Transformation of atrazine to hydroxyatrazine in the environment may be underestimated by current assessment schemes since immobilization and further transformation of the metabolite can render parent-to-daughter compound ratios unreliable. This study reports significant C and N isotope fractionation of atrazine in transformation to hydroxyatrazine by Chelatobacter heintzii, Pseudomonas sp. ADP, and Arthrobacter aurescens TC1 highlighting an alternative approach to detecting this natural transformation pathway. Indistinguishable dual isotope slopes big up tri, open (= delta(15)N/delta(13)C approximately epsilon(N)/epsilon(C)) for Chelatobacter heintzii (-0.65 +/- 0.08) and Arthrobacter aurescens TC1 (-0.61 +/- 0.02) suggest the same biochemical transformation mechanism despite different hydrolyzing enzymes (AtzA versus TrzN). With Pseudomonas sp. ADP (also AtzA) significantly smaller fractionation indicates masking effects by steps prior to enzyme catalysis, while a distinguishable big up tri, open = -0.32 +/- 0.06 suggests that some of these steps showed slight isotope fractionation. Abiotic reference experiments reproduced the pattern of biotic transformation at pH 3 (enrichment of (13)C, depletion of (15)N in atrazine), but showed enrichment of both (13)C and (15)N at pH 12. This indicates that the organisms activated atrazine by a similar Lewis acid complexation (e.g., with H(+)) prior to nucleophilic aromatic substitution, giving the first detailed mechanistic insight into this important enzymatic reaction.

Methane is a major product of anaerobic degradation of organic matter and an important greenhouse gas. Its stable carbon isotope composition can be used to reveal active methanogenic pathways, if associated isotope fractionation factors are known. To clarify the causes that lead to the wide variation of fractionation factors of methanogenesis from H 2 plus CO 2 (a CO2ÀCH4 ), pure cultures and various cocultures were grown under different thermodynamic conditions. In syntrophic and obligate syntrophic cocultures thriving on different carbohydrate substrates, fermentative bacteria were coupled to three different species of hydrogenotrophic methanogens of the families Methanobacteriaceae and Methanomicrobiaceae. We found that C-isotope fractionation was correlated to the Gibbs free energy change (DG) of CH 4 formation from H 2 plus CO 2 and that the relation can be described by a semi-Gauss curve. The derived relationship was used to quantify the average DG that is available to hydrogenotrophic methanogenic archaea in their habitat, thus avoiding the problems encountered with measurement of low H 2 concentrations on a microscale. Boreal peat, rice field soil, and rumen fluid, which represent major sources of atmospheric CH 4 , exhibited increasingly smaller a CO2ÀCH4 , indicating that thermodynamic conditions for hydrogenotrophic methanogens became increasingly more favourable. Vice versa, we hypothesize that environments with similar energetic conditions will also exhibit similar isotope fractionation. Our results, thus, provide a mechanistic constraint for modelling the 13 C flux from microbial sources of atmospheric CH 4 .

Compound-specific stable isotope analysis by gas chromatography-isotope ratio mass spectrometry (GC-IRMS) is increasingly used to assess origin and fate of organic substances in the environment. Although analysis without isotopic discrimination is essential, it cannot be taken for granted for new target compounds. We developed and validated carbon isotope analysis of atrazine, a herbicide widely used in agriculture. Combustion was tested with reactors containing (i) CuO/NiO/Pt operating at 940 degrees C; (ii) CuO operating at 800 degrees C; (iii) Ni/NiO operating at 1150 degrees C and being reoxidized for 2 min during each gas chromatographic run. Accurate and precise carbon isotope measurements were only obtained with Ni/NiO reactors giving a mean deviation delta delta(13)C from dual inlet measurements of -0.1-0.2% per hundred and a standard deviation (SD) of +/- 0.4% per hundred. CuO at 800 degrees C gave precise, but inaccurate values (delta delta(13)C = -1.3% per hundred, SD +/- 0.4% per hundred), whereas CuO/NiO/Pt reactors at 940 degrees C gave inaccurate and imprecise data. Accurate (delta delta(15)N = 0.2% per hundred) and precise (SD +/- 0.3% per hundred) nitrogen isotope analysis was accomplished with a Ni/NiO-reactor previously used for carbon isotope analysis. The applicability of the method was demonstrated for alkaline hydrolysis of atrazine at 20 degrees C and pH 12 (nucleophilic aromatic substitution) giving epsilon(carbon) = -5.6% per hundred +/- 0.1% per hundred (SD) and epsilon(nitrogen) = -1.2% per hundred +/- 0.1% per hundred (SD).

Global groundwater resources are constantly challenged by a multitude of contaminants such as aromatic hydrocarbons. Especially in anaerobic habitats, a large diversity of unrecognized microbial populations may be responsible for their degradation. Still, our present understanding of the respective microbiota and their ecophysiology is almost exclusively based on a small number of cultured organisms, mostly within the Proteobacteria. Here, by DNA-based stable isotope probing (SIP), we directly identified the most active sulfate-reducing toluene degraders in a diverse sedimentary microbial community originating from a tar-oil-contaminated aquifer at a former coal gasification plant. On incubation of fresh sediments with 13 C 7 -toluene, the production of both sulfide and 13 CO 2 was clearly coupled to the 13 C-labeling of DNA of microbes related to Desulfosporosinus spp. within the Peptococcaceae (Clostridia). The screening of labeled DNA fractions also suggested a novel benzylsuccinate synthase alpha-subunit (bssA) sequence type previously only detected in the environment to be tentatively affiliated with these degraders. However, carbon flow from the contaminant into degrader DNA was only B50%, pointing toward high ratios of heterotrophic CO 2 -fixation during assimilation of acetyl-CoA originating from the contaminant by these degraders. These findings demonstrate that the importance of non-proteobacterial populations in anaerobic aromatics degradation, as well as their specific ecophysiology in the subsurface may still be largely ungrasped.

The isotope enrichment factors () in Methanosaeta concilii and in a lake sediment, where acetate was consumed only by Methanosaeta spp., were clearly less negative than the usually observed for Methanosarcina spp. The fraction of methane produced from acetate in the sediment, as determined by using stable isotope signatures, was 10 to 15% lower when the appropriate of Methanosaeta spp. was used.Methane is a major product of the degradation of organic matter in anoxic environments like rice paddies, natural wetlands, and lake sediments. Its production from H 2 -CO 2 and acetate (ac) in natural systems has been quantified by stable isotope modeling (7,17). One of the input parameters needed is the carbon isotope fractionation of acetoclastic methanogenesis. To determine the fractionation factor (␣) in culture or in an environmental system, the ␦ 13 C of the methyl carbon of acetate (ac-methyl) must be known, since CH 4 is produced from the methyl carbon rather than the carboxyl carbon of acetate (5, 19). For Methanosarcina spp. isotope fractionation has been determined in pure culture (7,10,20) and has been verified in natural environments dominated by this group of acetoclastic methanogens (14). However, detailed data on isotope fractionation by Methanosaeta spp. are rare. Valentine et al. (18) reported a rather low fractionation factor (␣ ϭ 1.007) for thermophilic Methanosaeta thermophila. Similarly, the fractionation factor for mesophilic Methanosaeta concilii also seems to be lower (␣ ϭ 1.017) (A. Chidthaisong, S. C. Tyler, et al., unpublished results) than that for Methanosarcina spp. (␣ ϭ 1.021 to 1.027) (for a review see reference 18). A difference in isotope fractionation by the two acetoclastic methanogenic groups is possible, since they differ in their biochemical activation of acetate to acetyl coenzyme A (acetyl-CoA).To increase our knowledge of isotope fractionation by Methanosaeta spp., we grew M. concilii under defined conditions and determined the isotope enrichment factor (ε). We also determined isotope fractionation by acetoclastic Methanosaeta spp. in a natural environment, Lake Dagow sediment, in which only members of the Methanosaetaceae were detected (2, 8).Cultures (n ϭ 5) of M. concilii DSM 3671 were grown under N 2 -CO 2 (80:20) at 37°C in glass bottles (250 ml; Müller Krempel, Bülach, Switzerland) without shaking using carbonate-buffered (pH 7.1) mineral medium (DSM medium 334). Sodium acetate was added to an initial concentration of approximately 70 mM. For the total molecule the added acetate (ac) had a ␦ 13 C ac of Ϫ24.0‰, and for the methyl group the ␦ 13 C ac-methyl was Ϫ29.4‰. After inoculation with a 20% bacterial suspension in the late exponential phase (resulting in a final volume of 125 ml), several gas (0.4-ml) and liquid (2-ml) samples were removed and used for analysis of pH, concentration, and the carbon isotope composition of acetate and the products formed.Sediment samples from eutrophic Lake Dagow (Northern Brandenburg, Germany) were obtained on 9 November 2004 and 17 Aug...

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Background: The Cefic Mixtures Industry Ad-hoc Team (MIAT) has investigated how risks from combined exposures can be effectively identified and managed using concepts proposed in recent regulatory guidance, new advances in risk assessment, and lessons learned from a Cefic-sponsored case study of mixture exposures. Results: A series of tools were created that include: a decision tree, a system for grouping exposures, and a graphical tool (the MCR-HI plot). The decision tree allows the division of combined exposures into different groups, exposures where one or more individual components are a concern, exposures that are of low concern, and exposures that are a concern for combined effects but not for the effects of individual chemicals. These tools efficiently use available data, identify critical data gaps for combined assessments, and prioritize which chemicals require detailed toxicity information. The tools can be used to address multiple human health endpoints and ecological effects.