Stable Transformation Products Research Articles

Approach for detecting mutagenicity of biodegraded and ozonated pharmaceuticals, metabolites and transformation products from a drinking water perspective

Many pharmaceuticals and related metabolites are not efficiently removed in sewage treatment plants and enter into surface water. There, they might be subject of drinking water abstraction and treatment by ozonation. In this study, a systematic approach for producing and effect-based testing of transformation products (TPs) during the drinking water ozonation process is proposed. For this, two pharmaceutical parent substances, three metabolites and one environmental degradation product were investigated with respect to their biodegradability and fate during drinking water ozonation. The Ames test (TA98, TA100) was used for the identification of mutagenic activity present in the solutions after testing inherent biodegradability and/or after ozonation of the samples. Suspicious results were complemented with the umu test. Due to the low substrate concentration required for ozonation, all ozonated samples were concentrated via solid phase extraction (SPE) before performing the Ames test. With the exception of piracetam, all substances were only incompletely biodegradable, suggesting the formation of stable TPs. Metformin, piracetam and guanylurea could not be removed completely by the ozonation process. We received some evidence that technical TPs are formed by ozonation of metformin and piracetam, whereas all tested metabolites were not detectable by analytical means after ozonation. In the case of guanylurea, one ozonation TP was identified by LC/MS. None of the experiments showed an increase of mutagenic effects in the Ames test. However, the SPE concentration procedure might lead to false-positive results due to the generation of mutagenic artefacts or might lead to false-negative results by missing adequate recovery efficiency. Thus, these investigations should always be accompanied by process blank controls that are carried out along the whole ozonation and SPE procedure. The study presented here is a first attempt to investigate the significance of transformation products by a systematic approach. However, the adequacy and sensitivity of the methodology need to be further investigated. The approach of combining biodegradation and ozonation with effect-based assays is a promising tool for the early detection of potential hazards from TPs as drinking water contaminants. It can support the strategy for the evaluation of substances and metabolites in drinking water. A multitude of possible factors which influence the results have to be carefully considered, among them the selectivity and sensibility of the mutagenicity test applied, the extraction method for concentrating the relevant compounds and the biocompatibility of the solvent. Therefore, the results have to be carefully interpreted, and possible false-negative and false-positive results should be considered.

Transformation products and reaction kinetics of fragrances in advanced wastewater treatment with ozone

The reaction of the fragrance compounds 4,6,6,7,8,8-hexamethyl-1,3,4,7-tetrahydrocyclopenta[g] isochromene (HHCB), 1-(3,5,5,6,8,8-hexamethyl-6,7-dihydronaphthalen-2-yl)ethanone (AHTN), 1-tert-butyl-3,5-dimethyl-2,4,6-trinitrobenzene (musk xylene/MX), 1-(4-tert-butyl-2,6-dimethyl-3,5-dinitrophenyl)ethanone (musk ketone/MK), and 1-(2,3,8,8-tetramethyl-1,3,4,5,6,7-hexahydronaphthalen-2-yl)ethanone (OTNE) with ozone in tap water as well as waste water treatment plant (WWTP) effluents is described. Several transformation products are characterized by means of gas chromatography coupled to mass spectrometry. One transformation product (HHCB-Lactone) was confirmed by means of a true standard. Musk xylene and musk ketone do not react with ozone under the conditions used in this study. AHTN and HHCB reacted slowly to a multitude of transformation products, while OTNE reacted quickly to several stable transformation products. The reaction constants and half lives are used to predict removal efficiencies for full scale reactors.

6:2 Fluorotelomer sulfonate aerobic biotransformation in activated sludge of waste water treatment plants

The aerobic biotransformation of 6:2 FTS salt [F(CF 2) 6CH 2CH 2 SO 3 - K +] was determined in closed bottles for 90 d in diluted activated sludge from three waste water treatment plants (WWTPs) to compare its biotransformation potential with that of 6:2 FTOH [F(CF 2) 6CH 2CH 2OH]. The 6:2 FTS biotransformation was relatively slow, with 63.7% remaining at day 90 and all observed transformation products together accounting for 6.3% of the initial 6:2 FTS applied. The overall mass balance (6:2 FTS plus observed transformation products) at day 90 in live and sterile treatments averaged 70% and 94%, respectively. At day 90, the stable transformation products observed were 5:3 acid [F(CF 2) 5CH 2CH 2COOH, 0.12%], PFBA [F(CF 2) 3COOH, 0.14%], PFPeA [F(CF 2) 4COOH, 1.5%], and PFHxA [F(CF 2) 5COOH 1.1%]. In addition, 5:2 ketone [F(CF 2) 5C(O)CH 3] and 5:2 sFTOH [F(CF 2) 5CH(OH)CH 3] together accounted for 3.4% at day 90. The yield of all the stable transformation products noted above (2.9%) was 19 times lower than that of 6:2 FTOH in aerobic soil. Thus 6:2 FTS is not likely to be a major source of PFCAs and polyfluorinated acids in WWTPs. 6:2 FTOH, 6:2 FTA [F(CF 2) 6CH 2COOH], and PFHpA [F(CF 2) 6COOH] were not observed during the 90-d incubation. 6:2 FTS primary biotransformation bypassed 6:2 FTOH to form 6:2 FTUA [F(CF 2) 5CF = CHCOOH], which was subsequently degraded via pathways similar to 6:2 FTOH biotransformation. A substantial fraction of initially dosed 6:2 FTS (24%) may be irreversibly bound to diluted activated sludge catalyzed by microbial enzymes. The relatively slow 6:2 FTS degradation in activated sludge may be due to microbial aerobic de-sulfonation of 6:2 FTS, required for 6:2 FTS further biotransformation, being a rate-limiting step in microorganisms of activated sludge in WWTPs.

Aerobic biodegradation of [ 14C] 6:2 fluorotelomer alcohol in a flow-through soil incubation system

The aerobic biodegradation of [1,2- 14C] 6:2 FTOH [F(CF 2) 6 14CH 2 14CH 2OH] in a flow-through soil incubation system is described. Soil samples dosed with [1,2- 14C] 6:2 FTOH were analyzed by liquid scintillation counting, LC/ARC (liquid chromatography/accurate radioisotope counting), LC/MS/MS, and thermal combustion to account for 6:2 FTOH and its transformation products over 84 d. Half of the [1,2- 14C] 6:2 FTOH disappeared from soil in 1.3 d, undergoing simultaneous microbial degradation and partitioning of volatile transformation product(s) and the 6:2 FTOH precursor into the air phase. The overall 14C (radioactivity) mass balance in live and sterile treatments was 77–87% over 84-d incubation. In the live test system, 36% of total 14C dosed was captured in the airflow (headspace), 25% as soil-bound residues recovered via thermal combustion, and 16% as soil extractable. After 84 d, [ 14C] 5:2 sFTOH [F(CF 2) 5CH(OH) 14CH 3] was the dominant transformation product with 16% molar yield and primarily detected in the airflow. The airflow also contained [1,2- 14C] 6:2 FTOH and 14CO 2 at 14% and 6% of total 14C dosed, respectively. The other significant stable transformation products, all detected in soil, were 5:3 acid [F(CF 2) 5CH 2CH 2COOH, 12%], PFHxA [F(CF 2) 5COOH, 4.5%] and PFPeA [F(CF 2) 4COOH, 4.2%]. Soil-bound residues as well as conjugates between fluorinated transformation products and dissolved soil components were only observed in the live test system and absent in the sterile soil, suggesting that such binding and complexation are microbially or enzymatically driven processes. At day 84, 5:3 acid is postulated to be the major transformation product in soil-bound residues, which may not be available for further biodegradation in soil environment.

Ferrous hydroxy carbonate is a stable transformation product of biogenic magnetite

An ~1:1 mixture of ferrihydrite and nanocrystalline akaganeite (β-FeOOH; 10.15 nm) was incubated with Shewanella putrefaciens (strain CN32) under anoxic conditions with lactate as an electron donor and anthraquinone-2,6-disulfonate (AQDS) as an electron shuttle. The incubation was carried out in a 1,4-piperazinediethanesulfonic acid (PIPES)-buffered medium, without PO43- at circumneutral pH. Iron reduction was measured as a function of time (as determined by 0.5 N HCl extraction), and solids were characterized by X-ray diffraction (XRD), electron microscopy, and Mössbauer spectroscopy. The biogenic reduction of Fe3+was rapid; with 60% of the total Fe (FeTOT) reduced in one day. Only an additional 10% of FeTOT was reduced over the next three years. A fine-grained (~10 nm), cation-excess (CE) magnetite with an Fe2+/FeTOT ratio of 0.5-0.6 was the sole biogenic product after one day of incubation. The CE magnetite was unstable and partially transformed to micrometer-sized ferrous hydroxy carbonate [FHC; Fe2(OH)2CO3(s)], a rosasite-type mineral, with time. Ferrous hydroxy carbonate dominated the mineral composition of the three year incubated sample. The Fe2+/FeTOT ratio of the residual CE magnetite after three years of incubation was lower than the day 1 sample and was close to that of the stoichiometric magnetite (0.33). To the best of our knowledge, this is the first report of biogenic FHC, and was only reported twice in literature but in a very different context. Ferrous hydroxy carbonate appeared to form by slow reaction of microbially produced carbonate with Fe2+-excess magnetite. The FHC may be an overlooked mineral phase that explains the infrequent occurrence of fine-grained, biogenic magnetite in anoxic sediments.

Structure of "unknown" soil nitrogen investigated by analytical pyrolysis

Curie-point pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS) and in-source pyrolysis-field ionization mass spectrometry (Py-FIMS) were applied for the first time to the structural characterization of organic nitrogen in hydrolyzates and hydrolysis residues resulting from the classical 6M HCl hydrolysis of mineral soils. Two well-described soils of widely different origin (i.e., a Gleysol Ah and a Podzol Bh) were investigated. Py-GC/MS was performed using a nitrogen-selective detector to detect and identify N-containing pyrolysis in the hydrolyzate (e.g., pyrazole and/or imidazole, N,N-dimethylmethanamine, benzenacetonitrile, propane- and propenenitriles) and the hydrolysis residue (e.g., pyrroles, pyridines, indoles, N-derivatives of benzene, benzothiazol, and long-chain aliphatic nitriles). Moreover, temperature-resolved Py-FIMS allowed us to record the thermal evolution of the N-containing compounds during pyrolysis. These were characterized by a particularly high thermostability compared to their thermal release from whole soils. The combination of pyrolysis with mass spectrometric methods permitted analyses of the identities and thermal stabilities of complex nitrogen compounds in hydrolysis residues of whole soils, which cannot be done by wet-chemical methods. Pyrolysis-methylation GC/MS with tetramethyl-ammonium hydroxide (TMAH) as methylating agent enabled the identification of N,N-dimethylbenzenamine and so confirmed the identification of benzeneamine by Py-GC/MS in nonmethylated hydrolysis residues. The described N-derivatives of benzene and long-chain nitriles are usually not detectable by pyrolysis-mass spectrometry of plants and microorganisms. These compounds are characteristic of soils, terrestrial humic substances and hydrolysis residues and seem to be specific, stable transformation products of soil nitrogen.