Bioaugmentation of aerobic microbial granules with Pseudomonas putida carrying TOL plasmid

This paper describes results of a successful bioaugmentation experiment on aerobic granular sludge using Pseudomonas putida KT2442 cells bearing the TOL (pWWO) plasmid. The methodology was designed to monitor incorporation of the added donor cells into pre-existent microbial granules and the subsequent plasmid transfer to the autochthonous microbial community using shake flask microcosms. Expression of reporter proteins (GFP and DsRed) allowed in situ monitoring of donor cell attachment and plasmid transfer to the recipient cells using confocal laser scanning microscopy. Concomitant with donor integration and transconjugant proliferation in the granules, a significant increase in degradation of benzyl alcohol (used as sole substrate) was observed in the augmented microcosms. In contrast, control microcosms (with non-augmented granules) did not show any noticeable increase in the degradation of the substrate. This study shows that bioaugmentation of aerobic granular sludge via donor colonization and plasmid transfer is feasible for enhanced biodegradation.     

In situ assay with the midge Kiefferulus calligaster for contamination evaluation in aquatic agro-systems in central Thailand

The aims of this study were to verify the suitability of in situ tests using the tropical midge Kiefferulus calligaster and to evaluate the most sensitive endpoint for the assessment of aquatic pesticide contamination. In situ tests were carried out in freshwater drainage channels (farm channels) that supply vegetable crops and receive considerable pesticide spray drift, and at channels outside farms (main channels). Moreover a pesticide-free farm was used as reference site. The endpoints analysed were: survival of the larvae, body length increment, capsule width increment, cholinesterase activity and glutathione S-transferase activity. Seasonal change was investigated as rainy season and dry season. Deleterious effects were observed at some farms especially during the rainy season when farmers apply heavier doses of pesticides. However, high mortality rates observed in main channels suggest that these water bodies are also affected by other impacts besides pesticide use. This work shows the potential of the in situ assay with K. calligaster as a tool for the environmental quality assessment of tropical aquatic ecosystems.     

Removal and degradation of aromatic compounds from a highly polluted site by coupling soil washing with photocatalysis

The possible application of two environmental remediation technologies - soil washing and photocatalysis - to remove and decompose various aromatic pollutants present in excavated soils of a contaminated industrial site has been investigated. Aqueous solutions containing the non-ionic surfactant Brij 35 were used to extract the contaminants from the soil samples. The photocatalytic treatment of the obtained washing wastes, performed in the presence of TiO(2) suspensions irradiated with simulated sunlight, showed a slow abatement of the toxic compounds due to the relevant concentrations of organics in the waste. A neat improvement of the process performances, obtained by operating in the presence of added potassium peroxydisulfate, suggests a feasible treatment route.     

The effect of nitroaromatics' composition on their toxicity in vivo: novel, efficient non-additive 1D QSAR analysis

Novel 1D QSAR approach that allows analysis of non-additive effects of molecular fragments on toxicity has been proposed. Twenty-eight nitroaromatic compounds including some well-known explosives have been chosen for this study. The 50% lethal dose concentration for rats (LD50) was used as the estimation of toxicity in vivo to develop 1D QSAR models on the framework of Simplex representation of molecular structure. The results of 1D QSAR analysis show that even the information about the composition of molecules provides the main trends of toxicity changes. The necessity of consideration of substituents' mutual impacts for the development of adequate QSAR models of nitroaromatics' toxicity was demonstrated. Statistic characteristics for all the developed partial least squares QSAR models, except the additive ones are quite satisfactory (R2=0.81-0.92; Q2=0.64-0.83; R2 test=0.84-0.87). A successful performance of such models is due to their non-additivity i.e. possibility of taking into account the mutual influence of substituents in benzene ring which plays the governing role for toxicity change and could be mediated through the different C-H fragments of the ring. The correspondence between observed and predicted by these models toxicity values is good. This allowing combine advantages of such approaches and develop adequate consensus model that can be used as a toxicity virtual screening tool.     

Estimating naphthenic acids concentrations in laboratory-exposed fish and in fish from the wild

Naphthenic acids (NAs) are the most water-soluble organic components found in the Athabasca oil sands in Alberta, Canada, and these acids are released into aqueous tailing waters as a result of bitumen extraction. Although the toxicity of NAs to fish is well known, there has been no method available to estimate NAs concentrations in fish. This paper describes a newly developed analytical method using single ion monitoring gas chromatography-mass spectrometry (GC-MS) to measure NAs in fish, down to concentrations of approximately 0.1mgkg(-1) of fish flesh. This method was used to measure the uptake and depuration of commercial NAs in laboratory experiments. Exposure of rainbow trout (Oncorhynchus mykiss) to 3mg NAsl(-1) for 9d gave a bioconcentration factor of approximately 2 at pH 8.2. Within 1d after the fish were transferred to NAs-free water, about 95% of the NAs were depurated. In addition, the analytical method was used to determine if NAs were present in four species of wild fish - northern pike (Esox lucius), lake whitefish (Coregonus clupeaformis), white sucker (Catostomus commersoni), walleye (Sander vitreus) - collected from near the oil sands. Flesh samples from 23 wild fish were analyzed, and 18 of these had no detectable NAs. Four fish (one of each species) contained NAs at concentrations from 0.2 to 2.8mgkg(-1). The GC-MS results from one wild fish presented a unique problem. However, with additional work it was concluded that the NAs concentration in this fish was <0.1mgkg(-1).     

Discriminating the pH toxicity to Poecilia reticulata Peters, 1859 in the Dunas Lake (Camaçari, BA, Brazil)

Toxic potential of the pH reduction to fingerlings of Poecilia reticulata, through acute toxicity bioassays, as well as the influence of increased pH on the toxicity were assessed. Acid lake samples (Dunas Lake) were collected during 19 months, and assessed with following treatments: water at local pH (+/-3.0) and samples with modified pH to 3.5, 3.8, 4.0, 4.3, 4.6, 5.0, 5.5, 6.0, and 6.5. Culture water samples with pH reduced to 3.0 were also assessed. Newborn P. reticulata were exposed during 96h, and dead/immobile organisms were counted at various time intervals during exposure (short intervals in the beginning and long towards the end). Mean results of LT(50) and confidence intervals from the Dunas Lake and control water with reduced pH were 1.36 (+/-0.48)h, and 1.03 (+/-0.50)h, respectively, with no statistical difference. Samples with increased pH showed a significant reduction in toxicity, with no toxicity detected at pH 6.0 and higher. Relationship between pH and lethal toxicity for fingerlings of P. reticulata demonstrated that pH exerted a strong effect on the survival of this species at the Dunas Lake, explaining about 80% of the toxicity observed.     

Biochemical effects of clomazone herbicide on piava (Leporinus obtusidens)

This study aims to verify the effects of the clomazone concentration used in rice fields on acetylcholinesterase (AChE), thiobarbituric acid reactive substances (TBARS), protein carbonyl and catalase activity in tissues of piava (Leporinus obtusidens). LC(50)-96h was 5.0 mg L(-1) and the fish were exposed to 1/10 of LC(50)-96 h: 0.5 mg L(-1) of clomazone for 96 and 192h. The same parameters were also assayed after a recovery period of 192 h in clean water. AChE activity was reduced only in the brain and heart of fish exposed for 96 h. AChE activity was decreased in the brain, muscle and heart tissues after 192 h of exposure. After 192 h of recovery period, AChE activity remained diminished in brain and muscle and showed a decrease in eye. However, after 192 h of recovery, AChE activity in heart was recovered. Fish showed increased TBARS levels in brain at all experimental periods. TBARS levels decreased in liver and muscle tissues after 192 h of exposure. The increase in muscle TBARS persisted in fish transferred to clean water. Protein carbonyl in the liver was increased in all periods studied including the recovery period. Catalase activity was reduced during all periods. The present study demonstrates the occurrence of disorders in AChE, TBARS, protein carbonyl and catalase activity in piava. The results also show changes in fish after exposure to an environmentally relevant concentration of clomazone. Most effects observed persisted after the recovery period. Thus, these parameters may be used to monitor clomazone toxicity in fish.     

Regio- and stereoselective isomerization of hexabromocyclododecanes (HBCDs): kinetics and mechanism of gamma- to alpha-HBCD isomerization

Hexabromocyclododecanes (HBCDs) are high production volume chemicals (>20000 ty(-1)) used as flame retardants for plastics and textiles. Lately, we reported on the stereoselective isomerization of beta-HBCDs. Herein we present insights into the mechanism and kinetics of (+)gamma- to (+)alpha- and of (-)gamma- to (-)alpha-HBCD isomerization. Only two of the six bromine atoms migrated, indicating that rearrangements of gamma- to alpha-HBCDs are regio- and stereoselective as well. The apparent first-order isomerization rate constants increased from 0.0013 to 0.0031 to 0.0070 min(-1) at 120, 130, and 140 degrees C, respectively, corresponding to half-lives of 540, 230, and 99 min. Thus, a thermal treatment of materials containing gamma-HBCDs at temperatures >100 degrees C may induce the formation of alpha-HBCDs and, hence, may alter the diastereomeric ratio of a HBCD mixture. The inversion of vicinal dibromides in like-configurations (RR/SS) prevailed, whereas unlike-configurations (RS/SR) were not affected. An intramolecular, stereoselective migration of neighboring bromine atoms via a four-center transition state would explain the observed stereoisomer pattern and first-order kinetics. Despite the fact that vicinal dibromides in HBCDs prefer synclinal (gauche) conformations, antiperiplanar (staggered) conformations are assumed to facilitate concerted 1.2-shifts of both bromine atoms. A conformation analysis revealed that under kinetic control, only those bromine atoms in the more flexible part of the molecules are migrating, whereas those in the conserved triple-turn motive were not affected. Thus, this structural motive, common to all alpha-, beta-, and gamma-HBCDs, is more rigid and less reactive than the flexible part, containing the reacting dibromides in like-configurations.     

Simplified contaminant source depletion models as analogs of multiphase simulators

Four simplified dense non-aqueous phase liquid (DNAPL) source depletion models recently introduced in the literature are evaluated for the prediction of long-term effects of source depletion under natural gradient flow. These models are simple in form (a power function equation is an example) but are shown here to serve as mathematical analogs to complex multiphase flow and transport simulators. The spill and subsequent dissolution of DNAPLs was simulated in domains having different hydrologic characteristics (variance of the log conductivity field=0.2, 1 and 3) using the multiphase flow and transport simulator UTCHEM. The dissolution profiles were fitted using four analytical models: the equilibrium streamtube model (ESM), the advection dispersion model (ADM), the power law model (PLM) and the Damkohler number model (DaM). All four models, though very different in their conceptualization, include two basic parameters that describe the mean DNAPL mass and the joint variability in the velocity and DNAPL distributions. The variability parameter was observed to be strongly correlated with the variance of the log conductivity field in the ESM and ADM but weakly correlated in the PLM and DaM. The DaM also includes a third parameter that describes the effect of rate-limited dissolution, but here this parameter was held constant as the numerical simulations were found to be insensitive to local-scale mass transfer. All four models were able to emulate the characteristics of the dissolution profiles generated from the complex numerical simulator, but the one-parameter PLM fits were the poorest, especially for the low heterogeneity case.     

A strategy for aromatic hydrocarbon bioremediation under anaerobic conditions and the impacts of ethanol: a microcosm study

Increased use of ethanol-blended gasoline (gasohol) and its potential release into the subsurface have spurred interest in studying the biodegradation of and interactions between ethanol and gasoline components such as benzene, toluene, ethylbenzene and xylene isomers (BTEX) in groundwater plumes. The preferred substrate status and the high biological oxygen demand (BOD) posed by ethanol and its biodegradation products suggests that anaerobic electron acceptors (EAs) will be required to support in situ bioremediation of BTEX. To develop a strategy for aromatic hydrocarbon bioremediation and to understand the impacts of ethanol on BTEX biodegradation under strictly anaerobic conditions, a microcosm experiment was conducted using pristine aquifer sand and groundwater obtained from Canadian Forces Base Borden, Canada. The initial electron accepter pool included nitrate, sulfate and/or ferric iron. The microcosms typically contained 400 g of sediment, 600 approximately 800 ml of groundwater, and with differing EAs added, and were run under anaerobic conditions. Ethanol was added to some at concentrations of 500 and 5000 mg/L. Trends for biodegradation of aromatic hydrocarbons for the Borden aquifer material were first developed in the absence of ethanol, The results showed that indigenous microorganisms could degrade all aromatic hydrocarbons (BTEX and trimethylbenzene isomers-TMB) under nitrate- and ferric iron-combined conditions, but not under sulfate-reducing conditions. Toluene, ethylbenzene and m/p-xylene were biodegraded under denitrifying conditions. However, the persistence of benzene indicated that enhancing denitrification alone was insufficient. Both benzene and o-xylene biodegraded significantly under iron-reducing conditions, but only after denitrification had removed other aromatics. For the trimethylbenzene isomers, 1,3,5-TMB biodegradation was found under denitrifying and then iron-reducing conditions. Biodegradation of 1,2,3-TMB or 1,2,4-TMB was slower under iron-reducing conditions. This study suggests that addition of excess ferric iron combined with limited nitrate has promise for in situ bioremediation of BTEX and TMB in the Borden aquifer and possibly for other sites contaminated by hydrocarbons. This study is the first to report 1,2,3-TMB biodegradation under strictly anaerobic condition. With the addition of 500 mg/L ethanol but without EA addition, ethanol and its main intermediate, acetate, were quickly biodegraded within 41 d with methane as a major product. Ethanol initially present at 5000 mg/L without EA addition declined slowly with the persistence of unidentified volatile fatty acids, likely propionate and butyrate, but less methane. In contrast, all ethanol disappeared with repeated additions of either nitrate or ferric iron, but acetate and unidentified intermediates persisted under iron-enhanced conditions. With the addition of 500 mg/L ethanol and nitrate, only minor toluene biodegradation was observed under denitrifying conditions and only after ethanol and acetate were utilized. The higher ethanol concentration (5000 mg/L) essentially shut down BTEX biodegradation likely due to high EA demand provided by ethanol and its intermediates. The negative findings for anaerobic BTEX biodegradation in the presence of ethanol and/or its biodegradation products are in contrast to recent research reported by Da Silva et al. [Da Silva, M.L.B., Ruiz-Aguilar, G.M.L., Alvarez, P.J.J., 2005. Enhanced anaerobic biodegradation of BTEX-ethanol mixtures in aquifer columns amended with sulfate, chelated ferric iron or nitrate. Biodegradation. 16, 105-114]. Our results suggest that the apparent conservation of high residual labile carbon as biodegradation products such as acetate makes natural attenuation of aromatics less effective, and makes subsequent addition of EAs to promote in situ BTEX biodegradation problematic.