Contaminated environments in the subsurface and bioremediation: organic contaminants.

Due to leakages, spills, improper disposal and accidents during transport, organic compounds have become subsurface contaminants that threaten important drinking water resources. One strategy to remediate such polluted subsurface environments is to make use of the degradative capacity of bacteria. It is often sufficient to supply the subsurface with nutrients such as nitrogen and phosphorus, and aerobic treatments are still dominating. However, anaerobic processes have advantages such as low biomass production and good electron acceptor availability, and they are sometimes the only possible solution. This review will focus on three important groups of environmental organic contaminants: hydrocarbons, chlorinated and nitroaromatic compounds. Whereas hydrocarbons are oxidized and completely mineralized under anaerobic conditions in the presence of electron acceptors such as nitrate, iron, sulfate and carbon dioxide, chlorinated and nitroaromatic compounds are reductively transformed. For the aerobic often persistent polychlorinated compounds, reductive dechlorination leads to harmless products or to compounds that are aerobically degradable. The nitroaromatic compounds are first reductively transformed to the corresponding amines and can subsequently be bound to the humic fraction in an aerobic process. Such new findings and developments give hope that in the near future contaminated aquifers can efficiently be remediated, a prerequisite for a sustainable use of the precious-subsurface drinking water resources.

Determination of phenylurea herbicides in natural waters at concentrations below 1 ng l(-1) using solid-phase extraction, derivatization, and solid-phase microextraction-gas chromatography-mass spectrometry.

A procedure is presented which allows the ultratrace level determination of phenylurea herbicides (PUHs) in natural waters. Samples were enriched by solid-phase extraction (SPE) on Carbopack B and alkylated with iodoethane and sodium hydride to yield thermostable products. After derivatization, the aqueous samples were extracted and injected by SPME. The use of iodoethane instead of iodomethane allowed the differentiation between parent compounds and the N-demethylated metabolites. Limits of detection were between 0.3 and 1.0 ng/l for the parent compounds. Standard deviations below 10% were achieved for samples containing more than 4 ng/l in very different matrices including Nanopure water, lake water, and waste water treatment plant (WWTP) effluent. Moreover, the para-hydroxylated metabolite of diuron could be quantified with the same procedure. The presence of further metabolites was assessed qualitatively. Chromatography was stable over a large number of measurements even with dirty samples from WWTP effluent. The precision and sensitivity of the developed analytical method allowed the investigation of the fate of PUHs in lakes, their degradation during drinking water treatment and their transport within the North Sea.

Influence of solution composition and column aging on the reduction of nitroaromatic compounds by zero-valent iron.

Granular iron is used in reactive permeable barriers for the reductive treatment of organic and inorganic groundwater contaminants. The technology is well established, however, its long-term performance and the importance of the groundwater composition are not yet well understood. Here, the influence of chloride, nitrate, silicate, and Aldrich humic acid on the reactivity of Master Builder iron was studied under anoxic conditions using small packed columns and 2-nitrotoluene (2-NT) as a model contaminant. After initially complete reduction of 2-NT to 2-aminotoluene (2-AT) in the column, possibly under mass-transfer controlled conditions, the reactivity of the iron was found to decrease substantially. In the presence of chloride, this decrease was slowed while exposure to silicate resulted in a very quick loss of iron reactivity. Nitrate was found to interfere strongly with the effect of chloride. These observations are interpreted in terms of corrosion inhibition/promotion and competition. Our results suggest that reactive barrier performance may be strongly affected by the composition of the treated groundwater.

Rapid determination of the molecular weight distribution of total cellular fatty acids using chemical ionization mass spectrometry.

A new method for the qualitative and quantitative determination of the molecular weight distribution of total cellular fatty acids is described. The method includes a simple extraction-saponification followed by chemical ionization-mass spectrometric analysis of the saponifiable matter. This technique requires small quantities of cell material which, combined with the rapidity and precision of the analysis, makes it attractive to the biologist interested in changes in the fatty acid composition of growing cells. As an example, an application of this method to the fatty acid determination of marine diatoms at different growth stages is presented.

Linear free energy relationships used to evaluate equilibrium partitioning of organic compounds.

In environmental chemistry, one often wants to interpret or predict equilibrium partitioning of organic compounds between any two phases. Hence, one needs to understand the partition variability that stems from using different types of compounds and the variability that arises from looking at different natural phases, e.g. different soil organic matter. It is current practice in environmental chemistry to evaluate equilibrium partitioning with the help of double logarithmic correlations between the unknown partition constant and a well-known partition constant of the compounds, e.g., partitioning between natural organic matter and water or air is correlated with the octanol/water or octanol/air partition constant, respectively. However, these relationships (in the following called one-parameter LFERs) can only predict the compound variability within a single substance class. They supply no means to understand the variability between substance classes or the variability between different natural organic phases. The reasons for these limitations are that (a) the complete compound variability cannot be described by a single parameter because partitioning results from different kinds of interactions that vary independently from each other and (b) the specific properties of the studied phase are represented in the slope and intercept of the double logarithmic correlation and not in a variable parameter. In contrastto one-parameter LFERs, polyparameter LFERs are based on a concept that considers all interactions involved in partitioning by separate parameters. They allow for predicting the complete compound variability by a single equation, and they also provide the possibility to evaluate and predict the variability in the sorption characteristics of different natural phases. Thus future research in the field of environmental partition processes should focus on adapting and improving the more comprehensive polyparameter LFERs rather than trying to refine existing one-parameter LFERs.

Analysis of serum bile acids by capillary gas--liquid chromatography--mass spectrometry.

A method for quantitative analysis of serum bile acids by capillary gas--liquid chromatography--mass spectrometry is described. The main features of this method are a Grob-type barium carbonate/polyethyleneglycol 20,000 glass capillary column, an all-glass capillary interface, use of the lipophilic anion exchanger DEAP-Sephadex-LH-20 for purification of the serum extract, and chenodeoxycholic-11,12-d2 acid as internal standard. Linearity of the response (ratio of intensities of the fragment ions diagnostic for the bile acid to be measured and for the internal standard) was demonstrated for four different bile acids. The method is sufficiently sensitive for measurement of bile acids in serum of healthy humans.

Rapid microbial mineralization of toluene and 1,3-dimethylbenzene in the absence of molecular oxygen.

Up to 0.4 mM 1,3-dimethylbenzene (m-xylene) was rapidly mineralized in a laboratory aquifer column operated in the absence of molecular oxygen with nitrate as an electron acceptor. Under continuous flow conditions, the degradation rate constant (pseudo-first order) was >0.45 h. Based on a carbon mass balance with [ring-C]m-xylene and a calculation of the electron balance, m-xylene was shown to be quantitatively (80%) oxidized to CO(2) with a concomitant reduction of nitrate. The mineralization of m-xylene in the column also took place after reducing the redox potential, E', of the inflowing medium with sulfide to <-0.11 V. Microorganisms adapted to growth on m-xylene were also able to degrade toluene under denitrifying conditions. These results suggest that aromatic hydrocarbons present in anoxic environments such as lake sediments, sludge digestors, and groundwater infiltration zones from landfills and polluted rivers are not necessarily persistent but may be mineralized in the absence of molecular oxygen.

pH dependence of the partitioning of triphenyltin and tributyltin between phosphatidylcholine liposomes and water.

Triorganotin compounds are very toxic contaminants. The site of their basic mechanism of action of acute toxicity is the biomembrane. Liposome-water distribution ratios of triphenyltin and tributyltin were determined between pH 3 and pH 8 with the equilibrium dialysis method in the micromolar concentration range, which is the concentration range where acute toxicity is observed. In addition, biomembrane-water distribution ratios of tributyltin were determined with chromatophores of Rhodobacter sphaeroides that contain approximately 70% protein intercalated in the lipid bilayer. The liposome-water distribution of both compounds showed only weak pH dependence. For tributyltin, the apparent distribution ratio decreased from 4100 at low pH to 2000 at high pH, while this ratio decreased from 70 000 to 22 000 for TPT. The distribution ratio of the triorganotin cation exceeded that of the neutral hydroxo complex by a factor of 2. The distribution ratio of both the cation and the hydroxo complex of triphenyltin exceeded that of tributyltin by a factor of 10. It is postulated that the sorption of the cation is governed by complex formation with ligands in the phospholipids, presumably the phosphate group. The biomembrane-water distribution ratio of tributyltin was found to be lower than the liposome-water distribution ratio at high pH. The hydroxo complex appears to partition only to the lipid fraction of the biomembrane. Yet, at low pH the biomembrane-water distribution ratio exceeded the liposome-water distribution ratio, which is attributed to complex formation of the cationic species with ligands of the protein fraction.

Interaction of phenolic uncouplers in binary mixtures: concentration-additive and synergistic effects.

The uncoupling activities of 14 binary mixtures of substituted phenols and of 4 binary mixtures of phenols and anisols were investigated at different pH values. Experiments were performed with time-resolved spectroscopy on membrane vesicles (chromatophores) of the photosynthetic bacteria Rhodobacter sphaeroides. Phenols are known to destroy the electrochemical proton gradient in energy-transducing membranes by a protonophoric mechanism. Anisols do not have protonophoric activity but disturb membrane structure and functioning as a nonspecific baseline toxicant. It was postulated in the literature that, for certain substituted phenols, the formation of a dimer between the phenoxide and the neutral phenol may contribute significantly to the overall protonophoric activity. In 13 of 14 mixtures of substituted phenols but in none of the mixtures of phenols with anisols, such a dimer appears to be formed between two different mixture partners. An extended shuttle mechanism of uncoupling, which includes a term for the contribution of such a mixed dimer, provided a good description of all experimental data. Opposite speciation favors interaction and ortho substituents abate interaction, which adds evidence for the dimerformation via a hydrogen bond between the phenol-OH and the phenoxide. These findings are significant not only regarding the mechanism of protonophoric action but also for the risk assessment process of chemical mixtures in the environment. When assessing the effect of mixtures, concentration addition is regarded as a reference X concept to estimate effects of similarly acting compounds. The substituted phenols in this work act according to the same action mechanism of uncoupling. Nevertheless, the overall effect of four of the investigated mixtures, which exhibit stronger dimer formation as compared to the single compounds or for which the resulting dimer is intrinsically more active, exceeded the effect calculated according to concentration addition considerably. In future work, this synergistic effect observed in-vitro has to be validated in-vivo to deduce its implications for the risk assessment process.

Oxidation of dimethyl sulfide to dimethyl sulfoxide by phototrophic purple bacteria.

Enrichment cultures of phototrophic purple bacteria rapidly oxidized up to 10 mM dimethyl sulfide (DMS) to dimethyl sulfoxide (DMSO). DMSO was qualitatively identified by proton nuclear magnetic resonance. By using a biological assay, DMSO was always quantitatively recovered from the culture media. DMS oxidation was not detected in cultures incubated in the dark, and it was slow in cultures exposed to full daylight. Under optimal conditions, the second-order rate constant for DMS oxidation was 6 day mg of protein ml. The rate constant was reduced in the presence of high concentration of sulfide (>1 mM), but was not affected by the addition of acetate. DMS was also oxidized to DMSO by a pure strain (tentatively identified as a Thiocystis sp.) isolated from the enrichment cultures. DMS supported growth of the enrichment cultures and of the pure strain by serving as an electron source for photosynthesis. A determination of the amount of protein produced in the cultures and an estimation of the electron balance suggested that the two electrons liberated during the oxidation of DMS to DMSO were quantitatively used to reduce carbon dioxide to biomass. The oxidation of DMS by phototrophic purple bacteria may be an important source of DMSO detected in anaerobic ponds and marshes.

Anaerobic degradation of alkylated benzenes in denitrifying laboratory aquifer columns.

Toluene and m-xylene were rapidly mineralized in an anaerobic laboratory aquifer column operated under continuous-flow conditions with nitrate as an electron acceptor. The oxidation of toluene and m-xylene was coupled with the reduction of nitrate, and mineralization was confirmed by trapping 14CO2 evolved from 14C-ring-labeled substrates. Substrate degradation also took place when nitrous oxide replaced nitrate as an electron acceptor, but decomposition was inhibited in the presence of molecular oxygen or after the substitution of nitrate by nitrite. The m-xylene-adapted microorganisms in the aquifer column degraded toluene, benzaldehyde, benzoate, m-toluylaldehyde, m-toluate, m-cresol, p-cresol, and p-hydroxybenzoate but were unable to metabolize benzene, naphthalene, methylcyclohexane, and 1,3-dimethylcyclohexane. Isotope-dilution experiments suggested benzoate as an intermediate formed during anaerobic toluene metabolism. The finding that the highly water-soluble nitrous oxide served as electron acceptor for the anaerobic mineralization of some aromatic hydrocarbons may offer attractive options for the in situ restoration of polluted aquifers.

Isolation and characterization of a bacterium that mineralizes toluene in the absence of molecular oxygen.

A bacterium tentatively identified as a Pseudomonas sp. was isolated from a laboratory aquifer column in which toluene was degraded under denitrifying conditions. The organism mineralized toluene in pure culture in the absence of molecular oxygen. In carbon balance studies using [ring-UL-14C]toluene, more than 50% of the radioactivity was recovered as 14CO2. Nitrate and nitrous oxide served as electron acceptors for toluene mineralization. The organism was also able to degrade m-xylene, benzoate, benzaldehyde, p-cresol, p-hydroxy-benzaldehyde, p-hydroxybenzoate and cyclohexanecarboxylic acid in the absence of molecular oxygen.

Reduction of nitroaromatic compounds mediated by Streptomyces sp. exudates.

Exudates from Streptomyces griseoflavus Tü 2484 effectively mediated electron transfer between hydrogen sulfide and various nitrobenzenes. In general, pseudo-first-order kinetics were observed, except for the initial phase of the reaction at higher pH values. Under fixed pH and Dh conditions, linear free energy relationships were found between the logarithms of the reaction rate constants and the one-electron reduction potentials of the nitroaromatic compounds. No competition was observed between various compounds. Comparison of the results of this study with the results of experiments conducted with model quinones and an iron porphyrin suggest that the secondary metabolites cinnaquinone and dicinnaquinone, excreted by strain Tü 2484 on the order of 100 mg/liter, are responsible for the catalytic activity of the exudate. Further support for this hypothesis comes from the facts that the catalytic activity of the exudate became prominent only after the growth phase of the microorganisms and that the mediating substances have a molecular weight of less than 3,000.

Quantification of dissolved natural organic matter (DOM) mediated phototransformation of phenylurea herbicides in lakes.

For many important classes of pesticides including phenylurea herbicides (PUHs) and triazines, photosensitized transformation may be the only relevant elimination process in surface waters. In this study, the dissolved organic matter (DOM) mediated phototransformation of PUHs has been investigated in laboratory and field experiments. The results indicate that, in surface waters, the photosensitized transformation of PUHs may be significant and occurs primarily by an initial one-electron oxidation most likely involving excited triplet states of DOM (3DOM*) constituents. Using isoproturon and diuron as model compounds, it is shown that for a given DOM, quantum yield factors determined in the laboratory at a few selected wavelengths can be used to quantify the overall DOM- mediated phototransformation of a given PUH under sunlight irradiation. Furthermore, it is demonstrated that this process can be modeled for a given surface water, by applying the program GCSOLAR and a simple algorithm for cloud cover for quantification of average daily light intensities. Finally, the model has been successfully applied to predict vertical concentration profiles of isoproturon and diuron in a small lake in Switzerland. To our knowledge, this is the first study in which DOM-mediated phototransformation of organic pollutants has been quantitatively validated in the field.

Phenyl- and butyltin analysis in small biological samples by cold methanolic digestion and GC/MS.

A very efficient technique for the analysis of six butyl- and phenyltin compounds in biota samples has been developed. No special equipment is needed for sample preparation, which is based on cold methanolic digestion with subsequent aqueous ethylation and liquid-liquid extraction. For samples of only 40 mg of biological materials, method detection limits ranging from 4 to 52 ng/g were achieved using gas chromatography/mass spectrometry. Relative recoveries for the individual butyl- and phenyltins, referring to perdeuterated organotin analogues as internal standards, ranged from 96 to 107%. Organotin concentrations in insect larvae (Chironomus riparius) and a reference mussel tissue (CRM 477) were determined with excellent precision (RSD <5%), and the measured butyltins in CRM 477 were in good agreement with the certified values. Comparison with accelerated solvent extraction confirmed high accuracy, and application for a bioconcentration experiment with phenyltins demonstrated the robustness and suitability of the method for routine analyses. The procedure allows fast, reliable, and simple determination of organotin compounds in low-size biological samples, which was demonstrated for bioconcentration experiments.

Abiotic reduction of 4-chloronitrobenzene to 4-chloroaniline in a dissimilatory iron-reducing enrichment culture.

4-chloronitrobenzene (4-Cl-NB) was rapidly reduced to 4-chloroaniline with half-lives of minutes in a dissimilatory Fe(III)-reducing enrichment culture. The initial pseudo-first-order rate constants at 25 degrees C ranged from 0.11 to 0.19 per minute. The linear Arrhenius correlation in a temperature range of 6 to 85 degrees C and the unchanged reactivity after pasteurization indicated that the nitroreduction occurred abiotically. A fine-grained black solid which was identified as poorly crystalline magnetite (Fe(3)O(4)) by X-ray diffraction accumulated in the enrichments. Magnetite produced by the Fe(III)-reducing bacterium Geobacter metallireducens GS-15 and synthetic magnetite also reduced 4-Cl-NB. These results suggest that the reduction of 4-Cl-NB by the enrichment material was a surface-mediated reaction by dissimilatory formed Fe(II) associated with magnetite.

Sorption and desorption behavior of organotin compounds in sediment-pore water systems.

Sediments contaminated with organotin compounds (OTs), in particular triorganotins (TOTs), are abundant in areas with high shipping activities. To assess the possible remobilization of these highly toxic compounds from such sediments, a profound understanding of their sorption/desorption behavior is necessary. In this work the extent and reversibility of sorption of OTs to sediments has been investigated using contaminated freshwater harbor sediments and two certified OT containing marine sediments. Experiments conducted with perdeuterated OTs showed that sorption of OTs to sediments is a fast and reversible process involving primarily particulate organic matter (POM) constituents as sorbents. The organic carbon-normalized sediment-water distribution ratios (DOC, expressed in L/kgOC) determined in the laboratory were consistent with in-situ DOCs obtained from OT concentrations measured in sediment and pore water samples from two dated sediment cores. For both butyl- and phenyltin compounds the log DOC values were in the range of 4.7-6.1, and the following sequence was observed: DOC (tri-OT) > or = DOC (di-OT) > or = DOC (mono-OT). However, the differences were much less pronounced than would have been expected for hydrophobic partitioning of the corresponding compounds into POM. These results support our hypothesis from earlier work with dissolved humic acids that OT sorption to sediments occurs primarily by reversible formation of (innerspere) complexes between the tin atom and carboxylate and phenolate ligands present in POM. Because of the high DOC values (i.e. log DOC > or = 4) the diffusion of OTs from deeper sediments to the surface will be rather slow, and thus a major release from undisturbed sediments is not expected. However, because OTs readily desorb, any resuspension of contaminated sediments (e.g., by the tide, storms or dredging activities) will lead to enhanced OT concentrations in the overlaying water column. Furthermore, in contrastto polycyclic aromatic hydrocarbons (PAH) where large fractions may be tightly bound (in)to soot or other carbonaceous materials, OTs will be more readily bioavailable due to the fast and reversible sorption/desorption behavior.

Spherical clay conglomerates: a novel stationary phase for solid-phase extraction and "reversed-phase" liquid chromatography.

A new solid phase is presented to be used for the solid-phase extraction (SPE) of organic compounds from aqueous solutions and as a stationary phase for the separation of organic compounds in "reversed-phase" HPLC. The material consists of spherical clay conglomerates (SCCs) in the size ranges of 2-5, 5-10, and 10-20 µm. SCCs are especially well suited for the extraction and separation of aromatic compounds with electron-withdrawing substituents, because of the formation of specific electron donor-acceptor (EDA) complexes of such compounds with natural clay minerals. A series of nitroaromatic compounds (NACs), e.g., nitrophenols, and nitrotoluenes, served as probe substances for the characterization of the SPE with SCCs online coupled to a C18-HPLC-DAD system. Breakthrough volumes were > 1 L and method detection limits (MDLs) < 100 ng/L for compounds with moderate to high affinity towards clay minerals. The performance of the material is hardly affected by matrix effects and because of its excellent physical properties, i.e., regenerability and pressure-resistance, it meets the requirements for fully automated routine trace analysis of several primary pollutants, such as 6-methyl-2,4-dinitrophenol (DNOC) or 2,4,6-trinitrotoluene (TNT), in various natural waters. Offline SPE with SCCs was superior or equivalent to commercial SPE products for analysis of such compounds. Finally, SCCs are shown to be well suited as a stationary phase in reversed-phase HPLC. This opens a wide range of applications, e.g., as an easy and fast separation technique that is orthogonal to C18 reversed-phase HPLC.