Online Nano Solid Phase Extraction Fourier-Transform Ion Cyclotron Resonance Mass Spectrometry Workflow to Analyze Small Scale Gradients of Soil Solution Organic Matter in the Rhizosphere.

A new method combining online nano solid phase extraction coupled with Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) was developed to extract and analyze organic matter (OM) from microliter volumes of salt containing soil solution samples. This approach allows the reproducible analysis of only minute amounts of organic carbon (down to 10 ng C) without the need of further sample preparation. The new method was applied to unravel developing small-scale patterns of dissolved organic matter (DOM) in soil solutions of a soil column experiment in which Zea mays plants were grown for 3 weeks. Soil solution was sampled by micro suction cups from the undisturbed soil-root system once a week. Growth of the root system and, hence, position of individual roots relative to the suction cups was followed by X-ray computed tomography (X-ray CT). Our method makes it possible to resolve the chemical complexity of soil solution OM (up to 4300 molecular formulas from 2.5 µL sample). This allows to observe chemical gradients in the rhizosphere on a molecular level over time. The increasing influence of roots on soil solution OM is visible from higher molecular masses, an increasing degree of oxygenation and a higher fraction of formulas containing heteroatoms. The online nano solid phase extraction-FT-ICR-MS method provides novel insight into the processes affecting DOM in the rhizosphere, such as root exudation, microbial processes, and soil organic matter stabilization.

Ozonation products of carbamazepine and their removal from secondary effluents by soil aquifer treatment--indications from column experiments.

Ozonation is known as an efficient treatment to reduce the concentration of many trace organic compounds from WWTP effluents, but the formation of unknown and possibly persistent and toxic transformation products has to be considered. In this paper tertiary treatment of wastewater by the combination of ozone and soil aquifer treatment was investigated with respect to the removal of the antiepileptic drug carbamazepine (CBZ, 10 µg/L) and its transformation products. Batch tests and pilot experiments confirmed efficient removal of carbamazepine from secondary effluent by ozone. With typical ozone consumption of 0.7 mg O3/mg DOC0, approx. 50% of the transformed CBZ was detected as its primary product 1-(2-benzaldehyde)-4-hydro-(1H,3H)-quinazoline-2-one (BQM). Structure proposals and a formation pathway were elaborated for a total of 13 ozonation products of CBZ. In subsequent biological treatment BQM turned out to be more effectively biodegraded than CBZ. Its aldehyde group was quickly oxidized to a carboxylic acid (BaQM), which was removed in sand column experiments. Most of the minor ozonation products of CBZ persisted in sand column experiments with residence times of 5-6 days. Non-target screening of column effluent revealed no formation of persistent biotransformation products.

Determination of pesticide residues in wine by membrane-assisted solvent extraction and high-performance liquid chromatography-tandem mass spectrometry.

The determination of pesticides in food products is an essential issue to guarantee food safety and minimise health risks of consumers. A protocol based on membrane-assisted solvent extraction and liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) that allows the determination of 18 pesticides in red wine at minimum labour effort for sample preparation was developed and validated. Ten millilitres of wine were extracted using 100 µL of toluene filled in a non-porous polyethylene membrane bag which is immersed in the wine sample. After 150 min extraction under stirring, an aliquot of the extraction solution is analysed using HPLC-MS/MS. The limits of quantification ranged from 3 ng/L for Pirimicarb to 1.33 µg/L for Imidacloprid. Quantification by matrix-matched calibration provided relative standard deviations ≤16 % for most of the target pesticides. The linearity of calibration was given over three to four orders of magnitude, which enables the reliable measurement of a broad range of pesticide concentrations, and for each target pesticide, the sensitivity of the protocol meets the maximum residue levels set by legislations at least for wine grapes. Good agreement of results was found when the new method was compared with a standard liquid-liquid extraction protocol. In five wine samples analysed, Carbendazim and Metalaxyl were determined at micrograms per litre concentrations, even in some of the organic wines. Tebuconazol and Cyprodinitril were determined at lower abundance and concentration, followed by Spiroxamin and Diuron.

Modelling the degradation of micropollutants in wastewater: parameter estimation and application to pilot (laboratory-scale) MBR data in the case of 2,6-NDSA and BTSA.

The paper summarises the definition of an extended biokinetic model dedicated to micropollutant degradation in wastewater treatment and the parameter estimation methodology for this model. Additionally it describes results on experimental parameter estimation for two target micropollutants, naphthalene disulfonate (2,6-NDSA) and benzothiazole sulfonic acid (BTSA). Subsequently the parameterised model is applied to real operational data from two laboratory-scale (MBR) installations. The work presents the full chain of theoretical model development, model analysis and practical application to case study data for micropollutants.

A strategy for the assessment of hazardous substances in industrial effluents (IDA).

A strategy for the assessment of industrial wastewater discharges with regard to the major hazard parameters toxicity, persistence and bioaccumulation is presented. The modular design of the strategy named IDA (Industrial Discharge Assessment) allows us to successively determine the three hazard parameters. Starting with the parameter toxicity biological tests for acute and chronic as well as genotoxicity are performed. Next persistence in the aquatic environment is simulated by a biological degradation test. A method based on solid-phase extraction was developed to cover the parameter bioaccumulation by determination of the amount of potentially bioaccumulating substances. The strategy was applied to real wastewater samples to test its applicability. In one of the investigated discharges toxic and persistent but no potentially bioaccumulating substances were found to be present. The strategy IDA is generally applicable and provides reasonable results to allow for a deeper insight into the hazard potential of an effluent.

Removal of persistent polar pollutants through improved treatment of wastewater effluents (P-THREE).

The EU-project P-THREE started with the establishment of analytical methods for persistent polar pollutants (P3) and quality assurance, followed by screening of P3 in influents and effluents of known wastewater (WW) treatment plants (TP), receiving waters and tap water produced thereof in several European countries. A final selection of analytes for further studies has been performed. Model MBR reactors have been constructed and an optimisation on synthetic wastewater spiked with P3 (linear alkylbenzene sulfonates (LAS), naphthalene sulfonates) has been performed. An initial dynamic modelling of treatment processes has also started. Advanced oxidation process (AOP) treatment has been done in groundwater with isoproturon and nonylphenol ethoxylates (NPEO). The analysis of individual P3 and potential degradation products was also performed. An integrated systeme analysis of the WW treatment processes has also been initiated.

Occurrence of benzothiazoles in municipal wastewater and their fate in biological treatment.

A number of 2-substituted benzothiazoles that are known to be used as fungicides, corrosion inhibitors and vulcanization accelerators in industry have been analyzed in municipal wastewater and the effluents of activated sludge and membrane bioreactor (MBR) treatment over a three month period. All six analytes were regularly detected in the municipal wastewater by liquid chromatography-mass spectrometry and amount to a total concentration of 3.4 microg/L. Of these compounds benzothiazole-2-sulfonic acid (1,700 ng/L), benzothiazole (850 ng/L) and 2-hydroxybenzothiazole (500 ng/L) were most prominent. The source of the benzothiazole emission is yet unknown. Activated sludge treatment did not reduce total benzothiazole concentration significantly. Removals of the individual compounds ranged from 90% for 2-mercaptobenzothiazole and 70% for hydroxybenzothiazole to 40% for benzothiazole. The concentration of benzothiazole-2-sulfonic acid increased by 20%, whereas 2-methylthiobenzothiazole increased by 160% during activated sludge treatment, likely due to the methylation of mercaptobenzothiazole. Total benzothiazole removal in two parallely operated MBRs was significantly better (43%) than in the conventional activated sludge treatment. Namely benzothiazole and benzothiazole-2-sulfonic acid were more effectively removed. This first systematic study on the occurrence of benzothiazoles in municipal wastewater has shown that this is a relevant class of trace contaminants in municipal wastewater which is only incompletely removed in biological wastewater treatment. Emission from sewage treatment is dominated by the most polar benzothiazole-2-sulfonic acid. MBR treatment may reduce but cannot avoid this emission.

Direct ring fission of salicylate by a salicylate 1,2-dioxygenase activity from Pseudaminobacter salicylatoxidans.

In cell extracts of Pseudaminobacter salicylatoxidans strain BN12, an enzymatic activity was detected which converted salicylate in an oxygen-dependent but NAD(P)H-independent reaction to a product with an absorbance maximum at 283 nm. This metabolite was isolated, purified, and identified by mass spectrometry and (1)H and (13)C nuclear magnetic resonance spectroscopy as 2-oxohepta-3,5-dienedioic acid. This metabolite could be formed only by direct ring fission of salicylate by a 1,2-dioxygenase reaction. Cell extracts from P. salicylatoxidans also oxidized 5-aminosalicylate, 3-, 4-, and 5-chlorosalicylate, 3-, 4-, and 5-methylsalicylate, 3- and 5-hydroxysalicylate (gentisate), and 1-hydroxy-2-naphthoate. The dioxygenase was purified and shown to consist of four identical subunits with a molecular weight of about 45,000. The purified enzyme showed higher catalytic constants with gentisate or 1-hydroxy-2-naphthoate than with salicylate. It was therefore concluded that P. salicylatoxidans synthesized a gentisate 1,2-dioxygenase with an extraordinary substrate range, which also allowed the oxidation of salicylate.

Sulfophthalimide as major metabolite formed from sulfonated phthalocyanine dyes by the white-rot fungus Bjerkandera adusta.

The reaction products formed during the decolorization of the sulfophthalocyanine textile dyes Reactive blue 15 (RB15) and Reactive blue 38 (RB38) by the white-rot fungus Bjerkandera adusta were analyzed by high-performance liquid chromatography with diode array detection and with liquid chromatography-electrospray ionization-tandem mass spectrometry. Sulfophthalimides (SPI; 3 and 4) were identified as major metabolites by comparison with synthesized reference compounds. SPI was formed from both dyes in fungal cultures and by incubation with its purified manganese peroxidase and lignin peroxidase. Quantitative assessment of the SPI formed from RB15 accounted for approximately 60% of the theoretical amount.

Analysis of sulfophthalimide and some of its derivatives by liquid chromatography-electrospray ionization tandem mass spectrometry.

A system was developed for the separation of sulfophthalimide (SPI), sulfophthalamide (SPAM), sulfophthalamic acid (SPAA) and sulfophthalic acid (SPA) by ion-pair liquid chromatography and their detection by electrospray ionization tandem mass spectrometry (ESI-MS-MS). Except for SPAM, the 3- and 4-sulfo-isomers of the analytes were separated by HPLC using volatile tributylamine as ion-pairing agent. Initial fragmentations of the analytes in the negative mode involve losses of CO2 or HNCO or condensation via H2O or NH3 elimination. ortho-Effects of the sulfonate group were recognized in the fragmentation of the respective 3-sulfo-isomers and allowed us to assign the order of elution of the SPAA isomers. Quantitative analysis of these sulfonated aromatic compounds with MRM detection was elaborated and resulted in detection limits ranging from 1 pg for SPA to 13 pg for SPAA isomers and in limits of quantification of 2-10 microg/L for 5 microL volumes of injected tap water, municipal wastewater or industrial effluents up to salt contents of 0.5-1 g/L. The method was applied to study the isomer-specific chemical and microbial transformations of SPI, which was previously shown to be formed by white-rot fungi from sulfophthalocyanine textile dyes.

Exact mass measurements on-line with high-performance liquid chromatography on a quadrupole mass spectrometer.

Exact mass measurements were performed on-line with high-performance liquid chromatography on a quadrupole mass spectrometer. Compounds with molecular weights from 98 to 797, mainly aromatic sulfonates and sulfonamides, were analyzed with electrospray ionization in positive or negative mode. Internal mass calibration compounds were continuously added after separation. A Gaussian fit of the mass errors of 808 individual measurements (concentrations of 1-10 mg/L, 20-200 ng absolute on column) resulted in a mean error of 0.1 mmu (0.45 ppm) and a standard deviation sigma of 1.5 mmu (5.4 ppm). The 99.7% confidence intervals (3sigma) were +/-4.5 mmu (+/-16.2 ppm) for single mass measurements. Averaging 10 measurements further reduced the errors to less than +/-1.5 mmu (+/-5 ppm). Isobaric interferences with ions resulting from the mass calibrants were avoided by the use of complementary mass calibrants. The results were verified (differences below +/-4.5 mmu) with a LC/ oa-TOFMS. Limited mass range chromatograms were used to enhance selectivity in the analysis of mixtures. The method was applied to determine the elemental composition of a potential dye metabolite detected in anaerobically treated textile wastewater.

Concerted chemical and microbial degradation of sulfophthalimides formed from sulfophthalocyanine dyes by white-rot fungi.

3- and 4-sulfophthalimide (SPI) have recently been shown to be the major product formed by white-rot fungi upon decolorization of sulfophthalocyanine (SPC) textile dyes. To make use of this metabolic potential in textile wastewater treatment, the fungal breakdown products should be degradable by activated sludge. Here,the aerobic degradation of SPI was studied in die-away tests, and biodegradation intermediates and degradation products were analyzed by liquid chromatography-electrospray ionization-mass spectrometry. The degradation of SPI is initiated by a chemical hydrolysis to sulfophthalamic acid (SPAA) with half-lives of 8 and 40 h at pH 6.5 for 4- and 3-SPI, respectively. Then, 4-SPAA can be mineralized by aerobic mixed cultures, while 3-SPAA remained stable throughout the experiment (35 d). Analogously, the potential intermediate in 4-SPAA degradation, the 4-sulfophthalic acid, but not its 3-isomer, can be completely mineralized aerobically by mixed cultures. In all chemical and microbialtransformations of these aromatic sulfonates the 4-sulfo-isomer is more reactive than the 3-isomer. The triade of fission of the SPC-system by white-rot fungi to SPI, chemical hydrolysis of SPI to SPAA, and microbial degradation of SPAA offers a pathway to mineralize the major part of the SPC system of textile dyes, whether in a respective effluent treatment system or in the aquatic environment. More general, these results illustrate on a molecular level how white-rot fungi and bacteria may cooperate in mineralizing structurally complex colored substances.

Analysis of sulfonated naphthalene-formaldehyde condensates by ion-pair chromatography and their quantitative determination from aqueous environmental samples.

An ion-pair solid-phase extraction (IPE), ion-pair chromatography (IPC) procedure with fluorescence detection for the quantitative analysis of sulfonated naphthalene-formaldehyde condensates (SNFC) was developed, which provides full resolution of SNFC up to a degree of condensation n = 5 and partial resolution up to n = 15. Liquid chromatography-electrospray ionization-mass spectrometry confirmed that SNFC elute in the order of condensation. Response factors in fluorescence detection proved to be mass-constant, thereby allowing us to determine total SNFC amounts. With this IPC method, the weight- and the number-average molecular weights of these high-volume production chemicals (kiloton per annum), used as synthetic tanning agents, concrete plasticizers, and dispersants, can be determined. Recoveries in IPE range from 73 to 85% in river Rhine water and from 79 to 93% in tap water for n = 2 to n = 7 with limits of detection of 3-8 ng/L for individual homologues from 500 mL of water. The IPE-IPC procedure was applied to samples of secondary industrial effluents, river Rhine water, a river bank filtrate, and a groundwater sample. SNFC up to n = 6 were detected in the treated effluents. Total concentrations ranged from 208 micrograms/L in a secondary treated SNFC production effluent to < 1.4 micrograms/L in groundwater. These first analyses suggest a widespread occurrence of the lower oligomers of SNFC in the aquatic environment.

Determination of 2-substituted benzothiazoles of industrial use from water by liquid chromatography/electrospray ionization tandem mass spectrometry.

A method was developed for the analysis of 2-substituted benzothiazoles from wastewaters by reversed-phase liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS). While benzothiazole (BT), 2-amino- (ABT), 2-methyl-, 2-thiocyanomethylthio- and 2-methylthiobenzothiazole were separated with a methanol gradient acidified with formic acid and detected in the positive mode, 2-mercapto- and 2-hydroxybenzothiazole (OHBT) as well as benzothiazole-2-sulfonic acid (BTSA) were separated with the addition of ammonium acetate and detected in the negative ion mode. Detection limits range from 10 ng L(-1) for ABT to 200 ng L(-1) for OHBT after direct injection of 100 µL sample volumes, while BT requires 2.5 µg L(-1). The method is, thus, suitable for direct analysis in wastewaters. Its application to tannery effluent samples revealed, that, besides a 90-95% elimination of total benzothiazoles, the concentration of OHBT and BTSA increased during the biological treatment. Copyright 2000 John Wiley & Sons, Ltd.

Use of volatile amines as ion-pairing agents for the high-performance liquid chromatographic-tandem mass spectrometric determination of aromatic sulfonates in industrial wastewater.

The use of trialkylamines (triethylamine, N,N-dimethyl-n-butylamine, and tri-n-butylamine) as volatile ion-pairing agents for the high-performance liquid chromatographic separation of aromatic sulfonates was investigated. Negative ion electrospray tandem mass spectrometry was used to detect both singly and multiply charged analyte ions. Sensitivity of detection was strongly reduced by amine concentrations above 2.5 mmol l(-1) in the eluent. With tributylamine as ion-pairing agent 19 aromatic sulfonic acids could be determined using time-scheduled selected reaction monitoring. Lowest orders of detection range from 3 to 74 microgl(-1). Several naphthalenesulfonic acids were determined in dyeing baths and textile wastewater in concentrations ranging from 0.1 mgl(-1) to 2.1 mgl(-1). A degradation product of sulfonated azodyes was identified in the effluent of a laboratory treatment plant.