxidative enzymes or Mn oxides is the rate-limiting step. Currently, we are developing a method to detect NERs of sulfonamides based on high pressure size-exclusion chromatography coupled to soft ionization high resolution mass spectrometry (HPSEC-MS/MS) to detect sulfonamide NERs in organic matter isolates. The objectives of the fourth year of this project are (i) combining this HPSEC-MS/MS method with a sequential extraction procedure to detect sulfonamide NER in soils, and (iv) to study the relevant mechanisms of sulfonamide covalent bond formation in soil by selective sample manipulation. For these studies, 14C-labeled sulfamethazine will be used. The sequential extraction procedure starts with a pressurized liquid extraction (PLE) of sulfonamides. To release organic matter and sulfonamide NER from the PLE-extracted soil, we will evaluate two different extraction procedures and develop appropriate purification steps to make these extracts SEC-MS amendable. The relevant mechanisms in soils will be assessed by applying this method to soil samples, in which either Mn oxides are selectively removed, oxidative enzymes are inhibited or Mn oxides and free enzymes are added prior to incubation. Furthermore, the nucleophilic addition reaction itself will be targeted by addition of a competing nucleophile and addition of selected model hydroquinones to increase the pools of compounds available to form quinones. From a direct comparison of the different treatments we will get insights in the limiting factors and mechanisms driving the NER formation of sulfonamides in soils from both, macroscopic distribution among different fractions and application of the HPSEC-MS/MS method.