SUMMARY Lindane, the γ-isomer of hexachlorocyclohexane (HCH), is a potent insecticide. Purified lindane or unpurified mixtures of this and α-, β-, and δ-isomers of HCH were widely used as commercial insecticides in the last half of the 20th century. Large dumps of unused HCH isomers now constitute a major hazard because of their long residence times in soil and high nontarget toxicities. The major pathway for the aerobic degradation of HCH isomers in soil is the Lin pathway, and variants of this pathway will degrade all four of the HCH isomers although only slowly. Sequence differences in the primary LinA and LinB enzymes in the pathway play a key role in determining their ability to degrade the different isomers. LinA is a dehydrochlorinase, but little is known of its biochemistry. LinB is a hydrolytic dechlorinase that has been heterologously expressed and crystallized, and there is some understanding of the sequence-structure-function relationships underlying its substrate specificity and kinetics, although there are also some significant anomalies. The kinetics of some LinB variants are reported to be slow even for their preferred isomers. It is important to develop a better understanding of the biochemistries of the LinA and LinB variants and to use that knowledge to build better variants, because field trials of some bioremediation strategies based on the Lin pathway have yielded promising results but would not yet achieve economic levels of remediation.

During wastewater treatment, many organic micropollutants undergo microbially mediated reactions resulting in the formation of transformation products (TPs). Little is known on the reaction pathways that govern these transformations or on the occurrence of microbial TPs in surface waters. Large sets of biotransformation data for organic micropollutants would be useful for assessing the exposure potential of these TPs and for enabling the development of structure-based biotransformation prediction tools. The objective of this work was to develop an efficient procedure to allow for high-throughput elucidation of TP structures for a broad and diverse set of xenobiotics undergoing microbially mediated transformation reactions. Six pharmaceuticals and six pesticides were spiked individually into batch reactors seeded with activated sludge. Samples from the reactors were separated with HPLC and analyzed by linear ion trap-orbitrap mass spectrometry. Candidate TPs were preliminarily identified with an innovative post-acquisition data processing method based on target and non-target screenings of the full-scan MS data. Structures were proposed following interpretation of MS spectra and MS/MS fragments. Previously unreported microbial TPs were identified for the pharmaceuticals bezafibrate, diazepam, levetiracetam, oseltamivir, and valsartan. A variety of previously reported and unreported TPs were identified for the pesticides. The results showed that the complementary use of the target and non-target screening methods allowed for a more comprehensive interpretation of the TPs generated than either would have provided individually.

Stable isotope labeling of agricultural polyesters enables demonstration of their microbial utilization in soils.

The environmental safety of decabromodiphenyl ether (BDE-209), a widely used flame retardant, has been the topic of controversial discussions during the past several years. Degradation of BDE-209 into lower brominated diphenyl ether congeners, exhibiting a higher bioaccumulation potential, has been a critical issue. Here, we report on the degradation of BDE-209 and the formation of octa- and nonabromodiphenyl ether congeners under anaerobic conditions. Sewage sludge collected from a mesophilic digester was used as the inoculum and incubated up to 238 days with and without a set of five primers. Following Soxhlet extraction and a liquid chromatography cleanup procedure, parent compounds and debromination products were analyzed by GC/HRMS. In experiments with primers, concentrations of BDE-209 decreased by 30% within 238 days. This corresponds to a pseudo-first-order degradation rate constant of 1 x 10(-3) d(-1). Without primers, the degradation rate constant was 50% lower. Formation of two nonabromodiphenyl ether and six octabromodiphenyl ether congeners proved that BDE-209 underwent reductive debromination in these experiments. Debromination occurred at the para and the meta positions, whereas debromination at the ortho position was not statistically significant. All three nonabromodiphenyl ether congeners (BDE-206, BDE-207, and BDE-208) were found to undergo reductive debromination as well. No significant change of the BDE-209 concentration and no formation of lower brominated congeners was observed in sterile control experiments. To our knowledge, this is the first report demonstrating microbially mediated reductive debromination of BDE-209 under anaerobic conditions.

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The complexing agents benzotriazole (BT) and tolyltriazole (TT) are not only widely applied as anticorrosives, e.g., in aircraft deicer and anti-icer fluid (ADAF), but they are also used for so-called silver protection in dishwasher detergents. Due to their low biodegradability and limited sorption tendency, BT and TT are only partly removed in wastewater treatment. Residual concentrations of BT and TT were determined in ambient surface waters in Switzerland including 7 rivers which have distinct water flows and receive treated wastewater effluents at various dilution ratios. A maximum BT concentration of 6.3 microg/L was found in the Glatt River, and a maximum mass flow of 277 kg BT per week was observed in the Rhine River. In most cases, TT was about a factor 5-10 less abundant. During winter 2003/4, BT mass flows at 2 locations in the lower stretch of the Glatt River clearly indicated the input from nearby Zurich airport, where BT was applied as an anticorrosive ADAF component. BT concentrations measured in the three lakes Greifensee, Lake Zurich, and Lake Geneva were approximately 1.2, 0.1-0.4, and 0.2 microg/L, respectively. The observed environmental occurrences indicate that BT and TT are ubiquitous contaminants in the aquatic environment and that they belong to the most abundant individual water pollutants.

The main removal process for polar organic micropollutants during activated sludge treatment is biotransformation, which often leads to the formation of stable transformation products (TPs). Because the analysis of TPs is challenging, the use of pathway prediction systems can help by generating a list of suspected TPs. To complete and refine pathway prediction, comprehensive biotransformation studies for compounds exhibiting pertinent functional groups under environmentally relevant conditions are needed. Because many polar organic micropollutants present in wastewater contain one or several amine functional groups, we systematically explored amine biotransformation by conducting experiments with 19 compounds that contained 25 structurally diverse primary, secondary, and tertiary amine moieties. The identification of 144 TP candidates and the structure elucidation of 101 of these resulted in a comprehensive view on initial amine biotransformation reactions. The reactions with the highest relevance were N-oxidation, N-dealkylation, N-acetylation, and N-succinylation. Whereas many of the observed reactions were similar to those known for the mammalian metabolism of amine-containing xenobiotics, some N-acylation reactions were not previously described. In general, different reactions at the amine functional group occurred in parallel. Finally, recommendations on how these findings can be implemented to improve microbial pathway prediction of amine-containing micropollutants are given.

A wide variety of antibacterial compounds is rapidly oxidized by 03 and hydroxyl radical (*OH) during aqueous ozonation. Quantitative microbiological assays have been developed here or adapted from existing methods and utilized to measure the resulting changes in antibacterial potencies during O3 and *OH treatment of 13 antibacterial molecules (roxithromycin, azithromycin, tylosin, ciprofloxacin, enrofloxacin, penicillin G, cephalexin, sulfamethoxazole, trimethoprim, lincomycin,tetracycline, vancomycin, and amikacin) from 9 structural classes (macrolides, fluoroquinolones, beta-lactams, sulfonamides, dihydrofolate reductase inhibitors, lincosamides, tetracyclines, glycopeptides, and aminoglycosides), as well as the biocide triclosan. Potency measurements were determined from dose-response relationships obtained by exposing Escherichia coli or Bacillus subtilis reference strains to treated samples of each antibacterial compound via broth micro- or macrodilution assays and related to the measured residual concentrations of parent antibacterial in each sample. Data obtained from these experiments show that O3 and *OH reactions lead in nearly all cases to stoichiometric elimination of antibacterial activity (i.e., loss of 1 mole equivalent of potency per mole of parent compound consumed). The beta-lactams penicillin G (PG) and cephalexin (CP) represent the only clear exceptions, as bioassay measurements indicate that biologically active products may be formed in the reactions of these two compounds with both O3 and *OH. The active product(s) generated in the direct reaction of O3 with PG appear(s) to be recalcitrant to further transformation by O3, though any biologically active products formed in the reactions of CP with O3, or of either PG or CP with *OH, are apparently deactivated by further reactions with O3 or *OH, respectively. Thus, with few exceptions, it can be expected that municipal wastewater ozonation will generally yield sufficient structural modification of antibacterial molecules to eliminate their antibacterial activities, whether oxidation results from selective reactions with O3 or from relatively nonselective reactions with incidentally produced OH.